key: cord-1022151-t41imaw6
authors: QI, M.; Le Bert, N.; Chan, W.; Tan, M.; Hang, S. K.; Hariharaputran, S.; Sim, J. X. Y.; Low, J.; Ng, W. L.; Wan, W. Y.; Ang, T. L.; Bertoletti, A.; Salazar, E.
title: Favourable vaccine-induced SARS-CoV-2 specific T cell response profile in patients undergoing immune-modifying therapies
date: 2022-02-23
journal: nan
DOI: 10.1101/2022.02.21.22271127
sha: e382d1dab814b4d91d8e5e939272a81909881b8f
doc_id: 1022151
cord_uid: t41imaw6

Patients undergoing immune-modifying therapies demonstrate a reduced humoral response after COVID-19 vaccination, but we lack a proper evaluation of the impact of such therapies on vaccine-induced T cell responses. Here, we longitudinally characterised humoral and Spike-specific T cell responses in IBD patients who are on antimetabolite therapy (azathioprine or methotrexate), TNF inhibitors and/or other biologic treatment (anti-integrin or anti-p40) after mRNA vaccination. We demonstrated that a Spike-specific T cell response is not only induced in treated IBD patients at levels similar to healthy individuals, but also sustained at higher magnitude, particularly in those treated with TNF inhibitor therapy. Furthermore, the Spike-specific T cell response in these patients is mainly preserved against mutations present in SARS-CoV-2 B.1.1.529 (Omicron) and characterized by a Th1/IL-10 cytokine profile. Thus, despite the humoral response defects, the favourable profile of vaccine-induced T cell responses might still provide a layer of COVID-19 protection to patients under immune-modifying therapies.

Immune-modifying agents are the treatment of choice for different chronic inflammatory diseases of autoimmune origin. Antimetabolites (azathioprine and methotrexate) or biologics, such as TNF inhibitors (adalimumab and infliximab), anti-p40 (ustekinumab) or anti-integrin (vedolizumab and etrolizumab) antibodies, are used alone or in combination to reduce inflammatory events in the gut (i.e. Crohn's disease or ulcerative colitis), skin (i.e. psoriasis), joints (i.e. rheumatoid arthritis), or in multiple systems (i.e. systemic lupus erythematosus). While these agents reduce disease burden and improve quality of life (1) , they are broadly considered immunosuppressive. The COVID-19 pandemic and the necessity to implement widespread vaccination sparked debate and research on the impact of these chronic therapies on the immunogenicity of SARS-CoV-2 vaccination (2) (3) (4) .

Others have already shown that these therapies, particularly TNF inhibitors, reduce the ability of different COVID-19 vaccines (based on mRNA or Adenoviral vector) to produce Spike-specific antibodies (5) (6) (7) (8) and recognize SARS-CoV-2 variants including B.1.617.2 (Delta) (4) . Such results are expected, since reduced humoral responses to other vaccines (i.e. anti-pneumococcal, anti-HBV) was already demonstrated in patients similarly undergoing TNF inhibitor or other antimetabolite therapy (9-11), and since TNFalpha has been demonstrated to play an important role in the coordinate maturation of humoral immunity (12).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. While these T cells might not play a role in preventing infection, their ability to recognize and lyse virus-infected cells likely represents an important antiviral mechanism that might prevent the unchecked spread of SARS-CoV-2 in the infected host (24). However, the impact that the different immune-modifying therapies exert on vaccine-induced Spikespecific cellular immunity has only started to be analysed (25), with initial evidence of preserved cellular immunity levels at least immediately after vaccination.

In this manuscript, we therefore studied a cohort of patients with inflammatory bowel diseases (IBD) who are antimetabolites (AM), TNF inhibitor (TNFi) and/or other biologic treatment (anti-integrin and anti-p40), and we characterized both cellular and humoral vaccine-induced Spike-specific immunity. Spike-specific immune responses were analysed from pre-vaccination, up until 3 months following the second dose of COVID-19 mRNA vaccination (BNT162b2 or mRNA-1273). Importantly, we designed an . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint experimental plan to investigate not only the ability of vaccine to elicit "classical" Spikespecific T cell responses producing Th1 cytokines (IFN-γ and IL-2), but also the antiinflammatory/regulatory IL-10 cytokine. The rationale of such an experimental design was based on data demonstrating that TNFi therapy mediates induction of IL-10 in T cells that likely contribute to its ability to dampen inflammation (26, 27).

