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Transplantation Immunology

Transplantation is the replacement of an organ or other tissue, such as bone marrow, with organs or tissues derived ordinarily from a nonself source such as an allogeneic donor. Organs include kidney, liver, heart, lung, pancreas (including pancreatic islets), intestine, or skin. In addition, bone matrix and cardiac valves have been transplanted. Bone marrow transplants are given for nonmalignant conditions such as aplastic anemia as well as to treat certain leukemias and other malignant diseases. Transplantation immunology is the study of immunologic reactivity of a recipient to transplanted organs or tissues from a histoincompatible recipient. Effector mechanisms of transplantation rejection or transplantation immunity consist of cell-mediated immunity and/or humoral antibody immunity, depending upon the category of rejection. For example, hyperacute rejection of an organ such as a renal allograft is mediated by preformed antibodies and takes place soon after the vascular anastomosis is completed in transplantation. By contrast, acute allograft rejection is mediated principally by T lymphocytes and occurs during the first week after transplantation. There are instances of humoral vascular rejection mediated by antibodies as a part of the acute rejection in response. Chronic rejection is mediated by a cellular response. Histocompatibility is tissue compatibility as in the transplantation of tissues or organs from one member to another of the same species, an allograft, or from one species to another, a xenograft. The genes that encode antigens which should match if a tissue or organ graft is to survive in the recipient are located in the major histocompatibility complex (MHC) region. This is located on the short arm of chromosome 6 in humans (Figure 21.1 and Figure 21.2) and of chromosome 17 in the mouse. Class I and class II MHC antigens are important in tissue transplantation. The greater the match between donor and recipient, the more likely the transplant is to survive. For example, a six-antigen match implies sharing of two HLAA antigens, two HLA-B antigens, and two HLA-DR antigens between donor and recipient. Even though antigenically dissimilar grafts may survive when a powerful immunosuppressive drug such as cyclosporine is used, the longevity of the graft is still improved by having as many antigens match as possible. A histocompatibility locus is a specific site on a chromosome where the histocompatibility genes that encode

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histocompatibility antigens are located. There are major histocompatibility loci such as HLA in humans and H-2 in the mouse across which incompatible grafts are rejected within 1 to 2 weeks. There are also several minor histocompatibility loci with more subtle antigenic differences, across which only slow, low-level graft rejection reactions occur. Histocompatibility antigen is one of a group of genetically encoded antigens present on tissue cells of an animal that provoke a rejection response if the tissue containing them is transplanted to a genetically dissimilar recipient. These antigens are detected by typing lymphocytes on which they are expressed. These antigens are encoded in humans by genes at the HLA locus on the short arm of chromosome 6 (Figure 21.2). In the mouse, they are encoded by genes at the H-2 locus on chromosome 17 (Figure 21.3). Transplantation antigens are histocompatibility antigens that stimulate an immune response in the recipient that may lead to rejection. The minor histocompatibility locus is a chromosomal site of genes that encode minor histocompatibility antigens which stimulate immune responses against grafts containing these antigens. Minor histocompatibility antigens are molecules expressed on cell surfaces that are encoded by the minor histocompatibility loci, not the major histocompatibility locus. They represent weak transplantation antigens by comparison with the major histocompatibilty antigens. However, they are multiple, and their cumulative effect may contribute considerably to organ or tissue graft rejection. Graft rejection based on a minor histocompatibility difference between donor and recipient requires several weeks compared to the 7 to 10 d required for a major histocompatibility difference. Minor histocompatibility antigens may be difficult to identify by serological methods. Minor transplantation antigens: See minor histocompatibility antigens. H-Y is a Y chromosome-encoded minor histocompatibility antigen that may induce male skin graft rejection by females or destruction of lymphoid cells from males by effector cytotoxic T lymphocytes from females.

HY is the male-specific transplantation antigen. Females of some but not all inbred mouse strains can reject skin grafts from males of the same syngeneic strain. By contrast, male-to-male, female-to-female, and female-to-male grafts succeed. This indicated the presence of a minor histocompatibility antigen gene on the Y chromosome that was designated H-Y. H-Y is a weak transplantation antigen compared to the mouse MHC-designed H-2. Several H-Y epitopes have been identified in mice and one in humans. H-Y peptide epitopes are derived from several linked genes. Minor histocompatibility peptides: H antigens. Among minor antigens thus far identified are H-3 antigens, malespecific H-Y antigen, β2 microglobulin, and numerous others that have not yet been firmly established. Minor lymphocyte stimulatory (MIs) loci: See Mls antigens. Minor lymphocyte-stimulating genes: See Mls genes.

Minor lymphocyte-stimulating (Mls) determinants are characterized by their activation of a marked primary mixed-lymphocyte reaction (MLR) between lymphocytes of mice sharing an identical MHC haplotype. MHC class II molecules on various cell surfaces present Mls epitopes to naive T lymphocytes which mount a significant response. V β-specific monoclonal antibodies have facilitated the definition of Mls epitopes. Mls determinants activate T lymphocytes expressing selected β specificities. See also Mls antigens. Histocompatibility testing is a determination of the MHC class I and class II tissue type of both donor and recipient prior to organ or tissue transplantation. In man HLA-A, HLA-B, and HLA-DR types are determined, followed by cross-matching donor lymphocytes with recipient serum prior to transplantation. A mixed lymphocyte culture (MLC) was formerly used in bone marrow transplantation, but has now been replaced by molecular DNA typing. The MLC may also be requested in living related organ transplants. As in renal allotransplantation, organ recipients have their serum samples tested for percent reactive antibodies, which reveals whether or not they have been presensitized against HLA antigens of an organ for which they may be the recipient. Human leukocyte antigen (HLA) is the product of the MHC in humans that contains the genes that encode the polymorphic MHC Class I and Class II molecules as well as other important genes. HLA is an abbreviation for human leukocyte antigen. The HLA histocompatibility system in humans represents a complex of MHC class I molecules distributed on essentially all nucleated cells of the body and MHC class II molecules that are distributed on B lymphocytes, macrophages, and a few other cell types. These are encoded by genes at the MHC. In humans the HLA locus is found on the short arm of chromosome 6. This has now been well defined, and in addition to encoding surface isoantigens, genes at the HLA locus also encode immune

FIGURE 21.1 Human chromosome 6.












FIGURE 21.2 Short arm of human chromosome 6.

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K Class I

I-A Aβ3


I-E Aβ


Eα Class II






C2 Class III













Q7 Q8/9 Q10

Class I

FIGURE 21.3 H-2 complex on chromosome 17 of a mouse.

response (Ir) genes. The class I region consists of HLAA, HLA-B, and HLA-C loci, and the class II region consists of the D region which is subdivided into HLA-DP, HLA-DQ, and HLA-DR subregions. Class II molecules play an important role in the induction of an immune response, since antigen-presenting cells must complex an antigen with class II molecules to present it in the presence of interleukin-1 to CD4+ T lymphocytes. Class I molecules are important in presentation of intracellular antigen to CD8+ T lymphocytes as well as for effector functions of target cells. Class III molecules encoded by genes located between those that encode class I and class II molecules include C2, BF, C4a, and C4b. Class I and class II molecules play an important role in the transplantation of organs and tissues. The microlymphocytotoxicity assay is used for HLA-A, -B, -C, -DR, and -DQ typing. The primed lymphocyte test is used for DP typing. Uppercase letters designate individual HLA loci such as HLA-B and alleles are designated by numbers such as in HLA-B*0701. HLA Class III: See MHC genes and Class III MHC molecules. HLA locus refers to the major histocompatibility locus in man. Immunotyping: See immunophenotyping. w is the symbol for “workshop” that is used for HLA antigen and cluster of differentiation (CD) designations when new antigenic specificities have not been conclusively decided. Once the specificities have been agreed upon among authorities, “w” is removed from the designation.

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Polymorphism indicates the occurrence of two or more forms, such as ABO and Rh blood groups, in individuals of the same species. This is due to two or more variants at a certain genetic locus occurring with considerable frequency in a population. Polymorphisms are also expressed in the HLA system of human leukocyte antigens as well as in the allotypes of immunoglobulin γ and κ chains. Supratypic antigen: See public antigen. HLA-A is a class I histocompatibility antigen in humans (Figure 21.4). It is expressed on nucleated cells of the body. Tissue typing to identify an individual’s HLA-A antigens employs lymphocytes. HLA-B is a class I histocompatibility antigen (Figure 21.5) in humans which is expressed on nucleated cells of the body. Tissue typing to define an individual’s HLA-B antigens employs lymphocytes. HLA-C is a class I histocompatibility antigen in humans which is expressed on nucleated cells of the body. Lymphocytes are employed for tissue typing to determine HLA-C antigens. HLA-C antigens play little or no role in graft rejection. The human MHC class II region is the HLA-D region, which is comprised of three subregions designated DR, DQ, and DP. Multiple genetic loci are present in each of these. DN (previously DZ) and DO subregions are each comprised of one genetic locus. Each class II HLA molecule is comprised of one α and one β chain that constitute a heterodimer. Genes within each subregion encode a particular class II molecule’s α and β chains. Class II genes

FIGURE 21.4 Human class I histocompatibility antigen (HLAA0201) complexed with a decameric peptide from calreticulin (HLA-A0201). Human recombinant extracellular fragment expressed in E. coli; peptide synthetic based on sequence of human calreticulin.

that encode ι chains are designated A, whereas class II genes that encode β chain are designated B. A number is used following A or B if a particular subregion contains two or more A or B genes. Primed lymphocyte test (PLT): Lymphocytes previously exposed or primed to a certain antigen in a primary mixed lymphocyte culture will divide rapidly when reexposed to the same antigen. Using a primed cell, one can determine whether or not an unknown cell possesses the original stimulating antigen. Cells previously exposed to MHC class II HLA antigens can be used in HLA typing for HLA-D region antigens. It is an assay for the detection of lymphocyte-associated determinants (LAD). For this procedure, lymphocytes donated by a normal person can serve as responder cells against the antigens of a known cell type. The test is based on the secondary stimulation of the primed or sensitized lymphocytes. The original stimulator serves as a positive control. The response of the sensitized

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FIGURE 21.5 Class I histocompatibility antigen HLA-B*2705 complexed with nonapeptide arg-arg-ile-lys = ala-ile-thr-leu-lys (theoretical model).

cell to other cells measured by the incorporation of tritiated thymidine, by comparison with the control, may suggest sharing of HLA-D-associated antigens with the original stimulator cell if high stimulation values result. The HTC typing procedure, on the other hand, implies an antigenic determinant shared between the two cell types when there is little or no response. PLT is a positive typing procedure and has the advantage that homozygous donor cells are not required. Primed or sensitized cells can be prepared whenever they are needed and frozen for future use. These cells can be used to type unknowns within a period of 24 h. This eliminates the 5 to 6 d needed for a homozygous cell typing procedure. Primed lymphocyte typing (PLT) is a method to type for HLA-D antigenic determinants. It is a type of mixedlymphocyte reaction in which cells previously exposed to allogeneic lymphocytes of known specificity can be reexposed to unknown lymphocytes to determine their HLADP type, for example.

