Respuesta inmune citotóxica hiperactiva persistente en una cohorte española de individuos con COVID

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Resumen Introducción Métodos Resultados Discusión Declaración de disponibilidad de datos Declaración de Ética Miembros Contribuyentes del Grupo Multidisciplinario de Estudio de COVID19 (MGS-COVID) Contribuciones de autor Fondos Conflicto de intereses Nota del editor Expresiones de gratitud Material suplementario Referencias

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Artículo de Investigación Original Parte delantera. Immunol., 25 de marzo de 2022 | https://doi.org/10.3389/fimmu.2022.848886 (https://doi.org/10.3389/fimmu.2022.848886) TABLA DE CONTENIDO

Respuesta inmune citotóxica hiperactiva persistente en una cohorte española de individuos con COVID-19 prolongado: identificación de biomarcadores diagnósticos

Resumen

Introducción Métodos

Resultados Discusión

Declaración de Miguelde Galán (https://www.frontiersin.org/people/u/1152847)1† , Lorena Vigón disponibilidad datos (https://www.frontiersin.org/people/u/1269907)1† , Daniel Fuertes 2 , María Aránzazu Murciano-

Antón , de Guiomar Casado-Fernández (https://www.frontiersin.org/people/u/1678975) , Declaración Domínguez-Mateos 3 , Elena Mateos 1,4 , Fernando Ramos-Martín 1 , Vicente Planelles Ética 3

(https://www.frontiersin.org/people/u/361110)5 ,

1

Susana

Montserrat Torres

Miembros 1 (https://www.frontiersin.org/people/u/1278506) , Sara Rodríguez-Mora Contribuyentes del 1,4 Grupo (https://www.frontiersin.org/people/u/1341198) , María Rosa López-Huertas Multidisciplinario de (https://www.frontiersin.org/people/u/1341068)1,4*‡ y Mayte Coiras Estudio de COVID(https://www.frontiersin.org/people/u/199362)1,4*‡en nombre del Grupo Multidisciplinario de Estudio de 19 (MGS-COVID)

COVID-19 (MGS-COVID)

Contribuciones de 1 Unidad de Inmunopatología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, España autor 2 Escuela Técnica Superior de Ingeniería de Telecomunicación, Universidad Politécnica de Madrid, Madrid, España 3 Fondos Medicina de Familia, Centro de Salud Doctor Pedro Laín Entralgo, Madrid, España 4 Red de Centros de Investigación Biomédica en Enfermedades Infecciosas (CIBERINFEC), Madrid, España Conflicto de 5 División intereses de Microbiología e Inmunología, Facultad de Medicina de la Universidad de Utah, Salt Lake City, UT, Estados Unidos

Nota del editor

Long-COVID es un nuevo síndrome emergente a nivel mundial que se caracteriza por la persistencia

Expresiones de de signos y gratitud

síntomas de COVID-19 no resueltos más de 4 semanas después de la infección e incluso después de más de 12 semanas. Los mecanismos subyacentes de Long-COVID aún no están

Material suplementario definidos, pero

una respuesta inflamatoria sostenida causada por la persistencia de SARS-CoV-2 en santuarios de órganos y tejidos o el parecido con una enfermedad autoinmune están dentro de las Referencias hipótesis más consideradas. En este estudio analizamos la utilidad de varios parámetros demográficos, clínicos e inmunológicos como biomarcadores diagnósticos de Long-COVID en una cohorte de individuos españoles que presentaron signos y síntomas de este síndrome pasadas 49 semanas post-infección, en comparación con individuos que se recuperó completamente en las primeras 12 semanas después de la infección.+ células TEMRA, células CD8 ± TCRγδ + y células NK con fenotipo CD56 + CD57 + NKG2C + . La persistencia de estas poblaciones citotóxicas de larga duración se vio respaldada por niveles mejorados de Tregs CD4 + y la expresión del marcador de agotamiento PD-1 en la superficie de los linfocitos T CD3 + . Con el uso de estos parámetros inmunológicos y características clínicas significativas como letargo, dolor torácico pleurítico y lesiones dermatológicas, así como factores demográficos como el género femenino y O +tipo de sangre, un algoritmo Random Forest predijo la asignación de los participantes en el grupo LongCOVID con un 100 % de precisión. La definición de los biomarcadores de diagnóstico más precisos podría ser de ayuda para detectar el desarrollo de Long-COVID y mejorar el manejo clínico de estos pacientes. https://www.frontiersin.org/articles/10.3389/fimmu.2022.848886/full

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Introducción

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El virus emergente denominado síndrome respiratorio agudo severo coronavirus 2 (SARS-CoV-2) es responsable de la pandemia de la enfermedad por coronavirus 2019 (COVID-19), que ha causado 281 millones de infecciones en todo el mundo y más de 5,4 millones de muertes hasta la fecha ( 1 ). Estos números están aumentando rápidamente debido a nuevas variantes emergentes como Omicron ( 2 ).

The clinical outcome of COVID-19 is highly variable, ranging from asymptomatic or mild to a fatal disease. This variability is mostly dependent on how the immune system reacts during the primary infection, due to an exacerbated systemic inflammatory response that has been described in the most severe and critical forms of the disease that may lead to multiple organ dysfunction syndromes (3–5). Those patients who develop severe or critical COVID-19 usually present bilateral pneumonia, respiratory failure with low blood oxygenation, and acute TABLA DE CONTENIDO

respiratory distress syndrome (ARDS) (6). Other organs may also be compromised, leading to nervous and cardiac

system injuries that cause severe complications such Resumen

as ataxia, acute cerebrovascular disease, olfactory

dysfunction, blood hypercoagulability, or cardiomyopathy (7). The median hospital length of stay for patients with Introducción severe forms of COVID-19 ranges from less than 1 week to nearly 2 months, whereas the median stay at the

Métodos

intensive care unit (ICU) for those patients with critical COVID-19 varies from 1 to 3 weeks, being shorter for those

Resultados

patients who die from the disease (8). Those hospitalized patients who recover from the most severe forms of the

Discusión disease may suffer permanent sequelae (9, 10).