The ability to modify the functional profile of classical Th1 T cells can be of particular importance in SARS-CoV-2 infection. The presence and induction of IL-10/IFN-γ producing SARS-CoV-2 specific T cells is associated with asymptomatic SARS-CoV-2 infection (28) and hybrid immunity (29), while their absence has been reported in severe COVID-19 (30). The induction of such T cells endowed with anti-inflammatory potential might be advantageous in the asymptomatic control of SARS-CoV-2 infection.

Furthermore, due to the pervasion of the Omicron variant globally, the ability of vaccineinduced Spike-specific T cells to tolerate the amino acid mutations characteristic of the Omicron variant necessitates evaluation. We therefore tested directly ex vivo the impact that Omicron variant mutations exert on the Spike-specific T cells induced in IBD patients under different treatments.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint

During the study period, 83 patients had at least one blood sample analysed. Forty-three patients completed at least three visits while the rest were either lost to follow up or recruited after first or second vaccination. Of the 83 patients, 57 (69%) patients had Crohn's disease and 26 (31%) had ulcerative colitis. Fifty three (64%) IBD patients and 18 (36%) HC were male (p=0.003), while median ages (years) in IBD (39) and HC group (40.5) were similar (p=0.65). Forty (48%) patients were on TNFi and 43 (52%) patients were on other non-TNFi immunotherapy. Baseline characteristics including duration of IBD diagnosis (years), disease phenotype and behaviour, and vaccine taken between TNFi and non-TNFi groups were similar as shown in Table 1 . Among those undergoing TNF inhibitor therapy, 22 (55%) had an additional antimetabolite. Those on other non-TNFi consisted of 15 (35%) on an antimetabolite only, 7 (16%) on anti-p40 monotherapy and 9 (21%) on anti-integrin monotherapy. Those on an additional antimetabolite include 9 (21%) on anti-p40 (anti-IL-12/23) + AM and 3 (7%) on anti-integrin therapy + AM; those on anti-integrin + AM were excluded from analysis due to the lack of data points. Six patients were on concomitant steroid; 2 in the TNFi group and 4 in the non-TNFi group (p=0.74). Eight from the TNFi group and 6 patients from the non-TNFi group took the mRNA-1273 (Moderna) vaccine (p=0.66).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint

RBD IgG levels were quantified in response to COVID-19 mRNA vaccination both in the HCs and the IBD cohort ( Figure 1A ). In line with previous observations (5- 

The magnitude and function of the Spike-specific T cell response induced by vaccination in HC and IBD patients was characterized directly in whole blood. A pool of 15-mer peptides covering the immunogenic regions of the SARS-CoV-2 S-protein (S pool) was used to measure Spike-specific T cell responses (Table S1 ). The quantity of Th1 cytokines (IFN-γ and IL-2) secreted in the plasma after peptide stimulation was quantified . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint 8 after overnight incubation ( Figure 2A ). This rapid quantitative assay has been demonstrated to possess identical sensitivities/specificities of conventional ELISpot assays (31).

Before vaccination, whole blood supernatants of HC and IBD patients stimulated with S pool displayed median IFN-γ and IL-2 levels below threshold. Some whole blood supernatants from either cohort demonstrate cytokine production higher than We then analysed cytokine levels induced in IBD patients under different treatment regimens. Notably, the durability of potential for cytokine induction is demonstrated broadly in the IBD cohort ( Figure 3A) . Furthermore, as shown in Figure 3B , patients treated with TNFi alone demonstrated not only more stable but also higher levels of IFNγ and IL-2 responses than HC. Although few, we also noted that patients treated with anti-p40 biologics present high median levels of IL-2 production (n=7, GMean 229.6 pg/mL). Importantly, no specific treatment caused an inhibition of the quantity of IFN-γ and IL-2 3 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

