ORIGINAL RESEARCH published: 10 May 2022 doi: 10.3389/fimmu.2022.815041 Skewed Cellular Distribution and Low Activation of Functional T-Cell Responses in SARS-CoV-2 Non-Seroconvertors Athina Kilpeläinen 1,2, Esther Jimenez-Moyano 1, Oscar Blanch-Lombarte 1, Dan Ouchi 1, Ruth Peña 1, Bibiana Quirant-Sanchez 2,4,5, Raul Perez-Caballero 1, Anna Chamorro 6, Ignacio Blanco 2,7, Eva Martı́nez-Caceres 2,4,5, Roger Paredes 1,6,9,10, Lourdes Mateu 9,10, Jorge Carrillo 1,2,3, Julià Blanco 1,2,3,10, Christian Brander 1,2,3,8,10, Marta Massanella 1,2,3, Bonaventura Clotet 1,6,9,10 and Julia G. Prado 1,2,3* 1 IrsiCaixa AIDS Research Institute, Badalona, Spain, 2 Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain, 3 CIBERINFEC, ISCIII, Madrid, Spain, 4 Department of Cell Biology, Physiology, Immunology, Universitat Autònoma de Edited by: Barcelona, Cerdanyola del Vallès, Spain, 5 Immunology Department, Hospital Universitari Germans Trias i Pujol, Badalona, Anthony L. Cunningham, Spain, 6 Lluita contra la SIDA Foundation, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, 7 Clinical Genetics and Westmead Institute for Medical Genetic Counseling Program, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, 8 ICREA, Barcelona, Spain, Research, Australia 9 Infectious Diseases Department, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, 10 University of Vic–Central Reviewed by: University of Catalonia (UVic-UCC), Vic, Catalonia, Spain Shetty Ravi Dyavar, Adicet Bio, Inc, United States Nicola Cotugno, The role of T cells in the control of SARS-CoV-2 infection has been underestimated in favor Bambino Gesù Children’s Hospital of neutralizing antibodies. However, cellular immunity is essential for long-term viral control (IRCCS), Italy and protection from disease severity. To understand T-cell immunity in the absence of *Correspondence: antibody generation we focused on a group of SARS-CoV-2 Non-Seroconvertors (NSC) Julia G. Prado jgarciaprado@irsicaixa.es recovered from infection. We performed an immune comparative analysis of SARS-CoV-2 infected individuals stratified by the absence or presence of seroconversion and disease Specialty section: severity. We report high levels of total naïve and low effector CD8+ T cells in NSC. This article was submitted to Viral Immunology, Moreover, reduced levels of T-cell activation monitored by PD-1 and activation-induced a section of the journal markers were observed in the context of functional SARS-CoV-2 T-cell responses. Frontiers in Immunology Longitudinal data indicate the stability of the NSC phenotype over three months of Received: 14 November 2021 follow-up after infection. Together, these data characterized distinctive immunological Accepted: 29 March 2022 Published: 10 May 2022 traits in NSC including skewed cellular distribution, low activation and functional SARS- Citation: CoV-2 T-cell responses. This data highlights the value of T-cell immune monitoring in Kilpeläinen A, Jimenez-Moyano E, populations with low seroconversion rates in response to SARS-CoV-2 infection Blanch-Lombarte O, Ouchi D, Peña R, Quirant-Sanchez B, and vaccination. Perez-Caballero R, Chamorro A, Keywords: SARS-CoV-2, non-seroconvertor, cellular immunity, T cell subsets, immune activation, function Blanco I, Martı´nez-Caceres E, Paredes R, Mateu L, Carrillo J, Blanco J, Brander C, Massanella M, Clotet B and Prado JG (2022) Skewed Cellular Distribution and INTRODUCTION Low Activation of Functional The COVID-19 pandemic is caused by SARS-CoV-2, the newest coronavirus crossing into the T-Cell Responses in SARS-CoV-2 Non-Seroconvertors. human population. The pandemic accounts for millions of infected people and around 6 million Front. Immunol. 13:815041. deaths worldwide. Despite the scientific success in rapidly generating SARS-CoV-2 vaccines, the doi: 10.3389/fimmu.2022.815041 number of infected people and the burden on healthcare systems continues (1). Consequently, Frontiers in Immunology | www.frontiersin.org 1 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors continuous characterization of the functional features of immune proposed as a mechanism limiting disease severity in a fraction protection of SARS-CoV-2 infection are needed. of individuals (24, 25). The disease outcome of SARS-CoV-2 infection is associated with Phenotypic and functional characterization of cellular a wide degree of interindividual heterogeneity. These divergences immunity against SARS-CoV-2 in the absence of immune may be associated with the level of immunocompetence and the pathogenesis is essential to identify the determinants of immune tight balance between immune control and immunopathogenesis protection from infection and disease severity. To gain deeper after infection (2). Indeed, beneficial and detrimental aspects of the insights into the characterization of cellular immunity in the immune responses elicited by SARS-CoV-2 infection have been absence of pathogenesis and serconversion, we focused on a described. As detrimental aspects, secondary multi-organ group of SARS-CoV-2 Non-Seroconvertors (NSC) and complications and persistent symptomatology for months in compared their cellular immunity to individuals who approximately 10% of the total infected people or “long COVID” seroconverted. Lack of seroconversion in SARS-CoV-2 infection (3) have been associated with the persistence of inflammatory has been demonstrated in 2-17% of Convalescent individuals (25– profiles and tissue damage. As beneficial aspects, cellular and 28) and cellular responses have been observed in 41 to 78% of humoral immune responses have been linked to protection from these individuals (25, 27, 29). Currently, the functional disease severity against other coronaviruses such as SARS-CoV-1 characterization of T cell responses in NSC remains limited due (4) and the induction of B and T cell memory responses has been to the low numbers of individuals identified, lack of confirmation described in most of the individuals recovered from SARS-CoV-2 of SARS-CoV-2 infection by PCR, or specific focus in CD8+ T infection (5). cells. Functional T-cell responses was recently described in a small Cellular perturbations and immune profiles have been related number of seronegative convalescent COVID-19 patients (30). To to disease severity (6–8). Moreover, disease outcome have been gain more knowledge on this subset of individuals, we correlated with early signatures