Page 1


Piero Musiani and Guido Forni



Contents Foreword About the Authors 0 INTRODUCTION. Why we do have an immune system? 1 Immunity at the surface 2 Sentinel cells 3 Cells of innate immunity 4 Cytokines 5 The Major Histocompatibility Complex (MHC) 6 Peptide presentation by HLA glycoproteins 7 T cells 8 The thymic education of T cells 9 Activation of virgin T cells 10 T killer T cells 11 T helper cells 12 B cells 13 B Cell Receptor and antibodies 14 Generation of B and T Cell Receptor repertoires 15 Binding site - antigen interaction 16 The antibodies 17 Activation of B cells 18 Secondary lymphoid organs 19 Direct and indirect antibody activities 20 The Complement system 21 Monoclonal antibodies 22 Immune tolerance and controls of the immune response 23 Autoimmunity 24 Immune memory 25 Vaccines 26 Immunodeficiencies

Page 3 4 5 7 17 24 45 56 64 73 83 91 101 106 117 126 132 142 147 158 176 183 191 195 201 211 215 223 229



This book intends providing an introduction to immunology for undergraduate and graduate students. As we were engaged for a few years in teaching in a Master’s Degree the Medical University of Huè, Viet Nam, we (Piero Musiani and Guido Forni) debated at length what should be emphasized and, on the contrary, what could be left to an eventual personal investigation in a basic course of Immunology. Our first agreement was that the main emphasis should be on immunological strategies, while most of the molecular mechanisms could be left to student exploration. Molecular biology is now so pervasive that everyone easily swims in the sea of molecular mechanisms. Updated information on molecular pathways of the immune system can be obtained on internet without difficulty. By contrast, the peculiar logic of immune reactions is sometimes not obvious and not easy to grasp. A second shared view was that a schematic drawing could be more informative than a long text. Therefore this book is mainly based on text illustrations, while only an explanatory comment is reported in legends. Finally, we wish to thank exquisite Mrs. Jehanne Marchesi for the numerous corrections and suggestions.

A 2018 printed version of this book is available directly from Piccin Nuova Libraria, Padua, Italy ( or through Amazon, also in a Kindle version.


About the Authors

Piero Musiani, MD (Right in the picture) was a semi-professional basketball player, but then he became a Surgical Pathologist working first at the Catholic University, Rome and later at the University of Chieti, Italy. Initially his main experimental interest was on the thymus gland and secretory immunoglobulins. At the University of Chieti he played a pivotal role in the foundation of the Excellence Center on Aging (CeSI). The sophisticated equipment acquired by the CeSI allowed Piero’s team to combine a state of art pathological analysis with experimental data. On many occasions these sophisticated analysis led to a fresh interpretation of new immunological findings. In the 2014 Piero died in a motorbike accident while he was drawing the illustrations of this book. Guido Forni, MD (Left in the picture) is a National Member of the Accademia dei Lincei, Rome. From 1970 to 2011 he was Professor of Immunology at the University of Torino Italy. He has experience in basic and translational cancer immunotherapy, and has a considerable expertise in transgenic mouse models of cancer and DNA vaccination. His work at the University of Torino, Italy on the role of cytokines in the immune recognition and vaccines to prevent tumors is recognized world-wide. His laboratory devoted considerable effort towards acquiring new knowledge concerning the direct role of 11vaccine-induced antibodies on tumor growth, their ability to trigger cell mediated reactions and activate complement cascade, and to trigger a cell mediated immune memory to tumor antigens.


0. INTRODUCTION. Why we do have an immune system?

Fig. 0.1. WHY WE DO HAVE AN IMMUNE SYSTEM? The continuous pressure constituted by the invasion of microbes forced the mammalians to develop a sequence of defense mechanisms that progressively evolved as the second more complex system of our body. Never lasting interactions between defense mechanisms and microbes around us play a key role in controlling the pathogenicity of the microbes, i.e. their ability to spread and cause a disease. Mutualistic, commensal, pathogenic opportunistic and truly pathogenic microbes may become capable of causing a disease as soon as a body barrier breach or a jam of an immune mechanism takes place.


Fig. 0.2. A SYSTEM OF INTEGRATED STRATEGIES. The cells of the immune system communicate among them to detect invaders and trigger a complex defense reaction. An immune cell perceiving an intruder or an anomalous situation delivers signals that trigger the activation of several other immune cells. The flow of defense reactions passes from body barriers to the elicitation of a quick innate immunity reaction and to a later adaptive immunity reaction followed by a long lasting immune memory of the past battle. Body barriers (See Chapter 1) are always operative in inhibiting microbial invasions. Their peculiar features and the immune mechanism associated to them make invasions a very infrequent event. When it occurs, in a lapse of time of few minutes to a few hours a storm of alarm signals (See Figs. 1.4, 2.3), chemokines and cytokines (See Chapter 4) recruits and activates several and distinct cell populations of innate immunity. Commonly, this fast and furious reaction effectively overcomes the intruder (See Fig. 3.32). In the few occasions in which the invasion is not quickly repressed, innate immunity signals along with the diffusion of the intruder concur in triggering a different strategy of reaction, the adaptive immunity. This new defense strategy based on the precise (specific) reaction of T cells and antibodies against the molecular peculiarities of the intruder requires about one or two weeks to be operative. Thanks to their ability to recognize the intruder with high specificity, adaptive immunity mechanisms guide innate immunity cells towards a more efficient destruction of the invaders. Lastly, a successfully repressed invasion leaves a state of long lasting and specific immune memory (See Chapter 24). Memory T and B cells are quickly activated by a second arrival of the same intruder and become operative in a few days. Memory reactions are about 10 to 1000 times stronger than primary adaptive responses. In most of the cases the efficacy by which memory mechanisms control subsequent invasions are so strong and the reaction is so quick as to pass almost unnoticed.




Fig. 1.2. SKIN AND MUCOSAE. Mechanisms of isolation and protection.


Fig. 1.3. THE SKIN: A COMPLEX AND DYNAMIC BARRIER. The isolation and protection from the hostile environment provided by the skin is due to the combination of several distinct mechanisms. Physical barrier: The skin is constituted by three cell layers: the epidermis, the dermis and the subcutaneous tissue or hypodermis. The epidermis is constituted by a keratinized stratified epithelium, the outermost layer of which (Stratum corneum) is composed of a-nucleated keratinized epithelial cells (Horny cells) and epidermal lipids, both produced in the granular layer and secreted by the sebaceous glands. The external surface of the stratum is a dry, acidic lipidic rich, high salt surface. Epidermal lipids and the thigh junctions among epithelial cells form a waterproof barrier. Proliferation of germinal cells (epithelial stem cells) in the basal layer provides a steady renewal of epithelial cells that accompanies the continual desquamation of horny cells. Complete skin cell renewal takes place every 2-4 weeks. Desquamation is an effective way to remove microbes and substances adhering to the skin. Physical stress leads to an enhanced proliferation of epithelial cells and the formation of a ticker stratum corneum (Callous). In the few areas where the skin is particularly thick a stratum lucidum made by dead cells containing eleidin, a transparent protein, is evident between the stratum corneum and stratum granulosus. Skin responds to the damage due to UV radiations with the proliferation of

9 melanocytes and melanin secretion that may form a more effective barrier to UV radiations. This process is associated with an enhanced proliferation of epithelial cells replacing dead and damaged cells. Several microbes produce cytolytic proteins and peptide toxins in order to disrupt epithelial barriers and initiate a mucosal infection. The fatty cells on the subcutaneous tissue provide an effective thermal-insulation. The contraction of pili muscles leading to goose bumps and porous restriction reduces the dispersion of body heat. By contrast the sweat secretion and the subsequent water evaporation lower body temperature. Signals provided by cells damaged by wounds trigger dermal fibroblasts and epithelial cell proliferation leading to wound healing. Biochemical barrier: Epithelial cells secrete several antimicrobial peptides (See Fig. 1.4). The acidic pH of the skin (pH 4-4.5) impairs microbial proliferation. Acidic pH is due to salts, lactic acid in sweat and the resident microbial flora. In addition fatty acids secreted by sebaceous glands and sweat components (Lysozyme, see Fig. 1.5; Lactoferrin) are endowed with marked antimicrobial activity. Direct immune activity: Epithelial cells express a series of receptors that sense microbial invasion and penetration. The activation of these receptors induces the release of alarm signals (danger signals, chemokines, and cytokines, see Fig. 2.3 and Chapter 4) that elicit innate immunity reactions and play a key role in promoting and polarizing the following adaptive immunity. Intra-epithelial Langherhans cells (epidermis immature Dendritic Cells, DC, see Figs. 3.22, 3.23), dermal Dendritic Cells, macrophages and blood derived neutrophils (See Chapter 3) activated by factors released by epithelial cells ingest cell debris and kill microbes. These cells along with lymphocytes and epithelial cells secrete a large array of anti-microbial molecules (See Chapter 2). Sentinel activity: Mast cells (See Figs 3.6, 3.7), DC,neutrophils , macrophage, lymphocytes (See Chapters 7 and 12), Innate Lymphoid Cells (ILC) (See Fig. 2.7-2.9) and endothelial cells of blood vessels (See Figs. 3.32-3.33) are the further outpost of the innate and adaptive immunity. The large array of cytokines and pro-inflammatory factors released by these cells when an intruder is perceived trigger and bias both a local and a systemic innate and adaptive immune responses. In effect, the peculiar state of differentiation acquired by Antigen Presenting Cells and their migration to lymphoid organs trigger and modulates both antibody and cell mediated adaptive immune responses (See Figs. 7.1-7.4). Skin microbes can fall anywhere along the continuum between mutualism and pathogenicity. To control microbes that normally colonize the barrier surfaces such as those of the skin and gut innate NK cells (iNK cells) induce a form of immunity that not directly retaliate against the microbes, but instead aids tissue repair and microbe containment (See Figs. 7.19, 8.17). Microbial antibiosis: Numerous and diverse microbes (>1012, viruses, fungi and bacteria) referred to as skin microbiome reside on the epidermis and the hair follicles. These microbes metabolize skin proteins and lipids and producing bioactive molecules that inhibit skin infection by invading microbes, and are involved in the skin pH acidification. The body odor is produced by skin microbiome. Memory response: Epithelial stem cells respond more rapidly to a secondary microbial assault since they remember the primary assault by acquiring epigenetic changes (See Fig. 24.1). A loss of methylation and different chromatin accessibility allow a quicker transcription of the genes activated by the primary assault. REFERENCES: H Hammad & BN Lambrecht, Immunity, 2015,43: 29; YE Chen, MA Fischbach & Y Belkaid, Nature 2018,553:427.


Fig. 1.4. MOLECULES SECRETED BY EPITHELIAL CELLS. Barrier epithelial cells are the very first line of defense. They secrete both molecules with direct antimicrobial activity and danger signals alarming innate and adaptive immune responses. Often combinations of molecules released by epithelial cells program Antigen Presenting Cells to induce a Th2 cell mediated response (See Figs.11.11-11.12).

Fig. 1.5. THE LYSOZYME. An effective anti-microbial enzyme


Fig. 1.6. THE BRONCHIAL MUCOSA: A BALANCE BETWEEN FUNCTION AND PROTECTION. The respiratory tract is incessantly exposed to microbes and substances from inhaled air. The upper respiratory tract and trachea, bronchi and bronchioles are lined by a delicate mono-stratified ciliated epithelium shielded by mucus. Physical barrier: Ciliated epithelial cells of the respiratory tract are much thinner than the skin and therefore respiratory infections are the most common in the human population. The continuous beats of cilia move mucus and the microbes trapped on it towards the throat where it is swallowed. Mast cell (See Fig. 3.6) degranulation causes constriction of the muscular layer (bronchial constriction). Biochemical barrier: The mucus is a dense and sticky substance covering the respiratory tract mucosa. It is made by water and mucins, substances secreted by goblet cells and submucosal serous mucus glands. Goblet cells are scattered among the respiratory tract ciliated epithelial cells. They are present also in the mucosa of the digestive tract. Mucus contains lysozyme (See Fig. 1.5), antibodies, inorganic salts, lactoferrin and mucins. Innate Lymphoid Cells (ILC) activated by IL23, IL33 and TSLP secrete IL13, a cytokine that enhances mucus production by goblet cells (See Figs. 2.7, 2.8, and 11.13) Direct immune activity: Dendritic Cells (DC), granulocytes and macrophages ingest microbes and foreign substances and migrate to draining lymph nodes. Moreover, these cells along with lymphocytes and epithelial cells secrete a large array of anti-microbial molecules. Sentinel activity: Here too DC, macrophages, lymphocytes and Innate Lymphoid Cells (ILC) are the further outpost of adaptive immunity.


Fig. 1.7. THE MUCOSA OF THE LIPS AND MOUTH: A DELICATE PHYSICAL BARRIER. The lips and the oral cavity are lined by a mucous membrane consisting of a stratified squamous epithelium similar to that of the skin. However here the keratinized stratum corneum is scarce or absent. The underlying lamina propria is a thin connective tissue layer containing blood and lymphatic vessels, many nerve fibers and elements of the immune system. This kind of mucosa is an efficient physical barrier even if less protective than skin. Biochemical barrier: Several minor salivary glands are located in the submuco Direct immune activity, Sentinel activity and Microbial antibiosis are similar to those of the skin and the bronchial mucosa, furthermore the saliva produced by the salivary glands is endowed with digestive and antimicrobial action. The antimicrobial immune activity depends on lysozyme, lactoferrin, myeloperoxidase and antibodies.

13 Fig. 1.8. THE MUCOSA OF THE DIGESTIVE TRACT. The intestinal mucosa functions as a barrier interacting up dynamically with the external environment since it has to perform a complex management of food absorption, symbiotic bacteria and inflammation due to their incursion. In fact, the human intestine houses a vast consortium of symbiotic bacteria (up to 100 trillion microbial cells, >90% anaerobic bacteria, the gut microbiota) which are a potential risk to the host. In addition, gut mucosa plays a crucial role in the maintenance of immune tolerance to food compounds. Gut mucosa is constituted by intestinal glands lined by a monostratified epithelium formed by columnar absorbing cells. The inner portion of the glands contains mucus secreting goblet cells (See Fig. 11.13), endocrine cells and Paneth cells. Lymphoid cells and lymphatic nodules are in the sub mucosa. The intestinal epithelium is the most rapidly self-renewing tissue of the body: Cells have a life cycle of 34 days. Physical barrier: The glandular monostratified epithelium is much thinner than in the skin, and therefore intestinal infections are common. Peristaltic movements, forcing mucus mixed with digested food and fecal contents toward the anal end control bacterial growth by mechanical cleansing which dislodges and removes potential colonies of microbes. Biochemical barrier: The intestinal mucosa is protected from microbes by mucus layers produced by goblet cells. Abundant numbers of commensal microbiota are present in the outer mucus layer, while a firm inner mucus layer acts as a physical barrier maintaining the segregation of microbes and epithelial cells, thus reducing the risk of intestinal invasion. Paneth cells release into the lumen granules containing antimicrobial proteins, including lysozyme (See Fig. 1.5) and alpha defensins and other antimicrobial peptides. Direct immune activity: Dendritic Cells (DC, See Figs. 3.22-3.23), macrophages and neutrophils (Figs. 3.93.13) in the lamina propria ingest microbes and foreign substances and migrate to sub mucosal lymphatic nodules. Moreover, Paneth cells along with other lymphoid cells secrete a large array of antimicrobial molecules. Sentinel Innate Lymphoid Cells (ILC,) activated by IL1 and IL23 release IL17, IL22 and IL23 (See Fig. 2.8) and play an important role in direct the containment of microbiota incursions. Moreover, the combination of IL17 and IL22 enhances the production of anti-microbial molecules by Paneth cells. These diverse immune mechanisms temper the expansion of some microbial species and protect gut epithelium. Sentinel activity: Here too DC, macrophages, lymphocytes and ILC are a further outpost of adaptive immunity (See Figs. 7.1-7.3). Signals delivered by microbes of gut microbiota create complex interaction

14 between epithelial cells, DC, macrophages and ILC (See Chapter 3), influencing the functions of innate immunity, the local differentiation and polarization of T cells (See Fig. 11.3) and promoting local IgA class-switch and production. Microbial antibiosis: Besides being a constant potential danger, gut microbiota regulates various aspects of host body physiology including immune response, tolerance to food compounds, metabolic functions and behavior. This vast consortium of symbiotic bacteria ferments carbohydrates, synthesizes vitamins, prevents the growth of pathogenic species by competing for nutrition and attachment and producing toxins (bacteriocidins) that inhibit growth of other microbial species.

Fig. 1.9. THE RECTAL MUCOSA: AN EFFECTIVE PROTECTION PRONE TO VIRAL INFECTIONS. Its internal portion is constituted by a glandular mucosa similar to that of the intestine while the more external portion consists in a pluristratified epithelium. Physical barrier: Mechanical stress easily damages the thin internal portion of the mucosa which is often made more vulnerable by the enlargement of the hemorrhoidal plexus. As mechanical stress, cuts and micro-abrasions increase the risk of Human Immunodeficiency Virus (HIV) infection (See below and Fig. 26.5). Indeed, the vast majority of new HIV infections are acquired via the rectal and vaginal mucosa. Biochemical barrier: As for the mucosa of the intestinal tract. Direct immune activity: As for the mucosa of the intestinal tract. Sentinel activity: The HIV infection as well by other viruses is made easier by the fragility of the mucosa to mechanical stress. The HIV virions are captured by Dendritic Cells and macrophages that pass the virus to CD4+ T lymphocytes. Then, infected mucosal CD4+ T lymphocytes display a massive production of HIV virions. Microbial anti/biosis: As for the mucosa of the intestinal tract.


Fig. 1.10. THE MUCOSA OF THE GLANS PENIS AND VAGINA: A SIMILAR FRAGILE PROTECTION IN DIFFERENT ANATOMICAL SITES. These thin and fragile mucosae are the site of common infections including sexually transmitted infections. Physical barrier: In both the glans penis and vagina the mucosa is constituted by a pluristratified squamous epithelium similar to that of the skin. However here the stratum corneum is very scarce or absent. Here too the infection by the lymphotropic Human Immunodeficiency Virus (HIV, See Fig. 25.6) as well by numerous other viruses and microbes is made easier by the micro-abrasions and cuts due to the fragility of the mucosa. Biochemical barrier: No sebaceous or serous-mucus glands are present in the glands penis and vagina mucosa. The mucus covering the vaginal mucosa is produced by glands of the vulva and uterine cervix. Direct immune activity: The glands penis and vaginal mucosa produce lysozyme (See Fig. 1.5), lactoferrin, mannose-binding protein, and small antimicrobial peptides endowed with broad antimicrobial activity. A rapid influx of neutrophils takes place during infections. Sentinel activity: The epithelium lies over a loose connective tissue with fibroblasts and a few Dendritic and lymphoid cells. As described for the rectal mucosa, HIV virions are captured by Dendritic Cells and macrophages that pass the virus to CD4+ T lymphocytes. Microbial antibiosis: Whereas microbial antibiosis virtually does not take place on the glans penis, it has a major importance in the vagina which is colonized by commensal microbiota whose composition changes under the influence of hormones from the neonatal to the reproductive period and menopause. Vaginal lactobacilli, the most important constituent of vaginal microbiota, produce lactic acid and hydrogen peroxide which maintain the low vaginal pH which protects against infections.


Fig. 1.11. THE WAR ON BODY SURFACE. On our skin and mucosae an everlasting war is taking place against chemicals, microbes, parasites and foreign cells. Defense mechanisms outlined in the previous figures have to continuously face ingenious and sophisticated invasion strategies that are incessantly evolved by microbe. Sometimes these may seem made-up, like a science fiction tale, while they hide a dramatic medical problem. For example, the Malaria parasite exploits the female Anopheles mosquitos to fly from one person to another and to overcome, with the sting, the thick and otherwise impenetrable skin barrier. The female mosquito alights on the skin and penetrates the epidermis with its proboscis to pierce a small vessel in the dermis (Blue arrows). The mosquito's saliva contains anticoagulants to keep the blood from clotting. If the mosquito is infested by the malaria parasite, the bite may be followed by a malaria infection.Currently, this imaginative and apparently unlikely strategy allows the Malaria’s parasite to infect 250 million and kill one million persons every year.



Fig. 2.1. SENTINEL CELLS. Frequently micro-lesions as well as larger wounds of body barriers allow a massive arrival of microbes. Immune cells should recognize and respond to invaders at the earlier stages of incursion, before they are able to adapt to the new environment and expand. Various signs of invasion (alarm and danger signals, see Figs. 1.4, 2.3) delivered following the invasion, by dying cells and damaged tissues are perceived by distinct sentinel cells strategically located in the districts of body where the invasions are more common. Pattern Recognition Receptors (PRR) are receptors expressed by sentinel cells that sense the presence of several microbial structures evolutionary conserved and widely expressed by microbes of various kinds. Other PPR recognize molecules commonly associated to microbial invasion (the so called Pathogen-Associated Molecular Patterns, PAMP), cell damage or death (Damage-Associated Molecular Patterns, DAMP) (See Fig. 2.4). By changing conformation and expressing adhesion molecules (endothelial cells) and through the release of soluble factors these sentinel cells rapidly signal the invasion and recruit and activate various leukocyte populations of innate immunity (See Chapter 3) which concur to counteract the invasion in a quick and efficient way. In the Fig. 1. Epithelial cells (See Fig. 1.4); 2. Mast cells (See Fig.3.6); 3. Macrophages (See Fig. 3.16) and Dendritic Cells (See Fig. 3.22); 4. Granulocytes: Neutrophils (See. Fig. 3.9) and Eosinophils (See Fig. 3.14); 5. Large granular lymphocytes/Natural Killer (NK Cells, see Fig. 3.24); 6.Lymphocytes and Innate Lymphoid Cells (ILC, see Fig. 2.8); 7.Fibroblasts; 8. Endothelial cells (See Figs. 3.32).


Fig. 2.2. PATROLLING THE MICRO-ENVIRONMENT. In the dark of the interior of our body cells of innate immunity sense traces of microbial invasion, tissue damage, cellular stress, and alarm signals through a series of receptors called Pattern Recognition Receptors (PRR, See Fig. 2.4). These sensors provide a continuous surveillance for the presence of microbes and anomalous conditions. Other receptors on the membrane of innate immunity cells guide their homing inside our body (Chemokine receptors, See Fig. 4.23), their activation (Chemokine and cytokine receptors, See Chapter 4), their interaction with other cells (Adhesion molecules), their metabolism (Hormone receptors), their involvement in reactions triggered by Complement activation (Complement receptors, See Fig. 20.8) or by antibodies (Receptors for the Fc fragment of antibodies, See Figs. 19.7). The expression of the various membrane receptors may change during the various stages of the life of the cells of the innate immunity. The conformational modification of the receptor caused by its interaction with ligands triggers signal transduction pathways that modify the cell activation state. A continuous integration of myriad of signals perceived by the numerous membrane receptors modulates gene activation and guides differentiation, behavior and fate of immune cells. Ligands interacting with cell membrane receptors can be compared to language messages. They are meaningful only if the cell is able to perceive them, i.e. to express a receptor able to capture the signaling molecule (the ligand) and transduce the messages to the nucleus. Every population of immune cells expresses distinct repertoires of receptors, and therefore perceives and reacts to different environmental signals. There are also receptors able to capture the message but unable to transduce the signal to the nucleus. These decoy receptors remove or attenuate a specific message. Only a few families of the numerous membrane receptors expressed by natural immunity cells are shown here.


F i Fig. 2.3. A FEW ALARM SIGNALS RELEASED FOLLOWING A BODY BARRIER LESION. The release of these alarm signals quickly activates the inflammatory immune response. Necrotic cell death (Necrosis and Necroapoptosis) directly triggers a natural immunity reaction by releasing molecules belonging to the Damage Associate Molecular Patterns (DAMP). Moreover, peculiar signal combinations induce the activation and differentiation of macrophages towards M1 or M2 (See Fig. 3.19) and program Antigen Presenting Cells to activate a Th1 or a Th2 deflected T cell reaction (See Chapter 11).  The key role played by the Complement system in innate immunity is illustrated in Figs. 20.30-20.4; Cytokines are illustrated on Chapter 4; For reactive Reactive Oxygen Intermediates (ROI) see Figs. 3.21. /


Fig. 2.4. HOW A MICROBIAL INVASION AND LOCAL DAMAGE IS PERCEIVED. Pattern Recognition Receptors (PRR) sense various signals of local damage and invasion. Their expression on the cell membrane allows the detection of traces of invasion and alarm signals in the cell microenviroment (Upper panel). The expression of PPR on endosomes (Middle panel) or in the cytoplasm (Lower panel) permits to perceive the presence of microbes that live inside the cell (intracellular microbes: viruses, bacteria, protozoa…). LPS (lipopolysaccharide) is a bacterialendotoxin. Not methylated CpG, (Cytosine—phosphate—Guanine) dinucleotide: a basesequence typical of microbial DNA.


Fig. 2.5. REACTIVE CELL FUNCTIONS ACTIVATED BY PATTERN RECOGNITION RECEPTORS (PPR). PPR are expressed on the membrane of various sentinel cells. The interaction of a PPR with its ligand triggers both reactive activities specific for each cell and widespread reaction patterns. Inflammasome is a multimeric complex of caspases. Once assembled, inflammasomes permit the production of high amounts of very important pro-inflammatory cytokines (IL1, IL18…) (See Chapter 4).

Fig. 2.6. ALARMS REACTIONS ACTIVATED BY SENTINEL CELLS. Here we report the main activities set in motion by PPR expressed by sentinel cells associated with body barriers. Each of these activities is illustrated in more detail in the following figures. ILC, Innate Lymphoid Cells.

22 Fig. 2.7. INNATE LYMPHOID CELLS (ILC). ILC are a family of lymphoid cells with important sentinel and effector roles. All members of the ILC family have lymphoid cell morphology but lack the expression of peculiar surface molecules that identify other immune cells. In addition ILC do not express RAG-mediated recombined T cell receptor (TCR) nor the B cell receptor (BCR). Their activation is modulated by the peculiar combination of alarm signals and cytokines triggered by the invader. By quickly inducing and regulating an appropriate acute inflammatory response, ILC play a fundamental sentinel role.

Fig. 2.8. ILC: SUBSETS AND FUNCTIONS. As for T helper cells (See Chapter 11 ), the differentiation of ILC towards one subset or the other (trans-differentiation) is governed by the molecular features of the invader and by the combination of danger signals and cytokines released by the sentinel cells that first perceived the signs of the invasion. In response to these signals, ILC transcription factors bind to specific sequence motifs within the gene promoters. In this way one genetic program dominates while alternative programs are silenced.

23 Thus, in response to different stimuli, ILC differentiate in three major subsets (ILC1, ILC2 and ILC3) that provide a potent and early source of distinct cytokine sequences that tune up an appropriate innate immunity reaction against different invaders. ILC1: The acute inflammation triggered by the cytokines released by ILC1 is effective against intracellular microbes (viruses, bacteria, protozoa). ILC2: The reaction triggered by cytokines released by ILC2 is effective against worms, contribute to multiple homeostatic processes, dampen inflammation and tissue repair. Neuropeptides modulate the activaty of ILC2. ILC3: Cytokines released by ILC3 induce a rapid and effective reaction against extracellular bacteria and fungi. For acute inflammation, see Fig. 3.32; For M1 and M2 macrophages, see Fig. 3.19; For Th1 and Th2 see Chapter 11; For goblet cells see Figs. 1.6, 1.8, 1.9 and 11.13; For Paneth cells see Figs. 1.8 and 1.9; For neutrophils see Fig. 3.9; TSLP is the Thymus Stromal Lymphopoietin, a cytokine known to play an important role in the maturation of T cell populations through activation of Antigen Presenting Cells (See Chapter 11).

Fig. 2.9. DEFENSE REACTIONS. Here, listed, are the most relevant effector reactions activated by sentinel cells of innate immunity. Each of these activities is illustrated in more detail in the following figures. ILC, Innate Lymphoid Cells.


CHAPTER 3. CELLS OF INNATE IMMUNITY. Fig. 3.1. INNATE IMMUNITY: THE CELLS. Though immune memory is considered the hallmark of adaptive immunity, recent data show that even the cells of innate immunity can acquire a form of memory that allows them to respond with greater effectiveness against subsequent invasion.

Fig. 3.2. LEUKOCYTES PRESENT IN HUMAN BLOOD: THE WHITE BLOOD CELLS (WBC). Frequency of the main cell populations of human blood.


Fig. 3.3. ALL WHITE BLOOD CELLS (WBC) ORIGINATE FROM BONE MARROW. Panel A: The bone marrow is contained in flat bones and long bones such as tibias and femurs. There are two types of bone marrow: Red marrow, which consists mainly of hematopoietic tissue, and Yellow marrow, which is mainly made up of fat cells. Panel B: Hematopoiesis occurs in the extravascular spaces between the sinusoids. These spaces contain both the endosteal niche close to the bone lining osteoblasts (Ob in the Fig.) and the perivascular niche located around small sinusoidal blood vessels. Here Hematopoietic Stem Cells (HSC in the Fig.) are longlived, self-renewing cells which progressively differentiate into the various WBC. HSC located on the endosteal niche are more quiescent, whereas those located at the perivascular side display a more active self-renewal and differentiation. Panel C: The main blood source to the red bone marrow is provided by the nutrient artery which crosses the bone cortex and gives rise to numerous capillaries. These capillaries enter the medullary vascular sinuses forming a dense network of sinusoids through the medullary cavity. Osteoblasts (Ob in the Fig), macrophages (M in the Fig.), reticular cells producing the CXCL12 chemokine (Cell Abundant of CXCL12 Reticular, CAR in the Fig.), Nestin rich Mesenchymal Stem Cells (MSC in the Fig.), endothelial cells (See Fig. 3.32), and adipocytes (Ad in the Fig.) are components of the niches and their structural composition provides a specialized micro-environment that regulates HSC self-renewal and differentiation. During normal life the red marrow extravascular spaces are filled by HSC-derived hematopoietic cells. These //include myeloid and lymphoid cell precursors together with their cell progeny. Only mature leukocytes, erythrocytes and platelets are released into the sinusoids and enter in the circulatory blood. The bone marrow trabeculae provide a network of supportive frameworks. Adipocytes are dispersed throughout the marrow.


Fig. 3.4. FROM HEMATOPOIETIC STEM CELL TO MATURE BLOOD CELL. Diagram showing the developmental stages of hematopoiesis of lymphocytes, erythrocytes, other leukocytes and platelets. Erythrocytes and all white blood cells (WBC) derive from the myeloid progenitor. WBC (neutrophils, eosinophils, basophils), include monocytes and part of Dendritic Cells.

