SARS-CoV-2-specific T cell memory is sustained in COVID-19 convalescent patients for 10 months with successful development of stem cell-like memory T cells

Abstract

Memory T cells contribute to rapid viral clearance during re-infection, but the longevity and differentiation of SARS-CoV-2-specific memory T cells remain unclear. Here we conduct ex vivo assays to evaluate SARS-CoV-2-specific CD4⁺ and CD8⁺ T cell responses in COVID-19 convalescent patients up to 317 days post-symptom onset (DPSO), and find that memory T cell responses are maintained during the study period regardless of the severity of COVID-19. In particular, we observe sustained polyfunctionality and proliferation capacity of SARS-CoV-2-specific T cells. Among SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells detected by activation-induced markers, the proportion of stem cell-like memory T (T_SCM) cells is increased, peaking at approximately 120 DPSO. Development of T_SCM cells is confirmed by SARS-CoV-2-specific MHC-I multimer staining. Considering the self-renewal capacity and multipotency of T_SCM cells, our data suggest that SARS-CoV-2-specific T cells are long-lasting after recovery from COVID-19, thus support the feasibility of effective vaccination programs as a measure for COVID-19 control.

Fig. 1. SARS-CoV-2-specific IFN-γ responses over 10 months post-infection.

PBMC samples (n = 159) from individuals with SARS-CoV-2 infection (n = 87) and pre-pandemic PBMC samples (n = 8) from healthy donors (n = 8) were stimulated with OLPs of S, M, or N (1 μg/mL) for 24 h and the spot-forming units of IFN-γ-secreting cells examined by ELISpot. a Scatter plots showing the relationship between DPSO and IFN-γ responses. The black line is a LOESS smooth nonparametric function, and the gray shading represents the 95% confidence interval. b The composition of S-, M-, or N-specific IFN-γ responses among the total IFN-γ responses in each individual. c IFN-γ responses were compared between T1 (n = 49, 31–99 DPSO), T2 (n = 41, 100–199 DPSO), and T3 (n = 31, ≥200 DPSO). Data are presented as median and interquartile range (IQR). d, e IFN-γ responses were analyzed in longitudinally tracked samples (n = 103) from 39 individuals. d Scatter plots showing the relationship between DPSO and IFN-γ responses. e IFN-γ responses were compared between paired samples at two-time points (n = 15; t1, 31–100 DPSO; t2, ≥200 DPSO). Statistical analysis was performed using the two-sided Kruskal–Wallis test with Dunns’ multiple comparisons test (c) or the Wilcoxon signed-rank test (e). n.s, not significant.

Fig. 2. Kinetics of SARS-CoV-2-specific activation-induced marker (AIM)⁺ T cells.

PBMC samples (n = 146) from individuals with SARS-CoV-2 infection (n = 82) were stimulated with OLPs of S, M, or N (1 μg/mL) for 24 h. The frequency of AIM⁺ (CD137⁺OX40⁺) cells among CD4⁺ T cells and the frequency of AIM⁺ (CD137⁺CD69⁺) cells among CD8⁺ T cells were analyzed. Representative flow cytometry plots showing the frequency of AIM⁺ cells among CD4⁺ (a) or CD8⁺ (b) T cells. c Scatter plots showing the relationship between DPSO and the frequency of AIM⁺ cells among CD4⁺ (left) or CD8⁺ (right) T cells. The black is a LOESS smooth nonparametric function, and the gray shading represents the 95% confidence interval.

Fig. 3. Differentiation status of SARS-CoV-2-specific AIM⁺ T cells.