of soluble meditators including characterized SARS-CoV-2 T cell immunity in NSC in terms of growth factors, type-2/3 cytokines, type-1/2/3 cytokines, and the cellular landscape, activation and functional profile and chemokines in SARS-CoV-2 infection (9). High levels of compared to recovered individuals that seroconverted stratified cytokine and chemokine production upon infection, the so- by SARS-CoV-2 disease severity. Our analyses identify differential called cytokine storm has been observed in SARS, MERS and immunological traits in NSC. We observed skewed CD8+ T-cell SARS-CoV-2 infections, potentially leading to severe tissue distribution towards an increase in naïve populations in the damage (10). Patients suffering from severe COVID-19- absence of overt activation of functional SARS-CoV-2-specific T induced acute respiratory distress syndrome have been cell responses. Our current analyses provide additional described to display elevated levels of Interleukin-6 (IL-6), information on the characterization of T-cell immunity in the lymphopenia with low counts of CD8+ T cells, natural killer absence of SARS-CoV-2 seroconversion giving further insights of (NK) and naïve T helper cells, while B cells remained mainly the contribution T cells to viral control. Moreover, our data unaffected (11). Along with increased IL-6, elevated IL-10, IL-2 highlight the importance to monitor T-cell immunity in those and IFN-g peripheral blood levels have been described (12). populations with low levels of antibodies in response to SARS- Finally, higher plasma levels of IL2, IL7, IL10, GSCF, IP10, CoV-2 infection and vaccination. MCP1, MIP1A, and TNFa were observed in patients admitted to the ICU (13). On the other hand, neutralizing antibodies have been shown to predict severity and survival (14). Frequencies of MATERIALS AND METHODS SARS-CoV-2-specific CD4+CD40L+ T cells and Spike-specific B cells have been associated with anti-SARS-CoV-2 antibodies and Study Participants the magnitude of neutralizing activity in children (15). However, The KING extension cohort (N=403) is composed of 336 SARS- high neutralizing antibody titers do not positively correlate with CoV-2 infected individuals, including 120 individuals who less severe clinical outcomes (16, 17). required hospitalization. For the present study, we focused on Multiple studies support the relevance of cellular immunity in individuals with a SARS-CoV-2 infection diagnosis by PCR the control and prevention of SARS-CoV-2 infection. Antiviral without seroconversion (Non-Seroconverters, NSC, N=15), and CD4+ and CD8+ T-cell responses are key in the control of viral compared NSC with two groups of seroconvertors stratified by infections (18–20). Data from SARS-CoV-2 animal models disease severity (Mild-moderate, MM, N=15 and Severe, N=16) support the role of T-cell immunity as a correlate of protection (Figure 1A). The study groups were selected from the first wave from infection (21) and virus-specific CD4+ and CD8+ T-cell of the COVID-19 pandemic in Spain. In brief, the NSC were responses are considered key players in the resolution and long- defined as SARS-CoV-2 infected individuals by PCR diagnosis term protection from infection (22). The presence of SARS-CoV- with no detectable SARS-CoV-2 IgG, IgA and IgM by two 2 CD4+ and CD8+ T-cell responses up to 6 months after independent ELISAs (in-house and a commercially available infection in mild to moderate clinical course and persistent ELISA). All NSC displayed asymptomatic or symptomatic immunological alterations in the memory compartment have infection not requiring hospitalization. NSC represented 4.5% been described after SARS-CoV-2 infection (5, 23). Moreover, of the infected KING extension cohort. The first serology test was protection through pre-existent SARS-CoV-2 cross-reactive obtained a median of 63 Days from Symptom Onset (DfSO) and CD4+ T cell responses to other coronaviruses has been lack of seroconversion was confirmed a median of 94.5 DfSO. Frontiers in Immunology | www.frontiersin.org 2 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors A B C D E F G FIGURE 1 | CD4+ and CD8+ T-cell subset distribution in Non-Seroconvertors. (A) Flow chart of the study groups, including Non-Seroconvertors (NSC), Mild- moderate (MM), Severe (B, C) Cryopreserved PBMCs were cultured and surface stained with antibodies targeting CD4 and CD8 as well as T-cell lineage markers CD45RA, CCR7, CD27 to distinguish T-central memory cells (TCM), T effector memory cells (TEM), T transitional memory cells (TTM), T Naïve cells (TN) and T effector cells (TEF). Cells were pre-gated for lymphocytes and live cells. Whisker plots show the frequencies of CD4+ T-cell subsets and pie charts represent the frequency distribution of CD4+ and CD8+ T-cell subsets in each group (Panel B, C, respectively). Statistical analyses were performed using a multivariable regression analysis. Only significant values are indicated in the Figure. Linear regression was performed plotting relative frequencies of CD8+ TN (D) and CD8+ TEF (E) against the age of the study participants, as well as relative frequencies of CD8+ TN (F) and CD8+ TEF (G) against the Days from Symptom Onset (DfSO) to sample. Correlation analyses were performed using Spearman’s rank correlation test; r- and p-values are reported for each group. The MM were defined as SARS-CoV-2 infected individuals infected individuals diagnosed by PCR and/or SARS-CoV-2 diagnosed by PCR and/or SARS-CoV-2 IgG ELISA with IgG ELISA with symptomatic infection requiring asymptomatic or symptomatic infection not requiring hospitalization. The first serology test was obtained a median hospitalization. The Severe were defined as SARS-CoV-2 of 38.5 and 24 days DfSO in MM and Severe, respectively. The Frontiers in Immunology | www.frontiersin.org 3 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors clinical characteristics of the study groups are summarized in sample were used as standard. A pool of 10 SARS-CoV-2 negative Table 1. Biological samples were available for all study groups plasma samples, collected before June 2019, were included as the and additional longitudinal samples were available for 6 NSC a negative control. Samples were assayed at 1/100 dilution in blocking median of 93 DfSO. Also, we obtained samples from controls buffer for one hour at room temperature. The following reagents from the Catalan Blood