The HLA-DP subregion is the site of two sets of genes designated HLA-DPA1 and HLA-DPB1 and the pseudogenes HLA-DPA2 and HLA-DPB2. DP α and DP β chains encoded by the corresponding genes DPA1 and DPB1 unite to produce the DPαβ molecule. DP antigen or type is determined principally by the very polymorphic DPβ chain, in contrast to the much less polymorphic DPα chain. DP molecules carry DPw1–DPw6 antigens. The HLA-DQ subregion consists of two sets of genes designated DQA1 and DQB1, and DQA2 and DQB2. DQA2 and DQ B2 are pseudogenes. DQα and DQβ chains, encoded by DQA1 and DQB1 genes, unite to produce the DQαβ molecule. Although both DQα and DQβ chains are polymorphic, the DQβ chain is the principal factor in determining the DQ antigen or type. DQαβ molecules carry DQw1–DQw9 specificities. The HLA-DR subregion is the site of one HLA-DRA gene (Figure 21.6). Although DRB gene number varies with DR type, there are usually three DRB genes, termed DRB1, DRB2, and DRB3 (or DRB4). The DRB2 pseudogene is not expressed. The DR α chain, encoded by the DRA gene, can unite with products of DRB1 and DRB3 (or DRB4) genes which are the DR β-1 and DR β-3 (or DR β-4) chains. This yields two separate DR molecules, DR αβ-1 and DR αβ-3 (or DR αβ-4). The DR β chain determines the DR antigen (DR type) since it is very polymorphic, whereas the DR α chain is not. DR αβ-1 molecules carry DR specificities DR1–DRw18. Yet, DR αβ-3 molecules carry the DRw52, and the DR αβ-4 molecules carry the DRw53 specificity. W,X,Y boxes (class II MHC promoter) are three conserved sequences found in the promoter region of the HLA-DRα chain gene. The X box contains tandem regulatory sequences designated X1 and X2. Any cell that expresses MHC class II molecules will have all three boxes interacting with binding proteins, and decreased or defective production of some of these binding proteins can result in the “bare lymphocyte syndrome.” HLA-DR antigenic specificities are epitopes on DR gene products. Selected specificities have been mapped to defined loci. HLA serologic typing requires the identification of a prescribed antigenic determinant on a particular HLA molecular product. One typing specificity can be present on many different molecules. Different alleles at the same locus may encode these various HLA molecules. Monoclonal antibodies are now used to recognize certain antigenic determinants shared by various molecules bearing the same HLA typing specificity. Monoclonal antibodies have been employed to recognize specific class II alleles with disease associations. HLA-DM facilitates the loading of antigenic peptides onto MHC class II moleules. As a result of proteolysis of

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FIGURE 21.6 HLA-DR1 histocompatibility antigen.

the invariant chain, a small fragment called the class II-associated invariant chain peptide, or CLIP, remains bound to the MHC class II molecule. CLIP peptide is replaced by antigenic peptides, but in the absence of HLADM, this does not occur. The HLA-DM molecule must therefore play some part in removal of the CLIP peptide and in the loading of antigenic peptides. Minor lymphocyte stimulatory (MIs) loci: See MIs antigens. HLA-E is an HLA class I nonclassical molecule. HLA-F is an HLA class I nonclassical molecule.

HLA-G is a polymorphic class I HLA antigen with extensive variability in the α-2 domain. It is found on trophoblasts, i.e., placenta cells and trophoblastic neoplasms. HLA-G is expressed only on cells such as placental extravillous cytotrophoblasts and choriocarcinoma that fail to express HLA-A, -B, and -C antigens. HLA-G expression is most pronounced during the first trimester of pregnancy. Trophoblast cells expressing HLA-G at the maternal–fetal junction may protect the semiallogeneic fetus from “rejection.” Prominent HLA-G expression suggests maternal immune tolerance. HLA-H is a pseudogene found in the MHC class I region that is structurally similar to HLA-A but is nonfunctional due to the absence of a cysteine residue at position 164 in its protein product and the deletion of the codon 227 nucleotide. HLA nonclassical class I genes are located within the MHC class I region and encode products that can associate with β2 microglobulin. However, their function and tissue distribution are different from those of HLA-A, -B, and C molecules. Examples include HLA-E, -F, and -G. Of these, only HLA-G is expressed on the cell surface. It is uncertain whether or not these HLA molecules are involved in peptide binding and presentation like classical class I molecules. An extended haplotype consists of linked alleles in positive linkage disequilibrium, situated between and including HLA-DR and HLA-B of the MHC of man. Examples of extended haplotypes include the association of B8/DR3/SCO1/GLO2 with membranoproliferative glomerulonephritis, and of A25/B18/DR2 with complement C2 deficiency. Extended haplotypes may be a consequence of crossover suppression through environmental influences, together with selected HLA types, leading to autoimmune conditions. The B27 relationship to Klebsiella is an example. PCR amplification and direct sequencing help identify a large number of allelic differences and specific associations of extended haplotypes with disease. Extended haplotypes are more informative than single polymorphisms. Some diseases associated with extended haplotypes include Graves’ disease, pemphigus vulgaris, type I (juvenile onset) insulin-dependent diabetes mellitus, celiac disease, psoriasis, and autoimmune hepatitis. Linkage disequilibrium refers to the appearance of HLA genes on the same chromosome with greater frequency than would be expected by chance. This has been demonstrated by detailed studies in both populations and families, employing outbred groups in which numerous different haplotypes are present. With respect to the HLA-A, -B, and -C loci, a possible explanation for linkage disequilibrium is that there has not been sufficient time for the genes to reach equilibrium. However, this possibility is remote

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for HLA-A, -B, and -D linkage disequilibrium. Natural selection has been suggested to maintain linkage disequilibrium that is advantageous. If products of two histocompatibility loci play a role in the immune response and appear on the same chromosome, they might reinforce one another and represent an advantageous association. An example of linkage disequilibrium in the HLA system of man is the occurrence on the same chromosome of HLAA3 and HLA-B7 in the Caucasian American population. Lymphocyte defined (LD) antigens are histocompatibility antigens on mammalian cells that induce reactivity in a mixed-lymphocyte culture (MLC) or mixed-lymphocyte reaction. HLA disease association: Certain HLA alleles occur in a higher frequency in individuals with particular diseases than in the general population. This type of data permits estimation of the “relative risk” of developing a disease with every known HLA allele. For example, there is a strong association between ankylosing spondylitis, which is an autoimmune disorder involving the vertebral joints, and the class I MHC allele, HLA-B27. There is a strong association between products of the polymorphic class II alleles HLA-DR and -DQ and certain autoimmune diseases, since class II MHC molecules are of great importance in the selection and activation of CD4+ T lymphocytes which regulate the immune responses against protein antigens. For example, 95% of Caucasians with insulindependent (type I) diabetes mellitus have HLA-DR3 or HLA-DR4 or both. There is also a strong association of HLA-DR4 with rheumatoid arthritis. Numerous other examples exist and are the targets of current investigations, especially in extended studies employing DNA probes. Calculation of the relative risk (RR) and absolute risk (AR) can be found elsewhere in this book. Immunoinhibitory genes are selected HLA genes that appear to protect against immunological diseases. Their mechanisms of action are in dispute. HLA allelic variation is a genomic analysis that has identified specific individual allelic variants to explain HLA associations with rheumatoid arthritis, type I diabetes mellitus, multiple sclerosis, and celiac disease. There is a minimum of six α and eight β genes in distinct clusters, termed HLA-DR, -DQ, and -DP within the HLA class II genes. DO and DN class II genes are related, but map outside DR, DQ, and DP regions. There are two types of dimers along the HLA cell-surface HLA-DR class II molecules. The dimers are made up of either DRαpolypeptide associated with DRβ1-polypeptide or DR with DRβ2-polypeptide. Structural variation in class II gene products is linked to functional features of immune recognition leading to individual variations in histocompatibility, immune recognition, and susceptibility to disease.

There are two types of structural variations which include variation among DP, DQ, and DR products in primary amino acid sequence by as much as 35% and individual variation attributable to different allelic forms of class II genes. The class II polypeptide chain possesses domains which are specific structural subunits containing variable sequences that distinguish among class II ι genes or class II β genes. These allelic variation sites have been suggested to form epitopes, which represent individual structural differences in immune recognition. Interallelic conversion refers to genetic recombination between two alleles of a locus in which a segment of one allele is replaced with a homologous segment from another. HLA Class I and HLA Class II alleles are formed in this way. HLA oligotyping is a recently developed method using oligonucleotide probes to supplement other histocompatibility testing techniques. Whereas serological and cellular methods identify phenotypic characteristics of HLA proteins, oligotyping defines the genotype of the DNA that encodes HLA protein structure and specificity. Thus, oligotyping can identify the DNA type even when there is a failure of expression of HLA genes that render serological techniques ineffective. HLA tissue typing (Figure 21.7) is the identification of MHC class I and class II antigens on lymphocytes by serological and cellular techniques. The principal serological assay is microlymphocytotoxicity using microtiter plate containing predispensed antibodies against HLA specificities to which lymphocytes of unknown specificity plus rabbit complement and vital dye are added. Following incubation, the wells are scored according to the relative proportion of cells killed. This method is employed for organ transplants such as renal allotransplants. For bone marrow transplants, mixed lymphocyte reaction procedures are performed to determine the relative degree of histocompatibility or histoincompatibility between donor

FIGURE 21.7 HLA tissue typing.

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and recipient. Serological tests are largely being replaced by DNA typing procedures employing PCR methodology and DNA or oligonucleotide probes, especially for MHC class II typing. Class I typing involves reactions between lymphocytes to be typed with HLA antisera of known specificity in the presence of complement. Cell lysis is detected by phase or fluorescence microscopy. This is important in parentage testing, disease association, transfusion practices, and transplantation. HLA-A, -B, and -C antigens should be defined by at least one of the following: (1) at least two different sera if both are monospecfic, (2) one monospecific and two multispecific antisera, (3) at least three multispecific antisera if all multispecific are used. Class II typing detects HLA-DR antigens using purified B cell preparations. It is based on antibody-specific, complement-dependent disruption of the cell membrane of lymphocytes. Cell death is demonstrated by the penetration of dye into the membrane. Class II typing is more difficult than class I typing because of the variability of both B cell isolation and complement toxicity. At least three antisera must be used if all are monospecific; at least three antisera, must be used if all are monospecific; at least five antisera must be used for multispecifc sera. Antibody screening: Candidates for organ transplants, especially renal allografts, are monitored with relative frequency for changes in their percent reactive antibody (PRA) levels. Obviously, those with relatively high PRA values are considered to be less favorable candidates for renal allotransplants than are those in whom the PRA values are low. PRA determinations may vary according to the composition of the cell panel. If the size of the panel is inadequate, it may affect the relative frequency of common histocompatibility antigens found in the population. Tissue typing is the identification of MHC class I and class II antigens on lymphocytes by serological and cellular techniques. The principal serological assay is microlymphocytotoxicity using microtiter plates containing predispensed antibodies against HLA specificities to which lymphocytes of unknown specificity plus rabbit complement and vital dye are added. Following incubation, the wells are scored according to the relative proportion of cells killed. This method is employed for organ transplants such as renal allotransplants. For bone marrow transplants, mixed lymphocyte culture (MLC), also called mixed lymphocyte reaction, procedures are performed to determine the relative degree of histocompatibility or histoincompatibility between donor and recipient. Serological tests are largely being replaced by DNA typing procedures employing PCR methodology and DNA or oligonucleotide probes, especially for MHC class II typing. Sequence specific primer (SSP) technology is currently the method of choice in molecular typing.

FIGURE 21.8 Schematic representation of Ficoll-hypaque technique of cell separation.

FIGURE 21.10 A Hamilton syringe that is used to dispense lymphocytes into Terasaki plates for tissue typing.

FIGURE 21.9 The separation of lymphocytes from peripheral blood by centrifugation using Ficoll-hypaque.

Microlymphocytotoxicity is a widely used technique for HLA tissue typing. Lymphocytes are separated from heparinized blood samples by either layering over Ficollhypaque (Figure 21.8 and Figure 21.9), centrifuging and removing lymphocytes from the interface or by using beads. After appropriate washing, these purified lymphocyte

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preparations are counted, and aliquots are dispensed using a Hamilton syringe (Figure 21.10) into microtiter plate wells (Figure 21.11) containing predispensed quantities of antibody. When used for HLA testing, antisera in the wells are specific for known HLA antigenic specificities. After incubation of the cells and antisera, rabbit complement is added and the plates are again incubated. The extent of cytotoxicity induced is determined by incubating the cells with trypan blue, which enters dead cells and stains them blue, while leaving live cells unstained. The plates are read by using an inverted phase contrast microscope (Figure 21.12). A scoring system from 0 to 8 (where 8 implies >80% of target cells killed) is employed to indicate cytotoxicity. Most of the sera used to date are multispecific, as they are obtained from multiparous females who have been sensitized during pregnancy by HLA antigens determined by their spouse. Monoclonal antibodies are being used with increasing frequency in tissue typing. This technique is useful to identify HLAA, HLA-B, and HLA-C antigens. When purified B cell preparations and specific antibodies against B cell antigens are employed, HLA-DR and HLA-DQ antigens can be identified.

