Peripheral and lung resident memory T cell responses against SARS-CoV-2

Abstract

Resident memory T cells (T_RM) positioned within the respiratory tract are probably required to limit SARS-CoV-2 spread and COVID-19. Importantly, T_RM are mostly non-recirculating, which reduces the window of opportunity to examine these cells in the blood as they move to the lung parenchyma. Here, we identify circulating virus-specific T cell responses during acute infection with functional, migratory and apoptotic patterns modulated by viral proteins and associated with clinical outcome. Disease severity is associated predominantly with IFNγ and IL-4 responses, increased responses against S peptides and apoptosis, whereas non-hospitalized patients have increased IL-12p70 levels, degranulation in response to N peptides and SARS-CoV-2-specific CCR7⁺ T cells secreting IL-10. In convalescent patients, lung-T_RM are frequently detected even 10 months after initial infection, in which contemporaneous blood does not reflect tissue-resident profiles. Our study highlights a balanced anti-inflammatory antiviral response associated with a better outcome and persisting T_RM cells as important for future protection against SARS-CoV-2 infection.

Fig. 1. Functional characteristics of acute SARS-CoV-2-specific T cells.

a Comparison of the net frequency (background subtracted) of interferon (IFN)γ, CD107a, interleukin (IL)-4, and IL-10 expression in SARS-CoV-2-specific CD4⁺ and CD8⁺T cells in response to viral proteins (membrane (M), nucleocapsid (N), and spike (S)) between study groups (nonhospitalized n = 14 in orange; mild n = 20 in blue and severe n = 12 in pink). Statistical comparisons were performed using Kruskal–Wallis rank-sum test with Dunn’s multiple comparison test (two-sided): CD4⁺ IFNγ (M, p = 0.020 and p = 0.004; N, p = 0.011 and S, p = 0.020 and p = 0.007) and IL-10 (M, p = 0.035); CD8⁺ CD107a (M, p = 0.037), IL-4 (S, p = 0.004 and p = 0.003). b Donut charts summarizing the contribution of each function to the overall CD4⁺- and CD8⁺-specific T cell response by targeted viral protein and individual group of patients. Data represent the mean value of the net frequency of each function indicated by color code considering all patients, responders and nonresponders. Total response value (%) is shown under each pie chart and represents the cohort average of the overall net frequency considering all individuals and adding up all functions (nonhospitalized n = 14; mild n = 20 and severe n = 12). Statistical comparisons as performed in a are indicated with * and ** symbols. c Donut charts summarizing the distribution of individual functions among specific CD4⁺ and CD8⁺T cells to either the M, N, or S protein from the only fatal case within the severe COVID-19 group.

Fig. 2. T cell migratory patterns during acute SARS-CoV-2 infection.

a, b The frequency of various T cell subsets defined by CCR7 and CXCR3 within CD4⁺ (a) and CD8⁺T cells (b). Each dot represents one patient of a specific cohort, indicated by color code (normal donors n = 12; nonhospitalized n = 14; mild n = 20 and severe n = 12). Data are shown as individual patients and boxes and error bars represent median and interquartile range (IQR). Statistical comparisons were performed using Kruskal–Wallis rank-sum test with Dunn’s multiple comparison test (two-sided): CCR7^hCXCR3^d (p = 0.029 and p = 0.0007) and EM CXCR3⁺ (p = 0.002). c Representative flow cytometry plots gating the different CD8⁺T cell subsets in a nonhospitalized (top) and a severe patient (bottom). d, e Correlations between days to discharge since symptoms onset or interleukin (IL)-6 baseline levels and the frequency of CD8⁺ CCR7^hCXCR3^d (d) and CD8⁺ EM CXCR3⁺ (e) subpopulations. Two-sided spearman rank correlation (n = 46).

Fig. 3. Migratory patterns of acute SARS-CoV-2-specific CD4⁺T cells.

a Net frequency of interferon (IFN)γ, CD107a, interleukin (IL)-4 and IL-10 expression in SARS-CoV-2-specific CD4⁺ T cells based on CXCR3⁺CCR7⁺ and CXCR3⁺CCR7^- subsets for each individual patient (nonhospitalized n = 14; mild n = 20 and severe n = 12). Viral proteins are shown in color green (membrane protein, M), orange (nucleocapsid protein, N) and purple (spike protein, S). Dots connected by the same line represent the same individual. Statistical comparisons were performed using two-sided nonparametric Wilcoxon matched-pairs signed rank test to compare the two groups (CXCR3 + CCR7⁺ vs. CXCR3⁺CCR7⁻): nonhospitalized (IFNγ, p = 0.016 and p = 0.016; CD107a, p = 0.031; IL-4, p = 0.008; IL-10, p = 0.008, and p = 0.0005), mild (IFNγ, p = 0.006, p = 0.005, and p = 0.006; CD107a, p = 0.035; IL-4, p = 0.004, p = 0.008, and p = 0.002), and severe (IFNγ, p = 0.005, p = 0.024, and p = 0.039; IL-4, p = 0.042). b–d Correlation between the days to discharge since symptoms onset or IL-6 and the frequency of nucleocapsid or spike-specific CD4⁺ CXCR3⁺CCR7⁺ expressing IFNγ (b), CD107a (c), or IL-4 (d). e, f Correlation between IL-6 and the frequency of membrane-specific CD4⁺ CXCR3⁺CCR7^−/+ expressing IFNγ (e) or IL-10 (f). g Correlation between the viral load and the frequency of CD4⁺ EM CXCR3⁺ expressing IL-10 after PMA/Ionomycin stimulation. Two-sided spearman rank correlation (n = 46 for all correlations except for viral load (g), which corresponds to n = 33).

