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IL-10 suppresses T cell expansion while promoting tissue-resident memory cell formation during SARS-CoV-2 infection in rhesus macaques [1]

['Christine E. Nelson', 'T Lymphocyte Biology Section', 'Laboratory Of Parasitic Diseases', 'National Institute Of Allergy', 'Infectious Disease', 'National Institutes Of Health', 'Bethesda', 'Maryland', 'United States Of America', 'Taylor W. Foreman']

Date: 2024-07 

The regulation of inflammatory responses and pulmonary disease during SARS-CoV-2 infection is incompletely understood. Here we examine the roles of the prototypic pro- and anti-inflammatory cytokines IFNγ and IL-10 using the rhesus macaque model of mild COVID-19. We find that IFNγ drives the development of 18 fluorodeoxyglucose (FDG)-avid lesions in the lungs as measured by PET/CT imaging but is not required for suppression of viral replication. In contrast, IL-10 limits the duration of acute pulmonary lesions, serum markers of inflammation and the magnitude of virus-specific T cell expansion but does not impair viral clearance. We also show that IL-10 induces the subsequent differentiation of virus-specific effector T cells into CD69 + CD103 + tissue resident memory cells (Trm) in the airways and maintains Trm cells in nasal mucosal surfaces, highlighting an unexpected role for IL-10 in promoting airway memory T cells during SARS-CoV-2 infection of macaques.

Here we examine the roles of the prototypic pro- and anti-inflammatory cytokines IFNγ and IL-10 during SARS-CoV-2 infection of rhesus macaques. Whole body 18 FDG-PET-CT imaging showed that IFNγ promotes SARS-CoV-2 induced pulmonary disease and IL-10 dampens the size, activity, and duration of lung lesions induced after infection. We also find a major role for IL-10 in the regulation of SARS-CoV-2-specific T cell responses. Our data show that IL-10 limits the magnitude of the effector T cell clonal burst during the acute phase of infection. We also find that following clearance of the virus, IL-10 promotes the differentiation of lung effector T cells into CD69+CD103+ tissue resident memory cells.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.S. is a consultant for Gritstone Bio, Flow Pharma, Moderna, AstraZeneca, Qiagen, Avalia, Fortress, Gilead, Sanofi, Merck, RiverVest, MedaCorp, Turnstone, NA Vaccine Institute, Gerson Lehrman Group and Guggenheim. LJI has filed for patent protection for various aspects of T cell epitope and vaccine design work. All other authors have no competing interests to disclose.

Funding: D.L.B. is supported by the Division of Intramural Research/NIAID/NIH (1ZIAAI001294-04). The content of this publication does not necessarily reflect the views or policies of DHHS, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

In this rhesus macaque model of mild SARS-CoV-2 infection, we find that IL-10 and IFNγ have opposing effects on the development of lung lesions quantified with 18 FDG-PET/CT imaging without an appreciable effect on SARS-CoV-2 replication. We identify a key role for IL-10 in negatively regulating the clonal expansion of virus-specific CD4 and CD8 T cells. Unexpectedly, we also find that IL-10 drives the differentiation of newly recruited airway effector CD4 and CD8 T cells into tissue resident memory T cells (Trm) after resolution of the infection and has a role in maintaining Trm in the nasal mucosa.

Negative immune regulation also likely also has a key role in determining the outcome of coronavirus infection. In mice, the anti-inflammatory cytokine IL-10 has a protective role in coronavirus induced encephalitis [ 26 – 29 ]. In humans with SARS-CoV-2 infection IL-10 has been associated with severe COVID-19 in multiple studies [ 10 , 30 , 31 ] IL-10 is upregulated early in disease progression, and along with IL-6, is a predictive biomarker for poor COVID-19 outcomes [ 10 , 30 ]. However, a study in children reported that higher plasma IL-10 levels were correlated with decreased viral measurements in nasal aspirates [ 32 ]. The mechanistic role of IL-10 and IFNγ in the rhesus macaque model of mild SARS-CoV-2 infection has yet to be determined.