FIGURE 21.11 A Terasaki plate consisting of depressions in a plastic plate that contains predispensed antibodies to HLA antigens of various specificities and into which are placed patient lymphocytes and rabbit complement for tissue typing.

A cell tray panel (Figure 21.13) is used to detect and identify HLA antibodies. Patient serum is tested against a panel of known cells. The panel (or percent) reactive antibody (PRA) is the percent of panel cells reacting with a patient’s serum. It is expressed as a percentage of the total reactivity, i.e., %PRA = (No. of positive reactions/No. of cells in panel) × 100. This percentage is a useful indicator of the proportion of HLA antibodies of the patient. Patients may have preformed antibodies against class I or II HLA antigens. If these patients receive organs that possess the corresponding antigens, they will likely experience hyperacute or delayed rejection for class I or class II incompatibilities, respectively. In order to detect such incompatibilities before transplantation, a cross-matching procedure is performed. The conventional crossmatching procedure (Figure 21.14) for organ transplants involves the combination of donor lymphocytes with recipient serum. There are three major variables in the standard cross-match procedure that predominantly affect the reactivity of the cell/sera sensitization. These include (l) incubation time and temperature; (2) wash steps after cell/sera sensitization; and (3) the use of additional

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FIGURE 21.12 An inverted light microscope used to read Terasaki plates to determine tissue type.

TRAY CELL PANEL TYPING POS #CENTRL TEST ID RACE A B C BW 1 2 8 35 7 8 10571T H 6 1A 1 2 44 51 1 5 4 8 9891T C 1B 1 2 57 82 3 6 4 6 8 9884T B 1C 1 23 45 49 6 7 4 6 8 9898T B 1D 1 23 58 72 6 8 10356T B 4 6 1E 1 24 27 37 2 6 4 8 10990T O 1F 1 32 8 51 7 8 10367T C 4 6 2F 1 8 13 64 6 8 4 6 7109T H 2E 2 11 18 38 7 1 6606T C 4 6 2D 2 11 37 60 3 6 4 6 1 10567T C 2C 2 24 51 55 3 8 10988T C 4 6 2B 2 25 57 62 5 6 4 6 1 10359T C 2A 2 26 39 61 1 7 1 10549T O 6 3A 2 26 54 62 1 3 1 10361T O 6 3B 2 26 60 65 4 8 1 10570T O 6 3C 2 30 8 58 7 1 9899T B 4 6 3D 2 30 13 46 1 6 4 6 1 10352T O 3E 2 31 35 47 4 1 10547T C 4 6 3F 2 31 50 60 3 6 1 6688T C 6 4F 2 32 41 61 2 7 1 10568T H 6 4E

FIGURE 21.13 Cell tray panel showing positive reactions (8s) for HLA-A1 at tray positions 1A, 1B, 1C, 1D, 1E, 1F, 2F, and 2E, and a positive reaction for HLA-A24 at position 2B.

FIGURE 21.14 Crossmatching procedure.

reagents, such as antiglobulin in the test. Variations in these steps can cause wide variations in results. Lymphocytes can be separated into T and B cell categories for crossmatch procedures that are conducted at cold (4°C), room (25°C), and warm (37°C) temperatures. These permit the identification of warm anti-T cell antibodies that are almost always associated with graft rejection. Molecular (DNA) typing: sequence-specific priming (SSP) is a method that employs a primer with a single mismatch in the 3′-end that cannot be employed efficiently to extend a DNA strand because the enzyme Taq polymerase, during the PCR reaction, and especially in the first PCR cycles which are very critical, does not manifest 3′-5′ proofreading endonuclease activity to remove the mismatched nucleotide. If primer pairs are designed to have perfectly matched 3′-ends with only a single allele, or a single group of alleles, and the PCR reaction is initiated under stringent conditions, a perfectly matched primer pair results in an amplification product, whereas a mismatch at the 3′-end primer pair will not provide any amplification product. A positive result, i.e., amplification, defines the specificity of the DNA sample. In this method, the PCR amplification step provides the basis for identifying polymorphism. The postamplification processing of the sample consists only of a simple agarose gel electrophoresis to detect the presence or absence of amplified product. DNA amplified fragments are visualized by ethidium bromide staining and exposure to UV light. A separate technique detects amplified product by color fluorescence. The primer pairs are selected in such a manner that each allele should have a unique reactivity pattern with the panel of primer pairs employed. Appropriate controls must be maintained (Figure 21.14a). CREGs are crossreactive groups. Public epitope-specific antibodies identify CREGs. Public refers to both similar (cross-reactive) and identical (public) epitopes shared by more than one HLA gene product. CYNAP antibodies are cytotoxicity negative but absorption-positive antibodies that are concerned with HLA tissue typing. Most alloantibodies to public epitopes display

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FIGURE 21.14a Example of high-resolution DRBI typing using sequence-specific primer methodology. Molecular weight ladder of known base pairs is in the far left column for base pair sizing.

CYNAP when tested in complement-dependent cytotoxicity assays. Most alloantisera contain public or CREG antibodies, but they act operationally as “private” antibodies because of their CYNAP phenomenon. For this reason, the relative insensitivity of standard CDC, due to CYNAP, has been useful for detecting discrete gene products. Standard CDC is not the recommended procedure to define HLA molecule binding specificities. The antiglobulin-augmented CDC (AHG-CDC) more accurately defines the true binding capabilities of alloantisera than do complement-independent assays by overriding the CYNAP phenomenon. CDC is the procedure of choice for HLA antigen detection and HLA antiserum analysis. Haplotype designates those phenotypic characteristics encoded by closely linked genes on one chromosome inherited from one parent. It frequently describes several MHC alleles on a single chromosome. Selected haplotypes are in strong linkage disequilibrium between alleles of different loci. According to Mendelian genetics, 25% of siblings will share both haplotypes. CYNAP phenomenon: See CYNAP antibodies. Phenotype designates observable features of a cell or organism that are a consequence of interaction between the genotype and the environment. The phenotype represents those genetically encoded characteristics that are

expressed. Phenotype may also refer to a group of organisms with the same physical appearance and the same detectable characteristics. MHC haplotype refers to the set of genes in a haploid genome inherited from one parent. Children of parents designated ab and cd will probably be ac, ad, bc, or bd. Trypan blue is a vital dye used to stain lymphoid cells, especially in the microlymphocytotoxicity test used for HLA tissue typing. Cell membranes whose integrity has been interrupted by antibody and complement permit the dye to enter and stain the cells dark blue. By contrast, the viable cells with an intact membrane exclude the dye and remain as bright circles of light in the microscope. Dead cells stain blue. Trypan blue dye exclusion test is a test for viability of cells in culture. Living cells exclude trypan blue by active transport. When membranes have been interrupted, the dye enters the cells, staining them blue and indicating that the cell is dead. The method can be used to calculate the percent of cell lysis induced. The 2-mercaptoethanol agglutination test is a simple test to determine whether or not an agglutinating antibody is of the IgM class. If treatment of an antibody preparation, such as a serum sample, with 2-mercaptoethanol can abolish the serum’s ability to produce agglutination of cells, then agglutination was due to IgM antibody. Agglutination induced by IgG antibody is unaffected by 2-mercaptoethanol treatment and just as effective after the treatment as it was before. Dithiothreitol (DTT) produces the same effect as 2-mercaptoethanol in this test. Small “blues” are blue aggregates of acellular debris observed in clinical histocompatibility testing using the microlymphotoxicity test. It occurs in the wells of tissue typing trays and is due to an excess amount of trypan blue mixed with protein. This is a technical artifact. Serologically defined (SD) antigens are mammalian cellular membrane epitopes that are encoded by MHC genes. Antibodies detect these epitopes. Serological determinants are epitopes on cells that react with specific antibody and complement, leading to fatal injury of the cells. Serological determinants are to be distinguished from lymphocyte determinants, which are epitopes on the cell surface to which sensitized lymphocytes are directed, leading to cellular destruction. Although the end result is the same, antibodies and lymphocytes are directed to different epitopes on the cell surface. In a mixed-lymphocyte culture (MLC), lymphocytes from two members of a species are combined in culture where they are maintained and incubated for 3 to 5 d. Lymphoblasts are formed as a consequence of histoincompatibility between the two individuals donating the

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lymphocytes. The lymphocyte antigens of these genetically dissimilar subjects each stimulate DNA synthesis by the other, which is measured by tritiated thymidine uptake that is assayed in a scintillation counter. See mixed-lymphocyte reaction (MLR). MLC: Abbreviation for mixed-lymphocyte culture. In the mixed-lymphocyte reaction (MLR), lymphocytes from potential donor and recipient are combined in tissue culture. Each of these lymphoid cells has the ability to respond by proliferating following stimulation by antigens of the other cell. In the one-way reaction, the donor cells are treated with mitomycin or irradiation to render them incapable of proliferation. Thus, the donor antigens stimulate the untreated responder cells. Antigenic specificities of the stimulator cells that are not present in the responder cells lead to blastogenesis of the responder lymphocytes. This leads to an increase in the synthesis of DNA and cell division. This process is followed by introduction of a measured amount of tritiated thymidine, which is incorporated into the newly synthesized DNA. The mixed-lymphocyte reaction usually measures a proliferative response and not an effector cell killing response. The test is important in bone marrow and organ transplantation to evaluate the degree of histoincompatibility between donor and recipient. Both CD4+ and CD8+ T lymphocytes proliferate and secrete cytokines in the MLR. Also called mixed-lymphocyte culture. Mixed leukocyte reaction (MLR): See mixed-lymphocyte reaction (MLR). Homozygous describes containing two copies of the same allele. The homozygous typing cell (HTC) technique is an assay that employs a stimulator cell that is homozygous at the HLA-D locus. An HTC incorporates only a minute amount of tritiated thymidine when combined with a homozygous cell in the MLR. This implies that the HTC shares HLA-D determinants with the other cell type. By contrast, when an HTC is combined with a nonhomozygous cell, much larger amounts of tritiated thymidine are incorporated. Many variations between these two extremes are noted in actual practice. Homozygous typing cells are frequently obtained from the progeny of marriages between cousins. Homozygous typing cells (HTCs) are cells obtained from a subject who is homozygous at the HLA-D locus. HTCs facilitate MLR typing of the human D locus. Lymphocyte determinants are target cell epitopes identified by lymphocytes rather than antibodies from a specifically immunized host. Cross-match testing is an assay used in blood typing and histocompatibility testing to ascertain whether or not

31 Count 0 .1

1000 Negative Flow Crossmatch




FIGURE 21.15 Negative flow crossmatch.


1000 Positive Flow Crossmatch

FIGURE 21.17 Splits. FIGURE 21.16 Positive flow crossmatch.

donor and recipient have antibodies against each other’s cells that might lead to transfusion reaction or transplant rejection. Cross-matching reduces the changes of graft rejection by performed antibodies against donor cell surface antigens which are usually MHC antigens. Donor lymphocytes are mixed with recipient serum, complement is added, and the preparation observed for cell lysis. Flow cytometry can also be used to perform the crossmatching procedure. This method is highly sensitive (considerably more sensitive than the direct cytotoxicity method). Flow cross-matching is also faster and can distinguish antibodies according to class (IgG vs. IgM) and target cell specificity (T cells from B cells). It is a valuable procedure in organ and bone marrow transplantation and is particularly suitable to measuring antibodies against HLA class I antigens on donor T cells. False positives are rare and most errors are due to low sensitivity (lower antibody concentration). Flow cross-matching has the potential to be standardized and automated. The flow cytometry cross-matching method commonly utilizes F(ab´)2 antihuman IgG conjugated to fluorescein, and anti-CD3 for T cells conjugated to phycoerythrin. A two parameter display of anti-CD3 vs. IgG is generated. A positive flow crossmatch is defined as median channel shift values >40 (Figure 21.15 and Figure 21.16).