Declaración de The duration of the clinical signs and symptoms may also be very variable among those patients who do not disponibilidad de require hospitalization due to asymptomatic or mild COVID-19. In the case of short or acute COVID-19, all signs datos

and symptoms usually disappear from 10 days to 4 weeks after diagnosis (11). However, there is an increasing

Declaración de proportion of patients with Ética

mild COVID-19 in which the symptoms do not resolve completely after 4 weeks and

may last up to 12 weeks after the clinical onset, then termed Ongoing Symptomatic COVID-19, or even more than

Miembros 12 weeks, then Contribuyentes del termed Post-COVID-19 Syndrome (12). These new forms of the disease have been grouped under Grupo the term Long-COVID (13, 14), which describes those cases in which the signs and symptoms that continue or Multidisciplinario de develop after acute COVID−19 last from 4 to more than 12 weeks (12). Long-COVID may be developed by 1 in 10 Estudio de COVIDwho passed COVID-19 (15), and it includes both general and neuropsychiatric persistent symptoms such 19patients (MGS-COVID)

as cough, fatigue, muscle, and joint pains, insomnia, breathlessness, myalgia, or diarrhea. Other relevant

Contribuciones de symptoms may compromise the cardiopulmonary system, such autor

as dyspnea, pericarditis, or heart failure (13, 16,

17). Long-COVID may be developed not only by patients who were admitted at the ICU due to critical COVID-19 Fondos and now present long-term respiratory sequelae but also by patients with mild COVID-19 who did not require

Conflicto de hospitalization. intereses

Thedel causes Nota editorof Long-COVID are still uncertain, and in most cases, no clear evidence of organ damage is usually found (17). However, some host factors such as gender and age have been related to a higher susceptibility to

Expresiones de develop this prolonged syndrome. Women under 50 years of age seem to be more susceptible to present gratitud

unresolved COVID-19 (18), which indicates a potential role of sex hormones in Long-COVID (19). Another

Material plausible hypothesis could be viral persistence. Although suplementario

is known to occur in other RNA viruses such Referencias

the SARS-CoV-2 viral cycle is different, viral persistence

as the human immunodeficiency virus (HIV) or hepatitis C virus

(HCV), which may produce chronic infection with sustained activation of the immune system (20). Accordingly, prolonged viral shedding has been found in feces from patients with acute COVID-19 after negative PCR in respiratory specimens, suggesting that not only RNA of SARS-CoV-2 but also virions may persist in cells from the gastrointestinal tract (21, 22), likely in enterocytes and small vessels, as was described for the first SARS-CoV (23).

The presence of SARS-CoV-2 in the intestinal mucosa could cause intestinal damage that, along with systemic inflammation, may increase bacterial translocation, which would play an essential role in the sustained immune activation and cytokine release syndrome characteristic of COVID-19 (24). On the other hand, several symptoms described in individuals with Long-COVID are quite similar to those developed during autoimmune diseases such as fibromyalgia or chronic fatigue syndrome, which are also linked to persistent inflammation and exacerbated immune responses (25). Individuals with COVID-19 may develop autoantibodies against tissue-associated antigens and immunomodulatory proteins, both soluble such as interferon (IFN) type I or bound to the surface of the immune cells, which would also promote the persistence of the symptoms (26, 27).

In summary, the recovery from COVID-19 seems to be beyond hospital discharge or testing negative for SARSCoV-2 (28), but the causes for the subsequent perpetuation of the symptoms are still undetermined. To define the clinical guidelines for the prevention, diagnosis, follow-up, and rehabilitation of these individuals, it is necessary https://www.frontiersin.org/articles/10.3389/fimmu.2022.848886/full

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to gain a better understanding of the underlying mechanisms of Long-COVID. Our group previously determined INICIAR SESIÓN (HTTPS://WWW.FRONTIERSIN.ORG/PEOPLE/LOGIN) / REGISTRARSE (HTTPS://WWW.FRONTIERSIN.ORG/REGISTER) SOBRE(HTTPS://WWW.FRONTIERSIN.ORG/A that an inefficient cytotoxic response is a reliable biomarker for COVID-19 severity (4). Consequently, in this

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study, we evaluated the relative importance of this impaired cellular immune response, along with several demographic and clinical features, to develop the persistence of the symptoms and the evolution to Long-COVID by using a Random Forest algorithm.

Methods Study Subjects Blood samples from 50 individuals who had passed mild, symptomatic COVID-19 during the first pandemic peak in Madrid (Spain) (March–April 2020) TABLA DE CONTENIDO

were collected in the Primary Healthcare Center Doctor Pedro Laín

Entralgo (Madrid, Spain). The inclusion criteria were as follows: over 18 years old, have a positive RT-qPCR assay

Resumen

for SARS-CoV-2 in nasopharyngeal smear or positive titers of virus-specific IgG, and did not require

Introducción hospitalization while with

COVID-19. The subjects were subsequently classified into Long-COVID (n = 30) and

Métodos Recovered (n = 20). The Long-COVID group included those individuals who had passed mild COVID-19 and

referred the presence of at least 8 clinical signs and symptoms compatible with Resultados

Ongoing Symptomatic COVID-19

or Post-COVID-19 Syndrome according to the National Institute for Health and Care Excellence (NICE) guideline

Discusión

(12) for at least 4 to 12 weeks or more than 12 weeks after the clinical diagnosis of COVID-19. Individuals with

Declaración de Long-COVID were recruited in collaboration with the non-profit Spanish Association of Patients with Longdisponibilidad de COVID (Long-COVID-ACTS, Madrid, Spain). The Recovered group included those individuals who had passed datos

acute COVID-19, were homebound until medical discharge, and had the resolution of all signs and symptoms 2 to

Declaración de 4 weeks after the clinical diagnosis. Re-infection or vaccination previous to sampling was not reported by any Ética

participant. The most relevant clinical data of all participants with Long-COVID or completely recovered are

Miembros described in del Supplementary Tables 1, 2, respectively.

Contribuyentes Grupo Multidisciplinario de Estudio de COVID19 (MGS-COVID)

Ethical Statement

The individuals participating in this study were recruited at the Primary Healthcare Center Doctor Pedro Lain

Contribuciones de Entralgo (Alcorcón, Madrid, Spain). All participants gave informed written consent to participate, and their autor

anonymity was ensured by the current Spanish and European Data Protection Law. This study was conducted

Fondos

following the Declaration of Helsinki, and it was approved by the Ethical Committee of Instituto de Salud Carlos

Conflicto de III (IRB IORG0006384) (CEI PI 07_2021) and the Central Research intereses

Commission from the Health Counseling

(Comunidad de Madrid, Spain) (favorable report 20210008).

Nota del editor

Expresiones de gratitud

Cells

Material Peripheral blood lymphocytes (peripheral blood mononuclear cells (PBMCs)) and plasma were isolated from suplementario blood samples by centrifugation through Ficoll–Hypaque gradient (Pharmacia Corporation, North Peapack, NJ, Referencias USA) and cryopreserved until the moment of analysis. Due to the low quantity of samples, not all the parameters

could be determined in all participants. K562 cell line (ECACC 89121407) was kindly provided by Dr Cristina Eguizabal (Basque Centre Transfusions and Human Tissue, Álava, Spain), and Vero E6 (African green monkey kidney) cell line (ECACC 85020206) was kindly provided by Dr. Antonio Alcami (CBM Severo Ochoa, Madrid).