Type I and III interferons have a demonstrated role in control of SARS-CoV-2 infection [ 4 – 7 ]. The contribution of type II IFN, IFNγ, to protection or pathology during COVID-19 is less well understood. IFNγ and molecules induced by IFNγR signaling (e.g., CXCL10) have been associated with severe COVID-19 and the development of acute respiratory distress syndrome [ 8 – 18 ]. Elevated levels of IFNγ also strongly correlate with the development of multi-system inflammatory syndrome in children after SARS-CoV-2 infection [ 13 , 19 , 20 ]. In ACE2 transgenic mice, neutralizing IFNγ along with TNF reduced mortality of severe SARS-CoV-2 infection [ 12 , 21 ]. However, IFNγ may also contribute to host-protection. IFNγ has been shown to inhibit SARS-CoV-2 replication in vitro [ 22 ]. Administration of IFNγ to immunocompromised individuals with severe COVID-19 resulted in rapid declines in SARS-CoV-2 viral loads [ 23 ]. In the mouse model, IFNγ is required for non-specific protection against SARS-CoV-2 observed after intravenous inoculation with the tuberculosis vaccine Bacillus Calmette–Guérin (BCG) [ 24 , 25 ]. Thus, IFNγ could contribute to protection or lung pathology during SARS-CoV-2 infection depending on the context.

SARS-CoV-2 infection has a spectrum of clinical disease outcomes, ranging from asymptomatic to fatal. The severity of COVID-19 is largely determined by the degree of virus-induced damage and immune-mediated pathology [ 1 ]. However, the factors that prevent or promote pulmonary inflammation during SARS-CoV-2 infection are not well understood. Rhesus macaques experimentally infected with SARS-CoV-2 develop mild signs of disease and clear most of the virus within a couple weeks [ 2 , 3 ]. Accordingly, this species is a useful model for examining the mechanisms of effective viral control and well-controlled inflammatory response that occurs in most individuals with SARS-CoV-2 infection. Here we use rhesus macaques to examine the roles of prototypic pro- and anti-inflammatory cytokines IFNγ and IL-10, respectively, in host resistance to SARS-CoV-2 infection and the development of COVID-19 disease.

