Figure 3.

Functional characterization of in vitro-expanded cross-reactive T cells

(A) Gating.

(B) Representative plot of CD4/CD8 distribution in the CD3⁺ population and percentage of CD4⁺ cells (bar graph) in cells expanded from 3 donors.

(C) Representative plot of CD45RA/CD197 in the CD4⁺ population and summary of the percentage of naive (N), central memory (CM), effector memory (EM), and EM re-expressing RA (TEMRA) populations in expanded cells (n = 3).

(D) Representative ICS plots for IFN-γ, TNF-α, and IL-2 production, and CD107a mobilization, in the CD3⁺ population after re-stimulation of expanded cells with SARS-CoV-2 S pool (CoV-2 S) or peptides S_811–826 and S_816–831; positive responses shown in red boxes (>3-fold background).

(E) Visualization of the polyfunctional response using Simplified Presentation of Incredibly Complex Evaluations (SPICE) (Roederer et al., 2011): bar graph (means ± standard deviations) for each stimulating antigen (red for CoV-2, light green for S_811–826, dark green for S_816–831) and comparison to control (gray) (p < 0.05, Wilcoxon rank-sum test); pie and arcs show the combined contribution of each marker (pie slice colors correspond to colors shown at the bottom of the bar graphs).

(F) t-SNE analysis of concatenated data from 3 donors for stimulation with same antigens, showing density plots for each condition. Two gates (g1 and g2) were drawn, indicating major differences among stimulated and unstimulated samples. Histograms show IFN-γ, TNF-α, and CD107a in each gate. Representative density plots for responses of d0801 are shown (see also Figure S2).