and Tissue Bank before 2019 as were used as secondary antibodies: HRP conjugated (Fab)2 Goat methodological controls (controls, N=5). The information anti-human IgG (Fc specific) (1/20000), Goat anti-human IgM (1/ regarding age, sex and other epidemiological data was not 10000), and Goat anti-human IgA (alpha chain specific) (1/20000) available for controls because the Catalan Blood and Tissue (all from Jackson Immunoresearch). Secondary antibodies were Bank did not provide it. None of the study participants had incubated for 30 minutes at room temperature. After washing, received a COVID-19 vaccine during the course of the study. plates were revealed using o-Phenylenediamine dihydrochloride (OPD) (Sigma Aldrich) and the enzymatic reaction was stopped Determination of Anti-SARS-CoV-2 with 4N of H2SO4 (Sigma Aldrich). The signal was analysed as the Antibodies by Enzyme-Linked optical density (OD) at 492 nm with noise correction at 620 nm. Immunosorbent Assays The specific signal for each antigen was calculated after subtracting The presence of anti-SARS-CoV-2 antibodies in serum or plasma the background signal obtained for each sample in antigen-free samples was evaluated using two independent Enzyme-linked wells. The second ELISA was a commercially available IgM Immunosorbent Assays (ELISA). The first was an in-house and IgG class antibody ELISA against the SARS-CoV-2 NP developed sandwich-ELISA. Briefly, Nunc MaxiSorp ELISA plates (ImmunoDiagnostics, Hongkong). Briefly, serum samples were were coated overnight at 4°C with 50 ml of capture antibody (anti- diluted at 1:100 and incubated for 1 hour at room temperature. 6xHis antibody, clone HIS.H8; ThermoFisher Scientific) at 2 mg/ Anti-NP antibodies were captured by immobilized NP recombinant mL in PBS. After washing, plates were blocked for two hours at protein. After incubation, captured antibodies were measured by an room temperature using PBS containing 1% of bovine serum absorbance microplate reader at 450 nm. The antibody results were albumin (BSA, Miltenyi biotech). 50 ml (1 mg/mL in blocking expressed as an index value, calculated as the ratio of the OD value buffer) of the following SARS-CoV-2 derived antigens: S1+S2 for each sample to the OD value of the cut-off (0,200). The test was subunits of the Spike (S) protein and receptor-binding domain considered positive when the index value was ≥ 1.1, borderline (RBD, Sino Biological) were subsequently added and incubated when the index value was ≥ 0.9 to < 1.1, and negative when the overnight at 4°C. Each plasma sample was evaluated in duplicates at index value was < 0.9.2.3 Pseudovirus neutralization assay. a 1/100 dilution in blocking buffer for each antigen. Antigen free HIV-1 reporter pseudoviruses expressing SARS-CoV-2 S wells were also assessed in parallel for each sample in the same plate protein and Luciferase were generated. pNL4-3.Luc.R-.E- was to evaluate sample background. Serial dilutions of a positive plasma obtained from the NIH AIDS repository (Connor RI, Chen BK, TABLE 1 | Clinical characteristics of the study groups. NSC MM Severe p-value4 (n=15) (n=15) (n=16) NSC vs MM NSC vs Severe MM vs Severe Sex, Female, n (%) 14 (88) 11 (69) 4 (27) 0.801 0.005* 0.137 Age at diagnosis of SARS-CoV-2 median, [IQR1] 45 [40-53] 41 [29-48] 56 [48-70] >0.999 0.108 0.009* Days from positive PCR to sample, [IQR] 66 [43-130] 57 [21-173] 44 [30-85] >0.999 0.742 >0.999 DfSO to first positive PCR, [IQR] 6.5 [1-12.7] 7 [2-89] 8 [5-12] >0.999 >0.999 >0.999 DfSO to sample, [IQR] 79 [54.5-157.5] 55 [28.7-122.3] 53.5 [35.5-88.5] 0.590 0.272 >0.999 DfSO to second sample, [IQR] 92.5 [82.5-110.8] Confirmed Positive PCR, n (%) 16 (100) 7 (47) 7 (47) Positive serology, n (%) 0 (0) 15 (100) 16 (100) <0.0001* <0.0001* >0.9999 Plasma neutralizing titer, reciprocal dilution median, [IQR] <602 258 [60-378] 1276 [557-3158] 0.011* <0.0001* 0.010* Severe, n (%)3 0 (0) 0 (0) 16 (100) >0.999 <0.0001* <0.0001* Asymptomatic, n (%) 2 (13) 2 (13) 0 (0) >0.999 0.578 0.578 Co-morbidities, any comorbidity, n (%) 10 (67) 6 (40) 12 (75) >0.999 0.012* 0.084 HTA 1 (7) 0 (0) 6 (38) Obesity 0 (0) 0 (0) 6 (38) Allergies 3 (20) 4 (27) 2 (13) Asthma 1 (7) 0 (0) 2 (13) Primary Immunodeficiency 1 (7) 0 (0) 0 (0) Autoimmunity 1 (7) 0 (0) 0 (0) Others 7 (47) 5 (33) 9 (56) NSC, Non-Seroconvertors; MM, Mild-moderate. 1 [IQR]: inter-quartile range. 2 <60, below the limit of detection. 3 Severe presentation of SARS-CoV-2 requiring hospitalization. 4 Analysis performed by non-parametric ANOVA comparing all groups. *Statistical significance. Frontiers in Immunology | www.frontiersin.org 4 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors 195). SARS-CoV-2.SctD19 was generated (Geneart) from the full differentiated based on CD27 and CCR7 expression: naïve (TN: SARS-CoV-2 S gene sequence with a deletion of the last 19 C- CD45RA+, CD27+, CCR7+), effector (TEF: CD45RA+, CD27-, terminal codons (31), human-codon optimized and inserted into CCR7-, only for CD8+), central memory (TCM: CD45RA-, pcDNA3.4-TOPO. Expi293F cells were transfected using the CD27+, CCR7+), transitional memory (TTM: CD45RA-, CD27+, Expifectamine Reagent (Thermo Fisher Scientific, Waltham, CCR7-), and effector memory (TEM: CD45RA-, CD27-, CCR7-), MA, USA) with pNL4-3.Luc.R-.E- and SARS-CoV-2.SctD19 at as previously described (32, 33) (Figures S2A, B). To measure an 8:1 ratio, respectively. Control pseudoviruses were obtained proliferation, cryopreserved PBMCs were labeled with CFSE, by replacing the S protein expression plasmid with a VSV-G (Thermo Fisher –V12883, 0.5 mM) and cultured in the absence protein expression plasmid as reported previously (31). or presence of S and NP recombinant proteins (5 mg/mL, Supernatants were harvested 48 h after transfection, filtered at Sinobiological, China) and Staphylococcal enterotoxin B (SEB) 0.45 µm, frozen and titrated on HEK293T cells overexpressing (1 mg/mL, Sigma-Aldrich) as a positive control, for ten days at 37° WT human ACE-2 (Integral Molecular, USA). For the C in a 5% CO2 incubator. After incubation, PBMCs were surface neutralization assay, 200 TCID50 of pseudoviral supernatant stained for 30 min at room temperature (RT) with anti-human was preincubated with serial dilutions of heat-inactivated antibodies for CD3 (A700, clone UCHT1, BD), CD4 (A647, clone serum or plasma samples (ranging from 1/60 to 1/14580) for RPA-T4, BD), and CD8 (V500, clone