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Splits are human leukocyte antigen (HLA) subtypes (Figure 21.17). For example, the base antigen HLA-B12 can be subdivided into the splits HLA-B44 and HLA-B45. The term “split” is used to designate an HLA antigen that was first believed to be a private antigen but later was shown to be a public antigen. The former designation can be placed in parenthesis following its new designation, i.e., HLA-B44(12). A private antigen (Figure 21.18) is (1) an antigen confined to one MHC molecule; (2) an antigenic specificity restricted to a few individuals; (3) a tumor antigen restricted to a specific chemically induced tumor; (4) a low-frequency epitope present on red blood cells of fewer than 0.1% of the population, i.e., Pta, By, Bpa, etc.; and (5) HLA antigen encoded by one allele such as HLA-B27. A public antigen (supratypic antigen) is an epitope which several distinct or private antigens have in common (Figure 21.18). A public antigen such as a blood group antigen is one that is present in greater than 99.9% of a population. It is detected by the indirect antiglobulin (Coombs’ test). Examples include Ve, Ge, Jr, Gya, and Oka. Antigens that occur frequently but are not public antigens include Mns, Lewis, Duffy, P, etc. In blood banking, there is a problem finding a suitable unit of blood for a transfusion to

alleles, there is only a remote possibility that two unrelated persons would share the same pattern, i.e., about 1 in 30 billion. There is, however, a problem in deciphering the multibanded arrrangement of minisatellite RFLPs, as it is difficult to ascertain which bands are allelic. Mutation rates of minisatellite HVRs remain to be demonstrated, but are recognized occasionally. This method can be used in resolving cases of disputed parentage.

FIGURE 21.18 Public and private antigens.

Single locus probes (SLPs) are probes which hybridize at only one locus. These probes identify a single locus of variable number of tandem repeats (VNTRs) and permit detection of a region of DNA repeats found in the genome only once and located at a unique site on a certain chromosome. Therefore, an individual can have only two alleles that SLPs will identify, as each cell of the body will have two copies of each chromosome, one from the mother and the other from the father. When the lengths of related alleles on homologous chromosomes are the same, there will be only a single band in the DNA typing pattern. Therefore, the use of an SLP may yield either a singleor double-band result from each individual. Single-locus markers such as the pYNH24 probe developed by White may detect loci that are highly polymorphic, exceeding 30 alleles and 95% heterozygosity. SLPs are used in resolving cases of disputed parentage. Immediate spin crossmatch is a test for incompatibility between donor erythrocytes and the recipient patient’s serum. This assay reveals ABO incompatibility in practically all cases, but is unable to identify IgG alloantibodies against erythrocyte antigens. Orthotopic is an adjective that describes an organ or tissue transplant that has been in the site usually occupied by that organ or tissue. An orthotopic graft (Figure 21.20) is an organ or tissue transplant that is placed in the location that is usually occupied by that particular organ or tissue.

FIGURE 21.19 Multilocus probes.

recipients who have developed antibodies against public antigens. Multilocus probes (Figure 21.19) are used to identify multiple related sequences distributed throughout each person’s genome. Multilocus probes may reveal as many as 20 separate alleles. Because of this multiplicity of

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FIGURE 21.20 Orthotopic graft.

Heterotopic is an adjective that describes the placement of an organ or tissue graft in an anatomic site other than the one where it is normally located. A heterotopic graft is a tissue or organ transplanted to an anatomic site other than the one where it is usually found under natural conditions. For example, the anastomosis of the renal vasculature at an anatomical site that would situate the kidney in a place other than the renal fossa where it is customarily found. A graft is the transplantation of a tissue or organ from one site to another within the same individual or between individuals of the same or a different species. Heart–lung transplantation is a procedure that has proven effective for the treatment of primary respiratory disease with dysfunction of gas exchange and alveolar mechanics, together with a secondary elevation in pulmonary vascular resistance, and in primary high-resistance circulatory disorder associated with pulmonary vascular disease. A rescue graft is a replacement graft for an original graft that failed. Privileged sites are anatomical locations in the body that are protected from immune effector mechanisms because of the absence of normal lymphatic drainage. Antigenic substances such as tissue allografts may be placed in these sites without evoking an immune response. Privileged sites include the anterior chamber of the eye, the cheek-pouch of the Syrian hamster, and the central nervous system. Tissue allografts in these locations enjoy a period of protection from immunologic rejection, as the diffusion of antigen from graft sites to lymphoid tissues is delayed. Immune privilege alters the induction of immunity to antigens first encountered via privileged sites and also inhibits the expression of certain forms of alloimmunity in these same sites. Immunologically privileged sites are certain anatomical sites within the animal body that provide an immunologically privileged environment which favors the prolonged survival of alien grafts. The potential for development of a blood and lymphatic vascular supply connecting graft and host may be a determining factor in the qualification of an anatomical site as an area which provides an environment favorable to the prolonged survival of a foreign graft. Immunologically privileged areas include (1) the anterior chamber of the eye, (2) the substantia propria of the cornea, (3) the meninges of the brain, (4) the testis, and (5) the cheek pouch of the Syrian hamster. Foreign grafts implanted in these sites show a diminished ability to induce transplantation immunity in the host. These immunologically privileged sites usually fail to protect alien grafts from the immune rejection mechanism in hosts previously or simultaneously sensitized with

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donor tissues. The capacity of cells expressing Fas ligand to cause deletion of activated lymphocytes provides a possible explanation for the phenomenon of immune privilege. Animals with a deficiency in either Fas ligand or the Fas receptor fail to manifest significant immune privilege. Both epithelial cells of the eye and Sertoli cells of the testes express Fas ligand. Immune privilege is a consequence not only of the lack of an inflammatory response but also from immune consequences of the accumulation of apoptotic immune cells within a tissue. Immune cell apoptosis may be a signal to terminate inflammation. Apoptotic cell accumulation during an immune response could activate the development of cells that function to downregulate or suppress further immune activation. Immune privilege: See immunologically privileged sites. An immunologic barrier is an anatomical site that diminishes or protects against an immune response. This refers principally to immunologically privileged sites where grafts of tissue may survive for prolonged periods without undergoing immunologic rejection. This is based mainly on the lack of adequate lymphatic drainage in these areas. Examples include prolonged survival of foreign grafts in the brain. A semisyngeneic graft is a graft that is ordinarily accepted from an individual of one strain into an F1 hybrid of an individual of that strain mated with an individual of a different strain (Figure 21.21). Graft facilitation is a prolonged graft survival attributable to conditioning of the recipient with IgG antibody, which is believed to act as a blocking factor. It also decreases cell-mediated immunity. This phenomenon is related to immunologic enhancement of tumors by antibody and has been referred to as immunological facilitation (facilitation immunologique). Immunologic facilitation (facilitation immunologique) is the slightly prolonged survival of certain normal tissue allografts, e.g., skin, in mice conditioned with isoantiserum specific for the graft. Immunologic enhancement is the prolonged survival, conversely the delayed rejection, of a tumor allograft in a host as a consequence of contact with specific antibody. Both the peripheral and central mechanisms have been postulated. In the past, coating of tumor cells with antibody was presumed to interfere with the ability of specifically reactive lymphocytes to destroy them, but today a central effect in suppressing cell-mediated immunity, perhaps through suppressor T lymphocytes, is also possible. Enhancement is the prolonged survival, conversely the delayed rejection, of tumor or skin allografts in individuals

FIGURE 21.21 Semisyngeneic graft.

previously immunized or conditioned by passive injection of antibody specific for graft antigens. This is termed immunological enhancement and is believed to be due to a blocking effect by the antibody. Enhancing antibodies are blocking antibodies that favor survival of tumor or normal tissue allografts. An allograft is an organ, tissue, or cell transplant from one individual or strain to a genetically different individual or strain within the same species. Allografts are also called homografts (Figure 21.22). An allotransplant refers to the transplantation of an organ or tissue from one individual to another member of the same species. Fetus allograft: Success of the haplo-nonidentical fetus as an allograft was suggested in the 1950s by Medawar, Brent, and Billingham to rely on four possibilities. This proposal suggested that (1) the conceptus might not be immunogenic, (2) that pregnancy might alter the immune

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response, (3) that the uterus might be an immunologically privileged site, and (4) that the placenta might represent an effective immunological barrier between mother and fetus. Further studies have shown that transplantation privilege afforded the fetalplacental unit in pregnancy depends on intrauterine mechanisms. The pregnant uterus has been shown not to be an immunologically privileged site. Pregnancies usually are successful in maternal hosts with high levels of preexisting alloimmunity. The temporary status has focused on specialized features of fetal trophoblastic cells that facilitate transplantation protection. Fetal trophoblast protects itself from maternal cytotoxic attack by failing to express on placental villous cytotrophoblast and syncytiotrophoblast any classical polymorphic class I or II MHC antigens. Constitutive HLA expression is also not induced by known upregulators such as interferon y. Thus classical MHC antigens are not expressed throughout gestation. Extravillous cytotrophoblast cells selectively express HLA-G, a nonclassical class I MHC antigen which has limited genetic polymorphism. HLA-G might protect the cytotrophoblast population from MHC-nonrestricted

growth factor 3 (TGF-β). Fetal syncytiotrophoblast has numerous growth factor receptors. Thus an extensive cytokine network is preset within the uteroplacental tissue that offers both immunosuppressive and growth promoting signals. In humans, IgG is selectively transported across the placenta into the fetal circulation following combination with transporting Fcγ receptors on the placenta. This transfer takes place during the 20th to the 22nd week of gestation. Maternal HLA-specific alloantibody that is specific for the fetal HLA type is bound by nontrophoblastic cells expressing fetal HLA antigens. These include macrophages, fibroblasts, and endothelium within the villous mesenchyme of placental tissue, thereby preventing these antibodies from reaching the fetal circulation. Maternal antibodies against any other antigen of the fetus will likewise be bound within the placental tissues to a cell expressing that antigen. The placenta acts as a sponge to absorb potentially harmful antibodies. Exceptions to placental trapping of deleterious maternal IgG antibodies include maternal IgG antibodies against RhD antigen and certain maternal organ-specific autoantibodies. An allogeneic graft is an allograft consisting of an organ, tissue, or cell transplant from a donor individual or strain to a genetically different individual or strain within the same species.

FIGURE 21.22 Types of grafts.

natural killer (NK) cell attack. The trophoblast also protects itself from maternal cytotoxicity during gestation by expressing a high level of complement regulatory proteins on its surface, such as membrane cofactor protein (MCP;CD46), decay accelerating factor (DAF;CD55), and membrane attack complex inhibitory factor (CD59). The maternal immune system recognizes pregnancy, i.e., the fetal trophoblast, in a manner that results in cellular, antibody, and cytokine responses that protect the fetal allograft. CD56 positive large granular lymphocytes may be regulated by hormones in the endometrium that control their function. They have been suggested to be a form of NK cell in arrested maturation possibly due to persistent expression of HLA-G on target invasive cytotrophoblast. Contemporary studies have addressed cytokine interactions at the fetal–maternal tissue interface in pregnancy. HLA-G or other fetal trophoblast antigens have been postulated to possibly stimulate maternal lymphocytes in endometrial tissue to synthesize cytokines and growth factors that act in a paracrine manner beneficial to trophoblast growth and differentiation. This has been called the immunotrophism hypothesis. Other cytokines released into decidual tissue include colony stimulating factors (CSFs), tumor necrosis factor α(TNF-α), IL-6, and transforming

