Fig. 1. Detection of SARS-CoV-2-specific CD4⁺ T cell responses using combinatorial tetramer staining.

PBMC from DRB1*07:01 (DR7)⁺ subjects were stained with DR7-matched spike and nucleocapsid tetramers, magnetically enriched, and analyzed by flow cytometry. (A-C) Representative flow cytometry plots of live CD14^–CD20^–CD3⁺CD8^–CD4⁺ gated events with indicated tetramer⁺ gates for the 4 epitope-specific populations from a pre-pandemic negative control (A), a non-hospitalized subject (B), and a previously hospitalized subject (C). (D) Representative flow cytometry plots of combinatorial tetramer staining of live CD14^–CD20^–CD3⁺CD8^–CD4⁺ gated events (left column) and live CD14^–CD20^–CD3⁺CD8⁺CD4^– gated events (right column). (E) Summary of individual epitope-specific CD4⁺ T cell frequencies and combined tetramer⁺ cells per million CD4⁺ T cells. Uninfected n = 9, non-hospitalized n = 31, previously hospitalized n = 9. Statistics by Kruskal-Wallis and Dunn’s multiple comparisons tests. (F-H) Correlations between DR7:S310⁺ and DR7:S166⁺ (spike-specific) cell frequencies (F), between DR7:N305⁺ and DR7:N329⁺ (nucleocapsid-specific) cell frequencies (G), and between nucleocapsid-specific (combined DR7:N305⁺ and DR7:N329⁺) and spike-specific (combined DR7:S310⁺ and DR7:S166⁺) cell frequencies (H), with r and significance from Spearman correlation. (I) Comparison of timing of first time point sample collection of all non-hospitalized and previously hospitalized subjects in the MassCPR cohort. (J) Summary of circulating anti-S, anti-RBD, and anti-N IgG antibody levels from paired plasma samples. AU denotes arbitrary units. Statistics by Mann-Whitney tests. Solid horizontal lines indicate median values. Dotted horizontal line indicates limit of detection. *p < 0.05, **p < 0.01, ***p < 0.001. ns = not statistically significant.