RPA-T8, BD). FlowJo 10.0.7 1h at 37°C and then added to ACE2-overexpressing HEK293T was used to analyze flow cytometric data. The frequency of T-cell cells. After 48 h, cells were lysed with Britelite Plus Luciferase cytokine responders against S and/or NP proteins were defined by reagent (Perkin Elmer, Waltham, MA, USA), and luminescence the presence of at least one cytokine. In addition, the frequency of was measured for 0.2 s with the EnSight Multimode Plate Reader total T-cell responders against S and/or NP proteins were defined (Perkin Elmer). Data were fitted to a four-parameter logistic by the presence of at least one cytokine and/or AIM. We used a curve with variable slope using Graph Pad Prism software 0.2% cut-off value for positivity following background subtraction. (v8.3.0). IC50 values are expressed as reciprocal dilution. To determine the SARS-CoV-2 specific T-cell proliferation, we subtracted background signal obtained from unstimulated cells. Immunophenotype of SARS-CoV-2 T-Cell Single-cell analysis were performed using the statistical Responses and Proliferation Assays package R (v3.6.3). Cells were compensated and selected based To measure T cells and SARS-CoV-2 specific T-cell responses, on its antigen expression (TNF, IL-2 and IFN-g intensity) and cryopreserved PBMCs were stimulated in the absence or presence normalized before performing UMAP dimensionality reduction. of S and NP recombinant proteins (5 mg/mL, Sinobiological, We used FlowJo 10.0.7 for the graphical representation of the China) using as control Staphylococcal enterotoxin B (SEB) (1 single-cell analysis. mg/mL, Sigma-Aldrich), with CD28/49d co-stimulatory molecules (1 mg/mL, BD) for 17 h at 37°C in a 5% CO2 incubator. After Statistical Analyses incubation, PBMCs were treated with Monensin A (1 mg/mL, BD Descriptive and comparison tests were performed using Graph Golgi STOP, Thermo Fisher Scientific) for 6h at 37°C and stored Pad Prism, version 6 (GraphPad Software, Inc., San Diego, CA, overnight at 4°C. The next day, cells were stained with a USA). Non-parametric ANOVA (Kruskal Wallis) tests were used combination of T-cell lineage markers and functional markers for Groupwise comparisons and adjusted for multiple for immunophenotyping. In brief, cells were labelled with a comparisons (Dunn’s test). Wilcoxon matched-pairs signed- viability dye (APC-Cy7, Thermo Fisher Scientific) for 30 min at rank tests were used to compare parameters between sampling room temperature (RT) and surface stained for 30 min at RT with time points. Spearman’s rank correlation coefficient test was used anti-human antibodies for CD3 (A700, clone UCHT1, BD), CD4 for correlation analysis. In the analysis of T-cell subsets, median (FITC,clone OKT4, Biolegend), CD8 (V500, clone RPA-T8, BD), values within phenotypes were normalized to 100%. A CD45RA (BV786, clone HI100, BD), CCR7 (PE-CF594, clone multivariable regression analysis was used to compare groups, 150503, BD), CD27 (BV605, clone L128, BD), PD-1 (BV421, clone adjusting for age, gender, days from symptom onset, EH12.1, BD) and activation induced markers (AIM) CD25 (A647, comorbidities and comedications. The final model was clone BC96, Biolegend), OX40 (PE, clone Ber-ACT35, Biolegend) obtained by means of a stepwise variable selection procedure. and CD137 (PeCy7 clone 41BB, Biolegend). Afterwards, cells were For each model, the estimator with its confidence interval (CI) of fixed with Fix/Perm Buffer A (Thermo Fisher Scientific) for 15 95% was reported along with the p-value. The statistical package min at RT and stained intracellularly with Fix/Perm Buffer B and R (v3.6.3) was used for the analysis. antibodies for TNF (PE-Cy7, clone MAb11, BioLegend), IFN-g (BV711, clone B27, BD), and IL-2 (BV650, clone MQ1-17H12, BD) for 20 min at RT. Finally, cells were resuspended and fixed in RESULTS formaldehyde 1% and acquired on LSR Fortessa cytometer using FACSDiVa software (BD). Data analysis was performed using Non-Seroconvertors Present Skewed T FlowJo software version 10.0.7 (Tree Star, Ashland, OR, USA). Cell Distribution Towards Higher Naïve Specific gates were defined using fluorescence minus one (FMO) and Lower Effector CD8+ T Cells controls (Figure S1). Surface markers were measured in total CD4 Previous studies have demonstrated alterations in immunological + and CD8+ T cells. The CD4+ and CD8+ T-cell subsets were parameters following SARS-CoV-2 infection, including Frontiers in Immunology | www.frontiersin.org 5 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors lymphopenia with a marked decrease in CD8+ T cells and (Figure 2B). Despite the low activation, we observed CD4+ alterations in the frequency of CD8+ T-cell subsets compared to and to a lesser extent CD8+ T-cell proliferation in response to healthy donors (8, 34). We determined T cell subsets distribution S and NP in two out of two assessed NSC (Figure S3), supporting in NSC by staining for CD3, CD4 and CD8 markers and the functional capacity of antigen specific T-cells. differentiation markers (CD45RA, CCR7 and CD27) and The analysis of AIMs on CD8+ T cells did not reveal significant compared them MM and severe individuals (Figures 1A, S2A, differences between study groups despite a lower median B). We performed multivariate statistical analysis to account for frequency in NP CD137+ CD8+ T cells between NSC and the influence of differences in age, gender, DfSO to sample, co- Severe (0% vs 0.78%) (Figure 2B). In addition, to determine morbidities and treatments between groups. Analysis of CD4+ T- potential changes in activation over time we performed a cell subsets revealed lower levels of CD4+ TCM and TTM in NSC longitudinal follow-up of AIMs in six NSC individuals. As compared to MM (p=0.0347, p=0.0024 Figure 1B). No significant shown in Figure S2E, the frequencies of S and NP CD25+/ differences were observed regarding the distribution of any other OX40+ CD4+ T cells decreased or remained undetectable CD4+ T-cell subset between study groups (Figure 1B). between time points in all the NSC except for one individual, Interestingly, we observed marked differences in CD8+ T-cell who also displayed increased cytokine production in response to S. subsets, when comparing study groups (Figure 1C). Specifically, A comparable profile was observed for S and NP CD137+ CD8+ T NSC had significantly higher frequencies of CD8+ T naïve (TN, cells over time (Figure S2E). median 