Results

18FDG-PET/CT analysis of lung inflammation To investigate the role of pro- and anti-inflammatory cytokines in viral replication and pathogenesis during SARS-CoV-2 infection, we treated 15 male rhesus macaques (n = 5/group) with rhesus-modified, effector-silenced (LALA) monoclonal antibody targeting IL-10 (anti-IL-10 IgG1), a rhesus macaque IFNγR1-immunoglobulin fusion protein (rmIFNγR1-Ig), or isotype control (rhesus anti-DSP IgG1) by the intravenous (i.v.) route (Fig 1A). The LALA mutation prevents binding to Fc-gamma receptors and eliminates effector functions against antibody bound targets [33]. One day after treatment, animals were infected with a total of 2x106 TCID 50 of SARS-CoV-2/USA-WA-1 split between the intranasal (i.n.) and intratracheal (i.t.) routes. An additional dose of blocking reagent was given on day 3 post-infection. Anti-IL-10 and rmIFNγR1-Ig reagents were validated to efficiently block IL-10 and IFNγ signaling in vitro using functional cytokine signaling reporter cell lines (S1 Fig). Increased levels of circulating IL-10 after administration of the blocking antibody suggested the formation of antibody/cytokine complexes and persistent drug activity throughout the length of the study, however increased IFNγ was less apparent in the plasma of animals receiving the IFNγR-Ig fusion reagent (S1 Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 1. SARS-CoV-2 induced lung inflammation is increased with IL-10 blockade and decreased with IFNγ blockade. (A) Experimental design: Fifteen male rhesus macaques, with n = 5 per group: IgG isotype control, anti-IL-10, or anti-IFNγ (rmIFNγR1-Ig). Animals were treated with 10mg/kg of monoclonal antibody i.v. one day prior to infection and three days after infections with SARS-CoV-2/USA/WA-1 at a dose of 2x106 TCID 50 , administered intranasal (i.n.) and intratracheal (i.t.). Sampling was performed at the indicated timepoints. Individual animal IDs are indicated and used throughout. (B) 3D rendering of representative lung 18FDG-PET/CT images from baseline and 6 post infection from isotype control animal (DHDI). (C) Number of lesions per animal (left axis, points) and average number of lesions per group (right axis, grey bars). Significance calculated with individual t-test with Welch’s correction for lesions per animal. (D) Quantification of FDG uptake in standard uptake value (SUV) normalized to muscle, and volume of individual lesions (size of dot), based on volume of interest (VOI) > -550 Hounsfield units (HU) defined at days 2 or 6 post-infection. Significance was calculated with individual t-test with Welch’s correction for FDG uptake at day 6 between groups and Tukey’s multiple comparison test of day 2 vs. day 6 within each group. (E) Lesion score for individual lesions calculated as the sum of normalized max FDG uptake, normalized max Hounsfield’s units, and normalized max volume. Significance calculated with Dunn’s multiple comparison test. (F) Example PET/CT images showing pulmonary lymph node FDG signal from baseline, day 2, 6, 10, and 21–24 post-infection from isotype control animal DHNC. Orange arrows indicate lymph nodes and blue arrow indicates a lung lesion. (G) Quantification of metabolic activity of lymph nodes as measured FDG uptake in SUV, normalized to muscle. All time points post-infection were statistically significant over baseline by 2-way ANOVA and Tukey’s multiple comparison test. (H) Example PET/CT images with evidence of FDG signal from spleen, nasal turbinates, and tonsils from baseline and day 6 post infection. Animal IDs are embedded in image. DHBA and DHNC (isotype). DHMC (anti-IL10). (I) Quantification of change in FDG uptake (SUV) calculated as change from baseline for each animal with detectable signal from spleen, nasal turbinates, and tonsils. Significance calculated by 2-way ANOVA and Tukey’s multiple comparison test. (J) Plasma fibrinogen levels in mg/dL. Significance calculated with 2-way ANOVA and a Dunnett’s multiple comparison test. (K) Plasma C-reactive protein (CRP) in mg/L. Limit of detection >5mg/L. Significance calculated with a 2-way ANOVA and a Dunnett’s multiple comparison test. Panel A generated in part with BioRender.com. https://doi.org/10.1371/journal.ppat.1012339.g001 We monitored SARS-CoV-2 induced inflammation by 18Flurorine deoxyglucose (18FDG)-positron emission tomography/computed tomography (PET/CT) imaging of the head, chest, and abdomen. In control animals, we observed evidence of lung inflammation with increased density and 18FDG-avidity that peaked at day 2 after SARS-CoV-2 infection and which resolved by days 6–10 post-infection, consistent with previous findings (Figs 1B–1E and S2) [2,34,35]. Lung lesions were primarily peripheral ground glass opacities and consolidations, as well as peri-bronchial consolidations that were 18FDG-avid (SUV mean >1.5) [35]. Animals receiving IL-10 blocking antibody had an increased number of lesions that were more metabolically active on day 6 post-infection, as compared to isotype controls or rmIFNγR1-Ig treated animals (Fig 1C and 1D). Each lesion was given an intensity score based on the sum of the normalized maximum values for lesion size, FDG uptake, and density in Hounsfield’s units (HU) (Fig 1E). Lesions from anti-IL-10 treated animals had increased lesion intensity scores compared to controls. At day 6 post-infection when most of the inflammation had resolved in control animals, lesions in the anti-IL-10 treated animals still had significant 18FDG uptake and some lesions had increased in intensity from that observed at day 2 post-infection (Fig 1D). Conversely, animals that received rmIFNγR1-Ig tended to have decreased numbers, size, and density of lung lesions as compared to isotype controls or anti-IL-10 treated (Figs 1C–1E and S2). The few lesions that were present in the rmIFNγR1-Ig treated animals were also significantly less dense and metabolically active at day 6 compared to day 2 (Fig 1D). These data suggest that during SARS-CoV-2, IL-10 and IFNγ negatively or positively regulate pulmonary inflammation, respectively.