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Homologous is an adjective that describes something from the same source. For example, an organ allotransplant from one member to a recipient member of the same species, i.e., renal allotransplantation in humans. Allogeneic bone marrow transplantation: Hematopoietic cell transplants are performed in patients with hematologic malignancies, certain nonhematologic neoplasms, aplastic anemias, and certain immunodeficiency states. In allogeneic bone marrow transplantation the recipient is irradiated with lethal doses either to destroy malignant cells or to create a graft bed. The problems that arise include graft-vs.-host (GVH) disease and transplant rejection. GVH disease occurs when immunologically competent cells or their precursors are transplanted into immunologically crippled recipients. Acute GVH disease occurs within days to weeks after allogeneic bone marrow transplantation and primarily affects the immune system and epithelia of the skin, liver, and intestines. Rejection of allogeneic bone marrow transplants appears to be mediated by NK cells and T cells that survive in the irradiated host. NK cells react against allogeneic stem cells that are lacking self MHC Class I molecules and therefore fail to deliver the inhibitory signal to NK cells. Host T cells react against donor MHC antigens in a manner resembling their reaction against solid tissue grafts. Hemopoietic resistance (HR): Transplantation of allogeneic, parental, or xenogeneic bone marrow or leukemia cells

into animals exposed to total body irradiation often results in the destruction of the transplanted cells. The mechanism causing the failure of the transplant appears similar with all three types of cells. This phenomenon, designated hemopoietic resistance (HR), has a genetic basis and mechanism different from conventional transplantation reactions against solid tumor allografts. It does not require prior sensitization and apparently involves the cooperation between NK cells and macrophages, both resistant to irradiation. The NK cells have the characteristics of null cells; macrophages play an accessory cell role. The cooperative activity seems to represent in vivo surveillance against leukemogenesis. Homologous chromosomes are a pair of chromosomes containing the same linear gene sequences, each derived from one parent. Immunoisolation describes the enclosure of allogeneic tissues such as pancreatic islet cell allografts within a membrane that is semipermeable, but does not itself induce an immune response. Substances of relatively low mol wt can reach the graft through the membrane, while it remains protected from immunologic rejection by the host. Allogeneic inhibition is the better growth of homozygous tumors when they are transplanted to homozygous syngeneic hosts of the strain of origin than when they are transplanted to F1 hybrids between the syngeneic (tumor) strain and an allogeneic strain. This is manifested as a higher frequency of tumor and shorter latency period in syngeneic hosts. The better growth of tumor in syngeneic than in heterozygous F1 hybrid hosts was initially termed syngeneic preference. When it became apparent that selective pressure against the cells in a mismatching environment produced the growth difference, the phenomenon was termed allogeneic inhibition. Syngeneic preference is the better growth of neoplasms when they are transplanted to histocompatible recipients than when they are transplanted in histoincompatible recipients. See also allogeneic inhibition. Incompatibility refers to dissimilarity between the antigens of a donor and recipient as in tissue allotransplantation or blood transfusions. The transplantation of a histoincompatible organ or the transfusion of incompatible blood into a recipient may induce an immune response against the antigens not shared by the recipient in injurious consequences. Homograft is the earlier term for allograft, i.e., an organ or tissue graft from a donor to a recipient of the same species. Homograft reaction is an immune reaction generated by a homograft (allograft) recipient against the graft alloantigens. Also called an allograft reaction.

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FIGURE 21.23 Induction of tolerance to a xenogenic tissue graft.

Homograft rejection is an earlier term for allograft rejection, i.e., an immune response induced by histocompatibility antigens in the donor graft that are not present in the recipient. This is principally a cell-mediated type of immune response. Homotransplantation: Homograft, i.e., allograft transplantation. Heterograft: See xenograft. Heterogeneic: See xenogeneic. In transplantation biology, heterologous refers to an organ or tissue transplant from one species to a recipient belonging to another species, i.e., a xenogeneic graft. It also refers to something from a foreign source. A xenograft (Figure 21.23) is a tissue or organ graft from a member of one species, i.e., the donor, to a member of a different species, i.e., the recipient. It is also called a heterograft. Antibodies and cytotoxic T cells reject xenografts several days following transplantation. Xenogeneic is an adjective that refers to tissues or organs transplanted from one species to a genetically different species, e.g., a baboon liver transplanted to a human. Xenoantigen is an antigen of a xenograft. Also called heteroantigen. Xenoantibody is an antibody specific for xenoantigen. Xenoantibodies are antibodies formed in one species that are specific for antigens of a separate species. Xenoreactive refers to a T cell or antibody response to an antigen of a graft derived from another species. The T lymphocyte may recognize an intact xenogenic MHC molecule or a peptide from a xenogeneic protein bound a self MHC molecule. Xenotransplantation is organ or tissue transplantation between members of different species. An example of transplantation of tissues or organs from one species to another is a chimpanzee heart transplanted into a human recipient. It represents a possible substitute for the shortage

of human organs for clinical transplantation. Xenogeneic transplantation can involve concordant or discordant donors, according to the phylogenetic distance between the species involved. Natural preformed antibodies in a recipient specific for donor endothelial antigens that lead to hyperacute rejection of most vascularized organ transplants now occur in discordant species combinations. The immune response to a xenotransplant resembles the response to an allotransplant. However, there are greater antigenic differences between donor and host in xenotransplantation than in allotransplantation. Previously termed heterotransplantation. Xenozoonosis is a term that describes transmission of infection that might be the consequence of xenotransplantation. Infections resulting from xenotransplantation might involve infection of recipient cells with endogenous retroviral sequences from donor cells, giving rise themselves or after recombination with human endogenous retroviral sequences to previously unknown pathogenic viruses. Such new viruses might be pathogenic for other human beings in addition to the xenograft recipient. Zoonosis is a term that describes the general process of cross-species infection. Xenotype refers to molecular variations based on differences in structure and antigenic specificity. Examples would include membrane antigens of cells or immunoglobulins from separate species. A syngraft is a transplant from one individual to another within the same strain. Syngrafts are also called isografts. Syngeneic is an adjective that implies genetic identity between identical twins in humans or among members of an inbred strain of mice or other species. It is used principally to see transplants between genetically identical members of a species. An isograft is a tissue transplant from a donor to an isogenic recipient. Grafts exchanged between members of an inbred strain of laboratory animals such as mice are syngeneic rather than isogenic. Isogeneic (isogenic) is an adjective implying genetic identity such as identical twins. Although used as a synonym for syngeneic when referring to the genetic relationship between members of an inbred strain (of mice), the inbred animals never show the absolute identity, i.e., identical genotypes, observed in identical twins. Isologous means derived from the same species. Also called isogeneic or syngeneic. An antigen found in a member of a species that induces an immune response if injected into a genetically dissimilar member of the same species is termed an isoantigen.

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These are antigens carrying identical determinants in a given individual. Isoantigens of two individuals may or may not have identical determinants. In the latter case they are allogeneic with respect to each other and are called alloantigens. Since the individual red blood cell antigens have the same molecular structure and are identical in different individuals, they have been referred to in the past as isoantigens. This is only a descriptive term and should not be used, because two individuals may be allogeneic by virtue of the assortment of the antigens present on their red blood cells. An isoantigen is an antigen of an isograft. Isoantibody is an antibody that is specific for an antigen present in other members of the species in which it occurs. Thus, it is an antibody against an isoantigen. Also called alloantibody. Isoleukoagglutinins are antibodies in the blood sera of multiparous females and of patients receiving multiple blood transfusions that recognizes surface isoantigens of leukocytes and leads to their agglutination. Leukoagglutinin is an antibody or other substance that induces the aggregation or agglutination of white blood cells into clumps. A donor is one who offers whole blood, blood products, bone marrow, or an organ to be given to another individual. Individuals who are drug addicts or test positively for certain diseases such as HIV-1 infection or hepatitis B, for example, are not suitable as donors. There are various other reasons for donor rejection not listed here. To be a blood donor, an individual must meet certain criteria which include blood pressure, temperature, hematocrit, pulse, and history. There are many reasons for donor rejection, including low hematocrit, skin lesions, surgery, drugs, or positive donor blood tests. An organ bank is a site where selected tissues for transplantation, such as acellular bone fragments, corneas, and bone marrow, may be stored for relatively long periods until needed for transplantation. Several hospitals often share such a facility. Organs such as kidneys, liver, heart, lung, and pancreatic islets must be transplanted within 48 to 72 h and are not suitable for storage in an organ bank. Organ brokerage, or the selling of an organ such as a kidney from a living related donor to the transplant, recipient, is practiced in certain parts of the world but is considered unethical and is illegal in the U.S., as it is in violation of the National Organ Transplant Act (Public Law 98-507,3 USC). Adoptive immunity (Figure 21.24) is the term assigned by Billingham, Brent, and Medawar (1955) to transplantation

immunity induced by the passive transfer of specifically immune lymph node cells from an actively immunized animal to a normal (previously nonimmune) syngeneic recipient host. Adoptive immunization is the passive transfer of immunity by the injection of lymphoid cells from a specifically immune individual to a previously nonimmune recipient host. The resulting recipient is said to have adoptive immunity. Adoptive transfer is a synonym for adoptive immunization. The passive transfer of lymphocytes from an immunized individual to a nonimmune subject with immune system cells such as CD4+ T lymphocytes. Tumor-reactive T cells have been adoptively transferred for experimental cancer therapy. Leukocyte transfer: See adoptive transfer. Lymphocyte transfer reaction: See normal lymphocyte transfer reaction. Normal lymphocyte transfer reaction: The intracutaneous injection of an individual with peripheral blood lymphocytes from a genetically dissimilar, allogeneic member of the same species leads to the development of a local,

erythematous reaction that becomes most pronounced after 48 h. The size of the reaction has been claimed to give some qualitative indication of histocompatibility or histoincompatibility between a donor and recipient. This test is not used in clinical practice. A direct reaction is a skin reaction caused by the intracutaneous injection of viable or nonviable lymphocytes into a host that has been sensitized against donor tissue antigens. This represents a type IV hypersensitivity reaction, which is classified as a delayed-type reaction mediated by T cells. Reactivity is against lymphocyte surface epitopes. A skin graft uses skin from the same individual (autologous graft) or donor skin that is applied to areas of the body surface that have undergone third degree burns. A patient’s keratinocytes may be cultured into confluent sheets that can be applied to the affected areas, although these may not “take” because of the absence of type IV collagen 7 S basement membrane sites for binding and fibrils to anchor the graft. A skin-specific histocompatibility antigen is a murine skin minor histocompatibility antigen termed Sk that can elicit rejection of skin but not other tissues following transplantation from one parent into the other parent that has been irradiated and rendered a chimera by the previous injection of F1 spleen cells. The two parents are from different inbred strains of mice. The rate of rejection is relatively slow. Immunologic tolerance of F1 murine spleen cells to the skin epitope of the parent in which they are not in residence is abrogated following residence in the opposite parent. A split thickness graft is a skin graft that is only 0.25 to 0.35 mm thick and consists of epidermis and a small layer of dermis. These grafts vascularize rapidly and last longer than do regular grafts. They are especially useful for skin burns, contaminated skin areas, and sites that are poorly vascularized. Thick split thickness grafts are further resistant to trauma, produce minimal contraction, and permit some amount of sensation, but graft survival is poor.

FIGURE 21.24 Adoptive immunity.

FIGURE 21.25 Protocol for pancreas transplant.