Fig. 3.5. DIVERSIFICATION OF BLOOD CELLS. Our body produce about 300 billion of blood cells/day. This enormous production needs to have the flexibility to adapt to changes of demand caused by different pathological situations. Blood cell diversification rests on a series of nodal cell-fate decisions, i.e. choices between alternative gene expression programs in response to extracellular signals: one program is winning out while alternative programs are extinguished. The concurrence of various signals triggers the expression of a particular transcription factor that binds to specific sequence motifs within gene promoter, enhancer and silencer regions and recruits epigenetic regulators to modulate the activation status of a gene. The induced gene activation is transmitted subsequent cell generations as a permanent epigenetic modification. REFERENCE: E Laurent & B Gottgens, Nature 553, 418 2018


Fig. 3.6. BASOPHILS AND MAST CELLS: ULTRASTRUCTURAL (panels A and C) AND HISTOLOGICAL (panels B and D) ASPECTS. Basophils, along with eosinophils and neutrophils constitute a group of White Blood Cells (WBC) known as granulocytes, i.e. cells with many cytoplasmatic granules. Basophils: The name basophils derives from their ability to be stained by basic dyes. Basophils are smaller than other white blood cells; their nucleus is usually bi-lobed (two lobes) and the cytoplasm is often obscured by granules containing heparin and histamine. Basophils and mast cells derive from hematopoietic bone marrow precursors and share several functions while they differ in size, shape of the nucleus and amount of cytoplasmic granules. Mast cells: Mast cells are larger than basophils with irregular, elongated shapes and cytoplasmic extensions. The nucleus is round and the cytoplasm is packed with basophilic granules that may obscure the nuclear margin. Basophils mature in the bone marrow and stay in the blood whereas immature mast cells migrate in the lamina propria beneath the basal membrane of skin, conjunctiva, mouth, digestive tract and lung mucosa where they undergo final differentiation. Activity: Through Pattern Recognition Receptors (PRR, see Fig. 2.4), basophils and mast cells recognize invading microbes. Within seconds of activation they release the contents of pre-formed mediators present within cytoplasmic granules, an activity defined as degranulation. After that, these cells start to synthetize newly formed mediators. The released mediators increase local blood flow and vascular permeability, induce the recruitment of eosinophils, neutrophils and NK cells, increase mucus (See Fig. 1.6) production by goblet cells (See Figs. 1.6; 1.8; 1.9, 11.12, and 11.13) and intestinal mobility and act on smooth muscles to increase the expulsion of mucosal parasites. Basophils and mast cells are also involved in allergic reactions since their degranulation can be activated through the cross linking of IgE bound on their membrane (See Fig. 16.26, 19.10), and by constituents of Complement cascade adhering to Complement Receptors (See Fig. 20.8), immunocomplexes (See Fig. 19.4) and by direct injury.





Fig. 3.9. THE NEUTROPHIL: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. Neutrophils are phagocytes, capable of ingesting microorganisms or small particles. The name derives from their ability to be stained by neutral dyes. These cells derive from hematopoietic bone marrow stem cells (See Fig. 3.4), with a production 11 of 10 neutrophils/day (!): therefore they are the larger population of White Blood Cells. Neutrophil lifespan in the circulation is about 5.4 days while they survive for 1–2 days after migration into tissues. Neutrophils have a multilobated nucleus, and the cytoplasm contains small Golgi apparatus, sparse mitochondria and ribosomes and about 200 granules of three kinds: azurophilic granules (or primary granules), specific granules (or secondary granules) and gelatinase granules (or tertiary granules). The molecules contained in the granules are endowed with a strong antimicrobial activity but they can also damage normal and neoplastic cells. The massive release of granules outside the cell, with the death of the neutrophils, is called degranulation. Dead neutrophils are the major component of pus. Alternatively neutrophils phagocyte and kill microbes. Upon phagocytosis they produce nitric oxide (NO), activate NADH oxidase and generate O2- and other Reactive Oxygen Intermediates (ROI, See Figs. 2.5, 3.19, and 3.21). ROI production as well as the release of the toxic content of granules kills the ingested microbes. By the degranulation of granules neutrophils release an assortment of anti-microbial substances, enzymes, Properdin (See Fig. 20.4) and pro-inflammatory molecules (See Fig. 3.11). A further antimicrobial activity of neutrophils is the release of DNA nets that trap microbes in the microenvironment (See Fig. 3.13). Neutrophils are one of the first-responders of inflammatory cells to migrate towards the site of inflammation.


Fig. 3.10. NEUTROPHILS: THE CONTENT OF CYTOPLASMATIC GRANULES. The release of Properdin, a Complement constituent stored in secondary granules, enhances the lytic activity of Complement activated by the Alternative pathway (See Fig. 20.4).


31 Fig. 3.12. NEUTROPHILS: PHAGOCYTOSIS. A neutrophil develops cytoplasmic projections, called pseudopods (or pseudopodia) that engulf the microbe. After the formation of a phagosome containing the microbe there is the fusion of the phagosome with a lysosome to form a phagolysosome. Lysosomes are membrane-bound spherical organelles containing acid hydrolases, which are able to break down virtually all kinds of biomolecules in an acidic environment. A microbe in a phagolysosome is killed and digested by lysosomal enzymes. Indigestible and residual material is discharged by exocytosis.

Fig. 3.13. NEUTROPHILS: EXTRACELLULAR TRAPS. Once the invading microbes is perceived through several Pattern Recognition Receptors (PPR, see Fig. 2.4), neutrophils release Neutrophil Extracellular Traps (NET) which are webs of DNA covered with antimicrobial molecules (such as myeloperoxidase, neutrophil elastase, lactoferrin, gelatinase, cathepsin G, etc.) derived from primary azurophilic, secondary and gelatinase granules. After stimulation, the neutrophil chromatin undergoes decondensation, a process mediated by enzymes stored in the primary azurophilic granules which are relocated in the nucleus. The nuclear chromatin expands inside the cell and is mixed with anti-microbial factors of the granules. Finally the cell membrane breaks releasing NET. Microbes entrapped in NET are killed by oxidative and non-oxidative mechanisms.

32 Fig. 3.14. EOSINOPHILS: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. Eosinophils derive from haematopoietic bone marrow precursors but form only a minor population among White Blood Cells. Their nucleus is often bilobed (two lobes) and the cytoplasm displays several distinct granules stained with eosin dye. Specific or crystalloid granules: These granules are very numerous (more than 200/cell). They contain a core of crystallized Major Basic Protein, peroxidase, cationic proteins and a neurotoxin. All these proteins are toxic for helminths, parasites, microbes and tumor cells (See Fig. 11.13). Their release induces histamine release by mast cells (See Figs 3.6-3.7). Primary granules: These granules are less numerous and contain Charcot-Leyden crystal protein, a phospholipase. Small granules: These granules contain acid phosphatase, arylsulfatase B, catalase, and cytochrome lipid bodies (about 5/cell) contain arachidonic acid esterified into glycerophospholipids. Eosinophils bind antibodies on the surface of parasites and cancer cells, and then degranulate to release proteins and enzymes which disrupt the plasma membrane of parasites and tumor cells. Eosinophils also phagocytose and destroy bacteria but are less efficient than neutrophils.

Fig. 3.15. NEUTROPHILS AND EOSINOPHILS: MAIN FEATURES. Aged neutrophils and eosinophils modulate the expression of their chemokine receptors and home in the bone marrow where they die.


Fig. 3.16. MONOCYTES: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. Monocytes are the largest of all White Blood Cells (WBC) and have a large, single bean-shaped nucleus which gives them their name which means mononuclear leukocyte. The cytoplasm contains many lysosomes and lipid bodies. These cells derive from hematopoietic bone marrow precursors (See Fig. 3.4 and 3.5) and form a significant population among WBC. They stay for a few hours in the blood and then migrate into the tissues where they differentiate into macrophages.

Fig. 3.17. THE MONOCYTE: A LONG-LIVING CELL WHICH DIFFERENTIATES INTO DISTINCT SPECIALIZED CELLS. Monocytes and macrophages comprise a variety of subsets with diverse functions. Following the migration from blood vessel to tissues, monocytes differentiate into cells with a peculiar morphology and very specialized functions.

34 Fig. 3.18. THE TISSUE MACROPHAGE: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. Once monocytes exit from blood vessels and marginate into a tissue they differentiate in macrophages. These cells display an irregularly shaped nucleus. The cytoplasm with a few mitochondria contains lysosomes, phagosomes and phagolysosomes with residual bodies, which are undigested material. Macrophages often present numerous cytoplasmic projections, called pseudopods, indicating that these cells are actively engaged in phagocytosis.

Fig. 3.19. M1/M2 MACROPHAGES. A macrophage is a terminally –differentaited cell. When activated by microbes, interferons (IFN), tumor necrosis factor (TNF) and other pro-inflammatory Th1 cytokines (See Chapter 11) macrophages is termed M1 macrophage. By contrast, when a macrophage is activated by Th2 cytokines (IL4, IL13‌) and parasite products it is termed M2 macrophage (See Figs. 11.12 and 11.13). The names M1/M2 were chosen because, in their turn M1 and M2

35 promote Th1 and Th2 responses (See Chapter 11). However M1/M2 subdivision is a limited attempt to define the remarkable plasticity of macrophages. M1 MACROPHAGES acquire a high microbicidal activity, secreting pro inflammatory cytokines and Reactive Oxygen Intermediates (ROI). Activated M1 macrophages display a respiratory burst and an increased glycolysis. M2 MACROPHAGES display an anti-parasitic activity (See Figs- 11.12 and 11.13). Moreover M2 macrophages release molecules that work toward resolution of inflammation, promote cell proliferation, tissue repair, wound healing and angiogenesis. In addition M2 macrophages are implicated in allergy and asthma. M2 macrophages have low glycolysis rates but a marked fatty acid oxidation activity

Fig. 3.20. PHAGOCYTOSIS. Various forms of endocytosis are performed by all the body cells. However, macrophagocytosis is done in a professional way by tissue macrophages, Langherhans cells, immature Dendritic Cells, and -in a less intense way- by neutrophils and eosinophils. Microbes and foreign particles are perceived by distinct Pattern Recognition Receptors that trigger the emission of pseudopods that engulf the particle in a phagosome . In addition, macrophages digest their own parts (autophagy). Fragments of digested microbes, endocytosed proteins as well of those generated by autophagy can be presented as peptides on Class II glycoproteins of the Major Histocompatibility Complex (HLA in humans, see Chapter 5) and trigger a T cell response (See Chapter 9).


Fig. 3.21. M1 MACROPHAGES: MICROBE TRAPPING AND KILLING. Microbes are sensed by macrophages through a large array of Pattern Recognition Receptors (See Figs. 2.4) expressed on the membrane of long pseudopods stretched by the activated macrophage. Moreover, MAC 1 receptors on macrophage membrane interact with the C3b constituent of Complement cascade adhering to the membrane of a microbe (See Chapter 20). A lysosome containing various hydrolytic enzymes in an acidic matrix able to destroy proteins and nucleic acids fuses with the phagosome giving rise to a phagolysosome. On M1 activated macrophage NADPH oxidase assembled on the wall of phagolysosomes produces various Reactive Oxygen Intermediates (ROI). The combination of hydrolytic enzymes and ROI kills the majority of microbes picked up. Their debris are expelled from the cells or are presented as peptides on Class II HLA glycoproteins and trigger a T cell response (See Chapter 6). Activated M1 macrophages display an enhanced phagocytic activity, ROI production, killer activity and expression of the glycoproteins of the Major Histocompatibility Complex (HLA in humans).


FIG. 3.22. DENDRITIC CELLS (DC): ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. DC acquire and process antigens then migrate to lymph nodes and activate T cells, which in turn induce reactive immune responses (See Figs. 9.4 and 11.4). Immature DC differentiate from hematopoietic progenitors as well from monocytes. Immature DC are located in tissues in contact with the external environment such as the dermis and the lamina propria beneath the basal membrane of skin, conjunctiva, mouth, digestive tract and lung mucosa. In the skin immature DC are known as Langerhans cells. DC have an oval or lobulated nucleus. The cytoplasm contains a small Golgi apparatus, a moderate endoplasmic reticulum, some mitochondria, several lysosomes and a variable number of post Golgi vesicles that correspond to Class II HLA proteins containing organelles. When differentiated into mature DC, these cells become particularly efficient in presenting peptides of phagocytized proteins on Class II HLA glycoproteins and trigger a T cell response (See Chaper 9 and Fig. 11.4).

Fig. 3.23. DENDRITIC CELLS (DC): GENERAL FEATURES. The critical role played by DC in T cell activation is presented on Chapter 9 and Fig. 11.4)

38 Fig. 3.24. NATURAL KILLER (NK) CELLS: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. NK cells are a minor cell population present in the blood (10-15 %of circulating lymphocytes, not all White Blood Cells!), lymphoid organs and tissues. They differentiate from bone marrow progenitors under the influence of IL7 and IL15. As Innate Lymphoid Cells (ILC, see Fig. 2.8) NK cells are lymphocytes with a nucleus with the typical chromatin appearance of a mature lymphoid cell similar to that of T and B lymphocytes. However, NK cells are a little bit larger than T and B cells and display small cytoplasm with distinct azurophilic granules containing mainly perforin and granzymes (See Figs. 10.6 and 10.7), so these cells are also defined as a large granular lymphocytes. As ILC, NK cells do not express RAG-mediated recombined T cell receptor (TCR) nor the B cell receptor (BCR, see Chapter 14). Since NK cells do not express distinctive markers. Therefore, they are recognized through the combination of several markers co-expressed by other cells. Besides a killer activity, NK cells release several cytokines that have a major role in driving the inflammatory response and the normal formation of the trophoblast during pregnancy. Both killer activity and cytokine secretion are modulated by several cytokines released by the cells in the microenvironment (See Fig. Chapter 4).

Fig. 3.25. NK CELLS: CYTOKINES MODULATING KILLER ACTIVITY AND CYTOKINE RELEASE. The killer activity of NK cells (also known as cytotoxicity) rests on the release of perforin and granzymes and other granule contents on the membrane of the target cell (See Figs. 10.6 and 10.7). For cytokine release see Chapter 4.

39 Fig.3.26. WHY IS THE KILLER STRATEGY OF NK CELLS IS SO SPECIAL? Commonly immune cells are in a resting state. Signals of various kinds activate the cell and trigger the cell killer activity. By contrast, the killer program of NK cells is always active. Their killer activity is called natural precisely because it does not require activating signals to be turned on. Rather, this natural killer activity is usually blocked by various inhibitory signals delivered by body normal cells.

Fig. 3.27. THE BLOCKADE OF NATURAL KILLER ACTIVITY. The NK cell (Left in the Fig.) expresses both activating receptors (In red) and Killer Inhibitory Receptors (KIR) (In blue) whereas the target cell (Right in the Fig) expresses both normal inhibitory Class I HLA glycoproteins and other molecules that may trigger NK killer activity (In green).The integration of signals that the NK cell receives through KIR and activating receptors regulates the natural cytotoxicity of NK cells. KIR recognizing normal HLA Class I glycoproteins on the membrane of the target cells transduce signals that stop the killer program. Signals delivered by KIR are dominant and block the killer program even when activating receptors are triggering it. The most important inhibitory signals are delivered by KIR recognizing normal Class I glycoproteins of the HLA complex expressed on the membrane of the target cell (For HLA see Chapter 5). A human NK cell expresses on the cell membrane KIR of different kind that recognize various allelic glycoproteins coded by HLA-A, HLA-B, HLA-C and HLA-E genes (See Fig. 5.4)


Fig. 3.28. THE RELEASE OF THE NATURAL CYTOTOXICY. The NK cell (Left in the Fig) expresses both activating receptors (In red) and Killer Inhibitory Receptors (KIR) (in blue) whereas the target cell (Right in the Fig)does not expresses normal inhibitory Class I HLA glycoproteins but instead anomalous HLA glycoproteins and other activating molecules (In green and blue). The lack of inhibitory signals due to the poor expression or absence of normal Class I HLA glycoproteins on the cell membrane of the target cell allows activating receptors to unleash the NK lytic activity and cytokine release. On the cell membrane of NK cells a set of distinct activating receptors recognize Class I HLA glycoproteins that are not normally expressed by healthy cells and other membrane glycoproteins whose expression increases during neoplastic transformation, cell stress, senescence or microbial infection (See Fig. 5.18)

Fig. 3.29. NK CELLS: WHAT ACTIVATING RECEPTORS RECOGNIZE. A human NK cell expresses on the cell membrane different kind of activating receptors that recognize various molecules that are not expressed by happy and healthy cells. Therefore, their killer activity plays a critical role in the control of infections by intracellular microbes, anomalous cells, tumor progression and metastasis spread. For ADCC see Fig. 19.8.


Fig. 3.30. NK CELLS: ACTIVITIES MODULATED BY THE MULTIPLE CYTOKINE RELEASED. The roles played by NK cells through cytokine release are not less important than those of their killer activity.

Fig. 3.31. NK CELLS: ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC). The killer activity of NK cells can be guided by antibodies recognizing their target with high specificity. For details see Figs. 19.8 and 19.9.

42 Fig. 3.32. ENDOTHELIAL CELLS OF A POST CAPILLARY VENUE. Endothelial cells (EC) that line post-capillary venules are not just passive conduits for delivering blood. On the contrary EC guide White Blood Cell (WBC) migration to inflammatory sites. Postcapillary venules are minute vessels intermediate in structure and location between venules and capillaries. Their narrow lumen is lined with a single layer of EC surrounded by pericytes. The EC cells and pericytes are enveloped in a basal membrane. EC adhere to one another through junctional structures (tight junctions and adherent junctions) formed by transmembrane adhesive proteins. These junctions together with intercellular clefts create a barrier controlling the permeability of fluids and small molecules. Caveolae present as invaginations in the endothelial plasma membrane or as vesicles in the cytoplasm are involved in flow sensation and endocytosis. Weibel–Palade bodies contain the von Willibrand factor which is involved in blood coagulation and P-selectin, an adhesion molecule which plays a central role in the ability of inflamed EC to recruit passing leukocytes allowing them to extravasate.

Fig. 3.31. ACTIVATED ENDOTHELIAL CELLS: THE CONTROL OF WHITE BLOOD CELL (WBC) EXIT FROM BLOOD CAPILLARIES. Prostaglandins and histamine released by sentinel leukocytes, mast cells (See Figs. 3.53.7), and platelets increase permeability of post capillary venules by inducing endothelial cells and pericytes to widen inter-cellular spaces. In addition, once activated by inflammatory stimuli, cytokines and chemokines, the endothelial cells start to up-modulate the expression of adhesion molecules [Selectins; IntraCellular Adhesion Molecules (ICAM)]. The binding of these adhesion molecules to integrins expressed on the membrane of activated leukocytes (Rolling neutrophils in the Fig.) initiates a rolling adhesion of neutrophils to the vessel's wall. Chemokines and cytokines also up-modulate the neutrophil expression of membrane integrins which bind to ICAM on endothelial cells creating a firmer adhesion.

43 The exit of WBC from blood vessels (called diapedesis, trans endothelial migration or margination) is guided by further adhesion interactions involving molecules such as PECAM-1 which is expressed by endothelial cells at intercellular junctions. Neutrophils pass between endothelial cells by disrupting various cell-cell junctions. Alternatively, they exit passing through pores generated within activated endothelial cells. Thus, WBC recruitment and extravasation can be separated in four steps: a) Initial attachment and low-velocity rolling of WBC; b) Their arrest; c) The activation of leukocyte integrin expression; d) WBC transmigration and margination in the tissue.

Fig. 3.32. INNATE IMMUNITY: THE FAST AND FURIOUS RECRUITMENT OF WHITE BLOOD (WBC) CELLS TO THE BATTLE ZONE. In the cartoon the broken body barrier lined by a fibrin clot and platelets allows the microbe invasion. Alarm signals (See Chapter 2)and inflammatory chemokines (See Fig. 4.22) produced by damaged epithelial cells, dermal mast cells and sentinel lymphocytes lead to a fast and massive local recruitment of WBC followed by their activation by pro-inflammatory cytokines. This local acute reaction (acute inflammation) is denoted by five signs: dolor (Latin for pain), calor (heat), rubor (redness), tumor (swelling), and functio laesa (loss of functioning) which mostly depend on vessel dilatation and increase of vessel permeability. Vessel dilatation combined with the thickening of the blood due to the leaking out of plasma causes a slowing-down of blood flow rate. This leads leukocytes to stick to the vessel walls, especially in post capillary venules. Neutrophils are the first and the more numerous WBC that exit from blood vessels (extravasate) and enter the invaded tissues (marginate) moving along chemotaxic gradients.

44 Numerous neutrophils present in acute and suppurative inflammation massively release tissue damaging enzymes (degranulate) leading to microbes, cells and tissues necrosis and apoptotic death. Dead cells, secreted molecules and released nucleic acids give rise to pus. This fast and furious acute inflammation is an early and effective protective response against the invaders that triggers both the adaptive immune response and the regeneration of the damaged tissues. Wound healing and injury repair facilitate the resolution of inflammation by restoring barrier function, followed by tissue formation and remodeling. The storm of alarm signals and cytokines leading to the quick recruitment and activation of WBC along with a massive release of self antigens by the damaged tissues may trigger the induction of late autoimmune reactions (See Chapter 23). Nowadays, people take anti-inflammatory drugs to avoid pain and dampen the intensity of the acute inflammation.

Fig. 3.33. INNATE IMMUNITY: SOLUBLE ANTI-MICROBIAL MOLECULES. Several molecules endowed with a direct anti-microbial activity present in all body fluids make the survival of microbes inside our body difficult. The concentration of anti-microbial molecules is especially high in inflammatory edema (the tumor) due to the local influx of plasma from activated endothelia. CDCC, Complement Dependent Cellular Cytotoxicity: Complement components adhering on the surface of target cells or microbes interact with Complement receptors (See Fig. 20.8) on the cell membrane of granulocytes, macrophages and NK cells that become activated and kill the target cell or the microbe.


CHAPTER 4. CYTOKINES. Fig. 4.1. COMMUNICATION CODES WITHIN THE IMMUNE SYSTEM. In the dark of our body, various cells of the immune system interact with each other using a variety of different languages. Usually environmental signals are perceived through specific receptors expressed on the cell membrane. Some receptors are almost constantly present (constitutive) on the cell membrane during the life of the responder cells whereas other receptors are inducible, i.e. expressed only as a response to other signals or only during a peculiar stage of each cell life. Alarm signals and their receptors have been already considered in Chapter 2. The communication code based on cytokines is the subject of this Chapter, while the communication code based on MHC/HLA glycoproteins is discussed in Chapter 5. Finally the downstream signaling of various membrane receptors/ligands will be discussed considering various specific cell-cell interactions. In addition, all immune cells are subject to many hormonal influences. PPR, Pattern Recognition Receptors (See Figs. 2.4), TCR, T cell receptor (See Figs. 7.12).

Fig. 4.2. CYTOKINES: SELECTIVITY OF SIGNALS. The cell (a) secretes a cytokine in its micro-environment. The cytokine is perceived only by the surrounding cells expressing specific receptors for that cytokine (b). Following the capture of the cytokine this cell changes its behavior as dictated by signal transduced by the receptor. The other cells (c), while in contact with the secreted cytokine, does not express the specific receptor for this cytokine and do not perceive the cytokine message. The expression (or not) of the specific receptor makes selective a not-selective cytokine message.

46 Fig. 4.3. CYTOKINES: RELATED FAMILIES. Cytokines are molecules with many names. Commonly they are called Interleukins (IL), cytokines that interconnect (Inter: between) leukocytes (leuk)and activate (Greek, Kinos: movement) them. The progressive numbers of IL derive from their successive discovery. Somewhat arbitrarily, in other cases their name derives from the first biological activity attributed to the cytokine: Interferons (IFN), cytokines that were discovered for their ability to interfere with viral infections; Tumor Necrosis Factor (TNF), cytokines that were discovered for their ability to cause the necrosis of some tumors; Colony Stimulating Factors (CSF), cytokines that were discovered for their ability to promote the proliferation and differentiation of bone marrow progenitor cells towards granulocytes and macrophages; Transforming Growth Factors (TGF), cytokines that induce cell proliferation and neoplastic transformation.

Fig. 4.4. CYTOKINES: MAIN FEATURES. Cytokines are proteins secreted by an immune cell that modulates the behavior of other cells.

47 Fig. 4.5. CYTOKINES: PARACRINE SECRETION. The small amount of a cytokine secreted by a cell ( a in the Fig) is perceived only by the cells that are in its close micro-environment and express the specific receptor for that cytokine (cell b). The cytokine does not diffuse as a hormone because it is secreted in a very small amount and is rapidly and physiologically inactivated by specific inhibitors and quickly degraded in the kidneys.

Fig. 4.6. CYTOKINES: POLARIZED SECRETION. A small amount of a cytokine is secreted only in the synaptic area where the membrane of the secreting cell (Cell a in the Fig) interacts with the membrane of the cell that will receive the signal (Cell b).

Fig. 4.7. CYTOKINES: AUTOCRINE SECRETION. A cytokine can be secreted and utilized by the same cell. This happens when the cell secreting the cytokine also expresses on the cell membrane the specific receptor for that cytokine.

Fig. 4.8. CYTOKINES: ENDOCRINE SECRETION. In a few cases only a cytokine has an endocrine activity. For instance IL1 and IL6 secreted in the area invaded by microbes can reach the anterior hypothalamus and change the temperature setting and induce fever.

48 Fig. 4.9. COMMON FEATURES OF CYTOKINE RECEPTORS. The expression of cytokine receptors changes during the various stages of White Blood Cell (WBC) maturation and activation. Thus, the expression or not of a specific cytokine receptor confers selectivity to the not-selective cytokine message. In addition several WBC modulate the expression of cytokine decoy receptors. These receptors capture the cytokine but are unable to transduce an activation signal. Decoy receptors play an important role in inhibiting cytokine activity and controlling cytokine diffusion in the micro-environment.

Fig. 4.10. CYTOKINES: MAIN RECEPTOR FAMILIES. Cytokine receptors interact with the cytokine and transduce the signal to the nucleus. Often these receptors are made by two (dimers) or three (trimers) chains. Receptors of the common  chain family share the same signal transducing chain (CD 123). Also the receptors of the common  chain family share the same signal transducing chain (CD 131). Since the common  chain is a component of an important family of receptors, mutations of the common  chain gene causes a severe break down of the immune system (severe immunodeficiency) due to the failure of numerous distinct immune mechanisms. Since the gene for the common  chain is located on the X chromosome this immunodeficiency is called X-linked severe combined immunodeficiency (X-linked SCID, See Fig. 26.3).


Fig. 4.11. THE IL2 RECEPTOR. This important two-three chains receptor illustrates how a cytokine receptor varies during the progression of cell activation conferring selectivity to the cytokine message.

Fig. 4.12. THE POOR AFFINITY TWO CHAINS IL2 RECEPTOR. I. Resting lymphocytes express only the and the  chains of the IL2 receptor. Following the interaction with IL2, both chains transduce signals to the cell nucleus. However, the  chain does not bind IL2, while the  chain binds IL2 with very low affinity (For the concept of affinity see Fig. 15.5). Therefore, only anomalous very high concentration of IL2 in the lymphocyte microenvironment allows the binding of IL2 to the  receptor chain. Once bound to IL2, the chain assumes a new conformation.

Fig. 4.13. THE POOR AFFINITY TWO CHAINS IL2 RECEPTOR. II. Only the presence of an anomalous high concentration of IL2 in the lymphocyte micro-environment allows the binding of IL2 to the  chain. The new conformation acquired by the  chain bound to IL2 now permits its interaction with the  chain.


Fig. 4.14. THE POOR AFFINITY TWO CHAINS IL2 RECEPTOR. III. Both the  and  chain of the new complex transduce signals to the nucleus. The intra cytoplasmatic tails of both receptors are first phosphorylated (P in the Fig.) by the Janus kinases (JAK). Then the phosphorylated chains act as binding sites for the Signal Transducer and Activator of Transcription (STAT) factor (see Fig. 4.18).In this way high concentrations of IL2 directly activate T, B and NK cells.

Fig. 4.15. THE  CHAIN OF IL2 RECEPTOR. IV. A lymphocyte pre-activated by a ligand bound to antigen receptor and by membrane signals starts to express the  chain (CD25), a new chain of the IL2 receptor. The  chain binds IL2 at very low affinity and does not transduce activating signals.

Fig. 4.16. THE HIGH AFFINITY THREE CHAIN IL2 RECEPTOR. V. In the presence of IL2, the chain interacts with the  chain forming a new - receptor that binds IL2 with high affinity. The  receptor joining to the  chain forms a three chain high affinity IL2 receptor able to bind lL2 even when IL2 is present in low physiological concentrations. Then the  and  chains transduce the signals to the cell nucleus.


Fig. 4.17. THE HIGH AFFINITY THREE CHAIN IL2 RECEPTOR. VI. Following the interaction of the  and  chains with small, physiologic amounts of IL2, a trimeric --receptor is assembled. The cytoplasmatic tails of  and  chains are phosphorylated (P in the Fig.) by the Janus kinases.

Fig. 4.18. THE HIGH AFFINITY THREE CHAIN IL2 RECEPTOR. VII. Both in the two chain and three chain IL2 receptors, the phosphorylated tails of the and  receptor chains become the docking sites for STAT transcription factors. Bound STAT molecules are then phosphorylated by the Janus kinases (JAK). Dimers of phosphorylated STAT migrate to the nucleus where they activate the promoter of particular genes and trigger the progression of the cell cycle, cell proliferation and the formation of a cell clone.

Fig. 4.19. CYTOKINE RECEPTORS SHARING THE COMMON GAMMA CHAIN. The transmembrane receptor chain does not bind any cytokine while it acts as the main transducer chain for several distinct cytokine receptors. The  chain is encoded by a gene on the X chromosome. Since this chain is involved in the transduction of several important cytokine signals, the inactivation of the gene encoding the common  chain leads to the Xlinked Severe Combined Immunodeficiency (Xlinked SCID) (See Figs. 4.10 and 26.3).


Fig. 4.20. A FEW CYTOKINE CIRCUITS. This cartoon shows a few of the main cytokine messages delivered by M1 macrophages and Dendritic Cells (DC) when a microbial invasion has been perceived. Through the release of these various pro-inflammatory cytokines, the M1 macrophage orchestrates a complex multicellular inflammatory reaction. Moreover, each of the cells activated in this way releases its own repertoire of cytokines through which other cells (including macrophages and DC) are recruited, activated and induced to differentiate. The activation of endothelial cells leads to increase in adhesion molecule expression, blood flow with blood cell extravasation (See Fig.. 3.32).