18FDG-PET/CT analysis of extra-pulmonary inflammation We further investigated SARS-CoV-2 induced extra-pulmonary inflammation using 18FDG-PET/CT analysis of the pulmonary lymph nodes, spleen, nasal turbinates, and tonsils. 18FDG uptake in the pulmonary lymph nodes (pLN) was evident by day 2 post-infection and peaked at day 6–10 post-infection, with some lymph nodes retaining elevated 18FDG avidity through days 22–23 post-infection (Figs 1F, 1G and S2). There were no differences between treatment groups in the pLN PET/CT signal after infection (Fig 1G). We also observed increased 18FDG uptake in the spleens of isotype control and anti-IL-10 treated animals, that peaked at ~day 10 post-infection and reached statistical significance over baseline (Fig 1H and 1I). However, only 1 of 5 rmIFNγR1-Ig treated animals had splenic 18FDG uptake above baseline. The nasal turbinates also had evidence of modest 18FDG avidity that peaked at ~day 2–6 post-infection. However, these changes did not reach statistical significance over baseline, and there were no differences between treatment groups. Low levels of 18FDG uptake were observed in the tonsils of some animals at ~day 10 post-infection. However, no significant differences were observed between groups. We also measured circulating soluble markers of inflammation including fibrinogen and C-reactive protein (CRP). Plasma fibrinogen levels were higher in the anti-IL-10 treated animals compared to controls at days 2–10 post-infection, suggestive of increased coagulation (Fig 1J). CRP levels were also elevated in the anti-IL-10 treated animals at day 2 post-infection, as compared to controls (Fig 1K). Collectively, these data suggest that in the rhesus macaque model of mild disease, the lung is the primary site of inflammation following SARS-CoV-2 infection and IL-10 and IFNγ negatively and positively modulated markers of pulmonary inflammation, respectively.

SARS-CoV-2 replication kinetics and tissue distribution To determine whether IL-10 or IFNγ blockade influenced SARS-CoV-2 replication, we measured the kinetics of subgenomic RNA of the nucleocapsid gene (sgN) in bronchoalveolar lavage fluid (BAL), nasal swabs, throat swabs, as well as in tissues at necropsy. In the BAL, viral RNA levels were highest at day 3 post-infection and were below the limit of detection (L.O.D.) by days 7–14 (Fig 2A). In nasal and throat swabs, sgN levels peaked at ~2–3 days post-infection and were undetectable in most animals by ~14 days post-infection. We did not observe any statistical difference in viral RNA loads in the BAL or swabs after IL-10 or IFNγ blockade. PPT PowerPoint slide

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TIFF original image Download: Fig 2. IFNγ and IL-10 are not required to suppress SARS-CoV-2 replication. (A) Subgenomic RNA quantification of the N gene (sgN) of SARS-CoV-2 by RT-qPCR in copies/mL from bronchoalveolar lavage (BAL), nasal swabs, and throat swabs. (B) sgN copies/gram of tissue at necropsy (day 28–35 post-infection) from spleen, axillary lymph node (axLN), non-PET/CT avid pulmonary lymph nodes (norm. pLN), previously PET/CT hot pulmonary lymph nodes (prev. hot pLN), salivary gland (SG), tonsil, nasal turbinates (nasal turb.), normal lung sections (norm. lung), and previously PET/CT hot lung sections. For A the cutoff for RNA detection is 3,000 copies/mL. For B the cutoff is 2,000 copies/gram of tissue. Graphs show individual animals from samples taken at baseline, days 2, 3, 6, 7, 10, 14, and 22 post-infection, as well as necropsy. Significance calculated with a 2-way ANOVA and a Dunnett’s multiple comparison test. https://doi.org/10.1371/journal.ppat.1012339.g002 At necropsy (day 28 or 35 post-infection), no viral RNA was quantified above the L.O.D. in the spleen, axillary lymph nodes (axLN), pulmonary lymph nodes without previously detected PET/CT signal (norm. pLN), salivary gland (SG), or tonsil (Fig 2B). Viral RNA was detected in previously PET-hot pulmonary lymph nodes (prev. hot pLN) in 2 of 5 anti-IL-10 treated and 3 of 5 rmIFNγR1-Ig, but not in any of the isotype control treated animals; however, the differences between groups did not reach statistical significance. Viral RNA was also detected in the nasal turbinates of a subset of animals, with no differences between groups. Lung sections from previously PET-hot regions were isolated separately from uninvolved lung sections (normal lung). Viral RNA was not detected in any normal lung sections, and in only one of the previously PET-hot lung sections isolated from an rmIFNγR1-Ig treated animal. Altogether, there was little impact of either IL-10 or IFNγ blockade on SARS-CoV-2 peak viral RNA loads or viral RNA clearance in this model, regardless of changes in PET-activity.