Fig. 4. IL-10 expression in acute SARS-CoV-2-specific CD8⁺T cell subsets.

a Net frequency of interleukin (IL)-10 expression in CCR7⁺CXCR3⁻, CCR7⁺CXCR3⁺, CCR7^hCXCR3^d, and EM CXCR3⁺ subsets within CD8⁺ T cells after stimulation with any of the three viral proteins (membrane (M), nucleocapsid (N), and spike (S) proteins). Data are shown as median and upper range, where each dot represents an individual patient for each group (nonhospitalized n = 14; mild n = 20, and severe n = 12). Statistical comparisons were performed using Kruskal–Wallis rank-sum test with Dunn’s multiple comparison test (two-sided): nonhospitalized (N, p = 0.041) and mild (M, p = 0.003; N, p = 0.007 and S, p = 0.042). b, c Correlation between CD8⁺ EM CXCR3⁺T cells responding with any function (added net response for interferon (IFN)γ, CD107a, IL-4 and IL-10) against M peptides and viral load (b) and against N peptides and baseline IL-6 levels (c). Two-tailed spearman rank correlation (n = 33 for viral load and n = 46 for IL-6).

Fig. 5. Caspase-3 expression in T cells during acute SARS-CoV-2 infection.

a Frequency of caspase-3 expression in CD4⁺ and CD8⁺T cells after stimulation with membrane (M), nucleocapsid (N) or spike protein (S) and PMA/Ionomycin, in nonhospitalized (orange, n = 14), mild (blue, n = 20), and severe (pink, n = 12) COVID-19 patients. Data are shown as individual patients and boxes and error bars represent median and interquartile range (IQR). Statistical comparisons were performed using Kruskal–Wallis rank-sum test with Dunn’s multiple comparison test (two-sided): CD4⁺ (M, p < 0.0001 and p < 0.0001; PMA/Io p = 0.036) and CD8⁺ (S, p = 0.007 and p = 0.002). b Correlation between days to hospital discharge since symptoms onset, viral load or baseline interleukin (IL)-6 levels (pg/mL) and the net frequency (background subtracted) of caspase-3 in CD4⁺ and CD8⁺T cells after stimulation with the spike protein or PMA/Ionomycin. c Frequency of caspase-3 expression in CD8⁺ CCR7^hCXCR3^dT cells after stimulation in the same groups as in a. Statistical comparisons were performed using Kruskal–Wallis rank-sum test with Dunn’s multiple comparison test (two-sided): p = 0.026. d Correlations between clinical parameters and the net frequency of caspase-3 expression in CD8⁺ CCR7⁺ T cell subsets after stimulation. e Frequency of caspase-3 expression in IL-10-secreting SARS-CoV-2-specific CD4⁺ and CD8⁺T cells responding to the spike protein, the nucleocapsid protein or to PMA/Ionomycin in the same groups as in a. Statistical comparisons were performed using Kruskal–Wallis rank-sum test with Dunn’s multiple comparison test (two-sided): CD4⁺ (S, p = 0.003; PMA/Io, p = 0.036) and CD8⁺ (N, p = 0.013; PMA/Io, p = 0.004). f Correlation between clinical parameters and IL-10-expressing SARS-CoV-2-specific CD4⁺ or CD8⁺T cells, or after PMA/Ionomycin stimulation. g Correlation between viral load and the frequency of caspase-3 expression in basal CD107a⁺ degranulating CD4⁺T cells and between IL-6 and the net frequency of caspase-3 expression in CD107a-expressing CD4⁺ in response to N peptides. Two-tailed spearman rank correlation (n = 46 for all correlations except for viral load, which corresponds to n = 33).

Fig. 6. Functional analysis of lung-resident SARS-CoV-2-specific T cells.

a, b Flow cytometry plots showing the frequency of interferon (IFN)γ and degranulation (CD107a) by non-resident memory T cells (T_RM), T_RM or CD103⁺ T_RM in CD4⁺ from HL27 after spike stimulation and control (a) and in CD8⁺ from HL52 after nucleocapsid stimulation and control (b). c Heatmaps summarizing the net frequencies of IFNγ, CD107a, interleukin (IL)-4, and IL-10 SARS-CoV-2-specific CD4⁺ or CD8⁺ non-T_RM, T_RM, and T_RM CD103⁺ from seven different SARS-CoV-2 recovered patients. Cytokine production or degranulation are displayed as colors ranging from yellow to blue, based on the frequency, as shown in the key. d Net frequency of SARS-CoV-2-specific CD3⁺T cells in response to all viral proteins (membrane (M), nucleocapsid (N) and spike (S)) from from lung by non-T_RM, T_RM or CD103⁺ T_RM are shown as mean ± SEM (n = 7). Statistical comparisons were performed using Kruskal–Wallis rank-sum test with Dunn’s multiple comparison test (two-sided): p = 0.023. e Net frequency of double positive IFNγ/CD107a CD3⁺T cells from lung or blood after stimulation with membrane (M; green), nucleocapsid (N; orange), or spike protein (S; purple) (n = 7).

Fig. 7. SARS-CoV-2-specific T cells in lungs and blood of convalescent patients.

Total CD4⁺ and CD8⁺T cell net frequencies of interferon (IFN)γ, CD107a, interleukin (IL)-4, and IL-10 expression in SARS-CoV-2-specific T cells derived from lung or blood from the same patient. Each patient (n = 7) is labeled on the right and includes disease profile and sampling time: months (mo) after initial SARS-CoV-2 detection by PCR. Viral proteins are shown in color green (membrane protein, M), orange (nucleocapsid protein, N), and purple (spike protein, S).