53 Fig. 4.21. CHEMOKINES: GENERAL FEATURES. Chemokines (chemotactic cytokines) are classed in four families designated CC, CXC, CX3C and XC on the basis of the spacing of disulfide bonds. Chemokines of the CC family have two intra-chain disulfide bonds (In red in the Fig.) and the first two cysteine (C in the Fig.) residues are adjacent. Also chemokines of the CXC family have two intra-chain disulfide bonds but the first two cysteine residues are separated by a no conserved amino acid residue (X, red arrow). Chemokines of the CX3C family have two intra-chain disulfide bonds, and the two cysteines are separated by three spacer amino-acids. Finally those of the XC family have a single intrachain disulfide bond. The chemokines are referred to as CC, CXC, CX3C and XC chemokine ligands (CCL1, 2,..28; CXCL1, 2‌17; CC3L1; XC1, 2). The same abbreviations are used for their receptors (CCR1R.., CXC1R‌, CX3C1Rand XC1R..). Fig. 4.22. CONSTITUTIVE AND INFLAMMATORY CHEMOKINES. Constitutive chemokines play a central role in governing the homing of the various populations of White Blood Cells (WBC) and thus the general architecture of the immune system. Inflammatory chemokines released by sentinel cells in injured or infected tissues recruit locally reactive WBC. First inflammatory chemokines act on endothelial cells of blood capillaries and increase their expression of adhesion molecules (selectins, ICAM, Fig. 3.27). Then chemokines up-modulate the expression of integrins on WBC. In this way chemokines favor WBC adhesion to the activated endothelial cells and their exit from blood vessels. Once transmigrated out of the capillary, WBC are guided towards the highest chemokine concentration (chemotaxis, see Fig. 3.32).

54 Fig.4.23. CHEMOKINE RECEPTORS. Chemokine receptors (CCR, CXCR‌.) are expressed on the cell membrane of White Blood Cells (WBC). They are formed by seven transmembrane domain proteins coupled with G proteins. In this Fig., a prototypic chemokine receptor is drawn in blue. The gray arrow shows the chemokine binding site while the green arrow shows the G chain transduced signal.

Fig. 4.24.CHEMOKINE CIRCUITS. When macrophages, Dendritic Cells as well as other sentinel cells perceive a microbial infection they secrete a series of chemokines. These inflammatory chemokines are then selectively captured by specific receptors expressed on the membrane of several immune cells. The interaction between a chemokine and its receptor activates the gradient–dependent chemotaxis of the cell towards the area where the chemokine is secreted. In the Fig. the main cell populations activated by various CC and CXC chemokines are shown.

55 Fig. 4.25. TWO DISTINCT CHEMOKINE RECEPTORS MAY ACT AS CO-RECEPTORS FOR THE HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION. When the Human Immunodeficiency Virus (HIV) is bound to a CD4 cell membrane receptor, a domain of the gp120 on the HIV envelope changes conformation and binds the CCR5 and CXR4 chemokine receptors. This interaction is critical for the absorption of the HIV on the cell (See Figs. 26.6 and 26.7).

Fig. 4.26. THE -32 DEFECTIVE CCR5 CHEMOKINE RECEPTOR. A few persons inherit the -32 variant of the gene coding for the CCR5 receptor. The non-functioning CCR5 receptor encoded by this gene variant lacks 32 base pairs and therefore it does not bind the Human Immunodeficiency Virus (HIV) proteins. The HIV inability to bind the -32 variant hampers (when the -32 gene is in heterozygosis) or impedes (when it is in homozygosis) the HIV infection. The frequency of -32 variant of the CCR5 gene expression decreases from 14% in Northern Europe to 4% in Southern Europe, while its expression is rare or absent in Japan and Western and Central Africa.


CHAPTER 5. THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC). Fig. 5.1. THE MHC/HLA: I. A PECULIAR GENE CLUSTER. The genes of the MHC are those that vary the most from person to person: these genes are highly polymorphic. It is remarkable that this gene cluster is maintained substantially identical in all vertebrates. The reason why these genes did not undergo gene diaspora, a common genetic feature of other genes, is not yet clear.

Fig. 5.2. THE MHC/HLA: II. MAJOR FEATURES. Some HLA alleles are relatively common in the human population while others are very rare.




Fig. 5.5. FEATURES OF HLA GLYCOPROTEINS CODED FOR BY CLASS I GENES. I. This schematic drawing of the HLA Class I glycoprotein should be integrated with the scheme of the same glycoprotein on Fig. 5.6.

Fig. 5.6. FEATURES OF HLA GLYCOPROTEINS CODED FOR BY CLASS I GENES. II. This schematic drawing of the HLA Class I glycoprotein should be integrated with the scheme of the same glycoprotein on previous Fig.


Fig. 5.7. THE BETA (ď ˘)-2 MICROGLOBULIN. This non-HLA coded protein is an essential component of Class I HLA glycoproteins.

Fig. 5.8. THE HLA CLASS I GLYCOPROTEINS ARE POLYGENIC AND POLYMORPHIC. To be polygenic means that each person possesses several loci coding for slightly different HLA Class I glycoproteins. To be polymorphic means that in the human population for each of these HLA Class I loci there are several alleles.

Fig. 5.9. THE EXTENSIVE POLYMORPHISM OF PRINCIPAL HLA CLASS I GLYCOPROTEINS. The high number of alleles (polymorphism) present in the human population for HLA Class I gene (A, B and C) and Class II genes (DP, DQ and DR genes, see Fig. 5.14) makes it improbable to find two human beings expressing all the same HLA Class I and Class II glycoproteins. Of each of these numerous HLA-A, HLA-B and HLA-C alleles, every person expresses only two alleles, the one inherited from the mother and the one inherited from the father.




Fig. 5.12. FEATURES OF HLA GLYCOPROTEINS ENCODED BY CLASS II GENES. I. This schematic drawing of the Class II HLA glycoprotein should be integrated with the scheme of the same glycoprotein on Fig. 5.13

60 Fig. 5.13. FEATURES OF THE HLA HETERODIMER ENCODED BY CLASS II GENES. Ianthe  and  chains are made by two extracellular domains (alpha 1, alpha2; beta 1 and beta2), a transmembrane domain (S in the Fig.) and a short intra-cytoplasmatic tail (l in the Fig.).This schematic drawing of the Class II HLA glycoprotein should be integrated with the scheme of the same glycoprotein on Fig. 5.12.

Fig. 5.14. POLYMORPHISM OF PRINCIPAL HLA CLASS II GLYCOPROTEINS. The high number of alleles (polymorphism) present in the human population for DR-A and DR-B, DQ-A and DQ-B, and DP-A and DP-B genes makes it improbable to find two human beings expressing all the same HLA Class I and Class II glycoproteins. Of each of these numerous DR-A and DR-B, DQ-A and DQ-B, and DP-A and DP-B alleles, every person expresses two alleles only, one inherited from the mother and one inherited from the father.



Fig. 5.16. CO-DOMINANCE OF HLA ALLELES INHERITED FROM THE MOTHER AND THE FATHER: The creation of our molecular individuality.






Fig. 5.20. MAIN FEATURES OF CLASS III HLA GLYCOPROTEINS. In addition, several olfactory receptors are coded by Class II genes.

Fig. 5.21. A FEW CRITICAL OUTCOMES OF THE POLYMORPHISM OF HLA GENES. The variant of HLA genes we inherited by our mother and father affects several aspect of our life, some of which not yet fully defined. The kind of Class I and Class II grooves we have inherited allows (or not) the binding and presentation of certain peptides, a critical issue in the activation of T cells. To have inherited certain HLA variants allows (or not) to mount an efficient T cell reaction. Thus, some combinations of HLA genes could be especially protective against a particular microbial disease, and therefore propagate in the population of the world area where the disease is endemic. The expression or not of HLA glycoproteins inhibit or activate NK activity (See Fig. 3.26). Moreover, the intensity of our reaction to microbes depends not only by the grooves of Class I and Class II glycoproteins we have inherited but also by the levels of the soluble anti-microbial molecules coded by Class III genes. The intensity of these reactions also shapes our microbial flora (or microbiome, see fig. 1.4). In this way HLA polymorphism affects our individual smell. The individual smell and the polymorphism of olfactory receptors coded by Class III genes appear to bias the selection of sexual partners in mice and perhaps unconsciously contribute to this selection also in humans.



Fig. 6.1. HLA GLYCOPROTEINS ARE FLOATING ON THE CELL MEMBRANE OF HUMAN NUCLEATED CELLS. On the cell membrane, both Class I and Class II HLA glycoproteins display a protein fragment (a peptide) bound to them (In red in the Fig.).

Fig. 6.2. HLA GLYCOPROTEINS DISPLAY BOUND PEPTIDES. A. The majority of Class I HLA glycoproteins present on the cell membrane display a 8-10 amino acid long protein fragment (a peptide) entangled in the groove formed of alpha 1 and alpha 2 domains of the  chain. Also the majority of Class II HLA glycoproteins display a peptide entangled in the more flexible groove made by alpha 1 and beta1 domains of the  and  chains. Peptides in the groove of Class II HLA glycoproteins are longer (13 amino acids or more). Peptides associated to Class I HLA glycoproteins derive from proteins broken down in the cytosol whereas those associated to Class II HLA glycoproteins arise from proteins broken down by acidic hydrolysis in endocytic vesicles. B. Ribbon diagram of the structure of the HLA Class I groove. The floor is made by eight antiparallel beta strands while two alpha helices form the lips of the peptide binding groove.


Fig. 6.3. FROM WHERE ARE COMING THE PEPTIDES WHO END UP IN THE GROOVE OF CLASS I HLA GLYCOPROTEINS? Cell proteins are continuously renewed: old, surplus or altered proteins are destroyed and substituted by new synthetized proteins. The 10 x 106 ribosomes associated with the endoplasmic reticulum of a cell produce about 10 x 106 protein/minute. Autophagy is the dynamic physiological process of cellular housekeeping in which dysfunctional organelles, proteins and endocellular microbes are removed and degraded. Selective autophagy is a degradative pathway that controls the quality and abundance of proteins. Old, incorrect or surplus proteins are bound to the 100 x 106 ubiquitin molecules present in the cytoplasm and eliminated. Proteins bound by ubiquitin lose their conformational structure and interact with the lid of the proteasome. Ubiquitin is then removed and re-utilized while the protein introduced inside the proteasome is chopped into 8-9 amino acid long peptides.


Fig. 6.4. THE PROTEASOME. 26S proteasome structure as determined by electron microscopy (Left in the Fig.) and its schematic drawing (Right). The 26S proteasome is a cylinder-shaped particle consisting of a multi catalytic core, the 20S proteasome, and two regulatory complexes known as 19S lid or regulatory complexes. The 20S proteasome core consists of two outer rings, made up of seven different subunits with chymotryptic, tryptic and postglutamyl peptide hydrolytic activity.


Fig. 6.5. WHERE CYTOPLASMATIC PEPTIDES MET CLASS I HLA GLYCOPROTEINS? Cytoplasmatic peptides emerging from the proteasome are rapidly digested in amino acids by the aminopeptidases in the cytosol. However, a few lucky peptides are able to bind to particular transporters (Transporters associated with Antigen Processing, TAP1 and TAP2) on the membrane of the endoplasmic reticulum. TAP1 and TAP2 transporters are coded by Class II genes of the HLA complex. Lucky peptides reaching the endoplasmic reticulum through TAP1 and TAP2 transporters survive longer and may bind to the groove of nascent HLA Class I glycoproteins. In the endoplasmic reticulum the recently synthesized  chain of the HLA Class I glycoprotein binds a first chaperon protein, Calnexin. Subsequently, the  chain gets associated to the 2microglobulin, dissociates from Calnexin and binds Calreticulin another chaperon protein that sense nascent glycoproteins and Tapasin, a chaperon proteins that interacts with TAP transporters. The interaction of Tapasin with TAP1 and TAP2 transporter helps the interaction between the nascent glycoprotein and peptides. The loading of the peptide in the groove induces structure rearrangements in the nascent glycoprotein resulting in its dissociation from chaperon proteins. Other proteins scan the quality of newly formed peptide-Class I glycoprotein complex. By interacting with the structural elements that anchor the peptide in the groove (See Fig.6.6) they displace the peptides that have a modest affinity for the groove. Finally, Class I glycoproteins with a peptide in the groove leave the endoplasmic reticulum and move towards the cell surface. Also a small fraction of glycoproteins without the peptide in the groove reach the cell surface. These Class I glycoproteins can be loaded by peptides present in the body fluids. REFERENCE: A Blees et al., Nature 2017, 551:526.

68 Fig. 6.6. HOW THE LUCKY PEPTIDES BIND THE GROOVE OF CLASS I HLA GLYCOPROTEINS. The lucky peptides surviving a quick digestion to amino acids may bind the groove of recently formed Class I HLA glycoproteins. With high affinity. To remain associated with a Class I HLA groove a peptide has to establish multiple interactions (ionic interactions, hydrogen bonds) with the amino acids of the floor and the walls of the groove. The ability to bind or not, and the strength of the binding (the affinity) depends on both the structure of the groove and the amino acid sequence of the peptide. The groove of each Class I HLA allelic variant has a distinct amino acid sequence. Therefore grooves with different shape and electric charges can (or cannot) interact with the anchor amino acids of the peptide. Therefore, some Class I allelic variants bind certain peptides very well while other alleles are not able to do so. This genetic control of the ability to present a peptide is an issue of crucial importance since it is possible that a person having inherited certain Class I HLA alleles presents a particular peptide very well whereas another person does not present or poorly presents this particular peptide, having inherited a different Class I HLA allele. Moreover, the same peptide may bind the various grooves differently assuming a different conformation in order to adapt to the grove of distinct Class I HLA glycoproteins. The different conformations assumed by the Class I HLA glycoprotein and the bound peptide (HLA-p) are recognized by different receptors on the surface of T cells. Every groove of a Class I HLA glycoprotein can bind about 1000 different peptides. Moreover, every person displays multiple Class I HLA grooves (six at least considering only the HLA-A, -B, and –C glycoproteins inherited from the mother and the father). This promiscuity of the peptide-groove interaction and the multiplicity of distinct Class I HLA glycoproteins increase the individual probability to bind and present a particular peptide at least with one of the groves. In addition, the polymorphism of Class I HLA glycoproteins present in a population increases the probability that at least a few persons have a groove able to efficiently present a given peptide. A few variable portions of the variable (V) domain of the T Cell Receptor (TCR) interact with the peptide (Complementary determining region 1, CDR1); while other portions interact with the HLA glycoprotein (CDR2 and CDR3) (See Fig. 7.10, 7.12, 7.13 and 9.8).


Fig. 6.7. PEPTIDES PRESENTED BY CLASS I HLA SHOW WHAT IS GOING ON INSIDE THE CELL. On the cell membrane, the half-life of HLA glycoproteins bound to a peptide is about 6 hours. Newly made Class I HLA glycoproteins transit from the endoplasmic reticulum to the post Golgi vesicles moving toward the cell membrane. This mean that new HLA glycoproteins continuously display peptides derived from proteins recently destroyed in the cytoplasm.


Fig. 6.8.FROM WHERE ARE COMING THE PEPTIDES WHO END UP IN THE GROOVE OF CLASS II HLA GLYCOPROTEINS? While the synthesis of Class II HLA glycoproteins goes in parallel with that of HLA Class I, the peptides bound to their grooves have a different origin. In the endoplasmic reticulum, the  and  chains of HLA Class II glycoproteins are synthesized independently. The chaperon Invariant chain binds them while the Clip fragment seals their peptide groove. Therefore, cytosolic peptides transported by TAP 1 and TAP2 into the endoplasmic reticulum cannot bind to the groove of Class II HLA glycoproteins. The three molecular complexes made by the  and  chains of HLA glycoproteins and the Invariant chain transit from the endoplasmic reticulum to the post Golgi vesicles moving toward the cell membrane. During this journey these vesicles fuse with phagolysosomes.


Fig. 6.9. WHERE PEPTIDES FROM ENDOCYTED STRUCTURES MET CLASS II HLA GLYCOPROTEINS? During their journey towards cell membranes, post Golgi vesicles containing immature Class II HLA glycoproteins are guided by the Invariant chain to fuse with an acid phagolysosome containing peptides of the endocyted structures. The acidic pH and the action of peptidases cleave the Invariant chain leaving only the groove sealed by Clip. Subsequently, the association of nascent Class II glycoproteins with DM and DO chaperon proteins allows Clip dislodgment and favors the binding of peptides. DM and DO chaperon proteins are coded by additional Class II genes (See Fig. 5.19). Mature Class II glycoproteins displaying peptides from the endocyted structures are then inserted on the cell membrane. As the half-life of membrane HLA class II glycoproteins is of about 6 hours, peptides from the structures recently endocyted are displayed on the cell surface. The interaction established by the peptide with the floor and walls of the groove of Class II molecules are similar to those illustrated on Fig. 6.6. Here, however, the groove is made by two chains (the ď Ą and ď ˘ chains). It is longer and more flexible


Fig. 6.10. ENDOCYTED PEPTIDES END UP IN THE GROOVE OF CLASS II HLA GLYCOPROTEINS. A phagolysosome (Orange in the Fig.) containing peptides (Red) deriving from endocyted proteins fuses with a post-Golgi vesicle transporting newly synthetized HLA Class II glycoproteins towards the cell membrane. In the post-Golgi vesicle the Class II HLA glycoproteins interacting with the Invariant chain have the groove sealed by the Clip fragment of the invariant chain (A in the Fig). Phagolysosome acid proteases degrade the Invariant chain (B) and the Class II HLA glycoproteins with the groove still sealed by the Clip fragment (C) binds the DM glycoprotein. DM and DO (not shown) are other HLA Class II glycoproteins not expressed on the cell surface but only in the cytoplasmatic vesicles (See Fig. 5.19). The interaction with DM stabilizes the HLA Class II glycoproteins, permits the release of Clip (D) and the binding of the peptide.




Fig. 7.2. ADAPTIVE IMMUNITY. I: MAIN FEATURES. T and B cells are the key cells of the adaptive immunity. Roughly, in a person there are 1012 T and B cells. Each of those virgin T or B cell express many copies of an unique, individual receptor specific for a foreign molecule, which is called antigen. As almost every lymphocyte express a receptor different from that of the others, the total repertoire of different antigen receptors of T and B cells of a person exceed 1011.This means that the T and B population of a person is able to recognize more than 1011 different antigens. Fig. 7.3. ADAPTIVE IMMUNITY. II: CLONAL SELECTION. Adaptive immunity is a defense strategy based on: A. Cell membrane receptors recognizing with high precision (high specificity) the molecular features of their target (here defined as the antigen, red triangle in the Fig.); B. Proliferation (clonal expansion) of the lymphocyte whose receptor better interact with the antigen; C. Competition among lymphocytes expressing receptors able to bind the antigen with different strength (different affinity, see Fig. 15.5).


Fig. 7.4. CELLS OF ADAPTIVE IMMUNITY. T and B cells selectively interact with the specific cognate antigen at high precision (high affinity) and orchestrate the activation of multiple and complex defense reactions.

Fig. 7.5. THE SELECTIVE ACTIVATION OF A T AND B CELL. In the dark of the interior of our body, the life of T and B lymphocytes is modulated by a large series of membrane receptors perceiving environmental signals. Following the capture of its ligand, a receptor transduces the signal to the nucleus. The integration of multiple signals captured by distinct receptors modulates gene activation and cell activities. Unlike cells of innate immunity (See Fig. 2.2), every T and B cell expresses many copies of an unique individual antigen-specific receptor: the T Cell Receptor (TCR) and B Cell Receptor (BCR). The TCR expressed on the membrane of a virgin T cell are different from those of all the other virgin T cells. Similarly, the BCR on the membrane of a virgin B cell are different from those of all the other virgin B cells. The selectivity in the activation of a particular T cell rests on the interaction of its individual TCR with the cognate HLA-p complex (See Figs. 7.10, 7.12). On the other hand, the selective activation of a particular B cell rests on the interaction of its individual BCR with the cognate antigenic molecule (See Fig. 15.4). /Signals provided by the other receptors guide the T and B cell maturation, differentiation, and homing and concur to cell activation. Once they are selectively activated, T and B cells give rise to large clone of effector/memory cells, all expressing the same individual TCR (or BCR) of the cell initially activated (See Fig. 7.3).


Fig. 7.6. THE T CELL. Ultrastructural (A) and histological (B) aspects. A. The image derived from an electron micrograph shows a T cell with a round nucleus surrounded by a thin rim of cytoplasm containing several ribosomes, scattered mitochondria, scarce endoplasmic reticulum and very few lysosomes. Short microvilli are present at the cell surface. B. At light microscopy a T cell displays a round nucleus surrounded by an almost imperceptible rim of cytoplasm. T cells are morphologically identical to B cells. The antigen receptor expressed by T cells on the cell membrane is called T cell receptor (TCR). The TCR does not interact with foreign antigens in their natural conformation. The TCR binds only HLA glycoproteins presenting an antigen peptide in their groove (HLA-p, See Figs. 7.12 and 9.8).

Fig. 7.7. ORIGIN AND MATURATION OF ď Ą-ď ˘ T CELLS. As all White Blood Cells, T cells originate from bone marrow maturing passing through characteristic developmental stages (See Fig. 3.4).



Fig. 7.9. MAIN SUBSETS OF HUMAN T CELLS. The large - T cell population present in the human body can be subdivided in subsets on the basis of both their surface markers (the so called CD, See Fig. 21.7) and their main function. T cells expressing the CD4 marker (the CD4+ lymphocytes) interact with Class II HLA glycoproteins and peptides entangled in their groove (HLA-p). The majority of CD4+ T cells display helper activity (See Chapter 11). However, a few CD4+ T cells have instead a cytotoxic activity and kill HLA Class II positive target cells. On the contrary, T cells expressing the CD8 marker (the CD8+ lymphocytes) interact with Class I HLA-p. The majority of CD8+ T cells display cytotoxic activity (See Chapter 10). However, a few CD8+ T cells may have a T helper activity. Double negative (CD4-, CD8-) invariant NK cells display a TCR interacting with CD1 HLA Class I glycoprotein (See Figs. 5.18) presenting microbial lipids and glycolipids entangled in their groove. The immense diversity of the TCR repertoire and function of T cell populations ensures that t1he immune system can respond to almost any peptide in a high specific manner.


Fig. 7.10. ACTIVITY OF COMMON -T CELLS. The - T cell receptor (TCR) does not interact with foreign antigens but binds only HLA glycoproteins presenting an antigen peptide in their groove (HLA-p). T cells in circulation in the body tissues and in the lymphatic organs continuously interact with body cells. Once an interaction is established, the - TCR expressed on the T cell membrane probes Class I and Class II HLA glycoproteins and the peptides they present (HLA-p) on the surface of the target cells. Class II HLA p are scanned by the - TCR of CD4+ T cells while Class I HLA-p glycoproteins are scanned by the - TCR on CD8+ T cell membrane. Peptides generated from normal proteins are ignored (See chapter 22) whereas anomalous peptides will trigger T cell response.

Fig. 7.11. THE TCR OF - T CELLS. Both  and  chains are members of the Ig super family (See Fig. 13.5). Each chain is constituted by two globular extracellular domains, a transmembrane domain made by hydrophobic amino acids and a short intracytoplasmatic tail, unable to transduce the signal to the cell nucleus. The amino acid sequence of the most external domain (the V domain) is typical of each virgin T cell. This individual difference allows the TCR of each virgin T cell to react with a different HLA-peptide complex. By contrast the C domain is identical in all - T cells.


FIG. 7.12. THE -TCR OF CD3+, CD8+ T CELL SCANS THE CLASS I HLA GLYCOPROTEINS AND PEPTIDES IN THEIR GROOVE (HLA-p). A few portions of the variable (V) domains of both  and chains of the TCR (the complementary determining regions CDR1 and CDR2, see Figs. 7.13) should have a molecular conformation allowing a close interaction with the more external structures of HLA glycoproteins. A distinct portion of the TCR (the hypervariable portion, CDR3) should have a conformation allowing a close interaction with the shape of the peptide entangled in the groove of HLA glycoproteins. When the reciprocal molecular complementarity allows a high affinity interaction of the TCR with the HLA-p complex, activation signals are transduced to the nucleus of the T cell.

Fig. 7.13. WHAT A - T CELL RECOGNIZES. Two portions (CDR1 and CDR2 2) of the TCR mainly interact with the amino acids of the HLA molecule. The third portion, the CDR3, the more variable portion interacts with the peptide entangled in the groove of the HLA molecule. A similar three-component interaction guides the activation of all - T cells. iNK cell however, display a TCR interacting with CD1 HLA Class I glycoprotein (See Fig 5.18) presenting microbial lipids and glycolipids (and not peptides!) entangled in their groove.


Fig. 7.14. TCR SIGNAL TRANSDUCTION DEPENDS ON SEVERAL MONOMORPHIC CHAINS ( CHAINS and CD3 COMPLEX) ASSOCIATED TO THE TCR. When the TCR on T cell surface interacts with high affinity with the HLA-p complex, activation signals are transduced to the nucleus of the T cell. However, the TCR does not transduce directly the signal to the nucleus. High affinity interaction of the TCR with the HLA-p complex leads to the activation of four transducer chains [the (zeta) chains, and the three  (gamma), (delta) and (epsilon) chains defined as members of the CD3 complex, a typical marker of T cells].

Fig. 7.15. TRANSDUCTION OF THE TCR SIGNAL. High affinity interaction of TCR with the HLA-peptide (HLA-p) complex leads to the activation of the (zeta) chains, and the three  (gamma), (delta) and (epsilon) chains 11defined as members of the CD3 complex. The two tyrosines of a peculiar amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif) on the  chain and on the three CD3 chains are phosphorylated and form the docking site of the signaling Zap70 ( chain Associated Protein 70) (See Figs. 9.9, 9.10). Each CD3 chain expresses one ITAM, whereas the chains expresses three ITAM.




Fig. 7.17. SCHEMATIC DRAWING OF HOW CD3+ CD8+ T CELLS SCAN CLASS I HLA GLYCOPROTEINS AND PEPTIDES IN THEIR GROOVE. The CD8 co-receptor interacts with a monomorphic (invariant) sequence 3 domain of the  chain of class I HLA glycoprotein.

Fig. 7.18. SCHEMATIC DRAWING OF HOW CD3+ CD4+ T CELLS SCAN CLASS II HLA GLYCOPROTEINS AND PEPTIDES IN THEIR GROOVE. The CD4 co-receptor interacts with a monomorphic (invariant) sequence of the 2 domain of the  chain of a class II HLA glycoprotein

82 .

Fig. 7.19. INVARIANT NK T CELLS (iNK T cells) form a distinct but heterogeneous population of lymphocytes expressing a - T Cell Receptor with a limited variability (defined as invariant). iNKT cells predominantly are found at barrier sites. (For details see also Figs. 8.17).

Fig. 7.20. GAMMADELTA (-) T CELLS. The T cell receptor of this minor population of T cells is made by a gamma () and a delta () chain. (For details see also Fig. 8.16).


CHAPTER 8. THE THYMIC EDUCATION OF T CELLS. Fig. 8.1. T CELL SUB-POPULATIONS. T cells mature in the bone marrow. Then, in the absence of Notch fate signals, they exit from the bone marrow and are attracted to the thymus where their differentiation progresses. Each cell generates its peculiar, individual T Cell Receptor (TCR) and differentiate in one of the four populations of T cells: a) The most common -, CD3+ and CD4+ cell population; b) The less common -, CD3+ and CD8+ T cell population; c) The rare iNK T cells; d) The rare -, CD3+ T cells.

Fig. 8.2. THYMIC MATURATION OF T CELLS. Thymic stromal cells are connective cells that support the differentiation of T lymphocyte precursors arriving into the thymus from the bone marrow. Signals from thymic stromal cells trigger gene recombination events allowing each T cell to both acquire its individual T Cell Receptor (TCR) and differentiate in T helper (Th) and T killer (Tk) cell. Children born with a thymic defect may display a major immunodeficiency since normal T cell maturation is impaired (See Chapter 26).



Fig. 8.4. EDUCATION OF IMMATURE T CELLS ENTERING THE THYMUS. I. Early thymocyte differentiation takes place independently from interaction with HLA glycoproteins and the peptides in their groove (HLA-p) expressed on the surface of thymic epithelial-stromal cells.

Fig. 8.5. EDUCATION OF IMMATURE T CELLS ENTERING THE THYMUS. II. Until the double positive (CD4+, CD8+) stage T cell differentiation continues to take place independently from the interaction with HLA glycoproteins. By contrast, starting from this differentiation stage, the further differentiation of immature T cells (differentiation of thymocytes) is critically modulated by the interactions with HLA glycoproteins presenting peptides in their groove (HLA-p). The strength of signal transmitted by these interactions determines whether a developing T cell is selected to die or survive. TCR-HLA-p signaling strength below minimum (for example, non-functional TCR) causes death by neglect. By contrast, strong signals cause cell survival and cell proliferation. Signal strength is also influenced by the recruitment of T cell co-receptors (CD4 and CD8, See Figs 7.16 and 7.16).

85 Fig. 8.6. EDUCATION OF IMMATURE T CELLS ENTERING THE THYMUS. III. In the next educational step, T cells are selected for a low T Cell Receptor (TCR) signaling strength: selfreactive TCR induces cell death. NEGATIVE SELECTION. Thymocytes that have generated a TCR interacting at high affinity with self HLA glycoproteins presenting self peptides in their groove (HLA-p) undergo apoptosis (Central tolerance, See Figs.23.1, 23.2) or become a natural T regulatory (Treg) cell (See Fig. 22.12). Thus, apoptotic cell death becomes an important and dramatic occurrence since it involves the majority of thymocytes. The utmost importance of this negative selection of the TCR repertoire rests in the deletion of autoreactive cells with a TCR interacting at high affinity with self HLA-p. ROLE OF AIRE IN NEGATIVE SELELCTION. To provide a more broad negative selection of potentially autoreactive T cells, thymic epithelial cells express the Auto Immune Regulator (AIRE) gene. This gene encodes a transcriptional factor enabling epithelial thymic cells to express proteins that normally are expressed only by peculiar peripheral body tissues. SINGLE POSITIVE THYMOCYTES. Thymocytes that did not die by neglect and escaped negative selection by self HLA glycoproteins with self peptides in their groove (HLA-p) are CD4+ CD8+ double positive cells expressing a TCR that interacts with self HLA-p with low affinity. Depending on the HLA-p recognized by their TCR, double positive thymocytes become single positive: those with a TCR recognizing Class I HLAp glycoprotein will keep the CD8 co-receptor expression, whereas those with a TCR recognizing Class II HLA-p will keep CD4 co-receptor expression. EXIT FROM THE THYMUS. Following this complex and dramatic selection of the TCR repertoire, T cells emerging from the thymus express a TCR that recognizes self HLA-p at low affinity. The interaction of their TCR with self HLA-p is strong enough to transduce anti-apoptotic signals from the environmental cells but is not sufficient for T cell activation. These surviving cells enter the blood as mature virgin (naĂŻve) T cells. This cell population expresses a huge repertoire of different TCR since every T cell has generated its own TCR. A TCR binding self HLA-p at low affinity may interact with high affinity with self HLA glycoproteins expressing a foreign peptide or against foreign HLA glycoproteins. Foreign peptides and foreign HLA glycoproteins display only a few amino acid differences from self HLA-p and these differences may allow the establishment of a high affinity interaction with the TCR expressed by a virgin T cell.