Innate lymphocyte responses NK cells are important in early control of viral infections and have been shown to produce IFNγ in response to SARS-CoV-2 [38]. We investigated NK cells response in the peripheral blood mononuclear cell (PBMC) compartment and BAL. In NHP, NK cells can be defined as CD3-/CD8α+/CD8β-/NKG2A+ and divided into 4 distinct subpopulations based on the expression of CD16 and CD56 [39–42]. The population of CD16+/CD56- NK cells have been shown to be cytotoxic, with the expression of perforin and granzyme B [39]. CD16-/CD56+ NK cells are thought to be less cytotoxic and more likely to produce IFNγ [41]. In PBMCs, we observed a skewing of the NK population to a predominately CD16+/CD56- cytotoxic phenotype, which is consistent with previous reports (S4A and S4B Fig) [40]. In the BAL, CD16-/CD56+ cytokine producing NK cells were the most abundant. After SARS-CoV-2 infection, there was an increase in total NK cells that peaked at day 7 in isotype control samples in both the PBMCs and BAL (S4B Fig). All NK cell subsets in PMBCs had upregulated Ki67, a marker of cell-cycle/proliferation, by day 7 post-infection (S4C and S4D Fig). Ki67 upregulation was less robust in the BAL than in PBMCs. Granzyme B was differentially expressed by NK cell subsets, with CD16+/CD56- in both the PBMC and BAL having the highest expression of granzyme B (S4E and S4F Fig). All NK subsets in both PBMC and BAL upregulated granzyme B at day 3 in response to SARS-CoV-2 infection and had mostly returned to baseline levels by 4–5 weeks post-infection. Neither IFNγ nor IL-10 blockade had a substantial impact on the expansion of NK cells or their function after infection. These data suggest that NK cells in the blood and lungs respond to SARS-CoV-2 infection but are not highly dependent on IL-10 or IFNγ. Mucosal Associated Invariant T cells (MAIT cells) are innate-like lymphocytes that recognize 5-OP-RU, a small molecule produced during microbial riboflavin biosynthesis, presented by the MHC-I-like molecule MR1 [43]. In the context of viral infections, MAIT cells have been shown to be host protective via cytokine-driven, MR1-independent mechanisms [44–47]. MAIT cells have also been suggested to play a role in SARS-CoV-2 infection [48,49], so we next investigated the MAIT cell responses to SARS-CoV-2 in PBMCs and BAL. We observed an increase in Ki67 expression by MAITs in PBMCs at day 7 post-infection (S5A and S5B Fig). However, MAIT cell frequencies in PBMCs and BAL were relatively stable after SARS-CoV-2 infection (S5C Fig). At necropsy, we assessed MAIT cells in the spleen, lymph nodes, tonsil, and lung. We did not observe any changes in MAIT cell frequency in any compartment with either IL-10 or IFNγ blockade. However, there was an increase in Ki67+ MAIT cells in the PBMC and BAL on day 7 in the anti-IL-10 treated animals relative to controls (S5C Fig). These data suggest that, in rhesus macaques, MAIT cells become activated in response to SARS-CoV-2 but do not significantly expand in frequency. Moreover, IL-10 has a minor role in inhibiting MAIT cell proliferation during SARS-CoV-2 infection.

B cells and antibody responses Circulating anti-spike antibodies are correlated with protection against symptomatic SARS-CoV-2 infection provided by vaccines [50–53]. We investigated whether IL-10 or IFNγ blockade resulted in changes to B cells and SARS-CoV-2 specific antibody responses. The frequency of total B cells in PBMCs remained unchanged after SARS-CoV-2 infection in all treatment groups (S6 Fig). In the BAL, there was a statistically significant, albeit small, increase in total B cells at day 3 in the rmIFNγR1-Ig treatment group compared to isotype controls. At necropsy, there were no differences in the frequency of total B cells in tissue between treatment groups (S6 Fig). However, germinal center B cells (CD20+/BCL6+/Ki67+) were decreased in the previously PET-hot pLNs with IFNγ blockade (S6D Fig). Anti-spike and anti-RBD antibodies were detected in the plasma of all animals beginning as early as day 7 post-infection (S6E Fig). Virus-specific IgM responses peaked on ~day 14 post-infection and began to plateau or decline by day 28–35. Virus-specific IgG and IgA levels continued to increase throughout the length of the study and had not yet reached a plateau at the day 28–35 endpoint. Anti-IL-10 treated animals had higher levels of S-specific IgG and IgA on day 7 relative to the controls. However, by day 14 this difference was no longer significant, and overall, there were no substantial changes to the antibody responses with either IL-10 or IFNγ blockade. SARS-CoV-2 neutralizing antibodies were detected in 14 of 15 animals, apart from control animal DHMT, which also had the lowest anti-spike and RBD antibody titers at necropsy for all isotypes (S6F Fig). Plasma anti-RBD IgG and IgA levels correlated with live-virus neutralization titers at necropsy (S6G Fig). There were no statistically significant differences in neutralization titers between groups. Altogether, these data show that SARS-CoV-2-specific antibody responses are first detectable around day 7 and continue to increase over the first month of infection. While IFNγ may play a role in promoting GC responses in reactive pLNs, it has no major impact on the final development of serum antibody responses to SARS-CoV-2 infection in the observed timeframe. Additionally, while IL-10 blockade may accelerate the induction of serum antibody responses, it did not lead to sustained increases in SARS-CoV-2 specific antibody responses or increases in neutralizing antibody titers.