Antibodies and Flow Cytometry Conjugated antibodies CD3-APC, CD4-PercP, CD8-APC-H7, CD8-PercP-Cy5.5, CD16-PercP-Cy5.5, CD25-PECy5, CD56-FITC, CD57-PE, CD107a-PE-Cy7, CD127-FITC, CD158f-BV421, NKG2D-PECy7, NKp44-BUV395, and NKp46-BV650 were purchased from BD Biosciences (San Jose, CA, USA). PD-1-BV650, NKG2A-PE, and NKG2CAlexaFluor700 were obtained from R&D Systems (Minneapolis, MN, USA). TCRγδ-PE was obtained from BioLegend (San Diego, CA, USA). CD4+ and CD8+ T-cell memory subpopulations were quantified after staining with CCR7-FITC and CD45RA-PE-Cy7 (BD Biosciences) as follows: naïve (CD45RA+CCR7+), central memory (TCM) (CD45RA−CCR7+), effector memory (TEM) (CD45RA−CCR7−), and terminally differentiated effector memory (TEMRA) (CD45RA+CCR7−) cells. Data acquisition was performed in a BD LSRFortessa X-20 flow cytometer using FACS Diva software (BD Biosciences). Data analysis was performed with FlowJo_V10 software

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For intracellular staining of IFNγ, TNFα and granzyme B (GZB) in CD3+CD8+ T cells, PBMCs were treated for 4 h INICIAR SESIÓN (HTTPS://WWW.FRONTIERSIN.ORG/PEOPLE/LOGIN) / REGISTRARSE (HTTPS://WWW.FRONTIERSIN.ORG/REGISTER) SOBRE(HTTPS://WWW.FRONTIERSIN.ORG/A at 37°C with PepMix™ SARS-CoV-2 (NCAP) (JPT Peptide Technologies, Berlin, Germany), which contains 102

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peptides derived from the Nucleoprotein of SARS-CoV-2, to stimulate the cytotoxic activity of CD8+ T cells, in the presence of brefeldin A (BD Biosciences). Cells were then stained with anti-CD3-APC and anti-CD8-PercP. After fixation and permeabilization with IntraPrep Permeabilization Reagent (Beckman Coulter, Brea, CA, USA), cells were stained with antibodies against IFNγ-PE, TNFα-PE, and GZB-FITC (BD Biosciences) and then acquired and analyzed in a BD LSRFortessa X-20 flow cytometer (BD Biosciences) using FACS Diva (BD Biosciences) and FlowJo_V10 software (TreeStar).

RT-qPCR for Detecting SARS-CoV-2 RNA in Blood Viral RNA was extracted from the blood samples of all participants of the Long-COVID group using the QIAamp MinElute Virus Kit (Qiagen TABLA DE CONTENIDO

Iberia, Madrid, Spain). The presence of RNA of SARS-CoV-2 was determined in the

blood of all participants in this study following the protocol previously described (29), which is part of the Interim

Resumen

Guidance of the WHO for the diagnostic testing of SARS-CoV-2 (30).

Introducción Métodos

Pseudotyped SARS-CoV-2 Infection Assay

Resultados

One-cycle pseudotyped virus encoding SARS-CoV-2 S glycoprotein and the reporter gene renilla within an HIV-1

Discusión

Δenv genome (pNL4-3ΔenvRen) (31) was used to perform the infection assay in Vero E6 cells. As previously

Declaración de described, cDNA encoding G614 SARS-CoV-2 S glycoprotein (QHU36824.1) without the last 19 amino acids (32) disponibilidad de was cloned into pcDNA3.1 expression vector (4). A mutant clone introducing D614G change was generated by sitedatos

directed mutagenesis due to D614 viruses being the majority of the earliest variants detected in Spain within clade

Declaración de 19B (33). A monolayer of Vero E6 was infected with Ética

identical amounts of both D614 and G614 pseudoviruses (100

ng p24/Gag per p48 well). After 48 h of incubation, Vero cells were cocultured for 1 h with PBMCs isolated from

Miembros the blood of individuals from our cohort (1:2). Caspase-3 activity was measured in the monolayer as an indicator Contribuyentes del Grupo of cellular cytotoxicity by using the Caspase-Glo 3/7 Assay system (Promega, Madison, WI, USA).

Multidisciplinario de Estudio de COVID19 (MGS-COVID)

NK Cell Cytotoxicity Assay

Contribuciones de K562 cells that do not express the Major Histocompatibility Complex (MHC) class I molecules on the cell surface autor

(missing self) were used as classical targets of NK cells to evaluate their cytotoxic activity as previously described

Fondos

(4). Briefly, K562 cells were stained with PKH Red Fluorescence Cell Linker kit (Sigma-Aldrich Merck, Darmstadt,

Conflicto de Germany) and then cocultured with intereses

PBMCs (1:2). After 1 h, they were collected and stained with Annexin V

conjugated with fluorescein isothiocyanate (FITC) (Thermo Fisher, Waltham, MA, USA) to quantify early

Nota del editor

apoptosis by measuring the expression of phosphatidylserine on the cell surface by flow cytometry. BD

Expresiones de X-20 flow cytometer and FACS Diva software were used for data acquisition, and FlowJo_V10 LSRFortessa gratitud

software was used for data analysis.

Material suplementario

Random Forest Algorithm

Referencias

A Random Forest algorithm (34) was applied to evaluate the accuracy of those demographic, clinical, and immunological parameters that showed significant differences (p < 0.05) in the comparison with the Recovered group to predict the predisposition to develop Long-COVID. The selected parameters were as follows: demographic factors: female gender and O+ blood type; clinical factors: lethargy, pleuritic chest pain, dermatological injuries, mean body temperature, dyspnea, diarrhea, conjunctivitis, previous autoimmune diseases, and treatments during COVID-19 such as corticosteroids, antibiotics, and/or vitamin D; immune response factors: total NK cells (CD56+), CD3−CD56+CD16+, CD56+NKG2A−NKG2C+, and CD56+CD57+NKG2C+ subpopulations; CD3+PD-1+; total CD8+ T cells, CD8+ TEMRA, and CD8±TCRγδ+ subpopulations; CD4+ Tregs; and cytotoxic activity against NK target cells K562 and/or SARS-CoV-2 infected Vero E6 cells. A nested 5-fold cross-validation procedure for each competing algorithm was performed in order to avoid bias in the selection of training, testing, and validation sets, as previously described (35, 36). The relative importance for each feature in the categorization of patients was calculated by using the Gini Variable Importance Measure (VIM) method (37).

Statistical Analysis https://www.frontiersin.org/articles/10.3389/fimmu.2022.848886/full

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Statistical analysis was performed using Graph Pad Prism 8.0 (Graph Pad Software Inc., San Diego, CA, USA).

INICIAR SESIÓN (HTTPS://WWW.FRONTIERSIN.ORG/PEOPLE/LOGIN) / REGISTRARSE (HTTPS://WWW.FRONTIERSIN.ORG/REGISTER) SOBRE(HTTPS://WWW.FRONTIERSIN.ORG/A Quantitative variables were represented as the mean and standard error of the mean (SEM), and significance was

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analyzed using the unpaired, non-parametric Mann–Whitney test to compare between both Long-COVID and Recovered groups. Qualitative variables were represented as percentages, and significance was analyzed using Fisher’s exact test. p-Values (p) <0.05 were considered statistically significant in all comparisons.