Fig. 8.8. STRUCTURE OF THE HUMAN THYMUS. The thymus is practically divided into two histologically defined regions, the cortex and the medulla, each of which contains different thymic epithelial cells. In adults, T cell precursors enter the thymus at the cortico-medullary junction, and then begin a highly ordered differentiation program. Uncommitted progenitors, CD4-CD8- double-negative undergo a proliferative clonal expansion. T cell lineage commitment and the onset of T-cell receptor rearrangement and the transition from double negative to CD4+CD8+ double-positive cells occurs in the sub capsular cortex. Then, double positive cells migrate into the medulla where they differentiate into either CD4+ or CD8+ single-positive cells. Positive selection occurs mainly in the cortex, and requires cortical thymic epithelial cells, whereas negative selection occurs mainly in the medulla, and is mediated by medullary thymic epithelial cells and thymic DC. 98% of the thymocytes that enter or develop in the thymus die in the thymus by apoptosis during the complex educational process.

87 Fig. 8.9. HISTOLOGICAL SECTION OF THE THYMUS STAINED WITH HEMATOXYLIN AND EOSIN. The thymus is surrounded by a thin fibrous capsule. In the thymus two anatomically and functionally distinct regions can be identified, the outer cortex and the inner medulla. Fibrous trabeculae create numerous thymic lobules. The cortex appears darker because of the much higher number of precursor T cellsthymocytes than of medulla. The blue staining of the nuclei of the crowded thymocytes hides the endodermal-derived epithelial cells, forming a reticular meshwork. These cells contain long processes creating an interconnected network and a framework of supporting cells. In addition, the thymus harbors macrophages, mainly located in the cortex, and DC mainly located in the medulla. In their maturation, thymocytes move from the sub capsular region into the medulla. With ageing the thymus is progressively replaced by adipose tissue.

Fig. 8.10. HISTOLOGICAL SECTION OF THE THYMUS STAINED WITH AN ANTI-CD3 MONOCLONAL ANTIBODY. Staining with monoclonal antibodies (See Chapter 21) binding CD3 shows that the vast majority of the cells in the thymus are CD3 positive (CD3+) thymocytes. These cells are so numerous that they hide the reticular epithelial meshwork.

Fig. 8.11. HISTOLOGICAL SECTION OF THE THYMUS STAINED WITH AN ANTI-Ki-67 MONOCLONAL ANTIBODY. Antibodies binding Ki-67, a protein expressed by proliferating cells, disclose an intense proliferative activity. About a week after their arrival in the thymus, precursor T cells enter a phase of intense proliferation (positive selection) followed by a complex differentiating process. This process leads to the survival of only a minor population of mature T cells expressing a T Cell Receptor (TCR) able to bind with low affinity self HLA glycoproteins presenting self peptides.

88 Fig. 8.12. IMAGE OF A FRAGMENT OF HUMAN THYMUS OBTAINED BY SCANNING ELECTRON MICROSCOPY. I. The extremely numerous developing thymocytes (the spherical cells) occupy the small niches and interstices formed by the thymic epithelial cells. This and the following picture (See Fig. 8.13) well illustrate the close cell-to-cell contacts leading to the interactions of developing thymocytes and thymic cells.

Fig. 8.13. IMAGE OF A FRAGMENT OF HUMAN THYMUS OBTAINED BY SCANNING ELECTRON MICROSCOPY. II. The fragment of human thymus was washed in a tissue culture medium before the scanning electron microscopy procedure. When the majority of thymocytes are washed out, the extensive network of epithelial cells becomes evident. This and the previous picture show clearly the intimate and continuous interaction taking place between the membranes of developing thymocytes and thymus epithelial and lymphoid cells. These interactions form the basis of negative and positive selections of thymocytes

89 Fig. 8.14. STEPS OF THYMOCYTE EDUCATION. The education of T cells in the thymus is based on a complex series of massive cell deaths (negative selections) alternate to intense cell proliferations (positive selections). In the thymus, CD4-CD8-double-negative thymocytes which do not yet express the TCR or the co-receptors CD4 and CD8, undergo the rearrangements of –T Cell Receptor genes. Cells that rearrange successfully the TCR gene become CD3+. These cells also express both the CD4 and the CD8 co-receptors and are called double-positive cells (CD4+, CD8+). In the thymus cortex a first selection takes place: double positive thymocytes that have generated a - TCR unable to interact with self HLA glycoproteins presenting peptides in their groove (HLA-p) expressed on the surface of cortical epithelial cells die by neglect. By contrast, those that have generated a TCR interacting with self HLA-p are induced to proliferate (positive selection). Cells with a TCR binding HLA class I glycoproteins retain the expression of the CD8 but no longer express the CD4 and become CD3+, CD8+, CD4-. Cells with a TCR binding HLA class II glycoproteins retain the expression of the CD4 but no longer express the CD8, and become CD3+, CD8+, CD4+. In this way a double positive CD4+ and CD8+ cell becomes a single positive cell, CD4+ or CD8+. These single positive cells migrate to the thymus medulla. Here, the cells with a TCR binding self HLA-p with high affinity are induced to commit apoptosis. This negative selection is a dramatic event since the majority of single positive thymocytes die. The few surviving thymocytes express a TCR binding self HLA-p with low affinity. These thymocytes are induced to proliferate again and eventually migrate out to the periphery as mature virgin T cells.


90 Fig. 8.16. GAMMA-DELTA (- T CELLS. This minor population of T cells with a TCR resulting from the rearrangement of - genes originate from CD4- CD8- double negative thymocytes. The TCR of the majority of- T cells recognizes directly (i.e. without the presentation by HLA molecules) microbial metabolites, antigens rapidly displayed following infection and other forms of cell stress. Once activated, - T cells cell produce pro-inflammatory cytokines (IL17, IFN…) and chemokines. Other- T cells may acquire a cytolytic activity (See also Fig 7.20)

Fig. 8.17. iNKT CELLS. (See also Fig. 7.19) Another minor population of CD4+ CD8+ double positive thymocytes expresses a very limited T cell receptor repertoire (invariant TCR). The TCR of iNK T cells is positively selected to interact with CD1, a non-polymorphic Class Ib HLA glycoprotein (See Fig. 5.18) involved in the presentation of microbial lipids and glycolipids. At the barrier sites iNKT cells recognizing conserved molecules of commensal microbes (Nformyl peptides presented by Class Ib HLA glycoproteins) that normally colonize barrier surfaces such as those of the skin and gut do not directly retaliate against the bacterium, but instead aids tissue repair. In this way, the immune response elicited control microbial containemet rather than driving bacterial elimination which is usually accompanied by inflammation and tissue damage. REFERENCE: JL Linehan et al, Cell 2018, 172:784; P. Klenerman and G. Ogg, Nature 2019, 555:594.




Fig. 9.2. THE CONCURRENCE OF NUMEROUS SIGNALS IS REQUIRED TO ACTIVATE A VIRGIN T CELL. The activation of a virgin T cell is triggered by specialized cells, the Antigen Presenting Cells (APC). Only these cells provide the multiplicity of signals required for virgin T cell activation. This complexity of triggering signals secure against incorrect activation of T virgin cells. Once activated, a virgin T cell quickly generates a clone of effector/memory T cells that will persist almost indefinitely (See Chapter 24). Therefore, an erroneous T cell’s activation against self antigens can lead to a persisting and devastating autoimmune disease.

92 Fig. 9.3. ROLES OF ANTIGEN PRESENTING CELLS (APC) IN VIRGIN T CELL ACTIVATION. The conversion of an APC from a cell focused in antigen capture into a cell dedicated to antigen presentation to a T cell is clearly evident with Dendritic Cells (DC). Through the expression of Pattern Recognition Receptors (PPR, see Figs. 2.4) immature DC perceive the presence of foreign bodies. Their enhanced phagocytic activity allows an efficient antigen capture. Then, DC present the peptides derived from the captured foreign structure in the groove of Class II HLA glycoproteins. Antigen phagocytosis along with alarm and inflammatory signals induces the maturation of immature DC into mature DC. DC travel to the lymphatic organs where they home into T cell areas (See Figs. 17.5, 17.6 and 18.3). Fig. 9.4. ANTIGEN CAPTURE AND PRESENTATION TO T CELLS. Pattern Recognition Receptors (PPR) and Toll receptors (See Fig. 2.4) allow Antigen Presenting Cells (APC) to sense and capture foreign molecules in the body periphery. Then, alarm signals and pro-inflammatory cytokines induce APC maturation and mobilization. From the tissues where the antigen capture has taken place, mature APC migrate to local lymphatic vessels and then slowly travel towards the draining lymph node (Figs. 17.6, 17.6) where they present peptides of the captured antigen in the groove of their HLA glycoproteins.

93 Fig. 9.5. THE JOURNEY OF ANTIGEN PRESENTING CELLS (APC) ENDS IN THE T CELL AREAS OF DRAINING LYMPH NODES. Afferent lymphatic vessels collect and channel interstitial fluid and APC from the periphery to the sub capsular sinus of the lymph node (See Fig. 18.2). During their long journey to the lymph node, APC digest the captured antigen and present antigen peptides in the groove of their HLA glycoproteins (HLA-p).

Fig. 9.6. T CELL-ANTIGEN PRESENTING CELL (APC) INTERACTION. In lymphatic organs numerous T cells packed in the T cell area scan the HLA glycoproteins and the peptides (HLA-p) on the membrane of APC. A T cell first interacts with the APC through non-specific adhesion glycoproteins. Then, the TCR probes the HLA-p glycoproteins expressed on the APC membrane.

94 Fig. 9.7. FIRST ROLE OF ADHESION MOLECULES. The binding of adhesion molecules expressed by T cells with the corresponding molecules on Antigen Presenting Cells (APC) allows an initial T cell-APC interaction. Among the adhesion molecules expressed on T cell surface we have: Lymphocyte Function-Associated antigens (LFA-1 and LFA2/CD2), integrins connected with the cell cytoskeleton; The Intracellular Adhesion Molecule-3 (ICAM-3), a member of the Ig super family molecules (See Fig. 13.5). These adhesion molecules interact with other ICAM, LFA molecules, and C-type lectins (DC-SIGN, Dendritic Cell Specific Icam-3 Grabbing Non integrin) expressed on the APC cell membrane. These multiple and non-specific interactions lead towards the formation of temporary immunological synapse between the APC and the T cell. Fig. 9.8. TCR SCAN HLA GLYCOPROTEINS AND PEPTIDES ON ANTIGEN PRESENTING CELL (APC). Receptors and ligands involved in T cell activation cluster in the area of T and APC membrane contact. Here, the TCR expressed by T cells starts to probe HLA glycoproteins with peptides in their groove (HLA-p) displayed by the APC. In most of the cases, the TCR does not bind the HLA-p with high affinity and T cell and APC separate after a short period. By contrast, if the TCR binds HLA-p with high affinity, the LFA-1 adhesion molecule acquires a new conformation, binds ICAM-1 with higher affinity and the T cell remains in contact with the APC for several hours. During this prolonged cell-to-cell interaction, the APC and the T cell deliver and receive multiple signals leading toward the activation of the T cell. The high affinity binding of TCR with HLA-p is the central event on which rests the specificity of T cell activation. It is due to the spatial complementarity of TCR complementary determining regions (CDR1, 2 and 3, See Figs. 7.12 and 9.8) and HLA-p (See Fig. 7.13). A single amino acid difference may transform an activating, high affinity binding into a non-activating interaction. In the case of high affinity interaction, the TCR repeatedly binds the HLA-p, delivers activation signals and dissociates from the HLA-p. Signals coming from these repeated TCR bindings put the virgin T cell in state of initial activation. Accessory co-stimulatory signals are then required to further progress in the T cell activation, clonal expansion and differentiation. In the Fig. the alpha chain of the TCR is in dark blue; the beta chain of TCR in light blue; the alpha chain of Class I HLA in green; the beta-2 microglobulin in brown; the peptide on the HLA groove in red.

95 Fig. 9.9. T CELL RECEPTOR AND HLA GLYCOPROTEINS MOVE TO THE LIPID RAFT. Following the first high affinity interactions, TCR moves to a special microdomain of the membrane (the lipid raft, in orange in the Fig.) rich in saturated lipids and cholesterol. This move is critical for the transduction of TCR signals to the nucleus, since the Lck and Fyn Src kinases involved in T cell activation home in this area. During the multiple interactions between TCR and HLA glycoproteins presenting peptides in their groove (HLA-p), CD45, a tyrosine phosphatase, is activated. Activated CD45 removes phosphate groups of inactive Lck and Fyn. Once activated, Lck and Fyn phosphorylate the tyrosines of ITAM regions of the  chain and CD3. Then, phosphorylated ITAM act as docking site for ZAP 70 (the Zeta Chain Associated Protein kinase). Next, ZAP 70 activates LAT and SLP-76 adaptor molecules. In the Fig. Lck and Fyn are in black when inactive and in orange when active; yellow circles enclosing a P: phosphorylate ITAM domains. Fig. 9.10. TCR SIGNALING. The tyrosine kinase ZAP-70 phosphorylates scaffold proteins leading to the activation of phospholipase C-gamma. It moves to the cell membrane and cleaves phosphatidil inositol bi-phosphate in inositol tri-phosphate (IP3) and diaglicerol (DAG). IP3 causes the release of Ca2+ from endocelluar stores and opens Ca channels allowing the intracellular entrance of extracellular Ca2+. DAG diffusing into the cytoplasm activates protein kinase C  and Mitogen Activated Protein (MAP) kinases. MAP kinases are also activated by ZAP-70 kinase. Finally, the transcription factors of the NF-B, NFAT and AP-1 families trigger the activation of both the IL2 gene and the gene of the IL2 receptor  chain (CD25) (See Figs. 4.12-4.18 and 9.11).

96 Fig. 9.11. ACTIVATED T CELL. The final step in the activation of a T cell leads to the expression on the cell membrane of the IL2 receptor  chain (CD25), the formation of a trimeric high affinity IL2 receptor and the secretion of IL2. When CD25 is expressed on a T cell membrane, tiny amounts of the IL2 secreted by the T cell are autocrinally caught. Then, phosphorylated  and  chains of IL2 receptor transduce signals through the JAK- STAT pathway and induce T cell clonal proliferation and differentiation in effector/memory T cells (See also Figs. 4.12-4.18). Fig. 9.12. T CELL CO-STIMULATION BY CD4. For the activation of a virgin T cell, the high affinity interaction between TCR and HLA-p and the signal transduction schematically shown on Fig. 9.9 are not enough. Efficient signal transduction of TCR signals, T cell survival and differentiation require multiple accessory signals delivered by several costimulatory molecules and receptors. On the cell membrane of CD3+ CD4+ T cells the co-stimulatory molecules CD4 should bind a conserved domain of HLA Class II glycoprotein. This binding stabilizes the interaction of TCR with Class II HLA-p. Moreover, CD4 bound to Class II HLA-p activates Src kinases Lck and Fyn (see Fig.9.8) enhancing the phosphorylation of ITAM regions of the CD3 and  chains. The lack of these co-stimulatory signal induces a state of anergy (See also Fig. 23.7). Fig. 9.13. T CELL CO-STIMULATION BY CD8. On the cell membrane of CD3+ CD8+ T cells, the costimulatory molecule CD8 binds a conserved domain of HLA Class I glycoproteins. This binding stabilizes the interaction of TCR with Class I HLA-p. Moreover, CD8 bound to Class I HLA-p activates Src kinases Lck and Fyn enhancing the phosphorylation of ITAM regions of the CD3 and chains. The lack of co-stimulatory signals induces a state of anergy (See Fig. 23.7).

97 Fig. 9.14. T CELL CO-STIMULATION BY B7. B7.1 (CD80) and B7.2 (CD86) molecules are homodimers of the Ig superfamily (See Fig. 13.5) expressed by activated Antigen Presenting Cell (APC). Their interaction with the costimulatory receptor CD28 expressed on the cell membrane of T cells induces receptor phosphorylation. Phosphorylated CD28 activates phospholipase Cgamma to produce IP3.. In addition, signals delivered by TCR and CD28 induce the expression on T cell of the CD40 ligand (CD40L), another co-stimulatory molecule that binds CD40 receptors expressed by APC. The signals delivered by the activated CD28 are of critical importance for the clonal expansion of virgin T cells and their survival. Also in this case, the lack of B7-CD28 co-stimulatory signal induces a state of anergy (See Fig. 23.7).

Fig. 9.15. T CELL CO-STIMULATION BY CD40 LIGAND. Signals delivered by TCR, CD4 or CD8 and CD28 induce a pre-activated T cell to express on its cell membrane the CD40 ligand (CD40L) a trimeric membrane cytokine of the Tumor Necrosis Factor (TNF) family. This co-stimulatory molecule binds CD40, a receptor expressed by Antigen Presenting Cells. A ligand activated CD40 receptor delivers signals inducing APC to provide T cell co-stimulatory signals more efficiently and to increase their expression of HLA and B7 molecules and the secretion of cytokines (See Fig. 17.9). 17.9).

Fig. 9.16. T CELL CO-STIMULATION BY MEMBRANE TUMOR NECROSIS FACTOR (TNF). The expression of a trimeric form of the cytokine TNF-ď Ą on the cell membrane of activated APC is perceived by the TNF receptor II (TNFR II) expressed on the surface of T cells. On the contrary of what takes place with the co-stimulation by CD40L expressed by pre-activated T cells, the TNF expressed on APC cell membrane binds the TNF receptor on the T cell surface. Through the TRAF-2 (TNF Receptor Associated Factor) the TNFR II activates the AKT pathway promoting T cell survival, the NF-ď ŤB pathway enhancing T cell mitotic activity and the JNK and AP1 pathways enhancing IL2 secretion by T cells. All these transduction pathways have important roles in helping activation and clonal expansion of a virgin T cell.


Fig. 9.17. THE CO-STIMULATION OF A VIRGIN T CELL. When the TCR of a virgin T cell interacts with high affinity with HLA glycoproteins presenting peptides (HLA-p), multiple receptors and ligands co-stimulate T cell activation. These co-stimulatory signals have a critical importance in the full activation of a virgin T cell whereas they are disposable in the activation of effector/memory T cells. The interaction of TCR with an HLA-p in the absence of co-stimulatory signals induces T cell anergy: Anergic T cells normally circulate in the body but are unable to mount an immune response even when HLA-p recognition is accompanied by the correct co-stimulatory signals. The absence of a concomitant co-stimulatory signal may also drive Th cells1 to differentiate into Treg cells (trans-differentiation).


Fig. 9.18. THE SUPRA MOLECULAR ACTIVATION CLUSTER (SMAC). When TCR binds HLA glycoproteins presenting peptides (HLA-p) with high affinity, T cell and Antigen Presenting Cells (APC) establish a long lasting interaction that can be viewed as a prolonged cell to cell kiss.

A. The clustering in the area of cell-cell contact of molecules involved in T cell activation gives rise to a SMAC. SMAC molecules repeatedly interact and deliver signals. Then, are endocytosed and become reexpressed.

B. As the cell-cell interaction progresses, this continuously renewing molecular cluster acquires a characteristic spatial organization. On the T cell side TCR, CD3 and ď ş chains and the co-stimulatory CD4 or CD8 and B7 molecules occupy the central area (c-SMAC). Adhesion and co-stimulatory molecules (LFA-1, CD2-LFA-3; CD4 or CD8) home around it (p-SMAC). The adhesion molecule CD43, the CD44 marker and the tyrosine phosphatase CD45 gather in a more external area. On the APC side the corresponding ligands and receptors acquire a corresponding localization.


Fig. 9.19. STEPS OF VIRGIN T CELL ACTIVATION. Specialized sentinel cells (APC) in the body periphery sense, capture, digest the antigen and present antigen peptides in the groove of HLA glycoproteins. Activated by the antigen, danger signals and cytokines, APC travel to T cell areas of lymphoid organs where their HLA glycoproteins and peptides (HLA-p) are probed by the TCR of numerous T cells (See Figs. 9.4 and 9.5). If the TCR of one of these T cells binds their HLA-p with high affinity, the T cell reaches an initial pre-activation stage. Then, multiple co-stimulatory signals delivered by APC lead the T cell to express the ď Ą chain (CD25) of the IL2 receptor (See Figs. 4.12-4.18) and secrete IL2. The fully activated T cells proliferate giving rise to a clone of effector/memory cells whose differentiation is modulated by the cytokines secreted by the APC. In a week, an activated virgin T cell may generate more than 5 x 104 daughter cells. These activated effector/memory cells migrate throughout the body searching for target cells expressing the same peptide in the groove of the same HLA molecule. They exit from the spleen into blood, and from lymph nodes end Peyer’s patches into the lymph, travel in the circulatory fluid and they re-enter into lymphatics organs. This journey is guided by the chemoattractant sphingosine 1-phosphate (S1P), whose concentration is higher in the body fluids than in lymphatic organs, and by the S1P receptor, that is differently expressed by resting and activated T cells.



Fig. 10.1. THE KILLER T CELL. Once correctly activated, a virgin T cell quickly generates a clone of effector/memory T cells. Commonly, a virgin CD8+ cell generates a clone of effector/memory killer T cells (Tk cells), all with the same TCR as the virgin T cell.

Fig. 10.2. THE KILLER ACTIVITY OF EFFECTOR/MEMORY CYTOTOXIC T CELL. Effector/Memory Tk cells patrol the body in the search of the same HLA-p complex that triggered the activation of the progenitor virgin T cell. Following a first non-specific interaction (1), the high affinity binding of the T Cell Receptor (TCR) with an HLA-p displayed on the membrane of a cell (2) is sufficient to trigger their killer activity (3). During the short Tk–target cell interaction (the so called Kiss of Death) the Tk cell releases lytic molecules that cause the death of the target cells. While the target cell dies, the Tk cell detaches and is ready to engage another target (4)


Fig. 10.3. THE LYTIC SYNAPSE. Following a first, non-specific interaction due to an adhesion molecule, T Cell Receptors (TCR) of an effector/memory Tk cell repetitively scan the HLA-p expressed on the surface of the target cell. The target cell can be any cell of the body and not necessarily an Antigen Presenting Cell. If the TCR interacts with the HLA-p with high affinity, the signal transduced by the ď ş chain and the CD3 molecules triggers the killing program of the Tk cell.

103 Fig. 10.4. THE KISS OF DEATH. A temporary synapse takes place when the T Cell Receptor (TCR) of a T killer (Tk) cell interacts with high affinity with a complementary HLAp displayed by a target cell. During this short interaction, lasting about 5 minutes, the repeated TCR signals activate the killer program of the Tk cell. The Tk cell re-organizes microtubules and Golgi apparatus to have a localized and polarized exocytosis of perforins and granzymes in the area of cell to cell contact

Fig. 10.5. THE DYING OF THE TARGET CELL. The polarized secretion of perforin and granzymes and other lytic molecules stored within lytic granules in the cytoplasm of the T killer (Tk) cell leads to the death of the target cell. Membrane pores made by perforin allow granzymes to enter into the target cell and induce nuclear fragmentation and chromatin condensation. Dying apoptotic cells do not release their cellular constituents into the surrounding tissue since their fragments (apoptotic bodies) are always enclosed within a plasma membrane. These bodies are subsequently taken up by macrophages without production of inflammatory substances. Killing of virus infected cells by Tk cells is the most important mechanism of healing from viral diseases. The importance of this defense mechanism is illustrated by the fact that the evolution has endowed a variety of pathogenic viruses with the mean of blocking it. For example, Herpes virus simplex produces a protein that binds to TAP1 and TAP2 (See Fig. 6.5) and blocks the transport of peptides to endoplasmic reticulum. Cytomegalovirus encodes molecules which induce the degradation of Class I HLA glycoproteins by the proteasome.

104 .

Fig. 10.6. PERFORIN and GRANULYSIN. Lytic granules of T cells hold several cytolytic proteins including perforin, granzymes, granulisin and other lysosomial proteins.



Fig. 10.8. WHY IS IT THAT PERFORIN, GRANULYSIN AND GRANZYMES DO NOT KILL ALSO THE TK CELL? The membrane damaging proteins released by a Tk cell during the Kiss of Death could also lead toward autocrine damage of the Tk cell membrane. The unidirectional killing that takes place seems to be due to the simultaneous release of cathepsin B, Lamp-1 (CD107a) and other molecules that protect the Tk cell membrane in the area of the cell to cell contact. Cathepsin B is a protease. By adhering to the Tk cell membrane, its enzymatic activity degrades perforin and the other membrane damaging proteins.



Fig. 11.1. T HELPER CELLS. The activation of a virgin T cell depends on Antigen Presenting Cells (APC) displaying in the groove of HLA glycoproteins peptides of the captured antigen (HLA-p). Once correctly activated, a virgin CD4+ cell generates a clone of helper (Th) cells, all with the same TCR as the virgin cell. However, depending on the cytokines secreted by the APC and signals that are present during its activation, a virgin T cell generates a particular clone of effector/memory Th cells. In effect, in our body there are several Th cell sub-populations that modulate and bias the progression of the immune response by secreting distinct combinations of cytokines. These various Th cell populations are competing among themselves and the initial activation and expansion of a particular Th cell population inhibits the development of the other populations, polarizing the immune response being activated. The importance of the role of effector/memory Th cells in the regulation of immune responses is dramatically shown by patients infected by the Human Immunodeficiency Virus (HIV): the progressive decrease of CD4+ cells caused by the HIV infection results in a severe immunodeficiency incompatible with patient survival (See Fig.26.8).

107 Fig. 11.2. THE ANTIGEN PRESENTING CELL (APC) INDUCES AND BIASES Th CELL DIFFERENTIATION. Different antigens (bacteria, parasites, foreign or altered cells…) are recognized and captured by Antigen Presenting Cells (APC) through distinct cell membrane receptors (Pattern Recognition Receptors, PPR, see Fig. 2.4). These various PPR2transduce signals that induce APC to release distinctive cytokine combinations and express particular co-receptors on their cell membrane. For example, cytokines and co-receptors expressed by and APC following the capture of a bacterium are different from those expressed by an APC that has captured a parasite. The peculiar combination of cytokines and co-receptors expressed by an APC during HLA-p presentation to a virgin CD4+cell guides the polarization of the generated clone of Th cells towards Th1, Th2, Th17, Treg…(For Treg see Fig. 22.12). Fig. 11.3. ANTIGEN PRESENTING CELLS (APC) DECIDE THE TRANSDIFFERENTIATION OF EFFECTOR/MEMORY Th CELLS. APC bias the activation of the most appropriate immune reaction against the captured antigen by secreting a distinctive combination of cytokines and expressing particular co-receptors (For Treg see Fig. 22.12). In this way they guide the conversion from one Th cell type to another (transdifferentiation).


Fig. 11.4. ANTIGEN PRESENTING CELLS (APC) BIAS Th0 TRANSDIFFERENTIATION. Dendritic Cells and macrophages capture the invading antigen perceived through their Pattern Recognition Receptors (PPR), digest it and associate antigen peptides to Class II HLA glycoprotein. Moreover, the diferent knd of APC, the distinct PPR involve in antigen capture, the molecular structure of the captured antigen and the concomintant environmental signals concur to induce APC to release a particular combination of cytokines and to express a particular set of costimulatory molecules. In response to this combination of signals the Th0 cell transdifferentiate towards Th17, Th1 or Th2. Transdifferntiation means that the Th0 cell selects one of its different gene expression programs. One program is winning out while alternative programs are extinguished. Triggered transcription factors bind to specific sequence motifs whithin the promoter, enhancer and silencer gene regions and recruit also epigenetic regulators to modulate the activation state of the genes, activation state that is then transmitted to subsequent cell generations. REFERENCE: E Laurent and B Gottgens, Nature 2018,553:418.


Fig. 11.5. Th17 CELLS. Immediately after the capture of bacteria and fungi the Antigen Presenting Cells secrete TGBď ˘, IL23 and IL6, a combination of cytokines that bias the transdifferentiation of Th0 cells towards Th17. Also the epithelial cells lining the intestine and sensing tight attachment of microbes respond by producing proteins that guide the transdifferentiation of Th0 cells into Th17 cells. On the other hand, trans-differentiation of Th17 in Treg cells is a physiological mechanism leading towards the resolution of the inflammation. However, the high salt diet of the Western world may favor Th17 differentiation and thus contribute to both hyperthension and autoimmunity. REFERENCE: N Wilck et al, Nature 2018, 551:585.

Fig. 11.6. THE INFLAMMATORY RESPONSE INDIRECTLY ACTIVATED BY Th17 CELLS. IL17 and the various proinflammatory cytokines secreted by Th17 cells stimulate fibroblalsts, epithelial cells, endothelial cells and keratinocytes to release antimicrobial factors (See Fig. 1.4) and several cytokines and chemokines, including IL6, IL8, G-CSF and GM-CSF. These factors attract and activate neutrophils and macrophages on epithelial and mucosal surfaces.


Fig. 11.7. Th1 CELLS. The polarization of the cell clone deriving from the activated virgin Th cell towards Th1 rests on the combination of cytokines and membrane ligands expressed by the Antigen Presenting Cells displaying the complementary HLA-p. Following the capture of certain antigens, APC expresses distinct ligands of Notch receptors. Notch are a special family of receptors that specify cell differentiation and cell fate. The expression on the APC of the Delta ligand of Notch receptors favors the differentiation of the clone deriving from a virgin Th0 cell towards Th1 whereas the expression of Jagged, an alternative Notch ligand, favors clonal differentiation towards Th2cell.


Fig. 11.8. THE STRAIGHTFORWARD Th1 RESPONSE. The release of TNF, IFN and IL2 by Th1 cella triggers a complex and powerful immune response. These cytokines help B cell activation and drive activated B cells to produce opsonizin and Complement fixing antibodies endowed with a powerful anti-bacterial activity (See Figs. 19.11, 19.12). Macrophages activated by pro-inflammatory Th1 cytokines are defined M1 macrophages (See Fig. 3.18): their metabolism is dramatically enhanced. Macrophage phagocytic activity, their ability to digest ingested microbes, their secretion of anti-microbial substances such as nitric oxide (NO2), superoxide (O2-) and other Reactive Oxygen Radicals (ROS) are markedly increased.

Fig. 11.9. Th1 CELLS HELP THE EFFICIENT INDUCTION OF Tk CELL ACTIVITY. A Th1 cell activated by an Antigen Presenting Cell displaying the complementary Class II HLA-p and co-stimulatory molecules (B7, CD40…) secretes IL2 and IFNin its micro-environment. These cytokines are instrumental in promoting the efficient activation followed by enhanced survival of neighboring CD3+ C8+Tk cells which recognize the complementary Class I HLA-p.