Kinetics of SARS-CoV-2 specific T cell responses in the BAL and blood T cell responses likely have an important role in protection against SARS-CoV-2 infection in humans [54]. The kinetics of CD4 and CD8 T cells that recognize the major SARS-CoV-2 structural protein antigens of spike (S), nucleocapsid (N), and membrane (M) were measured by ex vivo peptide re-stimulation of the PBMCs and BAL cells. Consistent with our previous findings, SARS-CoV-2-specific T cell responses were first detected at day 7 post-infection in both the PBMCs and BAL. The S and N epitopes were immunodominant compared to M, and the frequency of virus-specific T cells in the BAL was ~5–50 fold higher as compared to the blood (Fig 4A–4C) [2]. SARS-CoV-2-specific CD4 and CD8 T cell responses were increased with IL-10 blockade, as compared to controls (Fig 4B). The area under the curve for the S-, N- and M-specific CD4 and CD8 T cell responses from each animal in PBMCs and BAL was calculated. The combined S-, N- and M-specific responses were summed, and the cumulative SARS-CoV-2-specific CD4 and CD8 T cell responses in BAL and PBMCs were significantly elevated with IL-10 blockade but were unchanged with IFNγ blockade, as compared to controls (Fig 4C). These data suggest that IL-10 limits early Ag-specific CD4 and CD8 T cell responses to SARS-CoV-2. These data highlight the need to quantify Ag-specific T cell response by flow cytometry, rather than relying solely on bulk transcriptomics. PPT PowerPoint slide

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TIFF original image Download: Fig 4. IL-10 blockade increases SARS-CoV-2-specific T cell responses in the blood and BAL fluid. (A) Representative flow cytometry plots of CD4+95+ and CD8+95+ T cells from the bronchoalveolar lavage (BAL) at day 14 post-infection responding to ex vivo peptide stimulation assay with SARS-CoV-2 15-mer peptide pools for spike (S), nucleocapsid (N), and membrane (M) proteins by production of IFNg and TNF production. Numbers in plots are the frequency of the gated cytokine+ population. (B) Quantification of frequency of antigen specific CD4+95+ and CD8+95+ responses in BAL at baseline (dpi 0), days 3, 7, 14, and necropsy (dpi 28 or 35), calculated by taking the frequency of IFNγ+ or TNF+ in the stimulated samples and subtracting the frequency in the matched unstimulated samples. Each animal is represented as a point and the mean as a line for each treatment group. Legend is in bottom right corner. Significance calculated by 2-way ANOVA with Dunnett’s multiple comparison test. (C) The mean and SEM of the area under the curve (AUC) for antigen-specific CD4 T cell responses (x-axis) and antigen-specific CD8 T cell responses (y-axis) responses in BAL and PBMC samples calculated from ex vivo peptide stimulation with spike (S), nucleocapsid (N), and membrane (M), as represented in B. The AUC was determined for dpi 0–28 and interpolated by linear regression for animals necropsied at day 35. The bottom graphs represent the sum of the AUC for the S-, N-, and M-specific CD4 and CD8 T cell responses, and statistics represent a Dunnett’s multiple comparison test for total AUC responses from treatment groups compared to isotype control. (D) Quantification of frequency of antigen specific CD4+95+ and CD8+95+ responses in spleen, peripheral lymph nodes (axillary, cervical, and/or inguinal lymph nodes), normal pulmonary lymph nodes (norm. pulm. LN), previously PET/CT hot pulmonary lymph nodes (prev. hot pulm. LN), normal lung sections (norm. lung), and previously PET/CT hot lung sections (prev. hot lung) at necropsy (dpi 28 or 35), calculated as in B. Each animal is represented as a point and the antigen as a shape. Significance calculated by 2-way ANOVA with Dunnett’s multiple comparison test. https://doi.org/10.1371/journal.ppat.1012339.g004 At necropsy, T cells responding to S, N, M and SARS-CoV-2 peptide megapools [55] were assessed in tissues. Low frequencies of Ag-specific CD4 and CD8 T cells were detected in the spleen and lymph nodes, with previously hot pLNs having the largest relative responses among the secondary lymphoid organs examined (Fig 4D). Normal and previously PET-hot lung specimens also had detectable populations of Ag-specific CD4 and CD8 T cells. We observed a small but statistically significant increase in the frequency of Ag-specific T cells in pLNs after anti-IL-10 treatment, with Ag-specific CD4 T cells in normal pLNs and Ag-specific CD8 T cells in previously hot pLNs both being increased compared to isotype control. These data are consistent with the elevated virus-specific CD4 and CD8 T cell responses observed in the BAL after IL-10 blockade. Furthermore, these data suggest that IFNγ has little role in regulating the expansion of SARS-CoV-2-specific T cells or their migration into the lungs and lower airways despite evidence of increases in T cell responses by transcriptomic analysis.