44.9% NSC vs 33.1% MM and 18.11% Severe; p=0.0119 Along with decreased T-cell functionality, changes in PD-1 and p=0.0250, respectively). Similarly, we observed significantly expression have been associated with severe COVID-19 (37). lower frequencies of CD8+ T effector cells (TEF) as compared with Consistently, our analysis of PD-1 demonstrated a significant MM and Severe (median 5.5% NSC vs 16.3% MM and 36% Severe; reduction in the frequency of PD-1 expressing T cells in NSC p=0.0263 and p=0.0025, respectively). The T-cell subset compared to Severe individuals in S- and NP-stimulated distribution observed in NSC was stable up to a median of 92.5 conditions (Figure 2D) in both CD4+ (S, p=0.0270 and NP, Days from Symptom Onset (DfSO) to this second sample p=0.0328) and CD8+ (S, p=0.0060, and NP, p=0.0258). Also, (Figures S2C, D). longitudinal follow-up revealed stable frequencies of PD-1 Then, we further investigated the association between the expression in CD4+ and CD8+ T cells in NSC a median of levels of CD8+ TN and TEF and age or DfSO to sample between 95.2 days (Figure S2F). Only we observed a decrease in the groups. Meanwhile, no significant correlation between age and frequency of PD-1+ CD4+ T cells following S-stimulation over frequencies of CD8+ TN nor TEF was found in NSC time (p=0.0313, Wilcoxon matched-paired signed-rank test, (Figures 1D, E). There was a significant negative correlation Figure S2F). Collectively, these data indicate lower levels of between frequency of CD8+ TN and age in MM and Severe AIMs in CD4+ T cells in response to S and low PD-1 expression groups (Spearman r=-0.6059, p=0.0107 and r=-0.6249, p=0.0107, in both CD4+ and CD8+ T cells in responses to S and NP SARS- respectively) (Figure 1D) and a significant positive correlation CoV-2 antigens as a specific immunological trait in NSC. between frequency of CD8+ TEF and age (r=0.5594, p=0.0324 and r=0.5556, p=0.0274) (Figure 1E) in the MM and Severe Functional SARS-CoV-2-Specific CD4+ groups. Also, no significant correlations were found between and CD8+ T Cell Responses in Non- DfSO to sample and CD8+ TN or TEF in any of the study groups Seroconvertors (Figures 1F, G). Taken together, these data indicate a skewed The importance of functional virus-specific T-cell responses in CD8+ T-cell subset distribution towards high levels of naïve and SARS-CoV-2 disease outcomes has been consistently reported low levels of effector cells independent of age in NSC. (17, 38). Furthermore, T-cell responses against S, NP and M SARS-CoV-2 proteins have been identified in Convalescent Non-Seroconvertors Display Lower Levels seronegative individuals (26, 27). To evaluate SARS-CoV-2 of T-Cell Activation-Induced Markers specific T-cell responses, we stimulated PBMCs with S and NP and PD-1 SARS-CoV-2 proteins and performed intracellular cytokine Next, we used PD-1 and activation-induced markers (AIM) to staining for IFN-g, IL-2 and TNF production (Figure 3). evaluate total T cell and SARS-CoV-2-specific T cell activation Analysis of S and NP-specific CD4+ T-cell responses revealed status (25, 35). AIMs have been used to identify antigen-specific antigen-specific cytokine production in all study groups CD4+ T cells via the detection of upregulated surface markers (Figure 3A). No significant differences were found in the following antigen stimulation (36). We assessed general T-cell frequency of S and NP-specific CD4+ T cells between NSC, activation and AIMs using antibodies directed against CD25, MM or Severe for any of the cytokines studied. However, MM OX40 and CD137 and PD-1 in unstimulated and S- and NP- displayed significantly lower NP-specific IFN-g production than antigen stimulated conditions (Figures 2A–C). We observed Severe individuals (p=0.0291). We observed lower median significantly lower expression of CD25+/OX40+ CD4+ T cells frequency of NP-specific CD4+ T cell responses in NSC vs in response to S in NSC compared to Severe (median 0.13% vs Severe individuals (0.11% vs 1.04% in TNF, 0.02% vs 0.36% in 0.82%, respectively, p=0.0358, Figure 2B). In addition, CD25+/ IFN-g and 0% vs 0.32% in IL-2, respectively; Figure 3A), despite OX40+ CD4+ T cells in response to NP were significantly lower no statistical differences. Similarly, S and NP-specific CD8+ T- in MM as compared to Severe (0.02% vs 0.85% p=0.0386) cell responses were present in all study groups in the absence of Frontiers in Immunology | www.frontiersin.org 6 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors A B C D FIGURE 2 | Expression of activation markers is lower in SARS-CoV-2 Non-Seroconvertors compared to seropositive individuals. PBMCs from Non-Seroconvertors and comparative study groups were stimulated for 17 hours with S and NP recombinant proteins and stained using antibodies directed against activation-induced markers (AIM; CD25, OX40, CD137) and PD-1, and analysed by flow cytometric analysis. Cells were pre-gated for lymphocytes and live cells. (A) Representative dot-plots of AIM expression in CD4+ and CD8+ T cells. (B) Bar graphs showing the expression of activation induced markers in CD4+ (CD25+/OX40+) and CD8+ T cells in response to S and NP (CD137+). Values from unstimulated cells were subtracted for each individual. (C) Representative dot-plots of PD-1 expression on CD4+ and CD8+ T cells. (D) Bar graphs of the expression of PD-1 on CD4+ and CD8+ T cells in unstimulated cells as well as following S and NP stimulation. Error bars represent the interquartile range and median values per group are shown. Statistical analysis was performed using Kruskal-Wallis non-parametric ANOVA adjusted for multiple comparisons, only significant differences are highlighted in the Figure. statistically significant differences between NSC and MM or We next assessed the polyfunctionality of S and NP-specific T Severe groups (Figure 3B). However, median frequencies of cells in NSCs and comparative study groups. Polyfunctionality IFN-g or TNF producing NP-specific CD8+ T cells were lower was assessed by Boolean gating (27). Visualization of cytokine in NSC vs Severe (0.08% vs 0.24% in TNF and 0.06% vs 0.25% for profiles in S and NP SARS-CoV-2-specific T cells was performed IFN-g, respectively). Significantly lower NP-specific IFN-g by UMAP analysis as represented in Figure S4. In response to S production was found between MM and Severe (p=0.0132) proteins, CD4+ T cells in NSCs displayed similar