Fig. 11.10. Th1 CELL: FEED-BACK ACTIVATION CIRCUITS. The Th1 biased immune response is markedly enhanced by the pro-inflammatory cytokines released by the cells recruited in the reaction. M1 macrophages, NK cells and Tk cells activated by IFNď §, TNF and IL2 released by Th1 cells not only acquire a powerful and differentiate killer activity but also start secreting a large array of pro-inflammatory cytokines that further enhance the reaction triggered by Th1 cells. triggered by Th1 cells.


Fig. 11.11.Th2 CELL. Both the factors released by barrier epithelial cells (See Figs. 1.4 and 1.5) and the features of certain captured antigens (worms, parasites‌) induce Antigen Presenting Cells (APC) to mount a Th2 cell mediated immune response. APC secrete IL4, IL10 and express the Jagged ligand of Notch receptors. Notch are a special family of receptors that specify cell differentiation and cell fate. The expression of the Jagged ligand favors the trans differentiation of the clone deriving from a virgin Th0 towards a peculiar Th2 cell. In addition, the secretion of IL10 by APC inhibits the clone polarization towards Th1.


Fig. 11.12. THE Th2 RESPONSE: A REFINED ANTI-PARASITE REACTION. The polarization of the cell clone derivingfrom activated virgin Th cell towards Th2 rests on the combination of cytokines (IL4‌) and ligands expressed by by the Antigen Presenting Cells (APC) and cytokines released by Innate Lymphoid Cells (ILC). Following the interception with certainintruders, APC expresse the Jagged ligand of Notch receptors tat favors the differentiation of the clone deriving from a virgin Th0 cell towards Th2.. IL4 and IL13 released by activated Th2 cells help B cell activation and drive activated B cells to produce IgG1 and IgE antibodies (See Figs. 16.24 and 16.25). Moreover, IL4 and IL13 secreted by activated Th2 cells induce a peculiar macrophage activation (alternatively activated macrophages or M2 macrophages). M2 macrophages (See Fig. 3.19) are different from M1 macrophages as they mostly release arginase which induces tissue remodeling and contractility of intestinal smooth muscles. IL13 intensifies the renewal of epithelial cells. The combination of IL4 and IL13 increases the production of mucus and induces the hyperplasia of mucus-secreting goblet cells. The elevated mucus production and smooth muscle hyper contractility triggered by IL4 and IL13 may result in worm expulsion.


Fig. 11.13. THE Th2 REACTION TO PARASITIC WORM INFECTION. Parasitic worms, also known as helmints are a major global health and social burden since nearly one-third of the human population is infected with these parasites, primarily in tropical regions. Therefore parasitic worms are among the most prevalent infectious agents in the world, and they are responsible for many debilitating diseases and syndromes. However, regions where helminth parasites are endemic record much lower prevalence of allergies and autoimmune diseases, suggesting that the immune reaction against parasites bias the Th0 cell response towards a Th2 prevalence and protects against some allergic syndromes. Sentinel phase: Following a worm infection epithelial cells of mucosal barriers (Tuft cells) secrete IL25 that activates Innate Lymphoid Cells (ILC) (See Fig. 2.8). Worms are also perceived by Antigen Presenting Cells (tissue macrophages and Dendritic Cells). Cytokines released by Innate Lymphoid Cells and Antigen Presenting Cells and the peculiar differentiation stage acquired by tissue macrophage (M2 macrophage differentiation, See Fig. 3.19) and Dendritic Cells concur to induce the differentiation of Th0 lymphocytes in Th2 cells. Effector phase: Activated Th2 cells orchestrate a complex and multicellular immune response to parasitic worms by releasing IL4 and IL13 cytokines that further induce M2 macrophage differentiation. a. Hostile micro-environment. Activated M2 macrophages secrete arginase, an enzyme of urea cycle that promote fibrosis and tissue repair. In addition, M2 macrophages produce extracellular matrix components that along with arginase causes tissue remodeling. Moreover, M2 macrophages further bias the differentiation of activated Th0 cells in Th2 cell. The combination of IL4, IL9 and IL13

116 cytokines released by Th2 and innate lymphoid cells induce the hyperplasia of mucus-secreting goblet cells, enhances mucus production and changes its composition. In addition, IL4, IL9 and IL13 cytokine combination triggers smooth muscle hyper contractility. This hostile micro-environment may result in the expulsion of worms. b. Direct killing. Tissue basophils and mast cells activated by Th2 cytokines enhance local blood supply and the extravasation of eosinophils. IL5 induces eosinophil chemotaxis and activation (See Fig. 3.14). Eosinophils directly attack worms by releasing cytotoxic molecules and Major Basic Protein. In addition, Th2 cells switch the production of IgE antibodies. IgE bound on the cell membrane of basophils and mast cells may induce their degranulation (See Fig. 16.26, 19.10). Moreover IgE bound on the surface of parasitic worms may impair worm activities and guide the killer activity of eosinophils (Antibody Dependent Cellular Cytotoxicity, ADCC) (See Fig. 3.31).

Fig. 11.14. THE FOLLICULAT T HELPER (TFh) CELL. T follicular cells (TFh) are a subset of CD4+ Th1 and Th2 cells characterized by their ability to home in the follicles of the lymphoid organs (See Figs.18.4-18-10). Their differentiation appears to be guided by the secretion of IL6 and elevated expression of ICOS ligands by the activating Antigen Presenting Cell (APC). ICOS ligands are members of the family of B7 co-stimulatory molecules expressed by APC, while ICOS (Inducible CO-Stimulator) is a receptor of the TNF receptor family expressed by T cells. IL6 and ICOS ligands induce Th cells to express the chemokine receptors that guide their homing at the interface of B cell follicle and T cell zone. For the role of TFh in B cell activation see Chapter 17.


12. B CELLS.

Fig. 12.1. B CELL ORIGIN AND ACQUISITION OF THE ANTIGEN RECEPTOR, THE SO CALLED B CELL RECEPTOR (BCR). For bone marrow differentiation of B cells see Figs. 3.4 and 3.5).

Fig. 12.2. ANTIGEN INDEPENDENT BONE MARROW PRODUCTION OF B CELLS. The first phase of B cell maturation is called Antigen Independent, since is taking place independently from any antigen stimulation.


Fig. 12.3. BONE MARROW MATURATION OF B CELLS. This cartoon shows a red bone marrow extravascular space between the sinusoids in the hemopoietic medullary cavity of a bone (See Fig. 3.3). Hemopoietic stem cells (HSC) are located close to both osteoblasts (In orange in the Fig.) and endothelial cells of sinusoids (In light purple). During differentiation, HSC move towards a particular kind of stromal cell (reticular stromal cells, in green). This contact induces the maturation to pro-B cell stage. Pro-B cells adjoining other stromal cells secreting interleukin 7 (IL7) mature in pre-B cells. Subsequently, pre-B cells leave stromal cells and express the cell membrane IgM B Cell Receptors (BCR): B cells at this stage of differentiation are called immature B cells. Then, these cells may express also an IgD BCR, exit the bone marrow, enter the blood and become peripheral virgin mature B cells.


Fig. 12.4. STROMAL CELLS GUIDE B CELL MATURATION. n some ways, this figure is a schematic representation of the events illustrated in Fig. 12.3. Cytokines and membrane molecules expressed by bone marrow stromal cells guide the progressive passage of Hematopoietic Stem Cells (HSC) into pro-B, pre-B and immature B cells. Then, immature B cells leave the bone marrow and home in various areas of lymphoid organs (transitional virgin B cell). During this maturation period and independently from any antigen stimulation, every maturing B cell acquires an unique individual BCR made by monomeric IgM. Every B cell expresses numerous copies (about 104) of BCR, all with the same binding site. As every maturing B cell generate a distinct binding site, different from that of the BCR of other maturing B cells, an extremely large repertoire of distinct binding sites is generated. The generatio of this extremely large repertoire of distinct binding sites is is made possible by a unique cut and paste of the genes encoding the binding site of the BCR. This cut and paste takes place in every B cell maturing in the bone marrow (See Chapter 14). This gene recombination is a difficult task, and very often B cells are unable to perform it successfully. In other cases B cells generate a binding site that binds self antigens expressed on the surface of bone marrow stromal cells. In this latter case, the B cell will try to change the binding site (receptor editing). If it is not possible, the B cells with a binding site that binds self antigens will become anergic (unable to mount an immune response) or will undergo apoptosis (See Chapter 22).


Fig. 12.5. GENERATION OF DISTINCT B CELL POPULATIONS. Signals delivered by bone marrow stromal cells on immature B cells and signals received in peripheral lymphoid organs by transitional virgin B cells (the first B cells which migrate from bone marrow to peripheral blood) influence transitional virgin B cell differentiation in B1 and B2 B cells. Signals received by Notch receptors on B B2 cell membrane induce their differentiation in Marginal Zone B (MZ B) cells. Notch are a special family of receptors that specify cell differentiation and cell fate (See Fig.11.7). Here the presence or the absence of Notch signalling plays a key role in the maturation of hematopoietic stem cells. In the absence of Notch signals, B2 cells differentiate into Follicular (Fo) B2 B cells. Then, the survival and homing of Fo B2 B cells in lymphoid organs is driven by the expression of the receptor for the B cell activating factor (BAFF), a cytokine of the TNF family produced in the lymphoid follicles.


Fig. 12.6. B1 B CELLS.These cells form a peculiar population of B cells that react against a variety of antigens and auto antigens (See Fig. 26.2). Once activated, B1 cells differentiate in plasma cells producing low affinity and poorly specific (cross-reactive) IgM. These antibodies provide an important first line of defence against a variety of microbes. However, antibodies reacting with self antigens can be implicated in several autoimmune pathologies. CD5+ B1 cells are also the normal counterpart of CD5 +B chronic lymphocytic leukaemia.


Fig. 12.7. ANTIGENS RECOGNIZED BY B1 B CELLS. The BCR of B1 B cells interact with low affinity with many different antigens, such as self antigens and common bacterial polysaccharides. B1 B cells produce the majority of natural immunoglobulin M (IgM) and A (IgA). These natural antibodies are largely encoded by germline immunoglobulin genes (not re-arranged genes, See Chapter 14). Because of their ability to bind different target antigens (poly-reactivity) and their ability to recognize large repetitive structures, B1 B cells provide a first line of defence against pathogens such as encapsulated polysaccharide-expressing bacteria in the gut mucosa and respiratory tract.












CHAPTER 13. B CELL RECEPTOR AND ANTIBODIES. Fig. 13.1.THE B CELL RECEPTOR (BCR) AND IMMUNOGLOBULIN (Ig). The binding site is the domain of BCR (and of Ig) interacting with the antigen. All BCR expressed by a B cell on the cell membrane and all the Ig that this cell will produce once activated and differentiated in plasma cells share the same binding site.

Fig. 13.2.BCR and IMMUNOGLOBULINS: BASIC STRUCTURE. The Fc fragment of BCR is longer than that of the Ig. It contains an extra transmembrane domain made by hydrophobic amino acids and a short intra-cytoplasmic tail (See Fig. 13.7).

Fig. 13.3.BCR AND IMMUNOGLOBULINS. MOLECULAR FEATURES. I. The binding site is a critical region of the BCR and Ig made by the NH2 terminus of both H and L chains. The extremely variable sequence of the amino acids of this region creates billions of different binding sites. This large repertoire of binding sites allows a few of them to establish a very precise (specific) interaction with the antigen. The repertoire of BCR is very large in the B cell population, however it should be noted that every B cell expresses BCR and produces Ig with a binding site of a unique specificity, the one randomly acquired among the billions of distinct binding sites expressed by the B cell populations (See Chapter 14).

127 Fig. 13.4. BCR AND IMMUNOGLOBULINS. MOLECULAR FEATURES. II. BCR and Ig are made by four chains, two identical H chains and two identical L chains. These chains are linked by inter-chain disulphide bridges (S-S). The L chain is made by two domains, the variable light domain (VL) and the constant light (CL) domain. The H chain is made by a variable domain (VH) and by three or four constant heavy domains (CH). The VL and VH form the binding site. The binding site of BCR and the Ig should interact with the natural conformation of unpredictable antigens. Therefore size and flexibility of the BCR are features of critical importance. By contrast these features have a limited importance with the T Cell Receptor (TCR) that constantly interacts only with HLA-p that displays little conformational variability. Fig. 13.5. BCR AND IMMUNOGLOBULINS: THE Ig SUPER-FAMILY. Several immune molecules are made by repeats of the Ig basic molecular structure of 110 amino acids and an S-S bridge. All the molecules made by replicas and slight modifications of this basic molecular structure are considered members of the Ig super-family. Numerous molecules of the Ig superfamily are expressed on the cell membrane by the cells of the immune system


Fig. 13.6. B CELL RECEPTOR (BCR) ON THE SURFACE OF A FoB2 B CELL. On the cell membrane, virgin B1 and MZ B cells express a BCR made only by monomeric IgM. By contrast, mature virgin FoB2 B cells have a BCR made by monomeric IgD and IgM. Note that IgM has an extra heavy chain domain and thus is longer than IgD (See Figs.13.8 and 16.18).


129 Fig. 13.8. THE ASSEMBLY OF A B CELL RECEPTOR (BCR). On a resting B cell, BCR are mobile monomers floating on the cell membrane. The BCR of virgin FoB2 B cells is composed by both monomeric IgM and IgD and the dimeric Ig alpha (Igď Ą) and Ig beta (Igď ˘) transducer molecules. By contrast, in the BCR of B1 and MZ B cells the BCR is made by IgM only, associated with the transducer Ig alpha and Ig beta. The specificity of the BCR signal rests on the ability of the binding site of IgD and IgM to interact with a specific antigen (Orange arrow in the Fig.). Then, the Ig alpha and Ig beta uniformly transduce the signal to the cell nucleus.

Fig. 13.9. THE BCR: SIGNAL TRANSDUCTION. When the binding site of a BCR encounters the specific antigen (in brown in the Fig.), BCR aggregate in micro-clusters that migrate to a lipid raft, a special microdomain of the membrane (Polar head groups are in orangeyellow) rich in saturated lipids and cholesterol. This move is critical for the transduction of BCR signals to the nucleus, since the Lck, Fyn and Blk Src kinases involved in B cell activation home in this area. CD45, a tyrosine phosphatase is the first to be activated. Activated CD45 removes phosphate groups of inactive Lck, Blk and Fyn and thus activates them. Once activated, Lck and Fyn phosphorylate the tyrosines of ITAM regions of the Ig alpha and Ig beta. In this drawing the cell membrane is depicted as two layers of lipids, their orange and light green polar head groups separate the yellow-orange hydrophobic tails from the aqueous cytosolic and extracellular environments.


Fig. 13.10. BCR SIGNALING. On the lipid raft, the ITAM of the intracellular portion Ig alpha and Ig beta phosphorylated by Lck, Fyn and Blk kinases act as a docking site for SYK. The SYK tyrosine kinase then phosphorylates scaffold proteins leading to the activation of phospholipase C-gamma. It moves to the cell membrane and cleaves phosphatidil-inositol bi-phosphate in inositol triphosphate (IP3) and diaglicerol (DAG). IP3 causes the release of Ca2+ from endocellular stores and opens Ca2+ channels allowing the intracellular entrance of extracellular Ca2+. DAG diffusing into the cytoplasm activates protein kinase C and Mitogen Activated Protein (MAP) kinases. These molecular events are sufficient to lead to the full activation of B1 and MZ B cells. By contrast, in FoB2 B cells the triggering of the transcription factors of the NF-kB, NFAT and AP-1 families activates the gene of IL2 alpha (ď Ą) chain receptors (CD25) (See Figs. 4.11-4.18 and 9.11) and the receptors of other cytokines, making the FoB2 B cells ready to receive the helper signals delivered by TFh cells.


Fig. 13.11. B CELL CO-STIMULATION BY COMPLEMENT. When C3b fragments of Complement cascade adhere on the antigen surface (See Chapter 20), B cell activation is greatly facilitated (co-stimulated). The C3b Complement fragments adhering on the antigen surface bind the Complement Receptor 2 (CR2, CD21) (See Fig. 20.8). CR2 in cooperation with CD19 and CD81 membrane proteins transduce signals that enhance the transduction of BCR activating signals. Many microbes directly activate the Complement cascade through the Lectinic (See Fig. 20.3) and Alternative pathway (See Fig. 20.4). These activation pathways are important mechanisms of natural immunity, quickly activated following a microbial invasion, which co-stimulate a specific B cell activation. Thus Complement, a key mechanism of natural immunity, plays an important role in the stimulation of antibody response.



Fig. 14.1. THE GENERATION OF THE REPERTOIRE OF BCR (and TCR) BINDING SITES. Each B cell generates a BCR with an individual binding site and expresses on the cell membrane around 104 BCR molecules all with this identical binding site. The acquisition of the individual BCR (and TCR, for T cells) comes from the recombination of three gene segments (V, D and J) that fuse to form a new unique coding gene. The mechanism of this gene rearrangement leading to the generation of the individual binding site is similar for the BCR and the T Cell Receptor (TCR) and it will be illustrated in detail for the BCR only. In both cases, however, less than two hundred genes recombine and code billions of BCR (and TCR) each with a different binding site. The rearrangement of BCR genes is taking place during B cell maturation in the bone marrow and is driven by signals delivered by stromal bone marrow cells, while the rearrangement of TCR genes is taking place during T cell maturation in the thymus and is driven by stromal and epithelial cells of the thymus (See Chapter 8). In order to understand the mechanisms resulting in the immense repertoire of different binding sites (roughly 1011) expressed by virgin B (and T) cells of our body we have first to realize that the binding site of the BCR is made by the variable domain of both the H (VH) and L (VL) chains. Rearranged H and L chains freely associated between them. Both the H and the L chain are coded by multiple genes: The constant region of the H chain (CH) is coded by a single gene, whereas the various domains of the VH region are coded by three genes, the V, the D and the J gene. Similarly, the constant region of the L chain (CL) is coded by a single gene, whereas the various domains of the VL region are coded by two genes, the V and J gene. The immense repertoire of BCR binding sites is the result of the combination among the various V, D and J genes coding the V region (Combinatorial diversity). Moreover, the junction of selected V with the selected D and J genes is an error-prone process allowing the formation of new DNA sequences (Junctional diversity). In the Fig. the antigen is shown as a green arrow while the area of binding site in orange.

133 Fig. 14.2. INVENTORY OF THE GENES CODING FOR THE BCR. The various domains of the VH regions are coded by three genes, the V, D and J gene. In our genome we have around 65 genes that may code the V domain, 27 that may code the D domain and 6 genes that may code the J domain. The domains of the VL region are coded by the V and J genes. In our genome we have 70 genes that may code the V domain and 9 genes coding the J domain. Each B cell maturing in the bone marrow randomly selects one of the various V, D and J genes to shape the VH and VL region forming the binding site. The CH and the CL regions are coded by a single gene. However, the CH gene can be coded by one of five genes (, , , ,  genes); the CL gene can be one of the two types, the  and  genes.

Fig. 14.3. WHERE ARE THE GENES CODING FOR THE BCR LOCATED? Genes coding for the H chain are on the human chromosome 14 (CR14), those coding for the L chain of  type on chromosome 2 (CR2) and those coding for the L chain of  type on chromosome 22 (CR22). Long introns are intermingled among these gene loci.

134 Fig. 14.4. RANDOM GENE COMBINATIONS RESULT IN A LARGE REPERTOIRE OF DIFFERENT BINDING SITES. A computation of the possible combinations among the various V, D and J genes shows that there are 320 possible VL regions and 10.530 VH regions. Supposing that any VH can be combined with any VL there are millions of different binding sites. During the antigen independent maturation driven by the molecules expressed and cytokines secreted by bone marrow stromal cells, every maturing B cell selects and combines these genes to make its own individual binding site. In the Fig. the antigen is shown as a green arrow while the area of binding site in orange.


Fig. 14.5. THE REARRANGEMENT OF GENES CODING FOR THE H CHAIN. Signals delivered by stromal bone marrow cells induce early pro-B cells to open the chromatin, to modify the histones and to express special enzymes and molecules (See Fig. 12.4) involved in the cutting and pasting (recombination) of the DNA, a process which takes place only during B cell maturation. In a random way, each pro-B cell selects one of the 27 D genes and attaches it to one of the 5 J genes. To do so, the double strand DNA must be cut, the two fragments should be pasted whereas the long DNA fragment between the two cuts is eliminated as an episome. Then, the pasted DJ genes should be attached to one of the 65 V genes. As before, the double strand DNA is cut, the two fragments are pasted whereas the long DNA fragment between the two cuts is eliminated as an episome. Therefore the rearranged DNA of the B cell is shorter than the germ line DNA, since it will miss long regions eliminated as episomes. Following a differential splicing, the mRNA codes an H chain with its own particular variable domain. C genes (Cď ­, Cď ¤, in pink) are two of the various genes coding the constat region of the H chain. In the Fig., L: The leader sequence.


Fig. 14.6. ASSESSMENT OF THE H CHAIN. The H chain coded by the V, D and J genes randomly rearranged should be tested before a pro-B cell proceeds to rearrange the genes of the L chain. In effect, in many cases the protein coded by the genetic sequence deriving from these gene rearrangements may not be functional. To test if the H chain is functioning, the new H chain is associated with an invariant surrogate L chain made by two segments: VpreB and Lambda5. The H chain (in green in the Fig.) associated with VpreB and Lambda5 (in red) is exposed on the cell membrane of pro-B cells. Here it spontaneously dimerizes with another H chain. The two H chains associated with VpreB and Lambda5 join another similar two H chains molecule and interacts with ligands expressed by bone marrow stromal cells. If everything is correct, the signals stemming from the interaction of the H chain associated with VpreB and Lambda5with stromal bone marrow cells are transduced to the nucleus by the Bruton’s tyrosine kinase (BTK). This signal blocks any further rearrangement of the genes coding for the H chain. On the contrary, if the H chain is unable to interact with stromal bone marrow cells, the residual genes on the same chromosome 14are rearranged again in the attempt to code a new function H chain. If also this further attempt fails, the cell rearranges the genes of the other allelic chromosome 14. If again the H genes are not correctly rearranged, the cell undergoes apoptosis. On the contrary, the pro-B cell finally able to express a correctly signaling H chain proliferates giving rise to a clone of pre-B cells (see Fig. 12.4) all expressing the same H chain. In this way, the H genes of only one of the two allelic chromosomes 14 are arranged to code the H chain (allelic exclusion). Then, each of these pre-B cells will start to rearrange independently the genes of the L chain and thus will generate a BCR with an individual binding site. A genetic defect of BTK causes the X-linked agammaglobulinemia, an immunodeficiency due to the inability of pro-B cells to mature in pre-B cells. This block impedes the antibody production (See Fig. 26.3).

137 Fig. 14.7. REARRANGEMENT OF THE GENES CODING FOR THE L CHAIN. Once the very difficult task of rearranging the genes of the H chain is achieved, the pre-B cell starts to rearrange the genes of the L chain. This is a less errorprone task since there is only one recombination event (V-J). Moreover, a pre-B cell can rearrange the V and J genes not only on the two alleles of chromosome 2 (ď Ť L chain) but also on the two alleles of chromosome 22 (ď Ź L chain). Here too, the first correct rearrangement of L genes blocks any further rearrangement of the L chain genes. In this way, only a chromosome displays a correct L gene rearrangement (allelic exclusion). The newly coded L chain substitutes VpreB e Lambda5 and the cell expresses a definitive BCR. At this maturation step VpreB e Lambda 5 genes are inhibited. The immature B cell expressing the definitive BCR down regulates the expression of adhesion molecules, exits the bone marrow and homes into peripheral lymphoid organs.

Fig. 14.8. REARRANGEMENT OF GENES CODING THE V DOMAINS OF H AND L CHAIN. The signals delivered by bone marrow stromal cells guide the nonhomologous recombination among the V, D and J genes coding the V domains of both the H (VDJ) and L (VJ) chains. These unique gene recombination events involve only genes that are flanked by particular invariant sequences (the Recombination Signal Sequences, RSS).

138 Fig. 14.9. THE RECOMBINATION SIGNAL SEQUENCES (RSS). RSS permitting gene recombination flank V genes at 3’, D genes at 5’ and 3’, and J genes at the 5’. The 12 base pair spacer allows the DNA helix to make one turn while the 23 base pair spacer allows it to make two turns. A gene flanked by a 12 base pair spacer can be joined only with a gene flanked by a 23 base pair spacer (12/23 rule). This 12/23 rule provides the specific orientation of gene recombination.

Fig. 14.10. RAG1 AND RAG2. Recombination of V, D and J genes is driven by RAG1 and RAG2 (Recombination Activating Gene coded proteins). These enzymes are expressed only during the pro-B cell maturation in the bone marrow.

A. RAG1 and RAG2 proteins along with the HMG1 protein (not shown) interact with RSS and pair a gene flanked by a 12 base pair spacer (In blue in the Fig.) with a gene flanked by a 23 base pair spacer (in green). Following this 12/23 rule, RAG1, RAG2 and HMG1 proteins pair the two Eptamers (7-7, in yellow) and the two Nonamers (9-9, in orange). Then, RAG1 and RAG2 cut the double stranded DNA at the end of the Eptamer sequence(zig zag in purple).

B. Magnification of the cutting of the double stranded DNA. The bases of D gene are in black; those of the Eptamer in red.



1. RAG1 and RAG2 enzymes cut the double stranded DNA in two places (In the Fig. zig-zag in purple). The long cut off portion of DNA is removed as an episome (in yellow).

2. The –OH groups of DNA bases at the end of the fragments form a hairpin loop (in blue) leaving a blunt double strand DNA break.

3. Several proteins (Ku factors, protein kinases DNA dependent, Artemis endonuclease‌) bind the blunted DNA breaks. Then, randomly Artemis nuclease (in pale blue) cuts one of the DNA filaments.

4. Following the Artemis cut, DNA bases that were located in one filament give rise to a new sequence (sequence P, Palindromic, in green) on the other DNA filament.


Fig. 14.12. THE JOINING OF DNA BREAKS. II. The joining of DNA breaks generates new coding sequences.

5. Following the random Artemis cut (Pale blue arrows in the Fig. 14.11), DNA bases located on one filament move to the other filament giving rise to new P coding sequences (in green). Moreover the enzyme Terminal Deoxynucleotidil Transferase (TDT) randomly adds new nucleotides in a template fre1e way at the 3’ terminus of single strand ends. This TDT activity gives rise to the N –new- regions (in red).

6. Finally, the DNA coding ends are ligated by the DNA ligase IV and XRCC4 protein while DNA polymerase and DNA repair enzymes remove and add nucleotides (in blue). The P and N regions and the activity of DNA repair enzymes create an enormous junctional diversity making about 1016 (!) new coding DNA sequences.


Fig. 14.13. THE BINDING SITE. The binding site encompasses the area of BCR that interacts with the antigen (Green arrow in the Fig.). It consists in framework and hypervariable sequences of amino acids at the NH2 terminus of both the H and L chain. The framework sequences are not too different among various B cells whereas hypervariable sequences are markedly different. These hypervariable sequences are those that directly bind the regions of the antigen establishing multiple and different bonds. These regions are defined Complementary Determining Regions (CDR) (See Figs. 7.12, 7.13, 9.8 and 15.1).




A. The binding site is made by the amino acids of the NH2 terminus of the H and L chains. The external loops (In light brown and pink in the Fig.) are called Complementary Determining Regions (CDR) since they interact with complementary conformations of the antigen. Somewhat similar CDR are evident on the TCR (See Figs.6.6, 7.11, 9.8, 15.4).

B. The variability of the amino acid sequences of the binding site is very high. In a person BCR and antibodies express over 1011 different binding site sequences. However, the variability of the amino acid sequences at the binding site is not constant: On both the H and L chains, ipervariable regions (in brown) where the variability of amino acid sequences is very high are alternated by conserved framework regions(in pink).

143 Fig. 15.2. WHAT IS IT THAT THE BINDING SITES BINDS TO? In most ca1ses, antigens are large molecules, large molecular aggregates, microbes or even foreign cells. In these cases the binding site of a BCR (and of an Ig) binds only a small portion of the antigen, the epitope. A large antigen expresses numerous identical and different epitopes. In most cases epitopes recognized by the binding site are made by an amino acid sequence. However, binding sites may interact with peculiar sugar and lipidic sequences.

Fig. 15.3. LINEAR AND CONFORMATIONAL EPITOPES. The epitope interacting with the binding site of a BCR and an antibody can be formed by a sequence of amino acids (linear epitope, upper panel) or by discontinuous amino acid sequences making a peculiar tridimensional structure (conformational epitopes, lower panel). In the Fig. in grey, a portion of the antigen; In light blue the amino acids sequences making linear and the conformational epitopes. These are the amino acid sequences interacting with the Complementary Determining Regions (CDR) of the binding site (See Figs.6.6, 7.11, 9.8, 15.1, 15.4).


Fig. 15.4. ANTIGEN-ANTIBODY INTERACTION. When a BCR (or an antibody) interacts with an antigen, the amino acids of the Complementary Determining Regions (CDR) of the binding site establish multiple non-covalent bonds with the epitope. The strength and the persistence of the interaction (Affinity) depend on the spatial (conformational) complementarity between the amino acid sequences of the CDR and the epitope. When there is a poor complementarity, the affinity is poor (low affinity). A good complementarity secures that the strength of the interaction is high and the interaction is more persistent (high affinity). However it should be noted that the interaction between the binding site and the epitope is reversible: its Association/Dissociation Constant (Affinity) rests on the strength and multiplicity of the non-covalent bonds occurring between the amino acids of the CDR and those of the epitope. In the Fig. in grey, the antigen; In blue the antibody. The complementary determining regions of the L and H chain are shown in light pink and yellow. The amino acid sequences of the epitope are in light blue.


Fig. 15.5. AFFINITY. The strength by which a single binding site of a BCR or an antibody interacts with an antigen expressing a single epitope (monovalent antigen) is evaluated by equilibrium dialysis. This test measures the amount of a monovalent antigen (in moles) required to bind a percentage (33-50%) of the binding sites of an antibody. The higher the Ig affinity for that special antigen, the lower the amount of antigen required to bind the determined percentage of the binding sites will be. To perform this equilibrium dialysis test, a small dialysis chamber full of a liquid medium with two compartments separated by a dialysis membrane is employed.

A. A small monovalent antigen (red dots) is added to the medium of one compartment of the dialysis chamber.

B. The small monovalent antigen goes through the dialysis membrane and reaches the same concentration in the two compartments of the dialysis chamber.

C. Antibodies (pale blue Y) are added to one compartment. These remain confined in the compartment since they are too large to cross the dialysis membrane.

D. The antigen bound by the antibodies is removed from the equilibrium, while the residual free antigen again reaches the same concentration in the two compartments. The difference in antigen concentration in the two compartments of the dialysis chamber is due to the amount of antigen bound by the binding sites that is removed from the equilibrium. The higher the antibody affinity for that special antigen the lower the amount of antigen required to bind the selected percentage of the antibody binding sites will be.