Results Cohorts of Participants Fifty individuals who passed natural infection by SARS-CoV-2 previous to vaccination were recruited for this study. were assigned to TABLA DEThey CONTENIDO

the Long-COVID group if they showed persistent clinical signs and symptoms from 4

to more than 12 weeks after diagnosis (n = 30), or to the Recovered group if they had resolved all signs and

Resumen

symptoms of COVID-19 in the first 4 weeks after diagnosis. The main demographic and clinical characteristics of

Introducción all participants are summarized in Tables 1, 2 and detailed in Supplementary Tables 1, 2. Most individuals in the Métodos Long-COVID group (86.6%) were female, whereas 55% of the participants in the Recovered group were female (p

= 0.0205). Median age at SARS-CoV-2 acute infection in the Long-COVID and Recovered groups was 42 Resultados (interquartile range (IQR) 37–46) and 45 years (IQR 28–57), respectively. Median time from the clinical onset to

Discusión

sampling was 49.7 weeks or 348 days (IQR 150–369 days) in the Long-COVID group versus 11.8 weeks or 83 days

Declaración de (IQR 73–99 days) in the Recovered group. Median length with signs and symptoms compatible with Long-COVID disponibilidad de at sampling was 49.7 weeks or 348 days (IQR 150–369 days) and 1.8 weeks or 13 days (IQR 0–49 days), datos

respectively. The most frequent Rh blood group was A+ in both cohorts, whereas O+ was present in 36.6% of the

Declaración de individuals from the Long-COVID-19 group and 5% from the Recovered group (p = 0.0160). During COVID-19, Ética

individuals from the Long-COVID group reported 38.2°C (± 0.66) of recurrent peaks of fever, in comparison with

Miembros 37.7°C (± 0.18) Contribuyentes del of peak fever during the first 10 days post-infection in participants from the Recovered group (p = Grupo 0.0156). Dyspnea was developed by the majority of participants from the Long-COVID group (86.6%) and 35% of Multidisciplinario de individuals from the Recovered group (p = 0.0002), whereas pleuritic chest pain was present in 76.6% and 25% of Estudio de COVIDindividuals, respectively (p = 0.0005). Additional signs and symptoms with statistical significance reported by 19the (MGS-COVID)

individuals from the Long-COVID group, in comparison with individuals from the Recovered group, were lethargy

Contribuciones de (80%, p < 0.0001), diarrhea (73.3%, p = 0.0037), dermatological injuries (63.3%, p = 0.0003), and conjunctivitis autor

(30%, p = 0.0073). Most participants from the Recovered group did not receive any drug for the treatment of Fondos COVID-19, whereas individuals from the Long-COVID group mostly received antibiotics (70%, p = 0.0012),

Conflicto de corticosteroids (50%, p < 0.0001), and/or cholecalciferol/vitamin D (40%, p < 0.0001). The most prevalent intereses

comorbidities in the group of Long-COVID were autoimmune diseases (30%, p = 0.0366), including psoriasis,

Nota del editor

rheumatoid arthritis, Hashimoto’s thyroiditis, mixed connective tissue disease (MCTD), and Raynaud’s disease,

Expresiones de versus one participant from the Recovered group (5%) who reported both gratitud

Sjögren’s syndrome and

antiphospholipid syndrome prior to SARS-CoV-2 infection.

Material suplementario Referencias TABLE 1

(https://www.frontiersin.org/files/Articles/848886/fimmu-13-848886-HTML/image_m/fimmu-13848886-t001.jpg) T able 1 Demographic and clinical data of all participants from the Long-COVID group and the Recovered group that were recruited for this study.

TABLE 2

(https://www.frontiersin.org/files/Articles/848886/fimmu-13-848886-HTML/image_m/fimmu-13848886-t002.jpg) T able 2 Clinical signs and symptoms reported by the participants from the Long-COVID group recruited for this study.

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Increased Levels of Tregs in the Long-COVID Group TABLA DE CONTENIDO

We did not find differences in the levels of total peripheral CD4+ T cells (Figure 1A) or the composition of CD4+

Resumen memory subpopulations between both

lymphocytes (CD3 Introducción

+

Long-COVID and Recovered groups (Figure 1B). The total level of

) expressing the immune exhaustion marker PD-1 on the cell surface was increased 1.5-fold in

the Long-COVID group, and this difference nearly reached statistical significance (p = 0.0506) (Figure 1C). The Métodos level of regulatory T cells (Tregs) (CD4+CD25+CD127low) was significantly increased 2.5-fold in the individuals

Resultados

from the Long-COVID group (p = 0.0007), in comparison with the Recovered group (Figure 1D). The flow

Discusión

cytometry gating strategy is shown in Supplementary Figure 1.

Declaración de disponibilidad de datos FIGURE 1

Declaración de Ética Miembros Contribuyentes del Grupo Multidisciplinario de Estudio de COVID19 (MGS-COVID) Contribuciones de autor

(https://www.frontiersin.org/files/Articles/848886/fimmu-13-848886-HTML/image_m/fimmu-13848886-g001.jpg) Figure 1 Analysis of CD3+ and CD4 + T-cell populations in PBMCs from the Long-COVID and Recovered groups. Total levels of CD4 + T cells (A) and CD4 + T-cell memory subpopulations (B) were analyzed by flow cytometry. Individual data are shown in a dot plot, and mean data are shown in the pie charts. (C) The expression of the exhaustion marker PD-1 was analyzed in CD3+ T cells. (D) The levels of CD4 + Tregs were also quantified by flow cytometry in both groups of individuals. Each dot in the graphs corresponds to one sample, and lines represent mean ± standard error of the mean (SEM). Statistical significance was calculated using non-parametric Mann–Whitney test. PBMCs, peripheral blood mononuclear cells.

Fondos Conflicto de intereses

Changes in CD8+ T-Cell Subpopulations in the Long-COVID Group

Nota del editor

Expresiones de of CD8+ T cells were increased 1.5-fold in the Long-COVID group, in comparison with Total levels gratitud

the Recovered

group (p = 0.0005) (Figure 2A). We also observed changes in the distribution of CD8+ T-cell memory

Material subpopulations, as the effector CD8+ TEMRA cells were increased 1.4-fold in the Long-COVID group (p = suplementario

0.00487), whereas the naïve CD8 T cells were reduced 1.4-fold, in comparison with the Recovered group

Referencias

(Figure 2B). Regarding the highly cytotoxic CD3+ T cells with TCRγδ, both CD8+TCRγδ+ and CD8−TCRγδ+ populations were increased 2.0-fold (p = 0.049) and 2.2-fold (p = 0.005), respectively, in the Long-COVID group in comparison with the Recovered group (Figure 2C). Stimulation of PBMCs with a pool of nucleoprotein peptides from SARS-CoV-2 showed no significant differences in the capacity of CD8+ T cells to release pro-inflammatory cytokines such as IFNγ and TNFα, as well as the serine protease GZB, between individuals with Long-COVID and the Recovered participants (Figure 2D). The flow cytometry gating strategy is shown in Supplementary Figure 2.