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Pancreatic transplantation (Figure 21.25) is a treatment for diabetes. Either a whole pancreas or a large segment

of it, obtained from cadavers, may be transplanted together with kidneys into the same diabetic patient. It is important for the patient to be clinically stable and for there to be as close a tissue (HLA antigen) match as possible. Graft survival is 50 to 80% at 1 year. Islets of Langerhans are groups of endocrine cells within the exocrine pancreas that consist of α cells that secrete glucagon, β cells that secrete insulin, and δ cells that secrete somatostatin. Islet cell transplantation is an experimental method aimed at treatment of type I diabetes mellitus. The technique has been successful in rats but less so in man. It requires sufficient functioning islets from a minimum of two cadaveric donors that have been purified, cultured, and shown to produce insulin. The islet cells are administered into the portal vein. The liver serves as the host organ in the recipient who is treated with FK506 or other immunosuppressant drugs. Bone marrow is a soft tissue within bone cavities that contains hematopoietic precursor cells and hematopoietic cells that are maturing into erythrocytes, the five types of leukocytes, and thrombocytes. Whereas red marrow is hemopoietic and is present in developing bone, ribs, vertebrae, and long bones, some of the red marrow may be replaced by fat and become yellow marrow. Bone marrow cells are stem cells from which the formed elements of the blood, including erythrocytes, leukocytes, and platelets are derived. B lymphocyte and T lymphocyte precursors are abundant. The B lymphocytes and pluripotent stem cells in bone marrow are important for reconstitution of an irradiated host. Bone marrow transplants are useful in the treatment of aplastic anemia, leukemias, and immunodeficiencies. Patients may donate their own marrow for subsequent bone marrow autotransplantation if they are to receive intense doses of irradiation. Bone marrow cells are stem cells from which the formed elements of the blood, including erythrocytes, leukocytes, and platelets are derived. B lymphocyte and T lymphocyte precursors are abundant. The B lymphocytes and pluripotent stem cells in bone marrow are important for reconstitution of an irradiated host. Bone marrow transplants are useful in the treatment of aplastic anemia, leukemias, and immunodeficiencies. Patients may donate their own marrow for subsequent bone marrow autotransplantation if they are to receive intense doses of irradiation. Bone marrow transplantation is a procedure used to treat both nonneoplastic and neoplastic conditions not amenable to other forms of therapy. It has been especially used in cases of aplastic anemia, acute lymphocytic leukemia, and acute nonlymphocytic leukemia. A total of 750 ml of bone marrow are removed from the iliac crest of an HLA-matched donor. Following appropriate treatment of

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the marrow to remove bone spicules, the cell suspension is infused intravenously into an appropriately immunosuppressed recipient who has received whole-body irradiation and immunosuppressive drug therapy. GVH episodes, acute graft-vs.-host disease (GVHD), or chronic GVHD may follow bone marrow transplantation in selected subjects. The immunosuppressed patients are highly susceptible to opportunistic infections. Autologous bone marrow transplantation (ABMT): Leukemia patients in relapse may donate marrow which can be stored and readministered to them following a relapse. Leukemic cells are removed from the bone marrow which is cryopreserved until needed. Prior to reinfusion of the bone marrow, the patient receives supralethal chemoradiotherapy. This mode of therapy has improved considerably the survival rate of some leukemia patients. Immunotoxin: The linkage of an antibody specific for target cell antigens with a cytotoxic substance such as the toxin ricin yields an immunotoxin. Upon parenteral injection, its antibody portion directs the immunotoxin to the target and its toxic portion destroys target cells on contact. Among its uses is the purging of T cells from hematopoietic cell preparations used for bone marrow transplantation. Immunotoxin is a substance produced by the union of a monoclonal antibody or one of its fractions to a toxic molecule such as a radioisotope, a bacterial or plant toxin, or a chemotherapeutic agent. The antibody portion is intended to direct the molecule to antigens on a target cell, such as those of a malignant tumor, and the toxic portion of the molecule is for the purpose of destroying the target cell. Contemporary methods of recombinant DNA technology have permitted the preparation of specific hybrid molecules for use in immunotoxin therapy. Immunotoxins may have difficulty reaching the intended target tumor, may be quickly metabolized, and may stimulate the development of antiimmunotoxin antibodies. Crosslinking proteins may likewise be unstable. Immunotoxins have potential for antitumor therapy and as immunosuppressive agents. Platelet-associated immunoglobulin (PAIgG) is present in 10% of normal individuals, 50% of those with tumors, and 76% of septic patients, and may be induced by GVHD. PAIgG is present in 71% of autologous marrow graft recipients and in 50% of allogeneic marrow graft recipients. Autologous is an adjective that refers to derivation from self. The term describes grafts or antigens taken from an individual and returned to the same subject from which they were derived. An autograft is a graft of tissue taken from one area of the body and placed in a different site on the body of the same individual, e.g., grafts of skin from unaffected areas to burned areas in the same individual.

Autologous graft refers to the donation of tissue such as skin or bone marrow by the same individual who will subsequently receive it either at a different anatomical site, as in skin autografts for burns, or at a later date, or as in autologous bone marrow transplants. Bone marrow chimera: The inoculation of an irradiated recipient mouse with bone marrow from an unirradiated donor mouse which ensures that lymphocytes and other cellular elements of the blood will be of donor genetic origin. They have been useful in demonstrating lymphocyte and other blood cell development. Stem cells have two unique biological features that include self-renewal and multilineage differentiation potential. In the past, stem cells were divided into two types that include the pluripotential stem cell and the committed stem cell. Pluripotential stem cells were the progenitors of many different hematopoietic cells, whereas the progeny of committed stem cells were of one cell type. “Committed stem cell” is now termed “progenitor cell.” Stem cells arise from yolk sac blood islands and usually are noncycling. They are not morphologically recognizable. Cell culture studies have yielded much information about hematopoietic precursor cells. Hematopoietic stem cells express the progenitor cell antigen CD34, which can be detected using monoclonal antibodies and by flow cytometry. A hematopoietic stem cell is a bone marrow cell that is undifferentiated and serves as a precursor for multiple cell lineages. These cells are also demonstrable in the yolk sac and later in the liver in the fetus. Hematopoietic stem cell (HSC) transplants are used to reconstitute hematopoietic cell lineages and to treat neoplastic diseases. A total of 25% of allogeneic marrow transplants in 1995 were performed using hematopoietic stem cells obtained from unrelated donors. Since only 30% of patients requiring an allogeneic marrow transplant have a sibling that is HLA-genotypically identical, it became necessary to identify related or unrelated potential marrow donors. It became apparent that complete HLA compatibility between donor and recipient is not absolutely necessary to reconstitute patients immunologically. Transplantation of unrelated marrow is accompanied by an increased incidence of GVHD. Removal of mature T lymphocytes from marrow grafts decreases the severity of GVHD but often increases the incidence of graft failure and disease relapse. HLA-phenotypically identical marrow transplants among relatives are often successful. HSC transplantation provides a method to reconstitute hematopoietic cell lineages with normal cells capable of continuous self-renewal. The principal complications of HSC transplantation are GVHD, graft rejection, graft failure, prolonged immunodeficiency, toxicity from radiochemotherapy

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given pre- and posttransplantation, and GVHD prophylaxis. Methrotrexate and cyclosporin A are given to help prevent acute GVHD. Chronic GVHD may also be a serious complication involving the skin, gut, and liver and an associated sicca syndrome. Allogenic HSC transplantation often involves older individuals and unrelated donors. Thus, blood stem cell transplantation represents an effective method for the treatment of patients with hematologic and nonhematologic malignancies and various types of immunodeficiencies. The in vitro expansion of a small number of CD34+ cells stimulated by various combinations of cytokines appears to give hematopoietic reconstitution when reinfused after a high-dose therapy. Recombinant human hematopoietic growth factors (HGF) (cytokines) may be given to counteract chemotherapy treatment–related myelotoxicity. HGF increase the number of circulating progenitor and stem cells, which is important for the support of high-dose therapy in autologous as well as allogeneic HSC transplantation. A chimera (Figure 21.26) is the presence in an individual of cells of more than one genotype. This can occur rarely under natural circumstances in dizygotic twins, as in cattle, who share a placenta in which the blood circulation has become fused, causing the blood cells of each twin to circulate in the other. More commonly, it refers to humans or other animals who have received a bone marrow transplant that provides a cell population consisting of donor and self cells. Tetraparental chimeras can be produced by experimental manipulation. The name chimera derives from a monster of Greek mythology that had the body of a goat, the head of a lion, and the tail of a serpent. Chimerism is the presence of two genetically different cell populations within an animal at the same time. Hematopoietic chimerism: A successful bone marrow transplant leads to a state of hematological and/immunological

FIGURE 21.26 Chimera.

chimerism in which donor type blood cells coexist permanently with host type tissues, without manifesting alloreactivity to each other. Usually incomplete or mixed hematopoietic chimerism are generated following bone marrow transplantation in which both host type and donor type blood cells can be detected in the recipient. In bone marrow transplantation, not only is immune reactivity against donor type cells an obstacle to bone marrow engraftment, there is also the problem of GVHD-mediated by donor T cells reactive against host antigens. See chimera. Radiation chimera: See irradiation chimera. An irradiation chimera is an animal or human whose lymphoid and myeloid tissues have been destroyed by lethal irradiation and successfully repopulated with donor bone marrow cells that are genetically different. Radiation bone marrow chimeras: Mice that have been subjected to heavy radiation and then reconstituted with allogeneic bone marrow cells, i.e., from a different mouse strain. Thus, the lymphocytes are genetically different from the surroundings in which they develop. These chimeric mice have yielded significant data in the investigation of lymphocyte development. Backcross refers to a crossing of a heterozygous organism and a homozygote. The term commonly refers to the transfer of a particular gene from one background strain/stock to an inbred strain via multigenerational matings to the desired strain. Breeding an F1 hybrid with either one of the strains that produced it. Corneal transplants (Figure 21.27) are different from most other transplants in that the cornea is a “privileged site.” These sites do not have a lymphatic drainage. The rejection rate in corneal transplants depends on vascularization;

if vascularization occurs, the cornea becomes accessible to the immune system. HLA incompatibility increases the risk of rejection if the cornea becomes vascularized. The patient can be treated with topical steroids to cause local immunosuppression. Certain anatomical sites within the animal body provide an immunologically privileged environment which favors the prolonged survival of alien grafts. The potential for development of a blood and lymphatic vascular supply connecting graft and host may be a determining factor in the qualification of an anatomical site as an area which provides an environment favorable to the prolonged survival of a foreign graft. Immunologically privileged sites include (1) the anterior chamber of the eye, (2) the substantia proproa of the cornea, (3) the menges of the brain, (4) the testis, and (5) the cheek pouch of the Syrian hamster. Foreign grafts implanted in these sites show a diminished ability to induce transplantation immunity in the host. These immunologically privileged sites usually fail to protect alien grafts from the immune refection mechanism in hosts previously or simultaneously sensitized with donor tissues. Leptin, the antiobesity hormone, is an endothelial cell mitogen and chemoattractant, and it induces angiogenesis in a cornea implant model. Endothelial cells express OB-Rβ, the leptin receptor. The allogeneic effect (Figure 21.28) is the synthesis of antibody by B cells against a hapten in the absence of carrier-specific T cells, provided allogeneic T lymphocytes are present. Interaction of allogeneic T cells with the MHC class II molecules of B cells causes the activated T lymphocytes to produce factors that facilitate B-cell differentiation into plasma cells without the requirement for helper T lymphocytes. There is allogeneic activation of T cells in the GVH reaction. Alloreactive is the recognition by antibodies or T lymphocytes from one member of a species cell or tissue antigens of a genetically nonidentical member. Alloreactivity is the stimulation of immune system T cells by non self MHC molecules attributable to antigenic differences between members of the same species. It represents

FIGURE 21.27 Corneal transplant.

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FIGURE 21.28 Allogeneic effect factor.

the immune response to an alloantigen based on recognition of allogeneic MHC. Allogeneic disease includes the pathologic consequences of immune reactivity of bone marrow allotransplants in immunosuppressed recipient patients as a result of GVH reactivity in genetically dissimilar members of the same species. Homologous disease: See allogeneic disease and graftvs.-host disease (GVHD). Alloimmunization is defined as an immune response provoked in one member or strain of a species with an alloantigen derived from a different member or strain of the same species. Examples include the immune response in man following transplantation of a solid organ graft such as a kidney or heart from one individual to another. Alloimmunization with red blood cell antigens in humans may lead to pathologic sequelae, such as hemolytic disease of the newborn (erythroblastosis fetalis) and in a third Rh(D)+ baby born to an Rh(D)− mother.

FIGURE 21.29 Types of skin graft rejection.

Allogeneic (or allogenic) is an adjective that describes genetic variations or differences among members or strains of the same species. The term refers to organ or tissue grafts between genetically dissimilar humans or unrelated members of other species. Alloantiserum is an antiserum generated in one member or strain of a species not possessing the alloantigen (e.g., histocompatibility antigen), with which they have been challenged, that is derived from another member or strain of the same species. A take is the successful grafting of skin that adheres to the recipient graft site 3 to 5 d following application. This is accompanied by neovascularization as indicated by a pink appearance. Thin grafts are more likely to “take” than thicker grafts, but the thin graft must contain some dermis to be successful. The term “take” also refers to an organ allotransplant that has survived hyperacute and chronic rejection. Engraftment is the phase during which transplanted bone marrow manufactures new blood cells. Graft rejection (Figure 21.29) is an immunologic destruction of transplanted tissues or organs between two members or strains of a species differing at the MHC for that species (i.e., HLA in man and H-2 in the mouse). The rejection is based upon both cell-mediated and antibodymediated immunity against cells of the graft by the histoincompatible recipient. First-set rejection usually occurs within 2 weeks after transplantation. The placement of a second graft with the same antigenic specificity as the first in the same host leads to rejection within 1 week and is

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FIGURE 21.29a Immunofluorescent “staining” of C4d in peritubular capillaries.

termed second-set rejection. This demonstrates the presence of immunological memory learned from the first experience with the histocompatibility antigens of the graft. When the donor and recipient differ only at minor histocompatibility loci, rejection of the transplanted tissue may be delayed, depending upon the relative strength of the minor loci in which they differ. Grafts placed in a hyperimmune individual, such as those with preformed antibodies, may undergo hyperacute or accelerated rejection. Hyperacute rejection of a kidney allograft by preformed antibodies in the recipient is characterized by formation of fibrin plugs in the vasculature as a consequence of the antibodies reacting against endothelial cells lining vessels, complement fixation, polymorphonuclear neutrophil attraction, and denuding of the vessel wall, followed by platelet accumulation and fibrin plugging. As the blood supply to the organ is interrupted, the tissue undergoes infarction and must be removed. Immunofluorescent “staining” of C4d in peritubular capillaries of renal allograft biopsies reveals a humoral component of rejection (Figure 21.29a).