Fig. 15.6. IN WHICH WAY DO THE AMINO ACIDS OF THE BINDING SITE INTERACT WITH THE EPITOPE? The chemical bonds resulting in the binding site-epitope interaction are weak and non-covalent. The presence of this kind of bond allows associations and dissociations between the binding site and epitope.

Fig. 15.7. STRENGTH OF THE INTERACTIONS. The strength of the multiple and diverse bonds between the amino acids of the binding site and the epitope is influenced on log scale by the distance between interacting structures (spatial proximity). Therefore a space proximity between the binding site and the epitope is of extreme importance for the force and persistence of the interaction.

Fig. 15.8. AFFINITY VS. AVIDITY. The strength by which a binding site interacts with a monovalent antigen is called affinity. However, a BCR (and antibodies) expresses two or more identical binding sites, each one of which has the same affinity for a given epitope. When a BCR (and an antibody) interacts with multiple identical epitopes on a multivalent antigen the binding strength is higher. The stronger binding’s strength resulting from multiple binding site–epitope interactions is called Avidity. In the Fig. in grey, a multivalent antigen; In blue the antibody. The complementary determining regions of the L and H chains are shown in light pink and yellow. The amino acid sequences of the epitope are in light blue.


CHAPTER 16.THE ANTIBODIES. Fig. 16.1. MEMBRANE BCR VERSUS SOLUBLE MONOMERIC IMMUNOGLOBULINS (Ig). As already show on Fig. 13.7, when a B cell is activated, it changes its genetic program and instead of producing BCR to be inserted on the cell membrane it produces and secretes a modified version of the BCR: The Antibody or Immunoglobulin (Ig) lacks the trans-membrane domain and the intra-cytoplasmatic tail. While structurally different, the BCR and the Ig produced by the same B cell share the same binding site. An activated B cell producing and secreting Ig acquires a distinct morphology and is defined as a Plasma cell (see Fig. 17.18). Fig. 16.2. Ig AS GAMMA GLOBULINS. The Ig secreted by activated B cells are so numerous that they form a distinct group of serum proteins, the gamma (ď §) globulins. Following an active immunization their concentration in serum increases significantly (red dotted line in the Fig.). This electrophoretic profile is obtained when electrical current is applied to serum proteins at pH 8.6 in a support medium. All the major serum proteins migrate towards the anode travelling in accord to the size and electrical charge and five major protein bands become evident. The diffuse band formed by gamma globulins shows that they are not exactly identical. What are the differences among them?

Fig. 16.3. MOLECULAR DIFFERENCES AMONG Ig. The diffuse electrophoretic band formed by Ig is due to their molecular differences. A distinct biological role corresponds to each of these structural differences.



Fig. 16.5. SCHEMATIC DRAWING OF THE MOLECULAR STRUCTURE OF THE FOUR IgG SUB-CLASSES. The difference in the location and the number of S-S cysteine bridges connecting the IgG chains markedly affect the flexibility of the molecule and its ability to bind two epitopes variously located in the space.

Fig. 16.6. DIRECT AND Fc MEDIATED ACTIVITY OF ANTIBODIES. Direct Ig activities: Numerous activities of antibodies are directly due to the Ig ability to bind the target antigen (See also Figs. 19.1-19.6) Indirect Ig activities: Several other important biological functions of Ig are mediated by the peculiar structure of the Fc fragment (See Figs 19.1, 19.719-12). The Fc of some Ig classes (IgM and IgG) activates the Complement cascade through the Classical pathway (See Fig. 19.11, 19.12) and interacts with specific receptors expressed on

149 the cell membrane of various immune cells. The peculiar structure of the Fc: a) Separates Ig in classes and sub-classes; b) Influences the half-life of the Ig; c) Allows Ig to form dimers or pentamers; d) Lets Ig diffuse into intravascular sites and cross the placenta and the gut mucosa; e) Allows the activation of the Complement cascade; f) Permits it to opsonize the target; g) Guides Antibody-Dependent Cellular Cytotoxicity (ADCC) (See Figs. 3.29; 19.9); h) Guides Antibody-Dependent Cellular Cytotoxicity (ADCC, See Figs. 3.29; 19.9); i) Guides cell activities; l) Decides cell survival or cell apoptosis.

Fig. 16.7. Fc RECEPTORS. Receptors for the Fc fragment (FcR) of the various Ig classes form a large family of molecules expressed on the surface of immune and epithelial cells. FcR are made by a single chain able to bind the Fc of the Ig and transduce the signal to the cell nucleus or by multiple chains. In most of the cases the receptor chain binding the Fc belongs to the super-family of Ig (See Fig. 13.5). Each FcR binds the Fc of a certain class (isotype) of Ig. A few FcR bind Ig at high affinity (10-10 M) and are able to immobilize a single Ig (upper panel) whereas other FcR bind the Fc at low affinity (10-6 M). In the latter case, several FcR are required to immobilize large Ig – antigen aggregates (immuno complexes, lower panel). The FcR transduce activating, inhibiting, survival or dead signals into the cell.

150 Fig. 16.8. THE BIOLOGICAL ROLE OF THE Fc GAMMA FRAGMENT. Several other important biological functions of IgG are mediated by the Fc fragment (See Fig. 13.2). The Fc interacts with specific receptors expressed on the cell membrane of various kinds of cells. A few of these receptors are of high affinity and strongly bind a single IgG. Numerous other receptors are of low affinity and cannot immobilize a single IgG. However a multiple of these weakly bonds can immobilize large immunocomplexes. Following the interaction with an IgG, FcR transduce the activating signal and trigger several cell effector functions. Fig. 16.9. THE TRANSPORT OF IgG ACROSS THE PLACENTA. The interaction of Fc with the FcRn receptor expressed on the placenta is instrumental for the passage of maternal IgG to the fetus. Mother IgG have an important protective role during the first three months of life. However, these antibodies may also cause major diseases: The passage of mother auto-antibodies against thyroid can cause thyroiditis in the fetus. Moreover, an Rh negative (Rh-) mother can transmit IgG reacting against the Rh blood group to the fetus and cause hemolytic disease (erythroblastosis fetalis) in an Rh positive (Rh+) fetus.




Fig. 16.12. IgA: SUB-CLASSES OF IgA.



Fig. 16.14. DIMERIC AND SECRETORY IgA. The joining chain (J chain), MW 15kD links two IgA or the five IgM monomers. At epithelial surfaces secretory IgA are an important contribution to gut barrier function (See Fig. 18.10).

Fig. 16.15. SECRETORY IgA: Secretory IgA play a critical role in the intersection between host immunity and microbiota, the entirety of microbes colonizing mucosal surfaces.

Fig. 16.16. SECRETORY IgA. The secretory component of secretory IgA (sIgA) protects the IgA from being degraded by proteolytic enzymes present on mucosal surfaces. Thus sIgA survive in the harsh gastrointestinal tract environment and provide protection against microbes present in body luminal spaces and secretions. sIgA coats and agglutinates microbes and antigens to prevent their direct interaction with the host.


Fig. 16.17. IgA DEFICIENCY.






Fig. 16.22. IgM: ANTIBODIES TO A, B, 0 GROUPS. These IgM are induced by a natural reaction against sugars of the cell wall of bacteria normally present in the gut and bronchi. The antibody response against carbohydrate antigens is of IgM class only since the majority of Th cells does not recognize carbohydrates and do not deliver isotype switching signals (See Fig. 17.13).






Fig. 16.26. THE TETRAMERIC FcR EPSILON. The interaction of IgE with the tetrameric Fcď Ľď€ receptor extends the life span of basophils and mast cells (Fig. 3.5) and makes these cells reactive against the antigen recognized by the IgE. The reaction elicited protects against parasites, nematodes especially. However, when the antigen recognized by an IgE is an innocuous antigen the reaction activated by the degranulation of basophils and mast cells is defined as allergic reaction. Allergic reactions can be localized or systemic. Localized reactions (immediate hypersensitivity) may cause itching of the eyes or involve skin, gastrointestinal tract and respiratory tract with clinical symptoms of various severity. Systemic degranulation of basophils and mast cells causes a potentially life-threatening reaction marked by swallowing and breathing difficulties, abdominal pain, vomiting, diarrhoea, hives, angioedema and dramatic decrease of the blood pressure.







Fig. 17.1. B1 B CELL ANTIBODY RESPONSE. The distinct antibodies produced by the three major B cell populations (B1, MZB, FoB2 B cells, See Fig. 12.5) sequentially counteract the invasion of microbes and antigens. In most instances, natural antibodies produced by B1 B cells (See Fig. 12.6 and Fig. 16.21) are already present before microbe or antigen invasion (Natural Antibodies). These natural antibodies are also present in persons who apparently had not been previously exposed to the corresponding antigen. In other cases, their production is boosted by the intruders. Natural antibodies are of IgM class. By interacting at low affinity with many distinct epitopes expressed by common bacterial polysaccharides (See Fig. 12.7) they provide a first line of defence against systemic blood-borne intruders. Some natural antibodies react at low affinity with self antigens. The role of these self-reactive natural antibodies is not yet defined. Perhaps they may have a homeostatic role and housekeeping functions, such as recognition and removal of senescent and altered cells. The IgM against A, B, 0 blood groups are an example of these natural IgM produced by B1 B cells (See Fig. 16.22).


Fig. 17.2. MARGINAL ZONE (MZ) B CELL ANTIBODY RESPONSE. When the invasion by microbes or antigens is not cleared by natural antibodies and cells of innate immunity, persisting intruders are drained to local lymph nodes (See Fig. 9.4). Here, the first reactive response is made by Marginal Zone B (MZB) cells homing in the marginal zone of the spleen and lymph nodes (See Fig. 12.9). BCR on the cell membrane of MZB cells recognize glycolipid and other microbial antigens bound to the C3b component of Complement cascade (See Fig. 20.2) and develop an early (< 3 days) IgM antibody production (See Fig. 12.10). These IgM bind their target epitopes at low affinity and form a second line of defence. Moreover, the immunocomplexes formed by these early IgM linked to the invading antigen are captured by Follicular Dendritic Cells (FDC) (Fig. 17.11) homing in the center of the follicle. The antigen trapped as immunocomplex by FDC plays a critical role in the induction of the high affinity antibody response made by B2 B cells.

160 Fig. 17.3. A SIMPLIFIED OUTLINE OF THE STEPS OF FoB2 B CELL ACTIVATION. The third line of defense against persisting intruders rests on the production of high affinity antibodies of various classes, the kind of reactive response made by activated FoB2 B cells. However, the activation of a FoB2 B cell is a complex process that requires the collaboration of Follicular Th cells and Antigen Presenting Cells (APC). These complex requirements are probably due to the important and persisting consequence of FoB2 B cell activation: a) The induction of a powerful, high affinity and long-lasting antibody response; b) The induction of an immune memory that may last several decades. These two outcomes may be catastrophic in the case of the induction of an erroneous immune response. The steps of FoB2 B cell activation summarized here are presented in detail in the following Figs. 1. The first step of FoB2 B cell activation rests on the arrival of the specific antigen in the spleen and lymph node. A soluble antigen with its natural conformation is drained to the B cell areas of lymph nodes and spleen (See Fig. 17.4). Here, antigen epitopes may be recognized by BCR on a FoB2 B cell membrane and prime the FoB2 B cell. 2. In the periphery, the antigen is also captured by APC (See Fig. 17.4). During their journey to the T cell areas of lymphoid organs, APC mature, digest the antigen and display antigen peptides associated to Class II HLA glycoproteins (HLA-p, See Chapter 6). Once in the T cell area, APC activate a Th cell with a TCR interacting at high affinity with HLA-p displayed on the APC membrane. An activated Th cell gives rise to an expanded clone of effector Th cells (See Chapter 9). 3. Following a first non-specific encounter, effector Th cells establish a long interaction with the antigenprimed FoB2 B cell. 4. A FoB2 B cell first primed by the antigen and then activated by effector Th cells proliferates and gives rise to a spherical area rich in blast cells (a Germinal Center). 5. Then, a Dark Zone made by proliferating B cell blasts (Centroblasts) and a Light Zone made by not proliferating B cell blasts (Centrocytes) becomes evident in the Germinal Center. 6. During successive migrations of Centroblasts to the Light Zone and Centrocytes to the Dark Zone, blast cells display hyper-mutation of the genes coding the binding site of their BCR, change the class of the antibody they produce (Isotype switch) and become Plasma cells. 7. Blast cells that have generated a hyper-mutated BCR interacting at poor affinity with the antigen displayed by Follicular Dendritic Cells die whereas those with a hyper-mutated BCR interacting with the antigen at high affinity survive and give rise to Plasma cells and long-living Memory B cells. A subsequent invasion by the same antigen triggers memory B cells to produce a quicker and more intense production of antibodies interacting with the antigen at high affinity.


Fig. 17.4. ANTIGEN PRIMED FoB2 B CELLS. Going into more detail through the phases of FoB2 B cell activation, when an invasion endures, the antigen in its native, conformation is drained to the lymphoid organ where it may be recognized by a FoB2 B cell expressing on the cell membrane a BCR able to interact at high affinity with antigen epitopes. The same intruder antigen is captured by Dendritic Cells, macrophages and other Antigen Presenting Cells (APC, in green in the Fig.) that shuttle it to the T cell areas of lymphoid organs. During their journey to lymphoid organs, APC mature, digest the antigen and associate a few antigen-derived peptides to the groove of Class II MHC glycoproteins. Once in T cell areas, APC present antigen peptides to Th cells (See Fig. 11.4).

162 Fig. 17.5. ANTIGEN PRIMING OF FoB2 B AND T CELLS. In lymphoid organs there are distinct areas where T cells (In the Fig. T cell area is in pale blue) and FoB2 B cells home (In the Fig. B cell area is in pale pink, see also Figs. 18.5 and 18.6). In B cell areas, FoB2 B cells form spherical aggregates (Primary follicles over a net of peculiar cells, the Follicular Dendritic Cells (See Figs 18.7 and 18.8). If an antigen interacts at high affinity with the BCR of a FoB2 B cell, the cell internalizes the BCR, processes the antigen and displays antigen peptides on the groove of MHC Class II glycoproteins (See Chapter 6). At the same time Antigen Presenting Cells (APC, in green) that have captured the same antigen in the periphery arrive in T cell areas. Here, APC presenting antigen peptides on the groove of HLA Class II glycoproteins activates TFh cells expressing the cognate TCR. The TFh cell activated by APC proliferates and gives rise to an expanded clone of effector TFh cells (intense blue).

Fig. 17.6. FULL ACTIVATION OF ANTIGEN-PRIMED FoB2 B CELLS. The antigen-primed FoB2 B cell starts to overexpress the chemokine receptor CCR7 and migrates towards the boundary with the T cell area attracted by chemokine ligands for CCR7 which are secreted by stromal and Dendritic Cells in the T cell area. This positioning of antigen activated FoB2 B cells at the interface with T cell area favors the scanning of HLA-p displayed on their cell membrane by the numerous effector TFh cells previously expanded upon the recognition of the same antigen presented by APC.


Fig. 17.7. THE FoB2 B CELL AS A PROFESSIONAL ANTIGEN PRESENTING CELL (APC). When the BCR of a FoB2 B cell interacts with the antigen, the B cell internalizes the BCR, processes the antigen as a professional APC, and presents antigen peptides in the groove of Class II HLA glycoproteins (HLA-p) (Figs. 6.8-6.10). Moreover, antigen-primed FoB2 B cells start to express special chemokine receptors and these cells are attracted at the interface of T-B cell areas. The positioning of antigen primed FoB2 B cells at the follicle-T zone interface favors their interaction with TFh cells. Initially, adhesion molecules establish multiple and not specific interactions between the leading towards the formation of a temporary immunological synapse between antigen primed FoB2 B and TFh cells (For details see Figs. 9.6 and 9.7). Once the two cells are in close contact, TCR on the membrane of TFh cell repeatedly scan the HLA-p displayed on the membrane of the FoB2 B cell. If TCR bind HLA-p with high affinity, the two cells remain in contact for several hours (See Fig. 9.8). During this prolonged interaction B cell acts as a professional APC: B cell not only displays on the groove of HLA Class II glycoproteins the peptides of the antigen initially bound by the BCR but also provides the required co-stimulatory molecules and receptors. While B cells are very effective APC they are also peculiar APC since they present only peptides of the antigen that has been specifically captured by their BCR. In the Fig. the antigen is in red, the BCR in orange and purple.


Fig. 17.8. THE TRIGGERING OF A FoB2 B CELL PROLIFERATION. Once a T and a B cell have established a first not specific interaction through adhesion molecules (in gray in the Fig.), TCR scan HLA class II glycoproteins and peptides (HLA-p) displayed on the cell membrane of the B cell. If TCR interact at high affinity with the HLA-p, several accessory molecules deliver a complex series of co-stimulatory signals to both the T and B cells. These accessory signals are the same as those delivered by Th cells and professional Antigen Presenting Cells (See Chapter 9). The final outcome of this prolonged Th and FoB2 B cell interaction is both the expression of a trimeric high affinity IL2 receptor (See Fig. 9.11 and 4.11-4.18) on the FoB2 B cell membrane and the secretion of IL2 by the Th cell.


Fig. 17.9. FoB2 B CELL-TFh CELL INTERACTION. ICAM, LFA and CD2 adhesion molecules (in gray in the Fig.) establish a first non-specific interaction between a FoB2 B cell and an effector Th cell resident in the follicle (TFh cell). Then the TCR on TFH cell membrane starts to scan HLA class II glycoproteins and peptides (HLA-p) on the cell membrane of the FoB2 B cell. At this point, the TFh cell secretes dopamine molecules that interact with the DRD1 dopamine receptor on the FoB2 B cell membrane. Dopamine induces the expression of ICOS (Inducible T cell costimulatory ligand). The interaction of ICOS with the newly expressed ICOS ligand enhances the expression of CD40 ligand (in red) REFERENCE: I Papa et al, Nature 2017, 547:318.

If TCR interact at high affinity with the HLA-p expressed by FoB2 B cell, CD3 and Zeta () chain molecules (See Fig. 7.15) transduce first activating signals to the TFh cell. By now, these interacting molecules move to a lipid raft (See Figs. 9.9 and 9.10). Here, TCR signals increases the expression of adhesion molecules making the FoB2 B–TFh cell interaction tight and persistent. The TCR - HLA-p interactions go along with the interactions of CD4 co-receptors with the monomorphic domain of the  chain of Class II molecules (Fig. 7.18). Additional co-stimulatory molecules transduce multiple signals enhancing both FoB2 B and TFh cell activation process. The CD40 ligand induced on TFh cell membrane binds the CD40 co-receptor on the surface of the FoB2 B cell (See Fig. 9.15). This CD40-CD40L interaction is of critical importance for triggering FoB2 B cell clonal expansion, gene hypermutation and immunoglobulin class switching (See

166 Fig. 9.15). CD40-CD40L interaction also induces (blue arrow) the over expression of molecules of the B7 co-stimulatory family (in green). The interaction of B7 molecules with the members of CD28 receptor family provides additional important co-stimulatory signals (See Fig. 9.14). The final outcome of this long TFh and FoB2 B cell interaction is both the expression of cytokine receptors on the FoB2 B cell membrane and the secretion of different combinations of cytokines by TFh cells (See Chapter 11). The IL2 released by TFh cells and captured by the high affinity IL2 receptor triggers B cell proliferation and induces its clonal expansion and the formation of a Germinal Center

Fig. 17.10. PROLIFERATION OF FoB2 B CELLS. Following a long-lasting interaction with TFh cells, antigenprimed FoB2 B cells are induced to proliferate by the IL2 secreted by TFh cells and captured by their high affinity IL2 receptor (See Figs. 4.11-4.16). Actively proliferating FoB2 B cells form a spherical aggregate of large blast cells (Germinal Center).Follicles in which a Germinal Center is evident are known as Secondary Follicles. The presence of Secondary Follicles shows that an immune response is going on. Germinal centers localize over a network of Follicular Dendritic Cells (FDC) (See Figs. 18.7, 18.8). FDC display antibody-antigen aggregates (immunocomplexes) on long elongations of their cell membrane. These immunocomplexes are made mostly by IgM, produced by Plasma cells (PC) deriving from MZ B cells during their earlier response to the same antigen (See Fig. 12.9-12.12). In the Fig. Antigen Presenting Cells are in green, B cells in pale pink, T cells in pale blue and the antigen is show as solid red circles


Fig. 17.11. SECONDARY LYMPHOID FOLLICLE. As the immune response continues, an affinity based B cell competition takes place to generate plasma cells producing antibodies reacting at high affinity with the antigen. A distinct Dark and a Light zone become evident in the germinal centers. The Dark Zone is made by packed blast cells in active proliferation (Centroblasts). Centroblasts undergo hyper-mutations of their BCR. Regularly, Centroblasts migrate from the Dark Zone to the Light Zone and become Centrocytes. Centrocytes are non-proliferating blast cells or blast cells proliferating to a low degree that express high levels of the hyper-mutated BCR. Centrocytes are apoptosis-prone cells which compete to get anti-apoptotic signals by the antigen displayed by Follicular Dendritic Cells. Only those Centrocytes that have acquired a hyper-mutated BCR interacting at high affinity with the antigen of the immunocomplexes on the surface of FDC win the competition and survive. Surviving Centrocytes re-enter in the Dark Zone and again become highly proliferating Centroblasts. Reiterated passages of blast cells from Light to Dark zone (Centroblasts-CentrocytesCentroblastsâ&#x20AC;Ś) are instrumental for the hyper mutations of the binding site of the BCR and the selection of cells that have acquired a hyper-mutated BCR interacting with the highest affinity with the antigen. Every time, a few blast cells do not re-enter the Dark Zone but differentiate into Plasma Cells (PC) secreting high amounts of antibodies.




Fig. 17.13. SWITCHING THE PRODUCTION OF IgM AND IgD TO IgG, IgA or IgE. Virgin FoB2 B cells produce IgM and IgD to be inserted into their cell membrane as BCR. Under the influence of cytokine combinations secreted by TFh cells, Centroblasts change the class of Ig they produce (Ig class switching) through a process of irreversible DNA recombination. Upstream from genes coding the constant part of the H chain (, , , ) there are conserved nucleotide stretches, called Switch regions (Shown as an orange star in the Fig.). Two switch regions are joined and the double strand DNA is open to allow the access of the enzyme called Activation Induced Deaminase (AID) that deaminates cytidines in the Switch regions. The lesions caused by AID deamination are converted into DNA double–strand breaks by general DNA repair factors. Through the combined action of other repair enzymes, the double strand DNA is cut at the two Switch regions. The DNA intervening between the Switch regions is excised and the free DNA ends are re-joined through a nonhomologous recombination mechanism. In this way the selected constant region is localized adjacent to the recombined VDJ.


Fig. 17.14. HYPERMUTATION OF THE GENES CODING THE BINDING SITE. This is another process of utmost importance which takes place in Centroblasts. Through the action of Activation Induced Deaminase (AID), the same enzyme responsible for the initiation of Ig Class Switching, cytosines in the rearranged genes segments coding for the Binding Site (VDJ/VJ), are deaminated to uracil. The anomalous presence of uracil in the DNA molecule triggers a series of repair mechanisms to respond to that damaged DNA sequence. These numerous mutations (hypermutations) can either increase or decrease the affinity of the binding site for the antigen.


Fig. 17.15. PROGRESSIVE INCREASE IN AFFINITY OF THE BINDING SITE (AFFINITY MATURATION). As the immune response continues, the affinity of Ig for the antigen increases. This increase in affinity rests on the combined action of hyper-mutations in the gene segments coding for the antibody binding site and a subsequent selection of Centrocytes expressing the hyper-mutated BCR. Centroblasts with a hyper-mutated BCR move from the Dark Zone to the Light Zone and become Centrocytes. Centrocytes express the hyper-mutated BCR at high density (Pink cells in the Fig.) and compete for the antigen (red circle) displayed as immunocomplexes by Follicular Dendritic Cells (FDC). Only Centrocytes that have acquired a BCR able to interact at higher affinity with the antigen win the competition, bind the antigen and receive anti-apoptotic signals by FDC. These Centrocytes either differentiate into Plasma Cells secreting the antibody or return to the Dark Zone and again, as Centroblasts, proliferate and hyper-mutate their BCR. After each round of hyper-mutations and competitive selection, the affinity and specificity of the BCR of surviving blasts continually increases (Affinity maturation). By contrast, those Centrocytes that have acquired a BCR interacting at poor affinity with the antigen are induced to die in the absence of surviving signals. The extremely numerous dead cells evident in Germinal Centers are rapidly eliminated by macrophages that acquire a characteristic histological aspect (Tingible body macrophages).


Fig. 17.16. STEPS OF FoB2 B CELL ACTIVATION. 1. The antigen in its natural conformation (In red in the Fig.) interacts with the BCR of a FoB2 B cell (red circle). 2. An antigen presenting cell (APC) displaying antigen peptides on Class II HLA glycoprotein is recognized by a TFh cell, which then generates a clone of activated effector T Fh cells (dark blue). 3. The antigen primed B cell expresses antigen peptides in the groove of Class II HLA glycoprotein (HLAp) and migrates to the boundaries of B (pink) and T (pale blue) cell areas. Here, HLA-p displayed by the FoB2 B cells may interact with the TCR of antigen-activated effector TFh cells. 4. As a result of this FoB2 B and TFh cell interaction, the FoB2 B cell starts to proliferate giving rise to aspherical area full of blast cells, the Germinal Center. 5. Progressively, both a Dark and Light Zone become evident in the Germinal Center. In the Dark Zone, blast cells (called Centroblasts) actively proliferate while BCR genes undergo hypermutations. The Centroblasts move to the Light Zone and differentiate in Centrocytes. Centrocytes compete for the antigen displayed as immunocomplexes by Follicular Dendritic Cells. Those Centrocytes expressing a BCR able to interact at high affinity with the antigen move to the Dark Zone and proliferate again. By contrast, those which have acquired a hypermutated BCR interacting poorly with the antigen undergo apoptotic death. 6. Reiterating passages of blast cells from the Light to the Dark Zone result in a progressive increase of the affinity of antibodies produced by Centrocytes. 7. At each stage of this reiterating Centrocyte - Centroblast recycling, a few blast cells differentiate into both Plasma Cells (PC) producing antibodies of various classes and into Memory Cells. The kind of antibody class produced by a Plasma Cell is then regulated by the combination of cytokines secreted by TFh cells.


Fig. 17.17. OUTCOMES OF FoB2 B CELL ACTIVATION. FoB2 B cells primed by the antigen and activated through a selective interaction with TFh cells start to proliferate as Centroblasts. Centroblasts hypermutate the binding site of their BCR, differentiate in Centrocytes and express numerous copies of the new BCR. Only those with a BCR able to bind the original antigen with higher affinity receive antiapoptotic signals, survive, hyper-mutate the binding site of their BCR again and proliferate. Rounds of Centroblasts-Centrocytes passages continually increase the affinity of the BCR. Progressively, Centrocytes expressing a BCR binding the antigen with high affinity differentiate into Plasma Cells and memory cells. At each round the Centrocytes with a hyper-mutated BCR binding the antigen at poor affinity die by apoptosis. Fig. 17.18. MAIN FEATURES OF PLASMA CELLS. Several Plasma cells migrate from the germinal center to the bone marrow where they survive for long times. Others migrate to the medullary cords of lymph nodes or into splenic red pulp. Plasma cells display absent or poor production of BCR but a massive production of secreted Ig. BCR H chains have a trans-membrane domain of about 25 amino acids and a short cytoplasmatic tail (See Fig. 13.7). The passage from transmembrane BCR to secreted Ig depends on alternative RNA splicing at two different adenilation sites.


Fig. 17.19. MAIN FEATURES OF MEMORY B CELLS. Following a subsequent arrival of the antigen, longlived memory B cells quickly differentiate in plasma cells producing large amounts of antibodies interacting with the antigen with high affinity and specificity. Memory B cells differentiate from a Centrocyte with a hyper-mutated BCR of a switched immunoglobulin class and inherit the genetic changes occurred in the Germinal Centers. Their activation (secondary immune response) rests on the cooperation with memory TFh cells and triggers the production of antibodies of the switched isotype reacting at high affinity with the antigen. The rapid and specific response of reactivated memory B cells effectively protects the body against subsequent infections by the same pathogen. These may be so mild as to be unnoticed. The protection afforded by vaccines rests on the induction and maintenance of memory B cells. The secondary antibody response made by the reactivation of memory B cells markedly differs from a primary response.


Fig. 17.20. KINETICS OF PRIMARY AND SECONDARY ANTIBODY RESPONSE. The antibody production is a protective mechanism which acts far away from the production site. During primary response, an initial protective response is based on the secretion of relatively poor affinity IgM. Slowly, a more efficacious response takes place. The switch of antibody classes leads to a more appropriate response at peculiar anatomical sites. Moreover, because of the hypermutations of the genes coding for the binding site, antibodies become highly specific and able to interact at high affinity with the antigen. A. Kinetics of the antibody class switch during a primary immune response. B. Comparison of the phases of a primary and a secondary antibody production. 1. Lag phase. In the primary response antibodies become detectable between 3-4 days to 2 weeks after antigen arrival, depending on the kind of antigen. Lag phase is shorter or virtually absent in the 111secondary response. 2. Log phase. The titer of antibodies increases exponentially doubling every 6 hours. This exponential increase lasts several days in the primary response. A shorter and more intense log phase is evident in the secondary response. 3. Steady stage. The amount of antibodies produced and catabolized is almost identical. In a secondary response this phase is reached quickly and lasts longer. 4. Decrease phase. It lasts much longer in the secondary response.


CHAPTER 18. SECONDARY LYMPHOID ORGANS. Fig. 18.1. SECONDARY LYMPHOID ORGANS. Usually microbes and antigens enter the body through the skin, gastrointestinal and respiratory tracts (See Chapter 1). Secondary lymphoid organs act as a series of filters monitoring the content of body fluids. The arriving antigen diffuses in the secondary lymphoid organs where it may directly interact with the BCR on the membrane of B cells or be captured by local Antigen Presenting Cells (APC) localized in T cell areas and around B cell follicles. Moreover, APC that have captured the antigen in the periphery at the site of entry travel to draining lymph nodes through lymphatic vessels (Fig. 9.4). T cells rapidly fan out to scan APC for HLA-p that match their specific T cell receptors and rapidly proliferate during the immune response. After an antigen challenge dividing B and T cells markedly increase lymphoid organ cellularity leading to their expansion. Tissue fluids are filtered by numerous small lymphoid aggregates and by larger lymphoid structures (Tonsils, Peyer's patches and Appendix) forming the Mucosa Associated Lymphoid Tissue (MALT). The blood is filtered by the spleen, the body's largest lymphatic organ. The spleen is also important for the elimination of aged blood cells. The interstitial fluid from peripheral body tissues is filtered by the lymph nodes. The various secondary lymphoid organs are anatomically different. However, they share a common basic microscopic architecture: A spherical B cell aggregate (B follicle) surrounded by a T cell area where numerous APC (macrophages, Dendritic Cellsâ&#x20AC;Ś) are present. The selective homing of APC, T and B cells in distinct areas of the lymphoid organs is guided by constitutive chemokines (See Fig. 4.22) released by stromal, Dendritic Cells and Follicular Dendritic Cells (FDC). Activation of an immune response may take place at the site of microbe or antigen invasion and in any district of the body. However, the specialized anatomical features of secondary lymphoid organs, the local interplay between chemokine, cytokine gradients and cell adhesion molecules markedly favor both cell to cell contacts and the interaction with the antigen. These features turn secondary lymphoid organs into a specialized site for the efficient activation of T and B cells.


