Figure 2.

SARS-CoV-2-Specific CD4⁺ T Cell Responses

(A) Representative flow cytometry gating of AIM⁺ (OX40⁺surfaceCD40L⁺) CD4⁺ T cells.

(B) Percentage of background subtracted SARS-CoV-2-specific total CD4⁺ T cells quantified by AIM after stimulation with MP_R (Non-Spike), S (Spike), M (Membrane), or N (Nucleocapsid) peptide pools in unexposed (n = 15), acute COVID-19 (n = 30) and convalescent COVID-19 (n = 15).

(C and D) Percentage of background subtracted combined MP_R, S, M, and N SARS-CoV-2-specific CD4⁺ T cells by AIM assay by (C) cohort and (D) by days PSO. Combined AIM responses were calculated as the sum of the CD4⁺ AIM response to background-subtracted individual peptide megapools. Statistics in (D) are reported for unexposed, convalescent, and acute samples.

(E and F) ICS of SARS-CoV-2-specific CD4⁺ T cells quantified by co-expression of (E) CD40L⁺IFNγ⁺ or (F) CD40L⁺IL-2⁺ after stimulation with SARS-CoV-2 peptide pools in unexposed (n = 8), acute COVID-19 (n = 14) and convalescent COVID-19 samples (n = 11).

(G and H) Cytokines IFNγ (G) and IL-2 (H) in the supernatants after stimulation with SARS-CoV-2 or CMV peptide pools in unexposed (n = 15), acute COVID-19 (n = 22), convalescent COVID-19 (n = 15), and CMV⁺ controls (n = 23). The black dotted line delineates background signal as determined by the unexposed controls.

(I) Representative flow cytometry of SARS-CoV-2-specific (OX40⁺CD40L⁺) CD4⁺ T cells (blue dots) overlaid on total CD4⁺T cells (black dots).

(J) Percentage of SARS-CoV-2-specific cT_FH cells in acute COVID-19 (n = 22) or convalescent COVID-19 (n = 15) samples that had a positive total CD4⁺ AIM response (> 0.04%) following stimulation with the SARS-CoV-2 S megapool (MP), or the total non-antigen-specific CXCR5⁺ CD4⁺ T cells in unexposed controls (n = 15, gray dots) (median displayed).

(K) Representative fluorescence-activated cell sorting (FACS) plots of CXCR3 and CCR6 staining in total cT_FH (CXCR5⁺CD4⁺ cells) in unexposed donors or S-specific AIM⁺ (OX40⁺CD40L⁺) CD4⁺ T cells in acute and convalescent donors.

(L and M) Frequency of (L) CXCR3 and/or CCR6 expressing S-specific AIM⁺ cells out of total CD4⁺ T cells in acute or convalescent samples or non-antigen-specific CXCR5⁺CD4⁺ cT_FH in unexposed samples (n = 15) and (M) CXCR5⁺ S-reactive AIM⁺ cells out of total CD4⁺ T cells in acute donors (n = 26 samples) or convalescent donors (n = 15 samples). Unless otherwise stated, the black dotted line indicates LOD; the green dotted line demarcates marginal responses as determined by unexposed donor responses. Pink dots denote samples where two or more peptide pools were not run due to cell numbers.

^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, NS = not significant. See also Figure S2, Table S4, and Table S5.