To confirm that COVID-19 mRNA vaccination induces Spike-specific CD4+ and CD8+ T cells in IBD patients undergoing immune-modifying therapy, IBD patient PBMCs (n=7) collected at different time points were stimulated with Spike peptides pool and analysed for expression of activation markers on gated CD4+ and CD8+ T cells. Clear populations of CD4+ and CD8+ T cells were visualized and quantified upon peptide pool stimulation in 7 out of the 7 tested IBD patients ( Figure 4A ).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint

We then analysed the impact of the mutations that characterize the Spike protein of the Omicron variant on the vaccine-induced Spike-specific T cells present in IBD patients ( Figure 4B ). Sample availability restricted our study to a subgroup of IBD patients (n=14).

Patient PBMCs were stimulated with three peptide pools covering the entire Spike protein (253 peptides) of the ancestral SARS-CoV-2 (Table S2) (Table S3) . We performed an IFN-γ ELISpot assay to quantify the frequency of SARS-CoV-2 specific T cells responding to conserved regions of the Spike protein, and to derive the frequency of responses altered by the variant-defining regions in the Omicron variant ( Figure 4B ). As already seen in healthy vaccinated individuals (20, 21), the Spike-specific T cell response to the Omicron variant is mainly preserved in the majority of patients irrespective of their treatment. Most Spike-specific T cells appear to target conserved regions, and an inhibition of more than 25% of the total Spike-specific T cell response due to Omicron mutations was observed in only 1 out of the 14 IBD patient sample tested.

Differences in the contraction kinetics of Spike peptide-induced IFN-γ and IL-2 detected in IBD patients undergoing TNFi therapy suggest that this treatment might modify vaccine-induced Spike-specific T cells. In addition, TNFi therapy has been shown to modify T cell function through expression of a transcriptional signature that upregulates IL-10 production in T cells (27). We therefore tested whether cytokine secretion profiles in whole blood supernatants after Spike-peptide stimulation contains not only classical . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint Th1 cytokines IFN-γ and IL-2, but also IL-10. The quantity of IL-10 detected in IBD treated patients and HC before and after two dose vaccination was measured ( Figure 5 ).

At both timepoints following first (D 21) and second dose vaccination (D 36), increased concentrations of IL-10 was detected in whole blood supernatants of HC and IBD patients relative to their respective pre-vaccination baselines. Furthermore, at 3 months after the second vaccine dose (D 115), no IL-10 could be detected in the majority of HC while we noticed a further increase in IL-10 induction in IBD patients ( Figure 5A ). This peak value (9.65 pg/mL), quantified at D 115, was low in comparison to corresponding IL-2 (61.5 pg/mL) and IFN-γ (31.5 pg/mL) responses in IBD patients.

To characterize the chronological evolution of cytokine profiles, we used UMAP to integrate quantified, log-transformed IL-10 data with IFN-γ and IL-2 for each donortimepoint ( Figure 5B ). UMAP projections of datapoints originating from pre-vaccination samples of either HC or IBD patients formed a distinct cluster. Moreover, the datapoints co-segregated following first dose (D 21) and two weeks after second dose vaccination (D 36), further highlighting the similarities of cellular responses between the two cohorts.

Notably, 3 months after completion of the two-dose regimen (D 115), the cytokine profiles diverge into distinct clusters, with IBD patient profiles coinciding with regions defined by higher IL-10, IFN-γ and IL-2.

To then confirm that Spike peptide pool stimulation induces IL-10 production in T cells, we performed direct ex vivo intracellular staining of T cells from TNFi treated patients ( Figure 5C ). Indeed, the low magnitude of cumulative IL-10 we observed in whole blood stimulated supernatants implied the identification of IL-10+ T cells to be a technically . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint 12 challenging feat, but a distinct population of IL-10+ cells was detected only in CD4+ T cells upon Spike peptide pool stimulation.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint

The attenuated humoral responses detected in SARS-CoV-2 vaccinated patients under different immune-modifying treatments, particularly in those treated with TNFi therapy, have generally been interpreted to imply reduced vaccine immunogenicity, fuelling debate on the possible increased risk of severe COVID-19 in patients treated chronically with these immune-modifying therapies (2, 3, 6, 7). Here, by studying IBD patients under various regimens and vaccinated with the prevailing Spike-based mRNA vaccines, we demonstrated that a Spike-specific T cell response is not only induced in IBD treated patients to levels similar to that in HC, but persists longer and at higher levels in the ones treated with TNFi.