characteristics (Figure 3B). Next, we evaluated the frequency of T-cell to Severe, while MM had higher frequencies of bifunctional responders across study groups defined by the presence of and lower of trifunctional cells (Figure 3C). On the other either S or NP stimulated CD4+ or CD8+ T cells positive for hand, CD8+ T cells in response to S displayed higher one AIM or cytokine. Overall, the frequency of SARS-CoV-2 frequencies of monofunctional cells in NSC (90.2%) and lower responders were 87% in NSCs, 73% in MM, 94% in Severe, and frequencies of bifunctional cells compared with the other two 20% in healthy controls. Distributed by SARS-CoV-2 proteins, groups. Negligible frequencies in CD8+ trifunctional cells were we observed a frequency of S responders in 73% of NSCs, 73% of observed in all groups (Figure 3C). In response to NP proteins, MM, 94% Severe, and 20% of healthy controls. The frequency of CD4+ T cells of NSCs displayed similar characteristics to MM NP responders was 53% in NSCs, 50% in MM, 86% in Severe, whereas Severe had a higher proportion of bifunctional cells and 0% in healthy controls. These data are consistent with (Figure 3D). Interestingly, NSC had the highest frequencies of previous studies demonstrating the presence of SARS-CoV-2 trifunctional NP-specific CD8+ T cells, accounting for 14.9% reactive T cells in seronegative SARS-CoV-2 convalescent compared with MM and Severe (Figure 3D) and a trend toward individuals and 50% of unexposed individuals (24, 25, 35). higher trifunctional cells was observed in NSC compared to Frontiers in Immunology | www.frontiersin.org 7 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors A B C D FIGURE 3 | Functional T cells of SARS-CoV-2 Non-Seroconvertors are present in NSC in response to Spike and Nucleocapsid. Cryopreserved PBMCs from SARS- CoV-2 Non-Seroconvertors (NSC) and comparative study groups, Mild-moderate (MM), Severe, and controls samples were stimulated for 17 hours with Spike (S) and Nucleocapsid proteins (NP). An unstimulated condition was used as a control and values were subtracted. Cells were pre-gated for lymphocytes and live cells. Production of IFN-g, IL-2, and TNF was monitored by Flow cytometric analysis following intracellular cytokine staining. (A, B) Left panels depict the gating strategy for analysis of cytokine expression and right panels represent the frequencies of IFN-g, IL-2, and TNF in CD4+ (A) and CD8 (B) T cells in the study groups in response to S and NP. Pie charts demonstrate the relative proportions of T cells producing one, two or three cytokines in response to S in CD4+ T cells and CD8+ T cells (C). Relative proportions of T cells producing one, two or three cytokines in response to NP in CD4+ T cells and CD8+ T cells (D). Statistical analysis was performed using non-parametric ANOVA (Kruskal Wallis) adjusted for multiple comparisons. Error bars represent the interquartile range and median values per group are shown. Only significant differences are indicated in the Figure. Severe (p= 0.0642, Mann-Whitney test). By contrast, no major DISCUSSION differences in mono and bifunctional CD8+ T cells between groups were observed (Figure 3D). These results demonstrate While unprecedented advances have been made for the approval the presence of functional SARS-CoV2-specific T cells responses of COVID-19 vaccines with a high degree of efficacy (39–42), in NSC. ensuring protection from infection and long-lasting immune Frontiers in Immunology | www.frontiersin.org 8 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors protection and remain challenges for current SARS-CoV-2 rapid viral control. In addition, the low levels of CD8+ TEF vaccines. Severe COVID-19 has been related to increased age, observed in NSC could partially be explained by the presence of high levels of inflammatory markers and seroconversion (28). polyfunctional CD8+ T cells, overcoming the need for a large Persistent seroconversion and higher neutralizing titers have proportion of effector cells. been found consistently in hospitalized SARS-CoV-2 infected We investigated the expression of AIMs in CD4+ T cells and individuals (16, 43). CD8+ T cells, and PD-1 in response to S and NP antigens. Data Non-Seroconvertors (NSC) present generally asymptomatic from AIMs identified lower median frequencies of S CD25+/ infection being present at a frequency of 2-17% among SARS- OX40+ CD4+ T cells and a trend towards lower CD137+ CD8+ CoV-2 Convalescent individuals (25–27). Thus, presence of NSC T cells in response to NP antigen in NSC compare with Severe. support the relevance of alternative immune mechanisms to The higher frequencies of AIM+ T cells observed in Severe are humoral responses important in limiting SARS-CoV-2 likely to detect cytokine profiles outside of IFN-g, IL-2 and TNF pathogenesis. While previous studies demonstrated the presence studied and T regulatory cells as previously described (36). Also, of SARS-CoV-2 specific T-cell responses in SARS-CoV-2 NSC (25, the pro-inflammatory environment associated with COVID-19 27), and SARS-CoV-2 specific CD8+ T-cell responses have been severity may be involved in the increased immune activation very well characterized in a small fraction of those (26), to our found. Moreover, we observed decreased levels of PD-1 in CD4 knowledge a complete characterization of the NSC immune + and CD8+ T cells in NSC compared to Severe following SARS- phenotype is still missing. Here, we have conducted a detailed CoV-2 antigen stimulation. Elevated activation and exhaustion immune characterization in SARS-CoV-2 individuals focusing in markers have been found in T cells of severe COVID-19 patients NSC and compare with MM and Severe in terms of T-cell subset (37). Aside from elevated activation and exhaustion markers such distribution, activation and functionality in response to SARS-CoV- as PD-1, decreased functionality has been observed in T cells from 2 antigens. severe COVID-19 patients (37). Recently, SARS-CoV-2 specific- In the context of the KING extension cohort, we identified a CD8+ T cells expressing PD-1 were shown to be functional rather 4.5% of NSC using a very conservative approach; confirmed than exhausted, as they also produced IFN-g (50). Therefore, while SARS-CoV-2 PCR positivity, double ELISA testing and the PD-1 expressing cells in Severe may be functional, our data sampling-time estimation to allow for seroconversion