FIGURE 2

(https://www.frontiersin.org/files/Articles/848886/fimmu-13-848886-HTML/image_m/fimmu-13848886-g002.jpg) Figure 2 Analysis of CD8+ T-cell populations in PBMCs from the Long-COVID and Recovered groups. Total levels of CD8+ T cells (A) and CD8+ T-cell memory subpopulations (B) were analyzed by flow cytometry. Individual data are shown in a dot plot, and mean data are shown in the pie charts. (C) The levels of CD8 ± TCRγδ + were also determined by flow cytometry in both groups of individuals. (D) Quantification of the release of pro-inflammatory cytokines IFNg and TNFa, as well as the serine protease GZB from PBMCs from individuals with Long COVID and the Recovered participants after stimulation with a pool of nucleoprotein peptides from SARS-CoV-2.

Each dot in the graphs corresponds to one sample, and lines represent mean ± SEM. Statistical significance was calculated using non-

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Fronteras | respuesta inmune citotóxica hiperactiva persistente en una cohorte española de pacientes con COVID-19 prolongado: identifica… parametric Mann–Whitney test. PBMCs, peripheral blood mononuclear cells.

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Increased Levels of NK Cells in the Long-COVID Group

TABLA DE CONTENIDO

The total levels of NK cells, characterized by the expression of the activation marker CD56, were increased 1.7-fold

Resumen

(p = 0.0005) in the Long-COVID group, in comparison with the Recovered group (Figure 3A). No significant

Introducción

differences were found in the expression of the immune exhaustion marker PD-1 in NK cells between both groups

Métodos (Figure 3B). The population of NK

cells expressing CD16 marker on the surface (CD3−CD56+CD16+) was

increased 1.7-fold (p = 0.032) in the Long-COVID group (Figure 3C, left graph), but no significant differences Resultados were found regarding the expression of the degranulation marker CD107a+ in this population (Figure 3C, right

Discusión

graph). There were no statistically significant differences either between both groups in NK cells not expressing

Declaración de CD16 marker disponibilidad de (Figure 3D) or in NKT cell populations (Figures 3E, F). The flow cytometry gating strategy is shown datos in Supplementary Figure 3.

Declaración de Ética Miembros FIGURE 3 Contribuyentes del Grupo Multidisciplinario de Estudio de COVID19 (MGS-COVID)

(https://www.frontiersin.org/files/Articles/848886/fimmu-13-848886-HTML/image_m/fimmu-13848886-g003.jpg) Figure 3 Analysis of NK and NKT cell subpopulations in PBMCs from the Long-COVID and Recovered groups. Total levels of CD56+ NK/NKT cells were analyzed by flow cytometry (A), as well as the expression of Contribuciones de the exhaustion marker PD-1 in CD56+ T cells (B). Total levels of NK cell subpopulation CD56+CD16+ over CD3− population or with the degranulation marker CD107a over this population CD3−CD56+CD16+ (C) and autor total levels of NK cell subpopulation CD56+CD16− over CD3− population or with the degranulation marker Fondos CD107a over this population CD3−CD56+CD16− (D) were also analyzed by flow cytometry. Analysis by flow cytometry of the total levels of NKT cell subpopulation CD56+CD16+ over CD3+ population or with the Conflicto de degranulation marker CD107a over this population CD3+CD56+CD16+ (E) and total levels of NKT cell subpopulation CD56+CD16− over intereses CD3+ population or with the degranulation marker CD107a over this population CD3+CD56+CD16− (F). Each dot in the graphs corresponds oneeditor sample, and lines represent mean ± SEM. Statistical significance was calculated using non-parametric Mann–Whitney test. PBMCs, Notatodel peripheral blood mononuclear cells.

Expresiones de gratitud Material suplementario

Activation Markers of NK Cells in the Long-COVID Group

Referencias

NK cell subpopulation expressing the activation marker NKG2C (CD56+NKG2A−NKG2C+) was increased 1.4-fold (p = 0.0149) in the Long-COVID group, in comparison with the Recovered group (Figure 4A, left graph), whereas no significant differences were found between both groups in the NK subpopulation with the inhibitory markers NKG2A (CD56+NKG2A+NKG2C−) (Figure 4A, middle graph) or KIR2DL5/CD158f (Figure 4A, right graph). There were no differences in the total levels of NK cells with the memory marker CD57 between both groups (Figure 4B, left graph), but the population of activated memory NK cells (CD56+CD57+NKG2C+) was increased 1.5-fold (p = 0.044) in the Long-COVID group, in comparison with the Recovered group (Figure 4B, right graph). The flow cytometry gating strategy is shown in Supplementary Figure 4.

FIGURE 4

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Figure 4 Analysis of the expression of NK cell markers in PBMCs from the Long-COVID and Recovered

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receptors NKG2A and KIR2DL5/CD158f on the surface of total CD56+ cells. (B) Analysis by flow cytometry of the expression of the activating receptor NKG2C and the memory marker CD57 on the surface of total CD56+ cells. Each dot in the graphs corresponds to one sample, and lines represent mean ± SEM. Statistical significance was calculated using non-parametric Mann–Whitney test. PBMCs, peripheral blood mononuclear cells.

Persistent Cytotoxic Activity in Peripheral Blood Mononuclear Cells From Individuals of the Long-COVID Group The cytotoxic activity of PBMCs from the individuals of the Long-COVID group against the classical target of NK TABLA DE CONTENIDO cells K562 was increased 2.3-fold (p <

0.0001), in comparison with the Recovered group (Figure 5A). The

cytotoxic activity of PBMCs was also tested against Vero E6 cells infected with Resumen

pseudotyped SARS-CoV-2. The

activation of caspase-3 was increased 1.7-fold (p = 0.0092) in the monolayer when they were cocultured with

Introducción

PBMCs from individuals of the Long-COVID group, in comparison with the Recovered group (Figure 5B).

Métodos

Resultados Discusión FIGURE 5 Declaración de disponibilidad de datos

(https://www.frontiersin.org/files/Articles/848886/fimmu-13-848886-HTML/image_m/fimmu-13848886-g005.jpg) Figure 5 Measurement of cytotoxic activity of PBMCs from the Long-COVID and Recovered groups. (A) Declaración de Diagram (left) and dot plot graph (right) of the assay for the quantification by flow cytometry of Annexin V Ética binding to K562 cells cocultured with PBMCs (1:2) from the Long-COVID and Recovered groups for 1 h (B) Diagram (left) and dot plot graph (right) of the assay for the quantification by chemiluminescence of caspase-3 Miembros activation in a monolayer of SARS-CoV-2-infected Vero E6 cells cocultured with PBMCs (1:2) from the LongContribuyentes del COVID and Recovered groups for 1 h Each dot in the graphs corresponds to one sample, and lines represent Grupo mean ± SEM. Statistical significance was calculated using non-parametric Mann–Whitney test. PBMCs, Multidisciplinario de peripheral blood mononuclear cells.

Estudio de COVID-

19 (MGS-COVID) Contribuciones de autor Fondos

Overall Aviremia in Individuals From the Long-COVID Group

Conflicto de intereses All individuals from the Long-COVID group showed an absence of detection of RNA from SAR-CoV-2 in blood,

except for one individual (Patient ID 26; see Supplementary Table 1) (3.33%) who showed amplification of RNA in Nota del editor blood at cycle threshold (CT) 32.97 (data not shown).