T cell responses in the nasal mucosa Immunity in the nasal mucosa is likely critical in protection from SARS-CoV-2 (re)infection in humans [56]. Previous work from Lim et al. showed that SARS-CoV-2 specific T cells can be detected in the nasal mucosa after breakthrough infection in previously vaccinated individuals [57]. However, in our previous work we were unable to detect SARS-CoV-2 specific T cells in the nasal mucosa of intranasally infected macaques [2]. To evaluate SARS-CoV-2-specific T cells in the human nasal mucosa, we obtained freshly resected samples of frontal, ethmoid, or maxillary sinuses from individuals undergoing surgical resection for inflammatory conditions unrelated to SARS-CoV-2 infection. Lymphocytes extracted from the nasal mucosa were restimulated with overlapping peptide pools derived from SARS-CoV-2 nucleocapsid and spike proteins or from CMV and EBV, as a positive control. SARS-CoV-2 specific CD4 or CD8 T cell responses were detected in 43% (6/14) of samples (Fig 5A). In comparison, we detected CMV/EBV-specific T cells in 100% (5/5) of samples, and frequencies tended to be higher than those observed for SARS-CoV-2 specific T cells. While the SARS-CoV-2 vaccination status and infectious history of the de-identified individuals is unknown in this study, it confirms that SARS-CoV-2 specific T cells in humans can reach the nasal mucosa. PPT PowerPoint slide