after suggest that T cells of NSC can exhibit functionality with lower infection. Moreover, we confirmed the true nature of NSC in PD-1 and AIM expression levels our study by longitudinal clinical follow-up by the absence of Complementary to AIMs, we extended the characterization of SARS-CoV-2 IgG, IgM and IgA seroconversion. Of note, the T-cell immunity to functional SARS-CoV-2-specific CD4+ and NSC were enriched in mild to moderate SARS-CoV-2 infection CD8+ T-cell responses by cytokine production against S and NP cases and a high frequency of females. The bias of an increase in antigens. Overall, we did not observe differences in cytokine the frequency of females observed may indicate a specific trait of production in CD4+ and CD8+ T cells in response to S and NP the NSC phenotype as suggested by previous studies (44). This proteins between groups, marked interindividual variability observation is consistent with previous studies suggesting robust found may account for the lack of statistical differences. T-cell responses in females and less severe SARS-CoV-2 Targeting of T-cell responses toward S-, M- and NP-proteins in infection and death (45–47). COVID-19 patient samples has previously been reported to be In this study, we characterized T-cell immunity in NSC and mainly uniform across disease severities (51). However, in NSC, S compared it with SARS-CoV-2 infected individuals with IgG and NP-specific T cells appear to have a balanced profile of IFN-g, seroconversion stratified by disease severity. In terms of T-cell IL-2 and TNF production, while Severe exhibits higher median immunophenotype, we observed a skewed distribution of CD8+ frequencies of TNF producing cells, likely related to cytokine T cell subsets towards a higher frequency of naïve (TN) and perturbations following severe disease (11–13). Although NSC lower frequency of effector (TEF) CD8+ T cells in NSC after displayed tendencies towards lower median frequencies of SARS- adjusting for age, DfSO to sample, co-morbidities and CoV-2-specific T-cell responses, we described a trend toward comedications. We found a significant negative correlation higher levels of IFN-g+ IL-2+ TNF+ NP-specific CD8+ T cells in between age and CD8+ TN in MM and Severe groups that did NSC. In agreement with our findings, CD8+ T-cells were also not hold in NSC. These findings are in line with previous found to be mainly directed toward the NP (52) indicating that observations indicating a decline in the frequencies of naïve T this type of response may be significant for long-term disease cells with age and the association between low levels of naïve protection. Our data is also in line with previous findings showing CD8+ and increased risk of severity in COVID-19 (17, 48). The NP-specific CD8+ T cells directed against the immunodominant skewed distribution of CD8+ TN and TEF observed in NSC B7/N105 epitope were detected at high frequency in pre-SARS- support specific characteristics associated with T-cell CoV-2 samples, as well as in acute and convalescent COVID-19. A homeostasis in this group. Thus, the higher levels of CD8+ TN predominantly naïve phenotype with high TCR plasticity was cells in NSC could be the result of a continuous thymic observed among these cells in agreement with the higher repopulation of the naïve compartment, as observed in HIV-1 frequency of Naïve CD8+ found in NSC (53). These findings viremic non-progressors individuals (49). High levels of CD8+ have additional meaning considering the worldwide expansion of TN could also allow for rapid and efficient priming and SARS-CoV-2 variants of concern for vaccine efficacy (54–56) expansion of SARS-CoV-2-specific CD8+ T cells favoring where further analyses of the quality of antigen-specific CD4+ Frontiers in Immunology | www.frontiersin.org 9 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors and CD8+ T-cell responses may be of crucial importance to Pujol PI-20-217. The patients/participants provided their written recognize emerging SARS-CoV-2 variants. Also, these data informed consent to participate in this study. highlight the lower overall low expression of AIMs under the presence of functional SARS-CoV-2 T-cell responses in NSC. Our study has some limitations. First, we found a high AUTHOR CONTRIBUTIONS frequency of females in NSC studied. However, sex differences associated the phenotype may be taken into account. Females Conceptualization, JP, AK, MM, BC, CB, JC, and JB. have been described to seroconvert at a lower rate than men (44). Methodology, EJ-M, AK, OB-L, DO, JC, RP-C, MM, EM-C, Second, we used S and NP proteins as opposed to optimal BQ-S, IB, and RPe. Validation, MM, JC, JB, RPe, LM, EM-C, BQ- peptides for the study of antigen-specific T cells, which S, IB, AK, and JP. Resources, JP, BC, MM, JC, JB, RPa, LM, and potentially may underestimate the frequency of SARS-CoV-2 AC. Formal analysis, EJ-M, AK, OB-L, and DO. Investigation, -specific T cells. However, recombinant Spike protein has AK and JP. Data curation, MM, JB, JC, EMV, BQ-S, EJ-M, AK, previously been used to detect antigen-specific T cells (57). OB-L, and DO. Software, DO. Visualization, AK, OB-L, DO, and Third, our functional study is limited to Th1-type cytokines JP. Writing – original draft preparation, AK and JP. Supervision, and does not necessarily address the overall functionality of JP. Writing – review and editing, AK, JP, EJ-M, RP-C, EM-C, JB, antigen-specific T cells including degranulation markers such as MM, OB-L, JC, BQ-S, CB, and DO. Project administration, JP. CD017a (58). Finally, the range of sampling time in NSC was Funding acquisition, JP and BC. All authors contributed to the higher than optimal in comparison with the other groups. article and approved the submitted version. However no statistically significant differences in sampling time were observed. Parameters such as DfSO to sample, age, co-morbidities etc. were accounted for in the statistical analysis FUNDING of T-cell subset distribution. Moreover, it is tempting to speculate that pre-existing T cell This study is supported in part by grants from National Health immunity derived from previous exposure to other Institute Carlos III (ISCIII) COV20/00660, PI17/000164 to JP and coronaviruses mediate the NSC phenotype given the reports on by the CIBER-Consorcio Centro de Investigació n Biomé dica en pre-existing immunity in seronegative and or uninfected