In contrast to the role of antibodies, T cells cannot prevent infection; instead, they excel in the clearance of intracellular pathogens either through recognition and lysis of infected cells or activating macrophages, and support B cell maturation (32). Furthermore, since coordination between humoral and cellular arms of immunity is likely to be essential for rapid viral control and reduced pathogenicity (17), we cannot claim that the increased T cell immunogenicity directly translates into better protective efficacy of vaccination in patients under immune-modifying therapies. Nevertheless, these patients, particularly those undergoing TNFi therapy, are clearly able to mount a robust Spike-specific cellular immunity. Additionally, TNFi therapy does not abolish, but only reduces production of antibodies after mRNA vaccination. Previous observations and our own data show this.

It is possible therefore to hypothesize that the presence of cellular immunity against Spike compensates for the observed humoral defect.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. There are some limitations in this study: the most important one being that the bulk of T cell experiments were performed not by direct measurement of T cell quantity, but by measuring cytokines secreted in whole blood after specific peptide stimulation. However, we provide direct evidence orthogonally that Spike-specific CD4+ and CD8+ T cells were induced by vaccination in IBD patients and visualized IL-10+ T cells. Nevertheless, while this method does not directly quantify the number of T cells, it provides a standardised . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint method well suited for longitudinal analysis of T cell responses in patients under different treatments. The simplicity of the assay reduces the inter-assay variability, and is directly performed on fresh whole blood, limiting the detrimental effects of freezing and thawing (43). Furthermore, since T cell functionality is analysed in whole blood, the immunemodifying therapies administered into the patients are present at therapeutic levels during the assay, mimicking, as we previously argued (44), more closely the situation in vivo. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint

This is a prospective, observational study conducted to assess both humoral and cellular . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint

Measurements were performed using the Abbott Architect i2000 automated analyser using the SARS-CoV-2 IgG II Quant assay, a chemiluminescent microparticle immunoassay (CMIA) for the quantitative detection of IgG targeting the receptor binding domain (RBD) of the S1 subunit of the spike protein of SARS-CoV-2. Results are expressed as AU/mL, where values ≥50.0 AU/mL are interpreted as positive.

We used a cytokine release assay (CRA) of whole peripheral blood stimulated using a SARS-CoV-2 spike-derived peptide (S) pool (Table S1) described previously (31). Freshly drawn whole blood (320µL; within 6 hours of venepuncture) was mixed with 80µL RPMI and stimulated with S pool peptides to a final peptide concentration of 2µg/mL or mixed with an equivalent amount of DMSO as control. Culture supernatants were collected 16 hours after culture and stored at −80°C until cytokine quantification. IFN-γ/IL-2 or IL-10 concentrations in plasma were quantified using an Ella machine (ProteinSimple) with microfluidic multiplex cartridges following the manufacturer's instructions. Background cytokine levels quantified from DMSO controls were subtracted from the corresponding peptide pool stimulated samples. The threshold for a positive response was set at 10 times the lower limit of quantification for each cytokine (IFN-γ = 1.7 pg/mL; IL-2 = 5.4 pg/mL; IL-10 = 5.8 pg/mL). A pseudocount of 1 pg/mL was applied to the dataset for logistic transformation. Subsequently, log-transformed concentrations of each cytokine in . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint all culture supernatants were projected onto UMAP space using 15 nearest neighbours (nn), min_dist of 0.2 and Euclidean distance.

(1:1) were separated by Ficoll-Paque density gradient centrifugation. PBMCs were frozen in FBS containing 10% DMSO and stored in liquid nitrogen until use.