Expresiones de gratitud

Application of Random Forest for the Evaluation of Diagnostic Biomarkers for Long-COVID

Material suplementario Referencias

An accuracy of 94% ± 4.90% was obtained for the 5 iterations of the outer loop of the nested K-fold cross validation for each competing algorithm (Figure 6A). As a result, all 30 patients (100%) in the Long-COVID group were correctly assigned to this group, whereas 17 individuals of 20 (85%) were correctly assigned to the Recovered group (Figure 6B). The Gini VIM method determined that clinical parameters such as lethargy, pleuritic chest pain, dermatological injuries, treatment with corticosteroids, immune factors (such as enhanced total levels of NK cells, CD8+ T lymphocytes, and Tregs), and increased cytotoxic activity were the most important variables to assign the individuals to the Long-COVID group (Figure 6C), whereas demographic parameters such as female gender and O+ blood type, or previous history of autoimmune disease, were within the variables with less importance.

FIGURE 6

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Figure 6 Application of Random Forest algorithm and Gini VIM method for the evaluation of the importance

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for Long-COVID. (A) Calculation of the accuracy for 5 iterations of the outer loop of the nested K-fold cross validation. (B) Confusion matrix confronting the conditions predicted by the algorithm and the true conditions for the correct assignment of the participants to the Long-COVID group or the Recovered group.

(C) Classification of the demographic, clinical, and immunological features with statistical significance according to their importance to predict the correct classification of the individuals to the Long-COVID group or the Recovered group. VIM, Variable Importance Measure.

Discussion

TABLA DE CONTENIDO

Long-COVID is emerging as a new relevant syndrome worldwide that is characterized by the maintenance of long-

Resumen

term signs and symptoms of COVID-19 that are not resolved in the first 4 weeks after the infection (12). Several

Introducción host and viral factors may be involved in the development of Long-COVID, although

the underlying basic

Métodos mechanisms are still unknown. To determine the most important host factors that could be used as diagnostic

parameters to evaluate the development of Long-COVID, we collected and analyzed essential demographic, Resultados clinical, and immunological characteristics in a Spanish cohort of individuals with signs and symptoms compatible

Discusión

with Long-COVID in comparison with individuals who recovered completely before 4 weeks after the infection. A

Declaración de Random Forest algorithm was applied to identify the most important variables to predict the susceptibility to disponibilidad de develop Long-COVID. datos Declaración Regardingdethe host factors that might be related to Long-COVID, some demographic characteristics such Ética

as age

and gender have been appointed as contributing host factors for an increased susceptibility (11, 19, 38). Before the

Miembros general vaccination against COVID-19, most patients admitted to the ICU due to a critical form of the disease were Contribuyentes del usually men over 65 years old (39). In contrast, most participants in our cohort of Long-COVID were women Grupo under 45 yearsdeold, which is in accordance with previous studies (18, 40). Rh blood O+ type was also significantly Multidisciplinario Estudio de COVID-in the individuals of the Long-COVID group, but we cannot rule out that most Spanish populations more frequent 19 (MGS-COVID) −

present this blood type (41). Although it has been reported that individuals with Rh blood type may be at lower

Contribuciones de risk of SARS-CoV-2 infection and development of severe illness or death autor

by COVID-19 (42, 43), other studies did

not find a significant impact of Rh on COVID-19 severity (44, 45). In our study, according to the Random Forest

Fondos analysis, both

female gender and Rh blood type were within the less important possible diagnostic factors for

Conflicto Long-COVID. de However, clinical signs and symptoms such as persistent lethargy, pleuritic chest pain, and intereses recurrent dermatological injuries showed more relative importance for the prediction of higher susceptibility to Nota del editor Long-COVID. Expresiones de the potential viral factors that might be involved in the development of Long-COVID, one hypothesis Considering gratitud

that should be taken into account is that SARS-CoV-2 might find mechanisms to persist in the organism after the

Material acute infection. In this case, Long-COVID syndrome would be a consequence of an unresolved SARS-CoV-2 suplementario

infection (46). A prolonged SARS-CoV-2 viral shedding in feces has been described several months after the

Referencias

COVID-19 diagnosis (21, 47). This suggests that SARS-CoV-2 may persist in sanctuaries such as the epithelial cells of the gastrointestinal tract or the tissue macrophages in the gut-associated lymphoid tissue (GALT) (48, 49), which would explain not only the continuous viral shedding but also the gastrointestinal symptoms developed by individuals with Long-COVID (50). In fact, alterations in the gut microbiota have been described in individuals with Long-COVID even 6 months after the infection (51). We cannot rule out this possibility in our cohort, as at least 70% of individuals with Long-COVID had been treated with broad-spectrum antibiotics. However, SARSCoV-2 could also persist in other anatomical sanctuary organs with immune privilege such as the central nervous system (CNS) (52) or in other organs and cells in which ACE2 receptor is highly expressed such as the intestine, kidneys, cardiac tissue, reproductive system, thyroid, gallbladder, and nasal mucosa (53, 54), which would also explicate the highly variable signs and symptoms that are associated with Long-COVID (55). The persistence of SARS-CoV-2 in the organism would also correlate with the recurrent peaks of fever reported by the participants from the Long-COVID group, which was significantly increased at 0.5°C in comparison with the individuals from the Recovered group, in which the fever stopped once the infection was resolved. Although more studies will be necessary to determine whether the virus may be found in the feces of these individuals, low SARS-CoV-2 viremia was detected in one participant from the Long-COVID group (Patient ID 26). This participant was a 44-year-old woman, A+, with previous history of asthma and chronic obstructive pulmonary disease (COPD) who was treated

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with azithromycin during the first days after COVID-19 diagnosis, had signs and symptoms compatible with LongINICIAR SESIÓN (HTTPS://WWW.FRONTIERSIN.ORG/PEOPLE/LOGIN) / REGISTRARSE (HTTPS://WWW.FRONTIERSIN.ORG/REGISTER)

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COVID at the time of sampling (21.4 weeks post-infection), and did not report a subsequent re-infection after the (https://www.frontiersin.org) first exposure to SARS-CoV-2. She informed about persistent neurological symptoms and diarrhea, which would support the possibility of a viral persistence in the CNS or in the GALT, with occasional viremia.

The persistence of SARS-CoV-2 or its viral products in the organism would then contribute to the increased antiviral, cytotoxic activity detected in individuals from the Long-COVID group, along with the associated

gastrointestinal symptoms, which were likely also due to the presence of activated immune cells in the GALT (56).