Fig. 18.2. THE LYMPH NODE: SCHEMATIC DIAGRAM. Lymph nodes are the meeting places for B cell and antigen, T cells and Antigen Presenting Cells (APC) and activated T and B cells. In our body there are thousands of lymph nodes strategically localized where antigen invasions are more common. In many cases they are aggregated in clusters. A lymph node is a complex, encapsulated structure present in mammals only. A lymph node weighing about 1 g contains around 2 x 107 lymphoid cells. Afferent lymphatic vessels collect and channel interstitial fluid into the subcapsular sinus (in pink). From here, the lymph is drained towards medullary sinuses. The lymph node is constituted of an outermost cortex where FoB2 B cells are organized into lymphoid follicles and inner paracortical areas made up mainly of T cells and APC as Dendritic Cells and macrophages. When an immune response involving B cells is underway, some of the primary follicles enlarge and display central areas of intense B cell proliferation called Germinal Centers and the follicles are known as secondary lymphoid follicles. About 2 x 107 lymphoid cell/hour leave the lymph node through the efferent lymphatic vessels: About 75% of these are T cells, 25% B cells, and only 1% macrophages and Dendritic Cells. 90% of the lymphoid cells leaving the lymph node were arrived through the blood and about 6% are locally generated cells.



A. Antigen Presenting Cells (APC): Free antigen (In red in the Fig.) and APC (Dendritic Cells, macrophagesâ&#x20AC;Ś, in green in the Fig.) arrive from peripheral body districts to the draining local lymph node through afferent lymphatics. Every hour, about one million cells arrive. Of these 75% are T cells, 6% B cells, and 15% are APC. In the presence of inflammatory cytokines, APC that have captured an antigen in the periphery decrease their adhesiveness to the tissues, migrate and localize in T cell areas. Immature Dendritic Cells (DC) in the body peripheral sites expresses surface receptors allowing the capture of microbes which are taken up by macropinocytosis and receptor-mediated phagocytosis (See Fig. 3.22). Then, DC leave the epithelium and migrate via the blind-ending afferent lymphatics to regional lymph nodes, where they arrive as fully matured DC expressing high levels of Class II HLA glycoprotein and efficiently present antigen peptides to TFh cells. In the T cell rich paracortical area of the lymph node mature DC select virgin specific TFh cells from those arrived from blood vessels (See below). DC stimulation of antigen-specific virgin TFh lymphocytes leads to their clonal expansion.

B. Lymphocytes: Once in the lymph node parenchyma, the afferent artery divides into smaller arterioles that become High Endothelium Venules (HEV) within the T cell areas. HEV consist of cuboidal endothelial cells with numerous lymphocytes within the walls. Blood lymphocytes, attracted by a combination of chemokine gradients produced by HEV and lymph node stromal cells increase their expression of L-Selectin, a homing receptor expressed on the cell membrane of virgin T and B cells, and cross the walls of HEV. In this way about 25% of virgin T and B cells exit (extravasate) in lymph node parenchyma. Only virgin lymphocytes extravasate since activated lymphoid cells do not express L-selectin anymore. Once in the lymph node parenchyma, other chemokine combinations segregate lymphocytes into the T and B cell area following a network of fibroblastic reticular cells: B cells move at the speed of 6 microns/minute while T cells at the speed of 12 microns/minute.



Fig. 18.5. THE LYMPH NODE: B CELL AREAS. Exploiting a monoclonal antibody (mAb, See Chapter 21) specific for a surface marker of B cells (the anti-CD20 mAb), B cells are selectively stained in brown. This technique (immune-histo-chemistry) shows the particular homing of B cells in lymph nodes where they give rise to spheroidal aggregates (follicles).

Fig. 18.6. THE LYMPH NODE: T CELL AREA. Exploiting a different monoclonal antibody (mAb, See Chapter 21) specific for a surface marker of T cells (the anti-CD3 mAb, See Fig. 7.15),T cells are selectively stained in brown. Immunohistochemistry shows that the majority of T cells localize around the B cell follicles. However, a significant population of T cells is also dispersed inside B cell follicles (TFh).



A. Exploiting a monoclonal antibody (mAb, See Chapter 21) specific to a surface marker highly expressed on the cell membrane of FDC (the anti-BLC mAb) a network of FDC becomes evident at the center of B cell follicles.

B. FDC display long cytoplasmatic elongations (dendrites). Several surface markers of both lymphoid and myeloid cells are expressed on FDC cell membrane. Moreover, FDC display numerous receptors for the Fc domain of antibodies of different classes (FcR, in blue in the Fig. ) and for Complement components (CD21, CD35, See Fig.). FDC also express numerous adhesion molecules (LFA-1 and other integrins, in gray) that favor their interaction with ICAM-1 and other adhesion molecules on the surface of B cells and Centrocytes.

C. Filiform dendrites of certain FDC develop multiple beads coated by immunocomplexes (iccosomes). D. Iccosomes are spherical bodies coated by immune complexes formed by antigen-antibodies trapped in follicular Dendritic Cells. Iccosomes can be shed from FDC and be captured by FoB2 B cells and Centrocytes. FDC do not derive from bone marrow, and TNF and Lymphotoxin play a crucial role in their development. Chemokines released by FDC guide the homing of T and B cells and are crucial for the normal architecture of secondary lymphoid organs.

181 Fig. 18.8. THE LYMPH NODE: ANTIGENPRESENTATION BY FOLLICULAR DENDRITIC CELLS (FDC). FDC capture the antigenantibody complexes (immunocomplexes) through their receptors for the Fc domain of antibodies (FcR). Antigens and immunocomplexes bound by Complement components (opsonized antigens; See Fig. 20.6) are captured by complement receptors (CR) (See Fig. 20.8). A captured antigen is not internalized but remains intact on the cell membrane. In this way, FDC act as longterm repositories of antigens to be presented to FoB2 B cells and Centrocytes and to maintain B cell memory. Centrocytes with a hyper-mutated BCR compete for the antigens displayed by FDC and the antiapoptotic signals they deliver (See Figs. 17.16 and 17.7).

Fig. 18.9. THE SPLEEN. Despite its complex structure, the basic microanatomy of the white pulp of the spleen is similar to that of the other secondary lymphoid organs: T cells are clustered around arterioles (PALS); FoB2 B cells give rise to spherical follicles, some of which may display Germinal Centers; B cell follicles are surrounded by peri-follicular areas rich in Antigen Presenting Cells (macrophages and Dendritic Cellsâ&#x20AC;Ś), non-circulating B cells and T cells. Follicular Dendritic Cells are at the center of B follicles while High Endothelial Venules are in the peri-follicular areas. As in lymph nodes and other secondary lymphoid organs, the homing of various cell populations is guided by chemokines.


Fig. 18.10. PEYER’S PATCHES. The Mucosa Associated Lymphoid Tissue (MALT) is formed by numerous small lymphoid aggregates and by larger encapsulated lymphoid structures (Tonsils) all sharing the basic micro-anatomy of the other secondary lymphoid organs. Peyer’s patches are 100-200 small lymph node-like structures lying beneath the gut mucosa. They consist of a central dome made by B cell follicles and Germinal Centres. TFh cells and Antigen Presenting Cells (APC), Macrophages, Dendritic Cells… occupy the area around the follicles. Peyer’s patches collect antigens and fluids under the epithelial gut surface. Moreover, specialized epithelial cells (Microfold cells or M cells) channel by transcytosis antigens and microbes from the gut lumen. Lymphoid cells enter the Peyer’s patches from High Endothelial Venules (HEV) and exit through efferent lymphatics to travel to mesenteric lymph nodes and, then, to the blood. Local cytokines (TGF-, TNF and Lymphotoxin, LT) and Dendritic Cells induce the differentiation of CD4+T cell into TFh cells. CD40 ligand and IL-21 from TFh cells induce the expression of Activation Induced Deaminase (AID) in B cells and promote IgA class-switch recombination (See Fig. 17.13). Dimeric IgA secreted by Plasma Cells (PC) generated in the Peyer’s Patches are trapped by the poly IgA receptor1 (pIgR1) and transported by transcytosis to the gut luminal surface. Following a proteolytic cleavage, dimeric IgA associated with the pIgR1 fragment (secretory IgA) are released into luminal spaces (See Figs. 16.14-16.16).





184 Fig. 19.3. VIRUS NEUTRALIZATION. Following a viral infection, antibodies are produced against many epitopes of viral proteins. A few of these antibodies may neutralize the virus while other antibodies may be ineffective. Frequently antibodies neutralize viral infectivity by blocking virus absorption. Other antibodies may prevent the uncoating of the virus genomes in endosomes or aggregate (agglutinate) virus particles. In addition, enveloped viruses may be lysed (destroyed) by Complement activated by anti-viral antibodies (indirect activity of antibodies).

Fig. 19.4. IMMUNOCOMPLEXES. An immunocomplex activates the Complement cascade. The adhering Complement fragments C4b and C3b bind to the CR1 Complement receptor (See Fig. 20.8) on the surface of red blood cells. In this way, red blood cells capture the immunocomplexes and transport them to the liver. Liver macrophages capture and destroy the immunocomplex without damaging the red blood cell.



Fig. 19.6. AGGREGATION AND INTERNALIZATION OF MEMBRANE MOLECULES AND RECEPTORS. Antibodies against cell membrane molecules and receptors cause their aggregation and internalization. Once they have interacted with the antibody, membrane molecules form several small aggregates (patches) that aggregate together (cap). The capped molecules are then internalized and temporarily the membrane becomes free from these molecules (stripped). Auto-antibodies directed against membrane receptors can cause major disease. For example, myasthenia gravis is caused by auto-antibodies stripping acetylcholine receptors. Another example is provided by monoclonal antibodies (See Chapter 21) against the membrane product of the Her-2 (neu) oncoantigen (Herceptin). Herceptin mediated aggregation and internalization of Her-2 receptors inhibit the proliferative activity of Her-2 positive neoplastic cells. Other antibodies do not aggregate membrane receptors but inhibit their function by blocking their binding site.

186 Fig. 19.7. IMMUNE PHAGOCYTOSIS: TARGET OPSONIZATION. A phagocytic cell can recognize a foreign body through a series of Pattern Recognition Receptors (See Fig. 2.4) which trigger the emission of pseudopods that engulf the particle in a phagosome (Fig. 3. 20) (Left side in the Fig.). However, the same target antigen is recognized and engulfed much better when it is coated by antibodies (opsonized antigen) (Right side, in blue). The Fc domain of the antigen-bound antibodies allows the cross linking of Fc receptors (FcR, in orange, mainly Fc gamma RI, CD64 and Fc alpha RI, CD89) on the phagocytic cell membrane. The signal delivered by cross-linked FcR activates macrophages and other phagocytic cells and increases the phagocytosis and the destruction of engulfed particles. The same antibodies not bound to their antigen (free antibodies) link the FcR with low affinity and cannot activate the phagocytic cell. A similar increase of phagocytosis (opsonization) is observed when the target antigen is coated by components of the Complement cascade. In this case, Complement receptors are involved in the opsonisation (See Fig. 20.8).

Fig. 19.8. ANTIBODYDEPENDENT CELLULAR CYTOTOXICITY (ADCC). The killer activity of an NK cell (See Fig.3.31 and 19.9), a macrophage, a granulocyte or a T cell can be activated and guided by receptors for the Fc domain of antibodies (FcR, in red in the Fig.). The cross-linking of various FcR on a killer cell membrane by the antibodies bound to antigens (triangles) on the surface of a target cell

187 triggers the cytolytic activity of the cell. This killer activity is dependent on antibodies (Antibody Dependent) but is fully mediated by the killer cell (Cellular Cytotoxicity). ADCC mediated by NK and T cells is an important defence mechanism against viral infections and tumor cells. ADCC mediated by eosinophils and neutrophils (Figs. 3.8-3.12) controls infection from parasites that are too large to be endocyted.

Fig. 19.9. THE Fc GAMMA RECEPTOR III (FcRIII, CD16). In numerous cases the ADCC (See Fig. 3.31 and 19.8) is mediated by the FcRIII. This important receptor binds the Fc domain of IgG1 and IgG3 antibodies. It is expressed on the cell membrane of NK cells, neutrophils, eosinophils and macrophages. Like the majority of receptors for the Fc, the FcRIII is a molecule of the Ig-superfamily (In dark red in the Fig.) (See Fig. 13.5). The cross-linking of multiple FcgRIII induces the phosphorylation of the ITAM sequences (See Fig. 7.14) present on the transducer and  chains. Phosphorylated ITAM sequences (P) provide docking sites for the activation of Zap 70 and Syk that begin the signal transduction.


Fig. 19.10. DEGRANULATION OF MAST CELLS AND GRANULOCYTES. Granulocytes, basophils and mast cells express a set of receptors for the Fc domain of the Ig (FcR) which bind IgE at high affinity (FcRIII, in purple in the Fig.).

In a few individuals FcR of basophil granulocytes and mast cells are bound to numerous IgE (in blue) specific for a special antigen (allergen). IgE remain bound for a very long time to cells that receive anti-apoptotic signals by the bound IgE. When the allergen binds IgE on FcR, the granulocyte basophil and the mast cell immediately degranulate, i.e. release the content of cytoplasmatic granules. Numerous mediators are suddenly released: vasoactive amines (histamine) and arachidonic acid metabolites (prostaglandins) and cytokines (TNF, IL-4…) (See Figs 3.6 and 3.7).


Fig. 19.11. CLASSICAL PATHWAY OF COMPLEMENT ACTIVATION. The Complement is a large family of proteins endowed with powerful and distinct biological activities (See Chapter 20). A few Complement proteins finalize (i.e. complement) the activity of IgM and IgG antibodies. Once bound to an antigen, the Fc domain of IgM and IgG interacts with C1q, a component of this classical Complement activation pathway. When the globular heads of C1q sense conformational changes of the Fc domain (In purple in the Fig.) of a single IgM or several IgG (at least two), C1q becomes activated and recruits C1r and C1s components of the Complement family. The complex C1q, r, s acquires a proteolytic activity and acts on the two subsequent components of Complement cascade, the C4 and C2. The complex C1q, r, s first cuts the inactive C4 into two active fragments, C4a and C4b. Then it cuts the inactive C2 into two active fragments, C2a and C2b. The C4b fragment exposes a reactive group that binds the antigen surface covalently. Subsequently, the fragment C2a binds C4b. The combined activity of C4b and C2a (C4b2a) acquires an important enzymatic activity called C3 convertase. C3 convertase cuts the inactive C3 into two active fragments (See Chapter 20).


Fig. 19.12. COMPLEMENT ACTIVATION: FINAL STAGES. The active group of C3b binds covalently to the surface of most antigens. Acting on C5, C3b generates C5a and C5b fragments. The final stages of Complement activation imply both lethal damage of the surface of the intruder cell (See Fig. 20.5) and the release of soluble Complement fragments (anaphylotoxins) that recruit a local inflammatory reaction (See Fig. 20.7). C5b initiates the assembly of other components of complement cascade and forms the Membrane Attack Complex (MAC), inserting it into the membrane of the target cell (See Fig. 20.5).


CHAPTER 20. THE COMPLEMENT SYSTEM. Fig. 20.1. THE COMPLEMENT SYSTEM: A CASCADE OF PROTEOLYTIC ACTIVITIES. The Complement system is a crucial component of the immune response to infection and tissue damage. Once activated Complement proteases cleave each other through precise cascade mechanisms. The Complement can be activated by IgM and IgG antibodies bound to an antigen. Alternatively, Complement can be directly activated by foreign sugars expressed on the surface of microbes. Thus, Complement is both a complement of specific activity of antibodies and a key defense mechanism of natural immunity. The final outcomes of Complement activation are: 1) The killing of the foreign intruder; 2) A facilitated phagocytosis (opsonization) of the intruder; 3) The recruitment of a local inflammatory reaction. Fig. 20.2. PATHWAYS OF COMPLEMENT ACTIVATION. The Complement cascade is activated by distinct sensor proteins. Through the classical activation pathway, Complement reactions are activated by IgM and IgG bound to an antigen. In this case, Complement reactions complement (i.e. complete, finalize) the specific reactivity of antibodies (See Figs. 19.11 and 19.12). Alternatively, specialized sensor Complement proteins directly perceive the presence of foreign molecules, mainly microbial sugars. All the three major pathways of Complement activation (the Classical pathway, the Lectinic pathway and the Alternative pathway) converge in the generation of a C3 convertase activity. Once a C3 convertase is generated, a common pathway (In brown in the Fig.) activates Complement effector functions.

192 Fig. 20.3. THE LECTINIC PATHWAY. The Complement components Mannose Binding Proteins (MBP) and Ficolins are typical Pattern Recognition Receptors (See Fig. 2.4). Following the recognition of foreign sugars these receptors trigger a rapid activation of the destructive and pro-inflammatory activities of Complement. As a result of this activation pathway Complement plays a central role in natural immunity. For the Classical pathway of Complement activation see Figs. 19.11 and 19.12) Fig. 20.4. THE ALTERNATIVE PATHWAY. In our body fluids C3 is spontaneously cleaved into the two fragments C3a and C3b. Normally the C3b fragment has a very short life. However, if microbes are present, C3b may bind the microbe. Once stabilized on the microbial surface, C3b binds Factor B, Factor D and Properdin and generates the Bb-C3 (C3bBb) convertase. Since the C3b fragment is generated continuously, the activation of Complement via the Alternative pathway takes place almost immediately following microbial invasion. In addition, when C3b is generated through the Classical or Lectinic pathway, its binding to Factor B, Factor D and Properdin on microbial surfaces triggers the Alternative pathway of Complement activation providing an important amplification of Complement activity. Properdin is another soluble Pattern Recognition Receptor (See Figs. 2.4) able to bind both microbes and C3b and, thus, to further amplify Complement activation. Properdin is stored in secondary granules of basophils, mast cells and in granules of neutrophils.

193 Fig. 20.5. THE MEMBRANE ATTACK COMPLEX (MAC). Complement activation through Classical, Lectinic and Alternative pathways triggers a common final cascade reaction that may result in the insertion of the MAC into the cell membrane and the subsequent death of the cell or microbe. C3 is the most abundant complement component (1.2 mg/ml in the human serum). A single C3 convertase (C4b2a or C3bBb) generates more than 1000 C3a and C3b fragments. Fig. 20.6. FACILITATION OF PHAGOCYTOSIS (OPSONIZATION). The Complement system does not always succeed in inserting Membrane Attack Complexes and making death holes in microbial surfaces. In numerous cases, the microbial surface remains intact even if covered by fragments of Complement components. However, Complement fragments adhering on cell membrane make the microbe (or a particulate antigen) much more susceptible to phagocytosis. In effect, as shown on Fig. 19.7, a phagocytic cell can recognize a target antigen through a series of Pattern Recognition Receptors (See Fig. 3.4) which trigger the emission of pseudopods to engulf the particle in a phagosome (Fig. 3.18) (left side). However, the same target antigen is recognized and engulfed 10100 times better when it is coated by antibodies or by a few Complement fragments (opsonized) (right side of the Fig.). The Complement fragments adhering on the microbeâ&#x20AC;&#x2122;s surface are recognized by many Complement receptors commonly expressed by phagocytic cells (See Fig. 20.8). The signals delivered by these receptors efficiently activate phagocytosis and the subsequent destruction of engulfed particles.

194 Fig. 20.7. INDUCTION OF A LOCAL INFLAMMATORY REACTION. These cleavage fragments of Complement components (anaphylatoxins) bind receptors expressed by granulocytes and macrophages and trigger a local inflammatory response. Acting on mast cells and, directly, on endothelial cells these Complement fragments increase vessel permeability, favor leukocyte extravasation and enhance the local accumulation (edema) of serum fluids rich in molecules of natural immunity, Complement and antibodies. These fluids are then drained to local lymph nodes where they may trigger a specific immune response. Fig. 20.8. COMPLEMENT RECEPTORS. The numerous Complement receptors (CR) mediate the multiple activities of Complement. The soluble cleavage fragments C2b, C3a, C4b and C5a bind receptors expressed by granulocytes, macrophages, mast cells and endothelial cells and trigger a local inflammatory response. By contrast receptors to fragments which adhere on the cell surfaces (C3b, C4b) trigger an efficient phagocytosis (opsonization), complement dependent cellular cytoxicity (CDCC) (See Fig. 3.33) and the capture and transport of immunocomplexes that have activated the complement cascade. CR1 expressed on a Follicular Dendritic Cell surface plays a key role in capturing immunocomplexes and in the antigen stimulation of Centrocytes (See Figs. 17.15 and 17.16). The CR2 receptor, by contrast, is an important co-stimulator of B cell response (See Fig. 13.11). Thus, the activation of Complement through the Lectinic and Alternative pathways provides an important costimulation of the antibody response.



Fig. 21.1. DYNAMICS OF A POLY-CLONAL IMMUNE RESPONSE. In general, an antigen displays several different epitopes (See Fig. 15.2). Each epitope is recognized by several B cells (a, b, c, d) expressing distinct BCR interacting with the same epitope with different affinity. Once activated, each of these B cells generates a clone of daughter cells all expressing the identical BCR During the evolution of an immune response a Darwinian competition among these clones takes place. Clones with a BCR that interacts better (with higher affinity) with the epitope win the competition and overcome poor affinity clones (See Fig. 17.16). Moreover, as time passes, B cells undergo BCR hyper-mutation (dark blue, orange clones) and may change the class of antibody they produced (isotype switch, See Fig. 17.13). Therefore, an immune response is a dynamic process, based on clonal expansion, clonal competition and clonal contraction. This means that the immune response mounted by an individual against an antigen, the titer of antibodies produced, their specificity for distinct epitopes, their affinity for the same epitope and the class of antibodies produced changes continuously. At the end of the immune response, only a few high affinity memory cells will survive (See Fig. 17.16).

196 Fig. 21.2. WHY ARE MONOCLONAL ANTIBODIES PRODUCED? An immune serum is a serum collected from an individual (a person, a mouse, a sheepâ&#x20AC;Ś) after immunization against an antigen. An immune serum contains a mixture of antibodies against the antigen used for the immunization. The titer of antibodies present in an immune serum, the percentage of antibodies of different classes and their affinity change continuously and can be markedly different in immune sera collected from various individuals immunized against the same antigen. By contrast a monoclonal antibody constantly displays the same specificity, affinity and isotype. Thus it provides a specific and invariant tool.

Fig. 21.3. HOW TO PRODUCE A MONOCLONAL ANTIBODY. The technology invented by George Kohler and CĂŠsar Milstein in 1975 (both were awarded the Nobel prize in 1984) is based on the fusion of a shortliving B cell obtained from a mouse immunized

197 against an antigen with a neoplastic plasma cell, potentially immortal but lacking an enzyme necessary to grow in a special culture medium. Thus both the B cells and these plasmacytoma cells cannot survive in the special culture medium. However, if a short-lived B cell fuses with a potentially immortal plasma cell, the resulting hybrid cells may acquire from the B cells the genes coding the specific BCR and the genes allowing the survival in the special culture medium. At the same time, the hybrid cell may also acquire from the plasma cell both the immortality and the machinery to produce and secrete high amounts of antibodies. Making millions of random cell-to-cell fusions it is possible to generate a significant number of these immortal hybrid cells (hybridoma cells) able to proliferate in the special culture medium and produce antibodies against the antigen used for the immunization.

Fig. 21.4. CELL-TO-CELL FUSION. There are several technologies to favor the fusion between a B cell and a plasma cell cultured together in a special selective medium. In all cases, the fusion is a random event which produces all possible kinds of hybridomas. The majority of cells will not survive the fusion shock. Moreover, independently from a successful integration of cell genomes, hybrids between B and B cells will undergo apoptosis. Also hybrids between plasma cells and plasma cells will die since they lack the gene necessary to survive in the special culture medium. Only hybrids between a B cell and the defective plasma cell have a chance to survive. Surviving B-plasma cell hybrids will then be placed in micro-culture wells in order to favor their clonal expansion.


Fig. 21.5. SELECTION OF HYBRIDOMAS PRODUCING THE DESIRED MONOCLONAL ANTIBODY. While previous maneuvers are mostly driven by casual events, the selection of the clones to be expanded is based on the intelligence of the researcher. To make this selection, a single hybrid cell will be placed in each micro-culture well. In a few of these wells the single hybrid cell survives and generates a clone of identical daughter cells producing the identical antibody or not producing any antibody. Accurate evaluation of the titer, the affinity and the class of the antibody produced in the various wells permits the selection of the best hybridoma producing the desired antibodies. Selected hybridomas are then expanded in larger cultures. Then, one can produce liters and liters of culture medium containing high amounts of the antibody produced by a large clone descending from a single hybrid cell (mono-clonal antibodies). A few cells of the selected hybridoma can be stored in liquid nitrogen for an almost indefinite period of time in order to re-start the clonal expansion and the production of the identical antibody on demand.

199 Fig. 21.6. FEATURES OF MONOCLONAL ANTIBODIES (mAb). Commonly mAb are produce with mouse cells. However there are mAb variously engineered to have part of the binding site or the constant domains of human origin. Humanization of murine antibodies is important when mAb are used in therapy since repeated administrations of mouse antibodies to a human patient may elicit the patient’s immune reaction. The patient antibodies against mouse immunoglobulins may neutralize the mAb activity. As mAb are specific and invariant tools that can be variously exploited in diagnosis and therapy, several new techniques (phage display, genetic engineering…) have been set up for their production. mAb conjugated with a fluorescent dye or an enzyme are currently widely used in immunohistochemistry (See Figs. 8.10, 8.11, 18.5-18.7). mAb conjugated with gold particles are exploited in immune-electron-microscopy. mAb conjugated with toxins, radioisotopes and drugs are used in therapy. Fig. 21.7. CLUSTERS OF DIFFERENTIATION (CD). Monoclonal antibodies (mAb) reacting against antigens expressed on the cell membrane of human leukocytes have been produced (and are still currently produced) in various parts of the world. So numerous mAb recognizing the same or different epitopes of the same membrane molecule with various affinities have been made available. An international standardization has allowed and is still allowing the molecule recognized by these groups (cluster) of mAb on the immune cell surface to be defined as Differentiation Antigen recognized by a cluster of mAb. Thus molecules on the surface of immune cells are now denoted by the acronym CD (a Differentiation molecule recognized by a Cluster of mAb) followed by a number (CD1, CD2, CD3, CD4…CD125…).


Fig. 21.8. TECHNOLOGICAL MANIPULATIONS OF MONOCLONAL ANTIBODIES (mAb). mAb can be variously engineered to better exploit their ability to bind a target antigen. The (Fab)â&#x20AC;&#x2122;2 fragment maintains the ability to bind two identical epitopes and to form antigen aggregates (immunocomplexes). When it is engineered to recognize two different epitopes [(Fab)â&#x20AC;&#x2122;2 bispecific] it can be exploited to put in contact two different cells, e.g. a killer cell and a target cell. A bi-specific diabody is a smaller version able to better diffuse in the body tissues. Diabodies, Triabodies and Tetrabodies are small interconnected antibody fragments able to bind two, three and four epitopes. Despite the removal of the constant domains, these small molecules may maintain the specificity of the original mAb. Alternatively they can be engineered to express different binding sites. Monovalent Fab is another small fragment of antibody that may freely diffuse in the body tissues. The scFv is an even smaller engineered molecule (Arrows indicate the binding sites; in purple peptides connecting antibody fragments.


CHAPTER 22. IMMUNE TOLERANCE AND CONTROLS OF THE IMMUNE RESPONSE. Fig. 22.1. IMMUNE TOLERANCE. The combination of several control mechanisms explains why the immune system reacts to a limitless variety of antigens without inappropriately attacking bodyâ&#x20AC;&#x2122;s own molecules. Self-tolerance is a first crucial developmental process that enables immune system to discriminate between self and not self molecules. Self tolerance is not genetically dictated. Instead it is learned! Distinct central and peripheral mechanisms contribute to the acquisition and maintenance of tolerance towards selfmolecules. A few of these mechanisms will be described here.

Fig. 22.2. CENTRAL TOLERANCE: THE DELETION OF SELF-REACTIVE T AND B CELLS. The generation of the large repertoire of BCR and TCR is based on a random and error prone gene recombination (See Chapter 14). Because of the casual nature of this process, numerous B and T cells express an antigen receptor interacting at high affinity with self antigens. These potentially dangerous autoreactive B and T lymphocytes have various differentiation options in order to not cause a harmful selfaggression. In any case, the final outcome of this control taking place during the generation of antigen receptors (Central Tolerance) is the disappearance or inactivation of the majority of

202 lymphocytes expressing a receptor reacting at high affinity with self molecules. However, because of our individual antigenic peculiarities, Central Tolerance to self-antigens shapes the repertoire of T and B cells differently in each person. Since immune tolerance is mostly learned during the development of T and B cells, any foreign antigen present in the body during the perinatal period and recognized by developing T and B cells may induce immune tolerance. While foreign and potentially able to elicit an immune reaction, various Central Tolerance mechanisms impede the elicitation of an immune reaction to these molecules.

Fig. 22.3. CENTRAL TOLERANCE OF IMMATURE B CELLS. B cells that have finally acquired their definitive individual BCR during their bone marrow differentiation (Immature B cells, see Figs.12.3 and 12.5) are cells prone to apoptosis. At this differentiation stage (Orange arrow in the Fig.) their survival depends on the strength of downstreamsignals triggered by BCR interacting with antigens expressed by surrounding cells (See Fig. 22.4). Both the attenuation of the signalling strength below minimum signal threshold (for example, in the case of a non-functional BCR) or hyper-activation of the signalling strength above maximum threshold (for example, in the case of BCR reacting at high affinity with the antigen) causes immature B cells apoptosis, and thus the disappearance of self-reactive B cells.