First-set rejection is an acute form of allograft rejection in a nonsensitized recipient. It is usually completed in 12 to 14 d and is mediated by type IV (delayed-type) hypersensitivity to graft antigens. Immunological rejection is the destruction of an allograft or even a xenograft in a recipient host whose immune system has been activated to respond to the foreign tissue antigens. Rejection is an immune response to an organ allograft such as a kidney transplant. Hyperacute rejection is due to preformed antibodies and is apparent within minutes following transplantation. Antibodies reacting with endothelial cells cause complement to be fixed, which attracts polymorphonuclear neutrophils, resulting in denuding of the endothelial lining of the vascular walls. This causes platelets and fibrin plugs to block the blood flow to the transplanted organ, which becomes cyanotic and must be removed. Only a few drops of bloody urine are usually produced. Segmental thrombosis, necrosis, and fibrin thrombi form in the glomerular tufts. There is hemorrhage in the interstitium and mesangial cell swelling; IgG, IgM, and C3 may be deposited in arteriole walls. Acute rejection occurs within days to weeks following transplantation and is characterized by extensive cellular infiltration of the interstitium. These cells are largely mononuclear cells and include plasma cells, lymphocytes, immunoblasts, and macrophages, as well as some neutrophils. Tubules become separated, and the tubular epithelium undergoes necrosis. Endothelial cells are swollen and vacuolated. There is vascular edema, bleeding with inflammation, renal tubular necrosis, and sclerosed glomeruli. Chronic rejection occurs after more than 60 d following transplantation and may be characterized by structural changes such as interstitial fibrosis, sclerosed glomeruli, mesangial proliferative glomerulonephritis, crescent formation, and various other changes. Second-set rejection is rejection of an organ or tissue graft by a host who is already immune to the histocompatibility antigens of the graft as a consequence of rejection of a previous transplant of the same antigenic specificity as the second, or as a consequence of immunization against antigens of the donor graft. The accelerated second-set rejection compared to rejection of a first graft is reminiscent of a classic secondary or booster immune response.

molecules present microbial proteins. The recipient professional antigen-presenting cells process allogeneic MHC proteins. The resulting allogeneic MHC peptides are presented, in association with recipient (self) MHC molecules, to host T lymphocytes. By contrast, recipient T cells recognize unprocessed allogeneic MHC molecules on the surface of the graft cells in direct antigen presentation. White graft rejection is an accelerated rejection of a second skin graft performed within 7 to 12 d after rejection of the first graft. It is characterized by lack of vascularization of the graft and its conversion to a white eschar. The characteristic changes are seen by day 5 after the second grafting procedure. The transplanted tissue is rendered white because of hyperacute rejection, such as a skin or kidney allograft. Preformed antibodies occlude arteries following surgical anastomosis, producing infarction of the tissue graft. ALG is an abbreviation for antilymphocyte globulin. ALS (antilymphocyte serum) or ALG (antilymphocyte globulin): See antilymphocyte serum. Antilymphocyte serum (ALS) or antilymphocyte globulin (ALG) is an antiserum prepared by immunizing one species, such as a rabbit or horse, with lymphocytes or thymocytes from a different species, such as a human. Antibodies present in this antiserum combine with T cells and other lymphocytes in the circulation to induce immunosuppression. ALS is used in organ transplant recipients to suppress graft rejection (Figure 21.30). The globulin fraction known as ALG rather than whole antiserum produces the same immunosuppressive effect. Antithymocyte globulin (ATG): IgG isolated from the blood serum of rabbits or horses hyperimmunized with human thymocytes is used in the treatment of aplastic anemia patients and to combat rejection in organ transplant recipients. The equine ATG contains 50mg/ml of

Second-set response is a term that describes the accelerated rejection of a second skin graft from a donor that is the same as or identical with the first donor. The accelerated rejection is seen when regrafting is performed within 12 to 80 d after rejection of the first graft. It is completed in 7 to 8 d and is due to sensitization of the recipient by the first graft. Indirect antigen presentation: In organ or tissue transplantation, the mechanism whereby donor allogeneic MHC

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FIGURE 21.30 Antilymphocyte globulin (ALG).

immunoglobulin and has yielded 50% recovery of bone marrow and treated aplastic anemia patients.

immunosuppressive drugs as cyclosporine, rapamycin, or FK506, or by monoclonal antibodies against T lymphocytes.

ATG is an abbreviation for antithymocyte globulin.

Host-vs.-graft disease (HVGD) is a consequences of humoral and cell-mediated immune response of a recipient host to donor graft antigens.

Orthoclone OKT3 is a commercial antibody against the T cell surface marker CD3. It may be used therapeutically to diminish T cell reactivity in organ allotransplant recipients experiencing a rejection episode; OKT3 may act in concert with the complement system to induce T cells lysis, or may act as an opsonin, rendering T cells susceptible to phagocytosis. Rarely, recirculating T lymphocytes are removed in patients experiencing rejection crisis by thoracic duct drainage or extracorporeal irradiation of the blood. Plasma exchange is useful for temporary reduction in circulating antibody levels in selective diseases, such as hemolytic disease of the newborn, myasthenia gravis or Goodpasture’s syndrome. Immunosuppressive drugs act on all of the T and B cell maturation processes. Mouse immunoglobulin antibodies: A total of 40% of human subjects may harbor heteroantibodies that include human antimouse antibodies (HAMA). HAMA in serum may induce falsely elevated results in immunoassays that involve mouse antibodies. This may represent a problem in organ transplant patients who receive mouse monoclonal antibodies such as anti-CD3, anti-CD4, and antiIL-2R for treatment.

Hyperacute rejection (Figure 21.32 to Figure 21.37) is due to preformed antibodies and is apparent within minutes following transplantation. Antibodies reacting with endothelial cells cause complement to be fixed, which attracts polymorphonuclear neutrophils, resulting in denuding of the endothelial lining of the vascular walls. This causes platelets and fibrin plugs to clock the blood flow to the transplanted organ that becomes cyanotic and must be removed. Only a few drops of bloody urine are usually produced. Segmental thrombosis, necrosis, and fibrin thrombi form in the glomerular tufts. There is hemorrhage in the interstitium, mesangial cell swelling, IgG, and IgM, and C3 may be deposited in arteriole walls. Hyperacute rejection is accelerated allograft rejection attributable to preformed antibodies in the circulation of the recipient that are specific for antigens of the donor.

Transplantation rejection (Figure 21.31) is the consequence of cellular and humoral immune responses to a transplanted organ or tissue that may lead to loss of function and necessitate removal of the transplanted organ or tissue. Transplantation rejection episodes occur in many transplant recipients, but are controlled by such

FIGURE 21.31 Rejection.

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FIGURE 21.32 Schematic representation of hyperacute graft rejection.

FIGURE 21.36 A high-power view of the same necrotic glomerulus shown in Figure 21.35. There are large numbers of polymorphonuclear leukocytes present. Extensive endothelial cell destruction is apparent. H&E stained section 50X. FIGURE 21.33 Hyperacute rejection of renal allotransplant showing swelling and purplish discoloration. This is a bivalved transplanted kidney. The allograft was removed within a few hours following transplantation.

FIGURE 21.37 Microscopic view of hyperacute rejection showing necrosis of the wall of a small arteriole.

FIGURE 21.34 A bivalved transplanted kidney showing hyperacute rejection. There is extensive pale cortical necrosis. This kidney was removed 5 d after transplantation.

These antibodies react with antigens of endothelial cells lining capillaries of the donor organ. It sets in motion a process that culminates in fibrin plugging of the donor organ vessels, resulting in ischemia and loss of function and necessitating removal of the transplanted organ. Acute rejection (Figure 21.38 to Figure 21.44) occurs within days to weeks following transplantation and is characterized by extensive cellular infiltration of the interstitium. These cells are largely mononuclear cells and include plasma cells, lymphocytes, immunoblasts, and macrophages as well as some neutrophils. Tubules become separated and the tubular epithelium undergoes necrosis. Endothelial cells are swollen and vaculoated. There is vascular edema, bleeding with inflammation, renal tubular necrosis, and sclerosed glomeruli.

FIGURE 21.35 Microscopic view of hyperacute rejection showing a necrotic glomerulus infiltrated with numerous polymorphonuclear leukocytes. H&E stained section 25X.

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Acute rejection is a type of graft rejection in which T lymphocytes, macrophages, and antibodies mediate vascular and tissue injury that may commence a week following transplantation. The response to the graft includes

FIGURE 21.38 Acute rejection of a renal allograft in which the capsular surface shows several hemorrhagic areas. The kidney is tremendously swollen.

FIGURE 21.41 Microscopic view of acute rejection showing interstitial edema. Mild lymphocytic infiltrate. In the glomerulus, there is also evidence of rejection with a thrombus at the vascular pole.

FIGURE 21.39 Acute rejection of a bivalved kidney. The cut surface bulges and is variably hemorrhagic and shows fatty degeneration of the cortex.

FIGURE 21.42 A higher magnification of the thrombus at the hilus of the glomerulus.

FIGURE 21.40 Microscopic view of the interstitium revealing predominantly cellular acute rejection. There is an infiltrate of variabily sized lymphocytes. There is also an infiltrate of eosinophils.

FIGURE 21.43 A trichrome stain of a small interlobular artery showing predominantly humoral rejection There is tremendous swelling of the intima and endothelium with some fibrin deposition and a few polymorphonuclear leukocytes.

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FIGURE 21.44 Immunofluorescence preparation showing humoral rejection with high-intensity fluorescence of arteriolar walls and of some glomerular capillary walls. This pattern is demonstrable in antiimmunoglobulin and anticomplement stained sections.

FIGURE 21.47 The wall of an artery in chronic rejection. There is obliteration of the vascular lumen with fibrous tissue. Only a slit-like lumen remains.

be characterized by structural changes such as interstitial fibrosis, sclerosed glomeruli, mesangial proliferative glomerulonephritis, crescent formation, and various other changes. Chronic rejection is a type of allograft rejection that occurs during a prolonged period following transplantation and is characterized by structural changes such as fibrosis with loss of normal organ architecture. The principal pathologic change is occlusion of arteries linking the graft to the host. This results from intimal smooth muscle cell proliferation and has been referred to as graft arteriosclerosis.

FIGURE 21.45 Renal allotransplant showing chronic rejection. The kidney is shrunken and malformed.