a-d PBMC samples (n = 146) from individuals with SARS-CoV-2 infection (n = 82) were stimulated with OLPs of S, M, or N (1 μg/mL) for 24 h and the expression of CCR7 and CD45RA was analyzed in AIM⁺ (CD137⁺OX40⁺) CD4⁺ (a, c) and AIM⁺ (CD137⁺CD69⁺) CD8⁺ (b, d) T cells. Gating strategies for identifying each memory subset among AIM⁺CD4⁺ (a) or AIM⁺CD8⁺ (b) T cells. Scatter plots showing the relationship between DPSO and the proportion of the indicated subsets among AIM⁺CD4⁺ (c) or AIM⁺CD8⁺ (d) T cells. e PBMC samples (n = 68) from COVID-19 convalescent patients (n = 59) were stimulated with OLPs of S, M, or N (1 μg/mL) for 24 h and the frequency of T_SCM (CCR7⁺CD45RA⁺CD95⁺) cells was analyzed in AIM⁺CD4⁺ (upper) and AIM⁺CD8⁺ (lower) T cells. Left, The gating strategy for identifying T_SCM cells. Right, Scatter plots showing the relationship between DPSO and the proportion of T_SCM cells among AIM⁺CD4⁺ or AIM⁺CD8⁺ T cells. The black line is a LOESS smooth nonparametric function, and the gray shading represents the 95% confidence interval (c, d, e).

Fig. 4. Frequency and differentiation status of SARS-CoV-2-specific MHC-I multimer⁺ T cells.

PBMC samples (n = 15) from individuals with SARS-CoV-2 infection (n = 11) were analyzed by flow cytometry. a Representative flow cytometry plots showing the ex vivo detection of SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells in the gate of CD3⁺ T cells. b Scatter plot showing the relationship between DPSO and the frequency of SARS-CoV-2 S₂₆₉ multimer⁺ cells among total CD8⁺ T cells. Samples from the same patient are connected by solid lines. The expression of CCR7, CD45RA, and CD95 was analyzed in SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells. IAV MP₅₈ multimer⁺ (n = 5) and CMV pp65₄₉₅ multimer⁺ (n = 6) cells from the PBMCs of healthy donors were also analyzed. Representative flow cytometry plots (c) show the proportion of the indicated subsets among multimer⁺ cells, and scatter plots (d) show the relationship between DPSO and the proportion of the indicated subsets among SARS-CoV-2 S₂₆₉ multimer⁺ cells. Samples from the same patient are connected by solid lines. Summary data showing the proportion of the indicated subsets among IAV multimer⁺ and CMV multimer⁺ cells are also presented (d). Horizontal lines represent median. e A representative flow cytometry plot (upper) and summary data (lower) showing the percentage of PD-1⁻TIGIT⁻ cells among SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T_SCM cells and total SARS-CoV-2 S₂₆₉ multimer⁺CD8⁺ T cells. Statistical analysis was performed using the two-sided Wilcoxon signed-rank test (e).

Fig. 5. Polyfunctionality and proliferation capacity of SARS-CoV-2-specific T cells.

PBMC samples (n = 90) from individuals with SARS-CoV-2 infection (n = 39) were stimulated with OLPs of S, M, or N (1 μg/mL) for 6 h. Intracellular cytokine staining was performed to examine the frequency of polyfunctional cells exhibiting positivity for ≥2 effector functions among SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells. a Representative flow cytometry plots showing the frequency of polyfunctional cells among CD4⁺ (left) and CD8⁺ (right) T cells. b Scatter plots showing the relationship between DPSO and the frequency of polyfunctional cells among SARS-CoV-2-specific CD4⁺ (upper) or CD8⁺ (lower) T cells. The black line is a LOESS smooth nonparametric function, and the gray shading represents the 95% confidence interval. c The fraction of polyfunctional cells among SARS-CoV-2-specific CD4⁺ (left) or CD8⁺ (right) T cells was compared between T1 (n = 17, 31–99 DPSO), T2 (n = 39, 100–199 DPSO), and T3 (n = 25, ≥200 DPSO). Data are presented as median and IQR. d Pie charts showing the fraction of cells positive for a given number of functions among SARS-CoV-2-specific CD4⁺ (left) or CD8⁺ (right) T cells. Each arc in the pie chart represents the indicated function. e CTV-labeled PBMCs (n = 18) obtained after 200 DPSO were stimulated with S OLP pool (1 μg/mL) for 120 h and the frequency of CTV^low and Ki-67⁺ cells among CD4⁺ and CD8⁺ T cells was analyzed. Representative plots (upper) and summary data (lower) are presented. Statistical analysis was performed using the two-sided Kruskal–Wallis test with Dunns’ multiple comparisons test (c) or the two-sided Wilcoxon signed-rank test (e). n.s, not significant.