203 Fig. 22.4. NEGATIVE SELECTION OF IMMATURE B CELLS. When the BCR of a B cell interacts at high affinity with an environmental antigen, downstream signals are initiated by the phosphorylation of Immunoreceptor Tyrosine-based Activation Motifs (ITAM) sequences in the cytoplasmatic tail of Ig alpha and Ig beta signaling chains (See Fig. 13.8). When these events take place in immature B cells, hyperactive downstream signaling initiated by a BCR interacting with high affinity with self antigens induces cell apoptosis. In this way, most of immature B cells expressing an autoreactive BCR are eliminated. By contrast, in mature B cells the same signaling pathway induces the expression of the ď Ą chain of the IL2 receptor (CD25) that is instrumental for B cell activation and clonal expansion (See Fig. 17.9). Fig. 22.5. B CELL UNRESPONSIVENESS AGAINST SELF ANTIGENS. The elimination of immature selfreactive B cells during their bone marrow differentiation is a crucial mechanism of tolerance, defined Central Tolerance (Fig. 22.3 and Fig. 22.4). However, this elimination of B cells with a BCR reacting at high affinity with self molecules (Central Tolerance) is never complete. Normally, mature autoreactive B cells that have slipped Central Tolerance do not produce dangerous high affinity auto-antibodies because are kept under control by several other mechanisms in the body periphery.


Fig. 22.6. T CELL UNRESPONSIVENESS AGAINST SELF ANTIGENS. During thymic education (See Chapter 8), lymphocytes that generate a T Cell Receptor (TCR) interacting at high affinity with self HLA glycoproteins (HLA-p) undergo apoptotic cell death (Negative selection, see Fig. 8.6). However, a few mature virgin T cells expressing a TCR reacting at high affinity with self molecules escape the negative selection. While these T cells are present in our body, normally self HLA-p are not targeted by their attack since, in most of the cases, HLA-p are just ignored. This Immune Ignorance of self antigens by selfreactive T cells is due to the fact that most self HLA-p are too scarcely expressed on the cell membrane of antigen presenting cells due to the enormous competition of self peptides for the groove of HLA glycoprotein (See Fig. 6.6). Thus, in most of the cases there are too few HLA glycoprotein expressing the target peptide to reach the T cell activation threshold. Consequently, T cells ignore them. Immune Ignorance is an important and common way to avoid autoimmune aggressions. However, viral infections, release of interferons and other pro-inflammatory cytokines may induce an over-expression of the HLA glycoprotein or self peptides and thus may overcome their Immunological Ignorance. This is one of the reasons why infectious diseases may result in autoimmune disorders. Mature virgin T cells may also not respond to an antigen and develop a state of anergy when the peptide is presented by cells unable to provide the co-stimulatory signals essential for T cell activation (See Figs. 22.5 and 22.6). Moreover, natural Treg cells recognizing the self HLA antigen presenting the self peptide at high affinity can block the T cell activation (See Fig. 22.12).


Fig. 22.7. ANERGY OF B AND T CELLS. The lack of reaction of mature B and T cells against a highly expressed target antigens recognized at high affinity by their membrane receptors is defined anergy. An anergic status takes place when the target antigen is first recognized without appropriate costimulatory signals.Following this first peculiar antigen encounter, the lymphocyte becomes functionally inactivated (anergic), while it remains alive for an extended period of time. Specialized Antigen Presenting Cells or the concurrence of various signals are required to trigger T and B cell activation. Since the majority of body cells display target antigens without co-stimulatory signals (See Figs. 9.14-9.17 and 22.5) the recognition of the cognate antigen presented by these cells induces a diffuse state of anergy instead of an autoimmune response. However, anergy is a temporary condition. A continuous binding of the target antigen in the absence of co-stimulatory signals is essential for the maintenance of anergy. Otherwise, anergy can be lost.


Fig. 22.8. T CELL STIMULATORY CO-RECEPTORS. The high affinity interaction of a TCR with HLA glycoprotein associate with the antigen peptide (HLA-p) is responsible for the crucial specificity of T cell activation (See Fig. 9.9-9.11). However, the activation of a virgin T cell requires a concurrent series of costimulatory signals (B7, CD40â&#x20AC;Ś See Fig. 9.14-9.17). The absence of these signals induces the anergy or apoptosis of the primed virgin T cell. Therefore, only a specialized population of cells, the so called Antigen Presenting Cells (APC, see Figs. 9.4-9.7) expressing co-stimulatory signals, is able to trigger the activation of a virgin T cell. By contrast, HLA-p on the surface of the majority of the body cells unable to provide concurrent costimulatory signals induces a state of T cell anergy (See Fig. 22.7).


Fig. 22.9. PHYSIOLOGICAL IMPORTANCE OF REGULATORY CHECK POINTS. CTLA-4 and PD-1 are members of the CD28 family of cell membrane receptors (See Fig. 9.14). Despite being highly homologous to CD28, surface expression of CTLA-4 and PD-1 on the cell membrane of CD4+ and CD8+ mature T cells differs markedly from that of CD28. CTLA-4 and PD-1 are not present on the cell membrane of resting T cells. Instead their expression is up-regulated later, following T cell activation. This late expression fits in well with the negative regulatory role that they play in T cell activation. In effect, CTLA-4 and PD-1 do not block the induction of the immune response of virgin mature T cells. On the contrary, these receptors play a critical role in regulating and dampening an ongoing immune response. Both receptors have an important role in inhibiting autoimmune activation of T cells. Several human tumors are able to dampen anti-tumor T cell reaction through the expression on their cell membrane the ligands of CTLA-4 and PD-1 receptors (See Fig. 22.11).

208 Fig. 22.10. CTLA-4 AND PD-1 INHIBITORY CORECEPTORS. Once activated, T cells express the CTLA-4 and the PD-1 co-receptors that bind B7 and PD1L ligands with higher affinity than CD28 co-receptor and deliver signals that dampen T cell activation. T cells transducing the signals delivered by CTLA-4 and PD-1 release inhibitory cytokines that block the activation of surrounding cells. The late expression of CTLA-4 and PD1 co-receptors allows the physiological regulation of the intensity and persistence of T cell immune response. The interaction of CTLA-4 and PD-1 co-receptors with their ligands serves as molecular brake, preventing hyperactivity of the T cells of the immune system and, in some cases, preventing autoimmunity (Regulatory or Inhibitory Check Points).

Fig. 22.11. THE BLOCKADE OF REGULATORY CHECK POINTS UNVEILS THE EFFECTIVENESS OF NATURAL IMMUNE RESPONSE TO CANCER CELLS. A large series of monoclonal antibodies (See Chapter 21) have been produced to interfere with regulatory immune check points. In several cancer patients, the administration of monoclonal antibodies to CTLA-4 (In red in the Fig.) or PD-1 receptors (In blue) expressed by activated T cells as well as monoclonal antibodies to PD-1 ligand (In black) expressed by tumor cells and infiltrating White Blood Cells (WBC) induce significant tumors shrinkage and significantly improve patient survival. Tumor shrinkage depends on the activity of tumor-infiltrating T cells that are no more inhibited by negative signals delivered by CTLA-4 and PD-1 co-receptors.


Fig. 22.12. T REGULATORY (Treg) CELLS. Treg cells expressing the transcription factor Foxp3 play an additional key role in maintaining tolerance to self-antigens, inhibiting autoimmunity and preventing immune responses from becoming uncontrolled. Several potentially lethal autoimmune diseases, allergies and food intolerance develop when Treg cells fail to limit the effector activity of autoreactive T cells that have escaped thymic negative selection or peripheral inactivation. CD4+Foxp3+Treg cells are 10-15 % of body CD4 cells. They can be separated in two distinct populations, natural and induced Treg. Natural Treg cells: For the thymic maturation of natural Treg cells see Fig. 8.6. CD25 is the  chain of the high affinity IL2 receptor (See Figs.4.11-4.18, 9.9 and 9.10). Foxp3 is a Forkhead-family transcription factor expressed by regulatory cells. Induced Treg cells: An antigen presenting cell (APC) displaying antigen peptides along with the secretion of a high amount of Transforming Growth Factor- (TGF-See Fig. 22.13) in the absence of IL-6 production triggers the differentiation of a virgin CD4+ T cell towards an Induced CD4+ Treg cell. On the other hand, the repertoire of cytokines released by an antigen presenting cells (APC) is dictated by the kind of antigen they have taken up. In many cases, the phagocytosis of microbes triggers a high production of TGF- and IL6 and switches the differentiation of virgin CD4+ T cells toward Th17 cells (See Figs. 11.5) and not toward CD4+ Treg cell. Induced Treg cells may trigger the production of indoleamine dioxygenase (IDO) by APC. IDO breaks down tryptophan, and the lack of tryptophan is an effective way to block the activation of the lymphocytes present in the micro-environment.



Fig. 22.14. IL10: A SUPPRESSOR CYTOKINE. Alone or in combination with TGF-, IL10 plays a central role in the negative regulation of immune responses. IL10 is directly involved in the induction of unresponsiveness to antigens introduced through oral and respiratory routes.


CHAPTER 23. AUTOIMMUNITY. Fig. 23.1. AUTOIMMUNITY. I. Autoimmunity is the immune aggression against oneâ&#x20AC;&#x2122;s normal own body molecules, cells and tissues. In the pathogenesis of autoimmunity concur genetic factors, peculiarities of individual immune system and microbial infections. A critical role is also played by the efficacy by which induced Treg cells (See Fig. 22.12) restrain the activation of pro-inflammatory Th17 cells (See Figs. 11.5, 11.6). Chronic inflammatory reactions to microbes driven by Th17 escaping Treg cell control may result in autoimmunity.

Fig. 23.2. AUTOIMMUNITY. II. The immune system is a collection of complex and sophisticated mechanisms. Therefore is not surprising that a few of these may fail to be appropriately controlled. These control mistakes often involve the discrimination between self and notself and allow the onset of selfaggressive immune reactions. The progressive extension of human life is accompanied by an increased incidence of control mistakes. At present in wealthy countries there is a large population of patients suffering from diseases due to aggression against self-antigens. Autoimmune diseases such as diabetes, multiple sclerosis, rheumatoid arthritisâ&#x20AC;Ś result from chronic autoimmune aggressions involving T and B cells recognizing multiple self epitopes. Self-reactive Th cells play a central role in autoimmune diseases because they orchestrate the reaction of several other cells of the immune system including B cells, T killer cells, and macrophages.

212 Fig. 23.3. FAILURE OF SELF TOLERANCE CAUSES AUTOIMMUNITY. During their maturation, T and B cells randomly generate their individual antigen receptor (TCR and BCR). The majority of these receptors react with self-molecules (See chapter 14). Therefore, various control mechanisms of selftolerance (See Chapter 22) remove or make unreactive (anergic) the B and T cells expressing an antigen receptor reactive to self-molecules with high affinity (autoreactive lymphocytes). In other cases, autoreactive lymphocytes fail to be activated because they encounter insufficient antigen stimulation (Immune ignorance, See Fig.22.6) or fail to receive concurrent costimulatory signals. Autoimmune reactions are due to a failure of these various control mechanisms caused by genetic and environmental factors.

Fig. 23.4. THE FAILURE OF REGULATORY MECHANISMS CAUSES AUTOIMMUNITY. Besides the mechanisms of tolerance, autoimmunity may arise from a failure of inhibitory checkpoints on which the regulation of immune responses depends (See Figs. 22.9 and 22.10). Major functional defects of the immune system (Immunodeficiencies) are often associated with autoimmune reactions.





Fig. 23.7. T AND B CELL AUTOIMMUNE DAMAGE. Often reactive T cells invade normal or damaged target tissues and induce a complex self-destructive inflammatory process.

Fig. 23.8. ANTIBODY AND COMPLEMENT AUTOIMMUNE DAMAGE. For details see: a- Fig. 20.5; b- Figs. 3.31, 19.8 and 19.9; c- Fig. 19.7; d- Fig. 19.6; e- The interaction of antibodies with the binding site of a membrane receptor can stimulate the receptor mimicking the ligand of the receptor ligand; f- Fig. 19.6; g- Fig. 19.4; hChapter 3; i- See Fig. 20.8; l- Fig. 20.8.



Fig. 24.1. THE IMMUNE MEMORY. The capacity to learn how to respond more efficiently to a second encounter with the same invader is an important and diffuse feature of the immune system.

Fig. 24.2. INFLAMMATORY MEMORY OF EPITHELIAL STEM CELLS. An inflammatory memory of the event in the form of changes to the chromatin facilitates the accessibility of selected groups of genes to the transcription machinery and enables cells to respond more rapidly and strongly to subsequent inflammatory challenge. REFERENCE: S Naik et al., Nature 2017, 550:475; X Dai, Nature 2017, 550:460.

216 Fig. 24.3 EPIGENETIC IMMUNE MEMORY OF MACROPHAGES. Temporary changes in the chromatin of macrophages increase the accessibility of their transcriptional machinery. Thus, macrophages respond more rapidly to subsequent inflammatory challenge. REFERENCE: R Medzihitov, Nature 2017, 550:460.

Fig. 24.4 IMMUNE MEMORY OF NK CELLS. The mechanisms of NK immune memory are not only based on possible epigenetic changes but also on a numerical expansion of the various NK cells expressing activating receptors interacting with anomalous molecules expressed by target cells (poly clonal expansion). REFERENCE: H Peng and Z Thian, Frontiers in Immunol, 2017,8:1143


217 FIG. 24.5. THE SPECIFIC MEMORY OF T AND B CELLS. When a microbe (or an antigen) enters our body for the first time, it involves T and B cells in a primary immune response. The T and B cells that are activated and expand are those that recognize the antigen with high affinity. When the microbe (or the antigen) is eliminated, this primary cell expansion slowly vanishes. However, immune responses involving the activation of T and B cells leave an important reactive state, called Immune memory, due to the persistence of a special lymphocyte population, the memory T and B cells. Thanks to the prolonged survival of memory cells, a re-infection by the same microbe (or a second arrival of the same antigen) elicits a secondary immune response due to the rapid and effective re-activation and clonal expansion of memory T and B cells.

Fig. 24.6. CELLULAR BASIS OF THE T AND B CELL MEMORY. Long-lived memory cells remaining after the resolution of an immune response can be quickly re-activated by the re-entry of the same antigen. The combination of a higher number of persisting memory cells (persistent clonal expansion, See Figs. 24.7 and 24.8), their lower threshold of activation and their quicker kinetics of reactivation provides a very efficient memory response. For memory cell activation the requirement for co-stimulatory signals is less demanding than for virgin T and B cells. In several cases, pro inflammatory cytokines (IL12, IL18, Interferons) produced by sentinel cells, granulocytes and macrophages can trigger memory cell proliferation.


Fig. 24.7. B CELL IMMUNOLOGICAL MEMORY IS LONG-LIVED. Following a primary immune response, antigen-activated B cells undergo asymmetrical division, wherein daughter cells mature into plasma cells (antibody-secreting cells) and long-lived memory B cells. Differentiation of activated B cells into plasma cells and memory B cells mostly rests on the expression of the protein Blimp-1. The expression of Blimp-1 controls the activation of many genes important for plasma cell differentiation, including genes inducing the formation of the secretory apparatus necessary for the production of large amounts of antibodies. In addition Blimp-1 inhibits cell proliferation and maintains the terminal differentiated plasma cells in a post-mitotic state. When Blimp1 expression is inhibited, activated B cells differentiate into memory B cells. The rapid kinetics of B cell activation and the intensity of the antibody production in memory response (secondary response) are shown on Fig. 17.20.


Fig. 24.8. T CELL IMMUNOLOGICAL MEMORY IS LONG-LIVED. Upon activation by an Antigen Presenting Cell (APC), a T cell generates a clone of Effector/Memory cells. This effector cell population is responsible of the reaction against the antigen. When the antigen is eliminated, progressively this population declines since these T cells, no more stimulated by the antigen, are facing apoptotic death (death by neglect). However, during later phases of the response, these effector/memory T cells undergo asymmetrical divisions, wherein the daughter cell proximal to APC differentiate again in effector/memory cells while the distal cell differentiate in long lasting memory T cells. These long-lasting memory T cells express different homing receptors responsible for their selective homing: Central memory T cells are attracted in lymphoid organs while Effector memory T cells are present in circulation and localize in peripheral body districts.

220 Fig. 24.9. WHAT CAN BE SAID ABOUT MEMORY T AND B CELLS? When virgin T and B cells differentiate into effector cells, their DNA methylation profile changes. Methyl groups are added to many genes associated with the virgin state whereas a loss of DNA methylation is evident at genes that encode key components of the effector response. These epigenetic changes maintained in memory cells enable them to rapidly become a proliferating effector cell upon a re-encounter with the same antigen. REFERENCE: RS Akondy et al. Nature 2017, 552:362; B Youngblood et al, Nature 2017, 552:404; KQ Omilusik and AW Goldrath, Nature 2017, 552:337

Fig. 24.10. WHY IS THE MEMORY RESPONSE SO TERRIFIC? The combination of a marked numerical cell expansion with the persistence of epigenetic changes makes the perfect conditions for an extremely effective long-lasting anamnestic immune response.


Fig. 24.11. HOW MANY MEMORY CELLS ARE REQUIRED FOR A SECONDARY IMMUNE RESPONSE? During T and B cell maturation we generate a large repertoire of TCR and BCR binding sites. This means that there is approximately a T (or B) cell with a specific binding in one every 1011 cell, an extremely low frequency. However, the frequency of lymphocytes reacting to an antigen is much higher since most of the antigens have many epitopes and each epitope is recognized with a different affinity by several virgin cells. Periodically memory cells display expansions (cell proliferation) and contractions (cell apoptosis). The rapid re-activation of these memory cells provides an enhanced response against the reentry of the same antigen. As time elapses, the frequency of memory cells slowly declines. When the frequency of memory cells specific for a given antigen is less than 1 every 700 000 cells, an antigen reentry does not more elicit a significant protective memory reaction.



Fig. 24.13. HOW TO RETAIN A GREAT IMMUNE MEMORY? Often, unnoticed antigen re-stimulations allow to keep an effective memory for long time. In the absence of these natural boosters, progressively the population of memory cells decreases and the memory reaction doesn't take place anymore. However, once a T and B memory was triggered, a timely booster induces a particularly effective memory that lasts much longer.


CHAPTER 25. VACCINES. Fig. 25.1. THE EXCEPTIONAL POTENTIAL OF IMMUNE MEMORY. The persistence of both the expanded population of the effector lymphocytes and a high high antibody titer that follows the recovery from an infectious disease accounts for the elimination of a subsequent invasion by the same intruder microbe with such an efficacy and rapidity that the re-infection generally goes unnoticed. A suggestive account of the potential of the immune memory was provided by Tucydides, an ancient Greek historian, when he describes the plague of Athens during the Pelonnesian war.

Fig. 25.2. ATTEMPS TO INDUCE AN IMMUNE MEMORY. Everr since become clear that the recovery from an infectious disease may confer a persistent protection against the risk of contracting again the same disease, human intelligence has been trying to induce a protective immune memory. An ancient method used in China for the prevention of smallpox, a disease that casused devastating epidemics, was to let children to inhale a dust obtained from the smallpox scabs. Variolization (also known as Inoculation) was another preventive method, practiced in the East,

224 especially in the Ottoman Empire but also known in Europe consisting in the introduction into superficial scratches made in the skin of powdered smallpox scabs from pustules of people affected by light cases of smallpox. After living for a while in Turkey, Lady Mary Wortley Montagu, an English aristocrat (16891762), sent letters to influential European personalities to promote this primitive way to induce a protective immune memory. However, a rational vaccination against small pox was first set up in England by Edward Jenner in 1796. Jenner, a country doctor, showed that the inoculation of purulent material obtained from bovine smallpox pustules protected humans from the infection by the human smallpox virus. Since the virus used by Jenner for his successful vaccination was obtained from a cow (vacca in Latin), this new bio-technology was called vaccination. Thanks to Jenner, empirical practices, often mixed with magic, become a rational procedure, even if the scientific basis of vaccination were ignored. About a century later, Louis Pasteur in France succeeded in making another important scientific quantum jump showing that vaccines based on living microbes that have been weakened so they can not cause disease can induce a significant and long-lasting protection. Beginning from the studies of Pasteur, the vaccines have progressively become a sophisticated and effective bio-technology.

Fig. 25.3. THE LESSON OF VACCINES. Against some microbes an effective protection depends only on the vaccineâ&#x20AC;&#x2122;s ability to elicit a T cell mediated immunity. Instead, against other microbes the vaccine should be able to elicit antibodies of certain classes (those able to activate the Complement cascade, those able to reach mucosal surfacesâ&#x20AC;Ś). Current vaccines are based on sophisticated technologies. In several cases the vaccine contains only the microbial epitopes that are critically important for the survival and proliferation of the microbe. Various attempts to elicit protective immunity against purified microbial epitopes showed that the simple injection of these foreign molecules is often not sufficient to elicit protective immunity. In many cases the target antigen should be administered in combination with particular substances (the adjuvant) that elicit the danger signals (See Figs. 2.3-2.4) required to trigger a concomitant reactivity of natural immunity and induce a local inflammatory response. Another lesson taught by the study of the vaccine is that to elicit and maintain a protective immunity the vaccine should be re-administered several times at determined intervals. These repeated vaccine administrations boost the immunity and allow a progressive selection and expansion of the T and B cell clones reacting against the microbe at higher affinity.


Fig. 25.4. KINDS OF VACCINES. A vaccine may be based on a living microbe whose ability to induce a disease has been disabled by culturing it under particular conditions or through genetic engineering (attenuated microbe). Other vaccines are based on microbes that have been killed in various ways but that maintain their immunogenicity. Several effective vaccines are based on toxoids, microbial toxins that have been modified in such a way as to have lost their ability to cause a disease while maintaining their immunogenicity. More sophisticated vaccines are based on the selection of fragments of a toxin or molecules on the surface of a microbe. In a few cases, these vaccines are based on a few small microbial epitopes (See Fig. 15.2). These fragments can be obtained directly from microbes or produced by genetic engineering. Other vaccines exploit the special ability of Dendritic Cells to present peptides to T cells. Since the antigens are taken up by Dendritic Cells and presented as peptide fragments associated to HLA glycoprotein, these vaccines activate the response of T cells only. As the presentation of endocyted antigens takes place mostly on Class II HLA glycoprotein, they elicit a particularly effective Th cell activation. DNA and RNA vaccines are molecularly deďŹ ned reagents that are easy to construct and modify. They consist in plasmids coding for the target protein. A few of these small plasmids injected in the muscle enter the cells and the coded antigen is expressed on the cell membrane. Damaged muscle cells and their fragments are drained to the local lymph node. There they directly prime cognate B cells or are taken up by macrophages and Dendritic Cells and presented to T cells. The consequent B-T cell interaction will lead to the activation of antibody response by B cells.


Fig. 25.5. ADJUVANTS. Adjuvants are a critical component of vaccines that markedly increase the vaccine capacity to elicit an immune response (antigen immunogenicity). Initially vaccines made use of live attenuated or killed and inactivated microbes that naturally express on their surface adjuvant molecules recognized by Patter Recognition Receptors (PPR) expressed on the cell membrane of the cells of innate immunity. By contrast, most vaccines developed in recent years are based on molecules or molecular aggregates rather than the whole microbe. These molecules have to be associated with adjuvants in order to trigger the activation of the cells of innate immunity. An adjuvant may act in many different ways. It may induce a slow release of the antigen, and thus it enhances the persistence of the antigen over a long period of time. It may also aggregate soluble antigens and thus favor their uptake by Antigen Presenting Cells (APC). An adjuvant may trigger the release of alarm signals (See Fig. 2.3) or may activate innate immunity cells and trigger the differentiation of Antigen Presenting Cells (APC) and Dendritic Cells (DC). A risk in the use of adjuvants lies in their ability to induce the immune recognition of a selfantigen and trigger an immune reaction against tolerated self-molecules causing autoimmunity and a devastating inflammation.


Fig. 25.6. VACCINE: KINDS, ADVANTAGES AND LIMITS. The various kinds of vaccines have distinct advantages and limitations. The selection of the best vaccine is based on a balance between its immunogenicity, cost, storage problems and kind of immunity elicited. World countries and the World Health Organization (WHO) have prepared detailed schedules indicating dose, times, boosters for the administration of compulsory and recommended vaccines for human and veterinary use.


Fig. 25.7. TRIUMPHS AND DEFEATS OF VACCINES. Vaccination is an exceptionally effective biotechnology of preventive medicine: Probably vaccination is the most successful biotechnology.

Fig. 25.8. OPINION POLLS AGAINST VACCINES: WHY? Efforts to make vaccines more effective and universally available clash with the passionate anti-vaccination reactions that slithers in the population of affluent countries. Until the last century, these movements were minorities and vaccination coverage tended to grow. At present, vaccine-opposing groups found the internet an effective vehicle to spread their positions and thereby we are witnessing a fall in vaccine coverage. The mass media emphasis on hypothetical side effects of vaccines triggers waves of collective fear that mainly concern the accusation of causing autism, adjuvant and preservative toxicity, and the weakening of the immune system caused by too many vaccines. While anti-vaccine movements spread their objections with militant enthusiasm, health authorities often appear unable to convincingly explain the fundamental importance of vaccines. No matter how authoritative the official documents are, it appears extremely difficult to wipe out the suspicion that these documents are the result of a concerned manipulation and global conspiracy.



Fig. 26.1. IMMUNODEFICIENCIES. The failure of some immune mechanisms may open the door to infections due to a particular class of microbes (extracellular vs. intracellular microbes, bacteria, virus or fungi) become recurrent. The kind of microbe causing these recurrent infections highlights the function of the failing mechanism.



Fig. 26.3. A FEW DEFECTIVE MECHANISMS OF INHERITED IMMUNODEFICIENCIES. Inherited Immunodeficiencies provide a dramatic illustration of the function of the various components of the immune system. Severe Immunodeficiencies due to the absence of granulocytes are incompatible with life. Due to their central role in the immune system, Immunodeficiencies due to a defective T cell or T and B cell maturation cause Severe Combined Immune Deficiencies (SCID). The common  chain is a transducer chain exploited by numerous distinct cytokine receptors (See Fig. 4.19). Therefore, the lack of a functioning common  chain causes a SCID due to the simultaneous inactivation of multiple immune mechanisms. As the common  chain gene is located on the X chromosome, the immunodeficiency caused by  chain gene mutations is known as an X linked SCID (XSCID). Genetic defects affecting only B cell maturation result in the absence of antibodies, a condition called a-gammaglobulinemia. The first immunodeficiency identified was an X-linked agammaglobulinemia caused by a defective Bruton tyrosine kinase (BTK) which blocks the maturation of B cells following H chain gene rearrangement (See Fig. 14.6). The recurrent infections by staphylococci and streptococci (microbes producing pus, pyogenic microbes) displayed by these patients shows the importance of antibody mediated microbe opsonization. Staphylococci and streptococci have a polysaccharide capsule that inhibits phagocytosis. However, when their surface antigens are bound by antibodies, they are picked up efficiently through the binding of the Ig Fc domain to Fc receptors (See Fig. 19.7). A similar clinical situation is caused by defect in Complement components impairing Complement-mediated opsonization of microbes (See Fig. 20.6).


Fig. 26.4. ACQUIRED IMMUNODEFICIENCIES. Acquired Immunodeficiencies may be due to factors of diverse kind. A decrease in body weight to less than 70% of recommended weight results in a severe immunodeficiency. In 2010 there were 925 million undernourished people. In 2013 undernutrition resulted in 469,000 deaths. Immunodeficiency disorders may result from aging, almost any prolonged serious disease and advanced cancer. Immunodeficiency may also result from several kinds of medical treatment (chemotherapy, radiation, immunosuppressive drugs administered after organ transplants, glucocorticoids). Immunodeficiencies may be the result of radioactive and nuclear accidents and several kinds of occupational exposure to immunosuppressive agents. Several viral infections result in systemic and local Immunodeficiencies.

232 Fig. 26.5. THE INFECTION BY THE HUMAN IMMUNODEFICIENCY VIRUS (HIV). Currently, more than 35 million people are infected worldwide, with million new diagnosis each year and 1.6 million deaths each year. The most prevalent route of HIV infection is across mucosal tissues. Because of the fragility of the barrier offered by the glans penis, vagina (See Fig. 1.10) and rectal mucosa (See Fig. 1.9), mechanical stress, cuts and micro-abrasions may open the door to HIV infection. Fig. 26.6. HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION OF CD4+ T CELLS. Anatomical localization and membrane receptor characteristics make tissue Antigen Presenting Cells (APC: Macrophages and Dendritic Cells) expressing both CD4 co-receptors and other co-receptors (DCSign…) one of the primary target of HIV. Then, infected APC present HIV peptides on the groove of HLA glycoproteins. CD4+ cells with a TCR which recognizes viral peptides establish a close interaction with HIV infected APC. The HIV exploits this close APC-T cell interaction to bind the CD4 glycoprotein on T cell membrane. Fig. 26.7. CD4 AND CHEMOKINE RECEPTORS ACTS AS HUMAN IMMUNODEFICIENCY VIRUS (HIV) BINDING SITES. Once gp120 is bound to the CD4, its conformation changes allowing its interaction with the CXCR4 and CCR5 chemokine receptors. Then, the fusogenic portion of gp41 allows the fusion of the membranes and subsequent entry of the viral capsid. A few persons express the -32 variant of the gene of the CCR5 receptor that does not bind the HIV gp120. The gp120 inability to bind the -32 variant hampers or impedes the HIV infection (See Fig. 4.25 and 4.26).


Fig. 26.8. FROM HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION TO ACQUIRED IMMUNODEFICIENCY (AIDS). In the early stage of the HIV infection, antibodies are produced against the virus (Serum conversion).

1. Once the HIV has entered the body, tissue Antigen Presenting Cells (Macrophages and Dendritic Cells) are the first cells to be infected (See Figs. 4.25-4.26).

2. Infected APC present HIV peptides on the groove HLA glycoproteins. 3. CD4+ Th cells with a TCR which recognizes viral peptides establish a close interaction with HIV infected APC. HIV exploits this interaction to bind CD4 and CXCR4 and CCR5 chemokine receptors on the membrane of CD4+ cells and infect them.

4. HIV infected CD4+ Th cells release virus particles that are now so numerous as to infect other CD4+ cells directly.

5. HIV infected CD4+ Th cells release HIV virions and die. Moreover, the fuosogenic domain of HIV stemming from the membrane of infected cells favors the formation of large syncytia of CD4+ Th cells. Lastly, the body mounts an immune reaction against the HIV and HIV infected cells. Tk cells recognizing viral peptides expressed by infected CD4+ Th cells kill them. Steadily a slow decrease in the number of CD4+ Th cells takes place for a period ranging from two to twelve years (or more). Then the equilibrium between destroyed CD4+ Th cells and those newly produced is lost and the number of CD4+ Th cells decreases progressively. Only when the number of CD4+Th cells is below 200 per square microliter, a severe combined immunodeficiency is acquired (AIDS).

Basic Immunology  

Basic Immunology by Piero Musiani and Guido Forni. A printed version of this book is available directly from Piccin Nuova Libraria, Padua,...

Basic Immunology  

Basic Immunology by Piero Musiani and Guido Forni. A printed version of this book is available directly from Piccin Nuova Libraria, Padua,...