FIGURE 21.46 Microscopic view of chronic rejection showing tubular epithelial atrophy with interstitial fibrosis and shrinkage of glomerular capillary tufts.

formation of antibodies and activation of effector T lymphocytes that mediate the process. Chronic rejection (Figure 21.45 to Figure 21.47) occurs after more than 60 d following transplantation and may

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Graft arteriosclerosis is characterized by intimal smooth muscle cell proliferation that occludes graft arteries. It may occur 6 to 12 months following transplantation and leads to chronic rejection of vascularized organ grafts. It is probably attributable to a chronic immune response to alloantigens of the vessel wall. It is also termed accelerated arteriosclerosis. The graft-vs.-host reaction (GVHR) is the reaction of a graft containing immunocompetent cells against the genetically dissimilar tissues of an immunosuppressed recipient. Criteria requisite for a GVHR include (1) histoincompatibility between the donor and recipient, (2) passively transferred immunologically reactive cells, and (3) a recipient host who has been either naturally immunosuppressed because of immaturity or genetic defect, or deliberately immunosuppresed by irradiation or drugs. The immunocompetent grafted cells are especially reactive against rapidly dividing cells. Target organs include the skin, gastrointestinal tract (including the gastric mucosa), and liver, as well as the lymphoid tissues. Patients often develop skin rashes and hepatosplenomegaly and may have aplasia of the bone marrow. GVHR usually develops within 7 to 30 d following the transplant or infusion of the lymphocytes. Prevention of the GVHR is an important procedural step in several forms of transplantation and may be accomplished by irradiating the transplant. The clinical

course of GVHR may take a hyperacute, acute, or chronic form as seen in graft rejection. GVH: See graft-vs.-host reaction and graft-vs.-host disease. Secondary disease is a condition that occurs in irradiated animals whose cell population has been reconstituted with histoincompatible, immunologically competent cells derived from allogeneic donor animals. Ionizing radiation induces immunosuppression in the recipients, rendering them incapable of rejecting the foreign cells. Thus, the recipient has two cell populations, its own and the one that has been introduced, making these animals radiation chimeras. After an initial period of recovery, the animals develop a secondary runt disease, which is usually fatal within 1 month. Posttransfusion graft-vs.-host disease is a condition that resembles postoperative erythroderma that occurs in immunocompetent recipients of blood. There is dermatitis, fever, marked diarrhea, pancytopenia, and liver dysfunction. Graft-vs.-host disease (GVHD) is a disease produced by the reaction of immunocompetent T lymphocytes of the donor graft that are histoincompatible with the tissues of the recipient into which they have been transplanted. For the disease to occur, the recipient must be either immunologically immature, immunosuppressed by irradiation or drug therapy, or tolerant to the administered cells, and the grafted cells must also be immunocompetent. Patients develop skin rash, fever, diarrhea, weight loss, hepatosplenomegaly, and aplasia of the bone marrow. The donor lymphocytes infiltrate the skin, gastrointestinal tract, and liver. The disease may be either acute or chronic. Murine GVH disease is called “runt disease,” “secondary disease,” or “wasting disease.” Both allo- and autoimmunity associated with GVHD may follow bone marrow transplantation. A total of 20 to 50% of patients receiving HLA-identical bone marrow transplants still manifest GVHD with associated weight loss, skin rash, fever, diarrhea, liver disease, and immunodeficiency. GVHD may be either acute, which is an alloimmune disease, or chronic, which consists of both allo- and autoimmune components. The conditions requisite for the GVH reaction include genetic differences between immunocompetent cells in the marrow graft and host tissues, immunoincompetence of the host, and alloimmune differences that promote proliferation of donor cells that react with host tissues. In addition to allogenic marrow grafts, the transfusion of unirradiated blood products to an immunosuppressed patient or intrauterine transfusion from mother to fetus may lead to GVHD.

sulfmethoxazole-trimethoprim, carbamazepine, and other agents. It may closely resemble erythema multiforme. Patients develop erythema, subepidermal bullae, and open epidermal lesions. They become dehydrated, show imbalance of electrolytes, and often develop abscesses with sepsis and shock. Toxic epidermal necrolysis may also be observed in a hyperacute type of graft-vs.-host reaction, especially in some babies receiving bone marrow transplants. Graft-vs.-leukemia (GVL): Bone marrow transplantation as therapy for leukemia. Partial genetic incompatibility between donor and recipient is believed to facilitate elimination of residual leukemia cells by T lymphocytes from the transplant. Parabiotic intoxication is the result of a surgical union of allogeneic adult animals. The course of immune reactivity can be modified to take a single direction by uniting parental and F1 animals. A hybrid recognizes parental cells as its own and does not mount an immune response against them, but alloantigens of F1 hybrid cells stimulate the parental cells leading to graft-vs.-host disease. The Simonsen phenomenon is a graft-vs.-host reaction in chick embryos that have developed splenomegaly following inoculation of immunologically competent lymphoid cells from adult chickens. Splenic lymphocytes are increased and represent a mixture of both donor and host lymphocytes. Acute graft-vs.-host reaction, the immunopathogenesis of acute GVHD, consists of recognition, recruitment, and effector phases. Epithelia of the skin (Figure 21.48 to Figure 21.52), gastrointestinal tract (Figure 21.53 to Figure 21.57), small intrahepatic biliary ducts, and liver (Figure 21.58 to Figure 21.60), and the lymphoid system constitute primary targets of acute GVHD. GVHD development may differ in severity based on relative antigenic differences between donor and host and the reactivity of donor lymphocytes against non-HLA antigens of recipient tissues. The incidence and severity of GVHD has been

GVH disease: See graft-vs.-host disease. Toxic epidermal necrolysis is a hypersensitivity reaction to certain drugs such as allopurinol, nonsteroidal antiinflammatory drugs, barbiturates, sulfonamides such as

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FIGURE 21.48 A diffuse erythematous to morbilliform rash in a child with acute graft-vs.-host disease (GVHD).

FIGURE 21.49 Diffuse erythematous skin rash in a patient with acute graft-vs.-host reaction (GVHR).

FIGURE 21.52 Papulosquamous rash in graft-vs.-host disease.

FIGURE 21.50 Histologically, there is an intense interface dermatitis with destruction of basal cells, particularly at the tips of the rete ridges, incontinence of melanin pigment, and necrosis of individual epithelial cells, referred to as apoptosis.

FIGURE 21.53 AND FIGURE 21.54 Gastrointestinal graftvs.-host disease in which there is a diffuse process that usually involves the ileum and cecum, resulting in secretory diarrhea. Grossly there is diffuse erythema, granularity, and loss of folds, and when severe, there is undermining and sloughing of the entire mucosa, leading to fibrinopurulent clots of necrotic material. Sometimes there is frank obstruction in patients with intractable-graft-vs.-host disease.

FIGURE 21.51 Histological appearance of the skin in disease with disruption of the basal cell layer, hyperkeratosis, and beginning sclerotic change.

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ascribed also to HLA-B alleles, i.e., an increased GVHD incidence associated with HLA-B8 and HLA-B35. Epithelial tissues serving as targets of GVHD include keratinocytes, erythrocytes, and bile ducts, which may express Ia antigens following exposure to endogeneous interferon produced by T lymphocytes. When Ia antigens are expressed on nonlymphoid cells, they may become antigen-presenting cells for autologous antigens and aid perpetuation of autoimmunity.

FIGURE 21.57 Histologically, graft-vs.-host disease in the gut begins as a patchy destructive enteritis localized to the lower third of the crypts of Lieberkuhn.

FIGURE 21.55 Stenotic and fibrotic segments alternating with more normal-appearing dilated segments of gut in graft-vs.-host disease.

FIGURE 21.58 The earliest lesions are characterized by individual enterocyte necrosis with karyorrhectic nuclear debris, the so-called exploding crypt, which progresses to a completely destroyed crypt as shown in the upper left-hand corner.

FIGURE 21.56 Sloughing of the mucosal lining of the gut in graft-vs.-host disease.

Cytotoxic T lymphocytes mediate acute GVHD. While most immunohistological investigations have implicated CD8+ (cytotoxic/suppressor) lymphocytes, others have identified CD4+ (T helper lymphocytes) in human GVHD, whereas natural killer (NK) cells have been revealed as effectors of murine but not human GVHD. Following interaction between effector and target cells, cytotoxic granules from cytotoxic T or NK cells are distributed over the target cell membrane, leading to perforin-induced large pores across the membrane and nuclear lysis by deoxyribonuclease. Infection, rather than failure of the primary target organ (other than gastrointestinal bleeding), is the major cause of mortality in acute GVHD. Within the

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FIGURE 21.59 Hepatic graft-vs.-host disease is characterized by a cholestatic hepatitis with characteristic injury and destruction of small bile ducts that resemble changes seen in rejection. In this section of early acute GVHD, there are mild portal infiltrates with striking exocytosis into bile ducts associated with individual cell necrosis and focal destruction of the bile ducts.

FIGURE 21.60 This liver section from a patient with GVHD demonstrates the cholestatic changes that evolve from hepatocellular ballooning to cholangiolar cholestasis with bile microliths, which signifies prolonged GVHD.

FIGURE 21.61 Chronic GVHD of the liver with pronounced inflammation and portal fibrosis with disappearance of bile ducts.

first few months posttransplant, all recipients demonstrate diminished immunoglobulin synthesis, decreased T helper lymphocytes, and increased T suppressor cells. Acute GVHD patients manifest an impaired ability to combat viral infections. They demonstrate an increased risk of cytomegalovirus (CMV) infection, especially CMV interstitial pneumonia. GVHD may also reactivate other viral diseases such as herpes simplex. Immunodeficiency in the form of acquired B cell lymphoproliferative disorder (BCLD) represents another serious complication of post-bone marrow transplantation. Bone marrow transplants treated with pan-T cell monoclonal antibody or those in which T lymphocytes have been depleted account for most cases of BCLD, which is associated with severe GVHD. All transformed B cells in cases of BCLD have manifested the Epstein-Barr viral genome. Chronic graft-vs.-host disease (GVHD) may occur in as many as 45% of long-term bone marrow transplant recipients. Chronic GVHD (Figure 21.61) differs both

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FIGURE 21.62 Venoocclusive disease (VOD) accompanying graft-vs.-host disease of the liver. On the left is early VOD with concentric subendothelial widening and sublobular central venules with degeneration of surrounding pericentral hepatocytes. There is deposition of fibrin and Factor VIII. On the right is a late lesion of VOD showing fibrous obliteration of the central venule and the sinusoids by combination of types 3, 1, and even type 4 collagen.

clinically and histologically from acute GVHD and resembles autoimmune connective tissue diseases. For example, chronic GVHD patients may manifest skin lesions resembling scleroderma; sicca syndrome in the eyes and mouth; inflammation of the oral, esophageal, and vaginal mucosa; bronchiolitis obliterans; occasionally myasthenia gravis; polymyositis; and autoantibody synthesis. Histopathologic alterations in chronic GVHD, such as chronic inflammation and fibrotic changes in involved organs, resemble changes associated with naturally occurring autoimmune disease. The skin may reveal early inflammation with subsequent fibrotic changes. Infiltration of lacrimal, salivary, and submucosal glands by lymphoplasmacytic cells leads ultimately to fibrosis. The resulting sicca syndrome, which resembles Sjögren’s syndrome, occurs in 80% of chronic GVHD patients. Drying of mucous membranes in the sicca syndrome affects the mouth, esophagus, conjunctiva, urethra, and vagina. The pathogenesis of chronic GVHD involves the interaction of alloimmunity, immune dysregulation, and resulting immunodeficiency and autoimmunity. The increased incidence of infection among chronic GVHD patients suggests immunodeficiency. The dermal fibrosis is associated with increased numbers of activated fibroblasts in the papillary dermis. T lymphocyte or mast cell cytokines may activate this fibroplasia, which leads to dermal fibrosis in chronic GVHD. OKT®3 (Orthoclone OKT®3) is a commercial mouse monoclonal antibody against the T cell surface marker CD3. It may be used, therapeutically, to diminish T cell reactivity in organ allotransplant recipients experiencing a rejection episode. OKT3 may act in concert with the complement system to induce T cell lysis, or it may act as an opsonin, rendering T cells susceptible to phagocytosis.

Venoocclusive disease (VOD) is a serious liver complication after marrow transplantation (Figure 21.62). Histopathology of early VOD reveals concentric subendothelial widening and sublobular central venules with degeneration of surrounding pericentral hepatocytes. At this early stage, there is deposition of fibrin and Factor VIII. Late lesions of VOD show fibrous obliteration of the central venule and

Copyright Š 2004 by Taylor & Francis

sinusoids by combination of type 3, 1, and even type 4 collagen. The clinical diagnosis of VOD is reasonably accurate based on the combination of jaundice, ascites, hepatomegaly, and encephalopathy in the first 2 weeks posttransplant. The incidence may be higher among older patients with diagnosis of AML or CML and with hepatitis. The mortality rate of VOD is relatively high at 32%.

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