Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19

Abstract

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4⁺ and/or CD8⁺ epitopes, including six immunodominant regions. Six optimized CD8⁺ epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8⁺ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.

Fig. 1. Memory T cell responses specific to SARS-CoV-2 virus proteins in 42 convalescent patients infected with SARS-CoV-2.

Of the 42 patients studied, 28 had mild symptoms while 14 showed severe symptoms. PBMCs were isolated and IFN-γ production was detected by ELISpot after incubation with SARS-CoV-2 peptides. a, Magnitude of IFN-γ T cell responses for each individual. Each bar shows the total T cell responses of each individual specific to all of the SARS-CoV-2 protein peptides tested. Each colored segment represents the source protein corresponding to peptide pools eliciting IFN-γ T cell responses. b, Breadth of T cell responses for each individual. The breadth of T cell responses was calculated by the number of peptide pools in the first-dimension (n = 29) cells that responded to spot-forming units. The experiments were repeated in 35 participants where sample availability permitted. Env: envelope protein

Fig. 2. Comparison of the magnitude and breadth of T cell responses specific to each viral protein between convalescent patients with mild symptoms and those with severe symptoms.

PBMCs were isolated and IFN-γ production was detected by ELISpot after incubation with SARS-CoV-2 peptides. a,b, Magnitude (a) and breadth (b) of T cell responses against each viral protein between those with mild symptoms (n = 28) and those with severe symptoms (n= 14). P values were as follows: overall: P = 0.002 for magnitude; P = 0.002 for breadth; spike: P = 0.021 for magnitude; P = 0.016 for breadth; membrane: P = 0.0003 for magnitude; P = 0.033 for breadth; ORF3a: P < 0.0001 for magnitude; P = 0.001 for breadth; ORF8: P = 0.011 for magnitude; P = 0.014 for breadth. Data are presented as medians with interquartile ranges. The Mann–Whitney U-test was used for the analysis and two-tailed P values were calculated. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001. NS, not significant.

Fig. 3. Correlation of T cell responses against SARS-CoV-2 with spike-, RBD- and NP-specific antibody responses.

a-c, Spike (a), RBD (b) and NP EPTs (c) in association with overall T cell responses. Red and black data points represent patients with severe and mild symptoms, respectively. n = 42. Spearman’s rank correlation coefficients (R) are shown. d, Comparison of spike EPTs (P < 0.0001), RBD EPTs (P < 0.0001) and NP EPTs (P = 0.0004) for patients with mild symptoms (n = 28) versus severe symptoms (n = 14). Data are presented as medians with interquartile ranges and a Mann-Whitney U-test was used for comparison. Two-tailed P values were calculated. ***P < 0.001; ****P < 0.0001.

Fig. 4. Distribution of SARS-CoV-2-specific CD4⁺ and CD8⁺ memory T cell responses.

Cytokine-producing T cells were detected by ICS after incubation with SARS-CoV-2 peptides. a,b, Flow cytometric plots representing CD4⁺ T cells (a) and CD8⁺ T cells (b) expressing IFN-γ (x axis), TNF (y axis) and/or IL-2 (y axis) upon stimulation with the respective SARS-CoV-2 peptide pools in examples of mild (left) and severe cases (right). c, Comparison of the relative proportion of SARS-CoV-2 peptide pool-reactive CD8⁺ T cells between mild (spike, n = 11; M/NP, n = 14; ORF/Env, n = 5; overall: n = 14) and severe cases (spike, n = 7; M/NP, n = 7; ORF/Env, n = 4; overall, n = 8). P values were as follows: P = 0.0268 (spike); P = 0.02 (M/NP); P = 0.0159 (overall). The SARS-CoV-2 peptide pool-reactive CD4⁺ or CD8⁺ T cells were identified with at least one of the three cytokines (IFN-γ, TNF and IL-2) detected. Data are shown as medians with interquartile ranges. The Mann-Whitney U-test was used for the analysis. Two-tailed P values were calculated. *P < 0.05.

Fig. 5. Cytokine profile of SARS-Cov-2-specific T cells.

The cytokine production of SARS-Cov-2-specific T cells was assessed by ICS after incubation with SARS-CoV-2 peptides. a, Pie charts representing the relative proportions of spike-, M/NP- and ORF/env-specific CD4⁺ and CD8⁺ T cells producing one (green), two (pink) or three cytokines (black) (out of IFN-γ, TNF and IL-2). b, Comparison of the frequency of multifunctional CD8⁺ T cells targeting spike and M/NP. The open circles and squares represent T cell responses in mild cases and severe cases, respectively. P values are as follows: P = 0.0037 (mild); P = 0.3823 (severe); P = 0.0231 (overall). c, Relative frequencies of CD4⁺ and CD8⁺ T cells expressing CD107a after antigen stimulation. The data shown are from 14 patients with mild symptoms and eight patients with severe symptoms. The Mann–Whitney U-test was used for the analysis. Two-tailed P values were calculated. *P < 0.05; **P < 0.01.

Fig. 6. Defined SARS-CoV-2-specific CD8 epitopes.

Examples of peptide–MHC class I pentamer staining ex vivo, with PBMCs (HLA-B*0702, -B*4001, -A*1101, -A*0101 and -A*0201) or with cultured cell lines (HLA-A*0301). Eleven donors were tested with positive pentamer staining.

Fig. 7. Memory phenotype and differentiation status of SARS-CoV-2-specific CD8⁺ T cells.

PBMCs were isolated and stained with peptide–MHC class I pentameric complexes and markers of T cell memory and differentiation. a, Representative fluorescence-activated cell sorting plots of gating for different cell subsets. b,c, Expression of memory markers (CCR7 and CD45RA) (b) and differentiation markers (CD27 and CD28) (c) on CD8⁺ pentamer⁺ T cells. n = 7 donors. Data are presented as means ± s.e.m.