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.