Robust T Cell Response Toward Spike, Membrane, and Nucleocapsid SARS-CoV-2 Proteins Is Not Associated with Recovery in Critical COVID-19 Patients

Abstract

T cell immunity toward SARS-CoV-2 spike (S-), membrane (M-), and nucleocapsid (N-) proteins may define COVID-19 severity. Therefore, we compare the SARS-CoV-2-reactive T cell responses in moderate, severe, and critical COVID-19 patients and unexposed donors. Overlapping peptide pools of all three proteins induce SARS-CoV-2-reactive T cell response with dominance of CD4⁺ over CD8⁺ T cells and demonstrate interindividual immunity against the three proteins. M-protein induces the highest frequencies of CD4⁺ T cells, suggesting its relevance for diagnosis and vaccination. The T cell response of critical COVID-19 patients is robust and comparable or even superior to non-critical patients. Virus clearance and COVID-19 survival are not associated with either SARS-CoV-2 T cell kinetics or magnitude of T cell responses, respectively. Thus, our data do not support the hypothesis of insufficient SARS-CoV-2-reactive immunity in critical COVID-19. Conversely, it indicates that activation of differentiated memory effector T cells could cause hyperreactivity and immunopathogenesis in critical patients.

Keywords: COVID-19; S/M/N protein-reactive T cells; SARS-CoV-2; critical COVID-19; immunity; immunopathology; nucleocapsid; spike.

Graphical abstract

Figure 1

SARS-CoV-2-Reactive T Cells Are Induced by the S-, M- and N-Proteins with Interindividual Patterns Peripheral blood mononuclear cells (PBMCs) isolated from 65 blood samples collected from 28 COVID-19 patients with moderate, severe, or critical disease and blood samples of 10 unexposed donors collected and cryopreserved before the COVID-19 pandemic were stimulated for 16 h with S-, M-, or N-protein OPPs. Antigen-reactive T cells were determined by flow cytometry and identified according to the gating strategy presented in Figure S2. Maximum values of each COVID-19 patient were compared to unexposed donors. (A) Representative plots of CD4⁺ T cells and CD8⁺ T cells after stimulation with S-, M-, and N-protein OPPs. Antigen-reactive CD4⁺ T cells were identified by CD154 and CD137 expression and antigen-reactive CD8⁺ T cells by CD137 expression and production of any cytokines out of IL-2, IFN-γ, TNF-α, and/or GrzB (CD137⁺ cytokine⁺). (B) Stimulation index (SI) of CD154⁺ CD137⁺ CD4⁺ T cells (SARS-COV-2-specific CD4⁺ T cells), CD137⁺ cytokine⁺ CD8⁺ T cells (SARS-COV-2-specific CD8⁺ T cells) and bifunctional and trifunctional CD154⁺ CD4⁺ and CD137⁺ CD8⁺ T cells. Bi- and trifunctional T cells were calculated by Boolean gating of IL-2, IFN-γ, TNF-α, IL-4, and GrzB production. SI was calculated by dividing the measured T cell subset response by the respective response in the DMSO control. Values >3 were considered detectable in the following analyses. The maximum value of each COVID-19 patient is depicted. Scatterplots show line at median; error bars represent the interquartile ranges. The statistical comparison was done with the Kruskal-Wallis test and the Dunn’s multiple comparisons test. p < 0.05 was considered significant. (C) Frequency of patient samples with detectable (SI > 3) CD4⁺ (left) and CD8⁺ (right) T cell responses in at least 1 sample after stimulation with S-, M-, or N-protein (total of 65 samples of 28 COVID-19 patients and 10 samples of 10 unexposed donors). (D) Venn diagrams of 28 COVID-19 patients and 10 unexposed donors with detectable (SI > 3) SARS-Cov-2-reactive CD4⁺ or CD8⁺ T cells after stimulation with S-, M-, or N-protein in at least 1 sample. A total of 27 COVID-19 patients and 4 unexposed donors showed CD4⁺ T cell reactivity and 21 COVID-19 patients and 3 unexposed donors showed CD8⁺ T cell reactivity toward at least 1 of the tested SARS-CoV-2-S-, M-, and N-proteins. See also Figures S1, S2, and S3 and Table S2.