Persistent epithelial damage due to a chronic inflammatory response has been appointed as a possible cause of Long-COVID (57). It is noteworthy that patients with critical COVID-19 who are admitted to the ICU usually present CD4+ cytopenia (4, 58), which makes it difficult to develop a complete, effective immune response against SARS-CoV-2. However, the individuals from the Long-COVID group in our study showed not only normal levels of CD4+ T cells in peripheral blood but also CD8+ lymphocytosis with an increased proportion of TEMRA cells,

TABLA DE CONTENIDO

which are highly differentiated effector cells essential for CD8+ function (59). This enhanced terminal

Resumen differentiation of memory CD8+ T cells pointed at the presence of a potent antiviral immune response that was

supported by the presence of increased levels of highly cytotoxic populations of CD8 Introducción

±

T lymphocytes expressing

TCRγδ. These cells have been related to a potent antiviral and antineoplastic response, and therefore, they are Métodos

currently the target of different strategies to reprogram their cytotoxic potential against specific targets such as

Resultados

viruses or cancerous cells (60, 61). Although the capacity of CD8+ T cells to produce pro-inflammatory cytokines

Discusión

and GZB was not significantly different between individuals with Long-COVID and those who completely

Declaración recovered,dethe sustained presence of these cytotoxic cell populations from the adaptive immune system indicated disponibilidad de that the participants from the Long-COVID group had developed a potent memory response against SARS-CoV-2 datos

that was still fully active after more than 49 weeks post-infection, whereas this response had waned in the

Declaración de Recovered group once the infection was cleared. The increased expression of exhaustion markers such Ética

as PD-1 in

CD3+ T lymphocytes from these individuals of the Long-COVID group could be a consequence of the continuous

Miembros activation of the immune system. The PD-1/PD-L1 axis is upregulated during acute viral infection and after Contribuyentes del infection with persistent RNA viruses such as HIV and HCV, and it has been related to viral evasion of the Grupo Multidisciplinario de and prolonged inflammatory responses (62). Moreover, the significant increase of Tregs observed immune system Estudio de COVIDin PBMCs from individuals of the Long-COVID group may indicate the failed attempt of the immune system to 19 (MGS-COVID)

control this persistent immune response.

Contribuciones de autor The long-term activation of the immune system in response to triggers such

as pathogens, vaccines, drugs, or

chemicals has been previously related to the development of autoimmune diseases due to the loss of tolerance Fondos (63). The link between the development of autoimmunity and a previous infection is not always clear, as occurs

Conflicto de with common autoimmune diseases such intereses

as lupus erythematosus, multiple sclerosis, type 1 diabetes mellitus, and

rheumatoid arthritis. However, the discovery of autoantibodies in individuals with Long-COVID may link this Nota del editor syndrome not only to a persistent viral infection but also to an immune dysregulation that would lead to a

Expresiones de sustained targeting of own cells (26, 64). The detection of high gratitud

levels of functional cytotoxic memory cells in

peripheral blood of the participants from the Long-COVID group may explain why several signs and symptoms of

Material this syndrome resembled those of an autoimmune disease. Some previous predisposition may be then suplementario

contributing since the most prevalent comorbidities showed by these individuals were autoimmune diseases,

Referencias

although they were not considered within the most accurate diagnostic biomarkers for Long-COVID in our Random Forest model. However, the hypothesis that a persistent memory cytotoxic response against SARS-CoV2 could be the cause for the development of Long-COVID was supported in our cohort by the presence not only of high levels of CD8+ TEMRA cells but also of NK cells expressing both memory (CD57) and activation (NKG2C) markers that did not waste and wane after SARS-CoV-2 infection was cleared, despite the presence of high levels of Tregs. Moreover, these immunological factors were considered the most important variables for the prediction of a higher susceptibility to develop Long-COVID in our Random Forest model.

The difference in the median time from the clinical onset to sampling in the Long-COVID group versus the Recovered group could be considered a potential limitation of our study. However, all participants were infected with SARS-CoV-2 during the same period of time that was the first pandemic peak in Spain (March to April 2020), and whereas all signs and symptoms of COVID-19 were completely resolved at the time of sampling (12 weeks post-infection) in all participants of the Recovered group, the individuals of the Long-COVID group still presented recurrent signs and symptoms of the disease more than 49 weeks post-infection. Therefore, all inclusion criteria were met in both groups.

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such as the CNS or tissue macrophages in the GALT, where it would be protected from clearance by the immune system. However, we also have to consider that Long-COVID shows characteristics of autoimmune disease and that infected individuals may be more susceptible to developing this syndrome due to the activation of an exacerbated memory immune response that cannot be adequately controlled by homeostatic mechanisms once the infection was cleared. Having a better understanding of the underlying mechanisms of Long-COVID could be helpful to prevent the development of this syndrome and to improve the clinical management of these patients.

According to our Random Forest model, 100% of the participants included in the Long-COVID group were correctly assigned to this group using parameters related to long-lasting cytotoxicity, which proved their validity as biomarkers to diagnose the development of this syndrome.

TABLA DE CONTENIDO

Data Availability Statement

Resumen

The original contributions presented in the study are included in the article/Supplementary Material. Further Introducción inquiries can be directed to the corresponding authors.

Métodos

Resultados

Ethics Statement

Discusión

Declaración de involving human participants were reviewed and approved by the Ethical Committee of Instituto de The studies disponibilidad de Salud Carlos III (IRB IORG0006384) (CEI PI 07_2021) and the Central Research Commission from the Health datos

Counseling (Comunidad de Madrid, Spain) (favorable report 20210008). The patients/participants provided their

Declaración de written informed consent to participate in this study. Ética Miembros Contribuyentes del Grupo Multidisciplinario de Estudio de COVIDAlonso Herrador1 , Pablo Amich 19Esther (MGS-COVID)

Contributing Members of the Multidisciplinary Group of Study of COVID19 (MGS-COVID) Alemany1 , Victoria Bosch Martos1 , Lorena Cordova-Castaño1 , Aurora

Expósito Mora1 , Javier García-Pérez2,3 , María Mercedes Gea Martinez1 , Alberto Gomez Bonilla1 , María Victoria

Contribuciones de Leon Gomez1 , Gema Lora Rey1 , Maria Luisa Muñoz Balsa1 , Javier Pérez Gonzalez1 , Sandra Pérez-Santos1 , Jose autor

Sanchez Hernández1 , Andrea Vinssac Rayado1

Fondos 1

Centrode de Salud Doctor Pedro Laín Entralgo, Alcorcón, Spain.

Conflicto intereses 2

AIDS Immunopathology Unit, National Center of Microbiology, Instituto de Salud Carlos III, Majadahonda,

Nota del editor

Madrid, Spain.

Expresiones de 3 Biomedical Research gratitud

Center Network in Infectious Diseases (CIBERINFEC), Spain

Material suplementario

Author Contributions

Referencias

MC and ML-H conceptualized the project. MG, LV, MC, and ML-H wrote the manuscript. LV, SR-M, FR-M, MT, GC, and EM processed and stored all blood samples. MM-A, SD-M, and MT identified, selected, and recruited the patients. MG, LV, GC, MT, EM, and SR-M performed the analytical experiments. LV and MG collected and analyzed the clinical data and laboratory results. DF and VP performed the Random Forest analysis. All co-authors read and approved the final version of the manuscript.