Red- (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia individuals (25, 26, 59). This would be in line with the e Innovació n and Unió n Europea – NextGenerationEU. The generation of a rapid T-cell response in the absence of funders had no role in study design, data collection and analysis, immune activation, it is also a plausible explanation of why the the decision to publish or drafting of the manuscript. OB-L was NSC can control SARS-CoV-2 in the absence of antibodies. supported by the grant for Catalan Government and the European Indeed, cross-reactive memory CD4+ T cells have been described Social Fund AGAUR-FI_B 00582 Ph.D. fellowship. This study has between SARS-CoV-2 responses and common cold received partial funding from Grifols and the crowdfunding coronaviruses in unexposed individuals (24). Cross-reactivity initiatives “https://www.yomecorono.com”, BonPreu/Esclat and to NP has previously been demonstrated in 30% of SARS- Correos. The funder was not involved in the study design, CoV-1 and SARS-CoV-2 unexposed individuals, and NP collection, analysis, interpretation of data, the writing of this amino acids 101-120 shared a high degree of homology to with article or the decision to submit it for publication. We thank NP amino acids in MERS-CoV, OC43 and HKU1 (60). “CERCA Programme/Generalitat de Catalunya” for institutional Collectively, these data strength our understanding of T-cell support and the Foundation Dormeur for financial support. immunity in the absence of seroconversion. Presence of skewed T-cell populations in the context of functional SARS-CoV-2 specific-T cells with low activation levels were specific traits of ACKNOWLEDGMENTS NSC. These findings further support the role of cellular We are deeply grateful to all participants and to the technical staff immunity in COVID-19 control, and the value of T -cell of IrsiCaixa for sample processing (L. Ruiz, E. Grau, R. Ayen, L. immune monitoring in populations with low seroconversion Gomez, C. Ramirez, M. Martinez, T Puig) and staff from the rates in response to SARS-CoV-2 infection and vaccination. Fight AIDS Foundation (R Toledo, A. Chamorro, J Puig). DATA AVAILABILITY STATEMENT SUPPLEMENTARY MATERIAL The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.815041/ full#supplementary-material ETHICS STATEMENT Supplementary Figure 1 | Gating strategy and control FMOs for the detection S and NP CD4+ and CD8+ T cells. (A) Gating strategy for CD4+ and CD8+ T cells, The studies involving human participants were reviewed and (B) Representative dot-plots summarizing the FMO strategy in CD4+ and CD8+ T approved by the Ethics Committee of Hospital Germans Trias i cells for TNF, IFN-g and IL-2 staining under several conditions FMO unstimulated, Frontiers in Immunology | www.frontiersin.org 10 May 2022 | Volume 13 | Article 815041 Kilpeläinen et al. Cellular Immunity in SARS-CoV-2 Non-Seroconvertors FMO in the presence of the positive control (SEB) and cells in the presence of Supplementary Figure 3 | Proliferation in CD4+ and CD8+ T cells of Non- antibodies and the positive control. seroconvertors and seropositive individuals with severe disease. (A) Representative dot-plots summarizing CFSE loss in CD4+ and CD8+ T cells following stimulation Supplementary Figure 2 | Longitudinal evolution of T-cell subsets and antigen with S and NP SARS-CoV-2 proteins in two NSC. Frequency of proliferating cells in specific responses in SARS-CoV-2 Non-Seroconvertors. T-cell markers were NSC and seropositve individuals in response to S and NP antigens. Values from assessed in two sampling timepoints in Non-Seroconvertors. (A) Gating strategy for unstimulated cells were subtracted for each individual (B). differentiation of subsets in CD4+ T cells, (B) and CD8+ T cells. Changes in T-cell subset distributions are represented by pie charts showing CD4+ and CD8+ T-cell subset distribution at sampling timepoint 1 and 2 (TP1 and TP2) (C) and graphs Supplementary Figure 4 | Cytokine expression in CD4+ and CD8+ T cells of representing the normalized subset values within phenotypes (D). Differences in Non-seroconvertors and seropositive individuals with severe disease. Single-cell Cytokine production and activation induced markers between TP1 and TP2 in analysis based on antigen expression (TNF, IL-2 and IFN-g intensity) using UMAP response to S and NP are shown in (E), and PD-1 expression in CD4+ and CD8+ T dimensionality reduction in CD4+ T-cells following stimulation with S (A) and NP (B) cells in unstimulated, S- and NP-stimulated cells at TP1 and TP2 are shown in (F). as well as CD8+ T cells stimulated with S (C) and NP (D). Who Develop Neutralizing Antibodies. Cell Rep (2021) 34(11):108852. REFERENCES doi: 10.1016/j.celrep.2021.108852 1. 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Age- this article, or claim that may be made by its manufacturer, is not guaranteed or And Sex-Associated Variations in the Sensitivity of Serological Tests Among endorsed by the publisher. Individuals Infected With SARS-CoV-2. JAMA Netw Open (2021) Feb 1;4(2): e210337. doi: 10.1001/jamanetworkopen.2021.0337 Copyright © 2022 Kilpeläinen, Jimenez-Moyano, Blanch-Lombarte, Ouchi, Peña, 45. Takahashi T, Ellingson MK, Wong P, Israelow B, Lucas C, Klein J, et al. Sex ́ Quirant-Sanchez, Perez-Caballero, Chamorro, Blanco, Martınez-Caceres, Paredes, Differences in Immune Responses That Underlie COVID-19 Disease Mateu, Carrillo, Blanco, Brander, Massanella, Clotet and Prado. This is an open- Outcomes. Nature (2020) 588:315–20. doi: 10.1038/s41586-020-2700-3 access article distributed under the terms of the Creative Commons Attribution 46. Jin J-M, Bai P, He W, Wu F, Liu X-F, Han D-M, et al. Gender Differences in License (CC BY). The use, distribution or reproduction in other forums is permitted, Patients With COVID-19: Focus on Severity and Mortality. Front Public Heal provided the original author(s) and the copyright owner(s) are credited and that the (2020) 8:152. doi: 10.3389/fpubh.2020.00152 original publication in this journal is cited, in accordance with accepted academic 47. Vahidy FS, Pan AP, Ahnstedt H, Munshi Y, Choi HA, Tiruneh Y, et al. Sex practice. No use, distribution or reproduction is permitted which does not comply with Differences in Susceptibility, Severity, and Outcomes of Coronavirus Disease these terms. Frontiers in Immunology | www.frontiersin.org 12 May 2022 | Volume 13 | Article 815041