Figure 2

SARS-CoV-2 Reactive T Cells Display a Higher M-Protein Reactivity in CD4⁺ T Cells and S- and N-Protein Reactivity in CD8⁺ T Cells From a total of 28 COVID-19 patients, 65 blood samples were drawn at 1 or at multiple time points after SARS-CoV-2 positive PCR tests. PBMCs were stimulated for 16 h with S-, M-, or N-protein OPPs. The gating strategy is presented in Figure S2. (A–F) Mean frequencies of samples of individual COVID-19 patients (n = 28). (A) CD154⁺ CD137⁺ CD4⁺ T cells (antigen-specific CD4⁺ T cells), and (B) IFN-γ-, (C) TNF-α-, (D) IL-2-, (E) IL-4-, and (G) GrzB-producing antigen-specific CD4⁺ T cells. The statistical analysis was performed with the Friedman test for non-parametric data and with Dunn’s multiple comparisons test. p < 0.05 was considered significant. Boxplots show medians and interquartile ranges. Whiskers and outliers were calculated with the Tukey method. (G–I) Correlation of M-, N-, and S-protein OPP-reactive (CD154⁺ CD137⁺) CD4⁺ T cells. The calculation was performed with Spearman’s rank correlation coefficient. (J–O) Mean frequencies per COVID-19 patient (n = 28). (J) CD137⁺ IL-2, IFN-γ, TNF-α, and/or GrzB (CD137⁺ cytokine⁺) CD8⁺ T cells (antigen-specific CD8⁺ T cells), and (K) IFN-γ-, (L) TNF-α-, (M) IL-2-, (N) IL-4-, and (O) GrzB-producing CD137⁺ CD8⁺ T cells. The statistical analysis was performed with the Friedman test for non-parametric data and with Dunn’s multiple comparisons test. p < 0.05 was considered significant. Boxplots show medians and interquartile ranges. Whiskers and outliers were calculated with the Tukey method. (P–R) Correlation analysis of M-, N-, and S-protein-reactive (CD137⁺ cytokine⁺) CD8⁺ T cells. The calculation was performed with Spearman’s rank correlation coefficient. See also Figures S1 and S2.

Figure 3

Critical COVID-19 Patients Maintain a Strong SARS-CoV-2 S-, M-, and N-Protein-Reactive CD4⁺ and CD8⁺ T Cell Response From a total of 28 COVID-19 patients, 65 blood samples were drawn at 1 or at multiple time points after COVID-19 diagnosis. The mean frequency was calculated for each individual COVID-19 patient. COVID-19 severity was assessed at the time of sampling as per the guidelines of the German Robert Koch Institute and the patients grouped according to their worst disease category (n = 7 moderate, n = 9 severe, and n = 12 critical samples). PBMCs were stimulated for 16 h with S-, M-, or N-protein OPPs and analyzed by flow cytometry. The gating strategy to identify SARS-CoV-2 S-, M-, or N-protein-reactive T cells is presented in Figure S2. (A–D) Frequencies of (A) CD154⁺ CD137⁺ CD4⁺ T cells (antigen-specific CD4⁺ T cells), and (B) IFN-γ-, (C) IL-2-, and (D) TNF-α-producing antigen-specific CD4⁺ T cells. (E and F) Frequencies of polyfunctional CD154⁺ CD4⁺ T cells. (E) Bifunctional and (F) trifunctional CD154⁺ CD4⁺ T cells were analyzed by Boolean gating of IL-2, IFN-γ, TNF-α, IL-4, and GrzB production. A detailed composition of bi- and trifunctional cells is presented in Figure S4. (G–I) Frequencies of antigen-specific CD4⁺ (G) T_EM, (H) T_CM, and (I) T_EMRA cells. (J–M) Frequencies of (J) CD137⁺ IL-2, IFN-γ, TNF-α and/or GrzB (CD137⁺ cytokine⁺) CD8⁺ T cells (antigen-specific CD8⁺ T cells), and (K) IFN-γ-, (L) IL-2-, and (M) GrzB-producing CD137⁺ CD8⁺ T cells. (N and O) Frequencies of polyfunctional CD137⁺ CD8⁺ T cells. (N) Bifunctional and (O) trifunctional CD137⁺ CD8⁺ T cells were analyzed by Boolean gating of IL-2, IFN-γ, TNF-α, IL-4, and GrzB production. Composition of bi- and trifunctional cells is presented in Figure S4. (P–R) Frequencies of antigen-specific CD8⁺ (P) T_EM, (Q) T_CM, and (R) T_EMRA cells. Statistical comparisons were done with 2-way repeated-measures ANOVA and with Tukey’s multiple comparison test. p < 0.05 was considered significant. Boxplots show medians and interquartile ranges. Whiskers and outliers were calculated with the Tukey method. See also Figures S1, S2, and S4– S6 and Table S1.