Funding This work was supported by the Coordinated Research Activities at the National Center of Microbiology (CNM, Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation), which is coordinated by Dr Inmaculada Casas (WHO National Influenza Center of the CNM); a generous donation provided by Chiesi España, S.A.U. (Barcelona, Spain); the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00); and the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2016-2020, Instituto de Salud Carlos III, European Region Development https://www.frontiersin.org/articles/10.3389/fimmu.2022.848886/full

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Fund (ERDF). The work of ML-H and SR-M is financed by NIH grant R01AI143567. The work ofSOBRE(HTTPS://WWW.FRONTIERSIN.ORG/A MT is supported INICIAR SESIÓN (HTTPS://WWW.FRONTIERSIN.ORG/PEOPLE/LOGIN) / REGISTRARSE (HTTPS://WWW.FRONTIERSIN.ORG/REGISTER)

by Instituto de Salud Carlos III (COV20_00679). The work of LV is supported by a pre-doctoral grant from

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Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). The work of FR-M is financed by the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00).

Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note TABLA DE CONTENIDO

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their

Resumen affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be

evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the Introducción publisher.

Métodos Resultados

Acknowledgments

Discusión

Declaración de We greatly appreciate all the participants for their contribution to this study. We also thank the excellent disponibilidad de secretarial assistance of Mrs Olga Palao. datos Declaración de Ética

Supplementary Material

Miembros Contribuyentes del The Supplementary Material for this article can be found online at: Grupo https://www.frontiersin.org/articles/10.3389/fimmu.2022.848886/full#supplementary-material Multidisciplinario de (https://www.frontiersin.org/articles/10.3389/fimmu.2022.848886/full#supplementary-material) Estudio de COVID19 (MGS-COVID) Contribuciones de autor

References

Fondos 1. WHO. Coronavirus (COVID-19) Dashboard. Available at: https://covid19.who.int/ (https://covid19.who.int/).

Google Conflicto de Scholar (http://scholar.google.com/scholar_lookup?author=WHO&journal=Coronavirus+(COVID-19)+Dashboard&) intereses 2. Kandeel M, Mohamed MEM, Abd El-Lateef HM, Venugopala KN, El-Beltagi HS. Omicron Variant Genome Evolution and Phylogenetics. J Med Virol (2021) 94(4):1627–32. doi: 10.1002/jmv.27515

Nota del editor

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9. Lopez-Leon Declaración de S, Wegman-Ostrosky T, Perelman C, Sepulveda R, Rebolledo PA, Cuapio A, et al. More Than 50 Long-Term Effects of COVID19: A Systematic Review and Meta-Analysis. Sci Rep (2021) 11:16144. doi: 10.1038/s41598-021-95565-8 disponibilidad de datos PubMed Abstract (https://pubmed.ncbi.nlm.nih.gov/34373540/) | CrossRef Full Text (https://doi.org/10.1038/s41598-021-95565-8) | Google Scholar (http://scholar.google.com/scholar_lookup?author=S+Lopez-Leon&author=T+Wegman-

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Métodos

Discusión

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TABLA DE CONTENIDO

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TABLA DE CONTENIDO

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Keywords: Long-COVID, cytotoxic immune response, immune exhaustion, CD8+ T cells, NK cells, Random Forest algorithm

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Citation: Galán M, Vigón L, Fuertes D, Murciano-Antón MA, Casado-Fernández G, Domínguez-Mateos S, Mateos E, Ramos-Martín F, Planelles V, INICIARM, SESIÓN (HTTPS://WWW.FRONTIERSIN.ORG/PEOPLE/LOGIN) / REGISTRARSE SOBRE(HTTPS://WWW.FRONTIERSIN.ORG/A Torres Rodríguez-Mora S, López-Huertas MR and Coiras M (2022) Persistent(HTTPS://WWW.FRONTIERSIN.ORG/REGISTER) Overactive Cytotoxic Immune Response in a Spanish Cohort of Individuals With Long-COVID: Identification of Diagnostic Biomarkers.

Front.

Immunol.

13:848886. doi: 10.3389/fimmu.2022.848886 (https://www.frontiersin.org) Received: 05 January 2022; Accepted: 25 February 2022; Published: 25 March 2022.

Edited by: Lucia Lopalco (https://loop.frontiersin.org/people/141624), San Raffaele Hospital (IRCCS), Italy Reviewed by: Ester Gea-Mallorquí (https://loop.frontiersin.org/people/937514), University of Oxford, United Kingdom Federica Facciotti (https://loop.frontiersin.org/people/500422), European Institute of Oncology (IEO), Italy Copyright © 2022 Galán, Vigón, Fuertes, Murciano-Antón, Casado-Fernández, Domínguez-Mateos, Mateos, Ramos-Martín, Planelles, Torres, Rodríguez-Mora, López-Huertas and Coiras. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) (http://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

TABLA DE CONTENIDO *Correspondence: Mayte Coiras, mcoiras@isciii.es (mailto:mcoiras@isciii.es); María Rosa López-Huertas, mrlhuertas@isciii.es (mailto:mrlhuertas@isciii.es)

Resumen †

These authors have contributed equally to this work and share first authorship

Introducción ‡

These authors have contributed equally to this work and share last authorship

Métodos

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or

those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer Resultados is not guaranteed or endorsed by the publisher.

Discusión

Declaración de disponibilidad de Write a comment... datos Declaración de Ética A dd Miembros Contribuyentes del MIRABA LA GENTE TAMBIÉN Grupo Factores asociados a la hospitalización o ingreso a cuidados intensivos en niños con COVID-19 en América Latina Multidisciplinario de (/articles/10.3389/fped.2022.868297/full) Estudio de COVIDEduardo López-Medina (https://loop.frontiersin.org/people/1164607/overview), German Camacho-Moreno 19(https://loop.frontiersin.org/people/1073437/overview), (MGS-COVID) Martin E Brizuela (https://loop.frontiersin.org/people/1429760/overview), Diana M Dávalos, Juan Pablo Torres (https://loop.frontiersin.org/people/1494145/overview), Rolando Ulloa-Gutierrez (https://loop.frontiersin.org/people/877391/overview), Pio

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Akt-Fas to Quell Aberrant T Cell Differentiation and Apoptosis in Covid-19 (/articles/10.3389/fimmu.2020.600405/full) Nota del editor Anthony J. Leonardi (https://loop.frontiersin.org/people/1049338/overview) and Rui B. Proenca

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PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma (/articles/10.3389/fimmu.2022.754645/full) Zhishang Meng (https://loop.frontiersin.org/people/1300208/overview), Yanzhu Chen (https://loop.frontiersin.org/people/1221906/overview), Wenyi Wu (https://loop.frontiersin.org/people/1352826/overview), Bin Yan (https://loop.frontiersin.org/people/1460888/overview), Lusi Zhang (https://loop.frontiersin.org/people/1360109/overview), Huihui Chen, Yongan Meng (https://loop.frontiersin.org/people/1675450/overview), Youling Liang (https://loop.frontiersin.org/people/1314302/overview), Xiaoxi Yao and Jing Luo (https://loop.frontiersin.org/people/1543278/overview)

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