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TIFF original image Download: Fig 5. IL-10 blockade does not rescue the lack of SARS-CoV-2-specific T cell responses in the nasal mucosa of rhesus macaques. (A) Representative flow cytometry plots of cytokine producing CD4+95+ or CD8+95+ T cells after ex vivo peptide stimulation with CMV and EBV peptide pools, SARS-CoV-2 peptide pools, or unstimulated samples from human nasal mucosa. Numbers in plots are the frequency of gated cytokine+ of activated CD4 or CD8 T cells. (B) Quantification of total antigen-specific T cells by cytokine+ (IFNγ+/TNF+) of total CD3+ T cells after stimulation with the indicated peptide pools. Number of samples with positive signal above background were calculated by subtracting the total cytokine+ T cells response in the unstimulated samples from the total cytokine+ response in the stimulated samples. Significance calculated with unpaired t-test. (C) Representative flow cytometry plots of CD4+95+ or CD8+95+ T cells responding to spike peptide pool from the nasal mucosa of isotype control rhesus macaque, DHDI, at necropsy (dpi 28). Numbers in plots are the frequency of the gated cytokine+ in stimulated or unstimulated samples. (D) Quantification of frequency of cytokine+ (IFNγ+ and/or TNF+) CD4+95+ or CD8+95+ T cells responding to spike peptide stimulation at necropsy (dpi 28 or 35) from stimulated and unstimulated samples from the nasal mucosa. Significance calculated with a 2-way ANOVA and Sidak’s multiple comparison test between stimulated and unstimulated samples. (E) (Left) Representative flow cytometry plots of i.v. stain and Spike tetramer (H-2Kb Spike 539-546 ) CD8+CD44+ T cells from the lung and nasal mucosa of mice infected with SARS-CoV-2 (B.1.351) at necropsy (dpi 30). Red shaded gate shows parenchymal (i.v. negative) Spike-specific CD8 T cells and grey shaded gate represents parenchymal (i.v. negative) non-antigen-specific CD8 T cells. (Right) Representative flow cytometry plots of CD69 and CD103 expression by parenchymal (i.v. negative) spike-specific (red dots) and non-specific (grey dots) CD8 T cells from the lung and nasal mucosa. Numbers in plots indicate the frequency within the quadrants. (F) Quantification of the frequency of parenchymal (i.v. negative) spike-specific CD8 T cells from the lung and nasal mucosa from mice infected with SARS-CoV-2 (B.1.351) at necropsy (dpi 30). Data shown from two separate experiments, squares represent one experiment and circles represent a second experiment. Nasal mucosa samples were pooled (n = 5) prior to staining and each experiment represented as one data point. (G) Quantification of the frequency of CD69-CD103- (grey bars), CD69+CD103- (dark blue bars), CD69-CD103+ (green bars), or CD69+CD103+ (turquoise bars), of parenchymal spike-specific CD8 T cells from the lungs and nasal mucosa of mice infected with SARS-CoV-2 (B.1.351) at necropsy (dpi 30). https://doi.org/10.1371/journal.ppat.1012339.g005 Given the discrepancy in the detection of SARS-CoV-2 specific T cells in humans versus macaques, we next asked if our IL-10 blockade regimen increased T cell responses in the nasal mucosa of infected macaques to detectable levels. We isolated the epithelial lining of the nasal passage, including the nasal turbinates, at 4–5 weeks post-infection and restimulated the extracted lymphocytes with the spike (S) peptide pool. S-specific T cells were not detected in the nasal mucosa of control animals, consistent with previous findings in this model (Fig 5C and 5D) [2]. Moreover, none of the animals receiving IL-10 or IFNγ blockade animals had detectable virus-specific T cells above baseline in the nasal mucosa. These data indicate that the lack of nasal SARS-CoV-2 specific T cells in macaques is not due to IL-10 mediated suppression. We next asked if the absence of SARS-CoV-2-specific T cell responses in the nasal mucosa is unique to rhesus macaques by examining responses in the nasal mucosa of SARS-CoV-2 infected mice. C57BL/6 mice were intranasally infected with SARS-CoV-2 beta strain (B.1.351). At necropsy, intravenous antibody labelling prior to necropsy was used to identify cells in the tissue vasculature. Virus-specific CD8 T cell responses in the nasal mucosa and lungs were quantified with Kb/Spike 539-546 tetramer. At day 30 post-infection, parenchymal spike-specific CD8 T cells were detected in both the lung and nasal mucosa (Fig 5E and 5F). SARS-CoV-2-specific T cells in the nasal mucosa had a highly tissue resident memory (Trm) phenotype, with ~50% of spike-specific T cell CD69+CD103+, compared to ~10% in the lung (Fig 5G). Therefore, SARS-CoV-2 infection generates spike-specific Trm in the nasal mucosa of mice and a subset of patients, but virus-specific responses are not detectable in the rhesus macaque model. To determine if rhesus macaques could generate Ag-specific T cells in the nasal mucosa in response to another pulmonary infection, we assessed T cells from the nasal mucosa of Mycobacterium tuberculosis (Mtb) infected animals. Three male rhesus macaques were infected with ~50 CFU of Mtb-H37Rv via endobronchial instillation, and Mtb-specific T cells from the nasal mucosa were quantified by intracellular cytokine staining after restimulation with Mtb peptide pools [58,59]. Mtb-specific CD4 and CD8 T cells were readily detected in the nasal mucosa of all infected rhesus macaques and ~50–65% of the IFNγ+TNF+ CD4 and CD8 T cells expressed CD69 (S7 Fig). Thus, SARS-CoV-2 specific nasal mucosa T cells are detected in humans and mice but not in macaques. This difference cannot be attributed to technical limitations of restimulation of nasal lymphocytes in macaques, as Mtb-specific T cells are readily detected in the nasal mucosa after infection. Moreover, IL-10 blockade boosted the clonal burst of virus-specific CD4 and CD8 T cells in the airways yet did not expand CD4 and CD8 T cells in the nasal mucosa to detectable levels.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012339

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