ELISpot plates (Millipore) were coated with human IFN-γ antibody overnight at 4℃. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint

We directly tested donor PBMCs by IFN-γ ELISpot for reactivity against the ancestral or Omicron variant Spike protein. To quantify total responses to the ancestral Spike, we used a 10-amino acid overlapping 15-mer peptide pool (SP-MP) covering the entire Spike protein listed in Table S2 (1273 amino acids). For Omicron variant Spike responses, we designed two peptide pools (Table S3) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint PE/Dazzle594, anti-CD134-PerCP-Cy5.5 and anti-CD-137-PE-Cy5 diluted in FACS buffer (RT for 25 minutes). Dead cells were excluded using the Fixable Yellow Live/Dead fixable cell stain kit (Invitrogen). After 2 more washes in FACS buffer, the cells were resuspended in PBS + 1% FA prior to analysis. The gating strategy is outlined in Figure   S1 , and the staining reagents used are outlined in Table S4 . Cells were then washed with Perm/Wash (BD) solution prior to intracellular staining with anti-IL-10-BV421 diluted in Perm/Wash. After 2 more washes in FACS buffer, the cells were resuspended in PBS + 1% FA prior to analysis. The gating strategy is outlined in Figure S1 , and the staining reagents used are outlined in Table S4 .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Data from flow cytometry was analyzed using FlowJo software (BD).

The study protocol was reviewed and approved by Institutional Research Board of Singhealth (IRB no: 2021/2398). All donors provided written consent for enrolment.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint

We also would like to thank Abigail Yeong,Tan Hui Fang and Stephanie Ren for coordinating the logistics of the study and follow-ups for all the study participants. Lastly we would like to thank all our patients who took their time off to participate in the study. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 23, 2022. 

STRIDE-II: An Update on the Selecting Therapeutic Targets in Inflammatory Bowel Disease (STRIDE) Initiative of the International Organization for the Study of IBD (IOIBD): Determining Therapeutic Goals for Treat-to-Target strategies in IBD

SARS-CoV-2 vaccination for patients with inflammatory bowel disease: a British Society of Gastroenterology Inflammatory Bowel Disease section and IBD Clinical Research Group position statement

SARS-CoV-2 vaccination for patients with inflammatory bowel diseases: recommendations from an international consensus meeting

Reduced antibody activity against SARS-CoV-2 B.1.617.2 delta virus in serum of mRNA-vaccinated individuals receiving tumor necrosis factor-α inhibitors

Infliximab is associated with attenuated immunogenicity to BNT162b2 and ChAdOx1 nCoV-19 SARS-CoV-2 vaccines in patients with IBD

Lower Serologic Response to COVID-19 mRNA Vaccine in Patients With Inflammatory Bowel Diseases Treated With Anti-TNFα

International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity

Mapping and role of T cell response in SARS-CoV-2-infected mice

Effector T cells control lung inflammation during acute influenza virus infection by producing IL-10

Prior SARS-CoV-2 infection rescues B and T cell responses to variants after first vaccine dose

Association of Prior SARS-CoV-2 Infection With Risk of Breakthrough Infection Following mRNA Vaccination in Qatar

TNF-α Blockers Showed Prophylactic Effects in Preventing COVID-19 in Patients with Rheumatoid Arthritis and Seronegative Spondyloarthropathies: A Case-Control Study

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint Geometric means (GMean) and number of data points (n) are indicated below each group . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint 40 IFN-γ and IL-2 quantities measured from each donor-timepoint. Top three panels display each point filled according to log10-transformed cytokine quantities (pg/mL). Bottom four panels display points filled according to study cohort at the respective timepoint. A shape is drawn enclosing a region mostly containing points with IL-10 values greater than 10 pg/mL. (C) Intracellular cytokine staining of donor PBMCs in CD3-or CD3+ (CD4+ and CD8+) compartments to identify IL-10+ cells with or without stimulation with Spike peptides . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Anti-p40+AM 9 (20.9) Anti-integrin+AM 3 (6.98)Corticosteroid therapy (N%) 2 4 0.74 † † Chi-squared test . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint 43 * Wilcoxon signed rank test . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.21.22271127 doi: medRxiv preprint