Figure 4

T Cell Reactivity Does Not Differ between Viral PCR-Positive and -Negative Patients Analysis of T cell responses of patients who cleared SARS-CoV-2 (n = 11) and patients who did not clear SARS-CoV-2 according to RT-PCR and were not discharged from the hospital during the observation period (uncleared, n = 7). In the cleared group, a sample before and after viral clearance was analyzed (initial and follow-up sample, respectively). In the uncleared group, the first (initial) and the last (follow-up) available sample were analyzed. PBMCs were stimulated for 16 h with S-, M-, or N-protein OPPs and analyzed by flow cytometry. The gating strategy to identify SARS-CoV-2 S-, M-, or N-protein-reactive T cells is presented in Figure S2. (A) SI of CD154⁺ CD137⁺ CD4⁺ T cells (antigen-specific CD4⁺ T cells), and CD137⁺ IL-2, IFN-γ, TNF-α, and/or GrzB (CD137⁺ cytokine⁺) CD8⁺ T cells (antigen-specific CD8⁺ T cells). SI was calculated by dividing the measured T cell subset response by the respective response in the DMSO control. Scatterplots show line at median; error bars represent interquartile ranges. The statistical comparison was done with 2-way repeated-measures ANOVA and with Sidak’s multiple comparisons test. p < 0.05 was considered significant. (B–E) Comparison of the frequencies of CD154⁺ CD137⁺ CD4⁺ T cells, CD137⁺ IL-2, IFN-γ, TNF-α, and/or GrzB (CD137⁺ cytokine⁺) CD8⁺ T cells, and bifunctional and trifunctional CD154⁺ CD4⁺ T cells. Bi- and trifunctional CD154⁺ CD4⁺ T cells were calculated by Boolean gating of IL-2, IFN-γ, TNF-α, IL-4, and GrzB production. The statistical comparisons were done with 2-way repeated-measures ANOVA and with Sidak’s multiple comparison test. p < 0.05 was considered significant. Boxplots show medians and interquartile ranges. Whiskers and outliers were calculated with the Tukey method. (F–I) Relative changes of the follow-up sample compared to the initial sample. A value of 1 indicates no change as compared to the initial sample, lower values indicate a reduction, and values >1 indicate an increase in the frequency of the subset; base 10 logarithmic scale. The statistical comparison was done with the Kruskal-Wallis test and Dunn’s multiple comparisons test. p < 0.05 was considered significant. Boxplots show medians and minimum and maximum values. See also Figures S1, S2, and S7 and Table S3.

Figure 5

T Cell Reactivity Does Not Differ between Patients with Non-critical Disease, Patients Who Recovered from COVID-19, and Patients Who Died Analysis of T cell responses in samples of patients who recovered from critical COVID-19 (n = 5) during critical disease (initial) and shortly before discharge from the intensive care unit (follow-up), compared to samples of critical COVID-19 patients who died (n = 6) and to samples of non-critical COVID-19 patients (n = 15). PBMCs were stimulated for 16 h with S-, M-, or N-protein OPPs and analyzed by flow cytometry. The gating strategy to identify SARS-CoV-2 S-, M-, or N-protein-reactive T cells is presented in Figure S2. (A) SI of CD154⁺ CD137⁺ CD4⁺ T cells (antigen-specific CD4⁺ T cells) and CD137⁺ IL-2, IFN-γ, TNF-α, and/or GrzB (CD137⁺ cytokine⁺) CD8⁺ T cells (antigen-specific CD8⁺ T cells). The SI was calculated by dividing the measured T cell subset response by the respective response in the DMSO control. Scatterplots show line at median; error bars represent interquartile ranges. The statistical comparison was done with 2-way repeated-measures ANOVA and with Sidak’s multiple comparisons test. p < 0.05 was considered significant. (B–E) Comparison of the frequencies of CD154⁺ CD137⁺ CD4⁺ T cells; CD137⁺ IL-2, IFN-γ, TNF-α, and/or GrzB (CD137⁺ cytokine⁺) CD8⁺ T cells; and bifunctional and trifunctional CD154⁺ CD4⁺ T cells. Bi- and trifunctional CD154⁺ CD4⁺ T cells were calculated by Boolean gating of IL-2, IFN-γ, TNF-α, IL-4, and GrzB production. The statistical comparisons between non-critical and recovered critical were done with 2-way repeated-measures ANOVA and with Sidak’s multiple comparison test. p < 0.05 was considered significant. Boxplots show medians and interquartile ranges. Whiskers and outliers were calculated with the Tukey method. (F–I) Relative changes of the follow-up sample compared to the initial sample. A value of 1 indicates no change as compared to the initial sample, lower values indicate a reduction, and values >1 indicate an increase in the frequency of the subset; base 10 logarithmic scale. The statistical comparison was done with the Kruskal-Wallis test and with Dunn’s multiple comparisons test. p < 0.05 was considered significant. Boxplots show medians and minimum and maximum values. See also Figures S1 and S2 and Table S4.