Figure S6.

SARS-CoV-2 Infection Induces Durable, Functional Spike-Reactive CD4⁺ T Cells, Related to Figure 3

(A) Flow cytometry sorting strategy for naive, T central memory (T_CM), and T effector memory (T_EM) cells from HC and CoV2⁺ PBMCs at Visit 1 and Visit 2 before 5-6 days of culture with autologous monocytes and SARS-CoV-2 spike protein or vehicle.

(B) Representative flow cytometry gating on PMA/Ionomycin-activated PBMCs for cytokine expression by antigen experienced (non-CD45RA⁺CCR7⁺) CD4⁺ T cells subset into CCR6^+/− T effector cells (Teff, CXCR5^-) and circulating T follicular helper cells (cTfh, CXCR5⁺).

(C) Representative flow cytometry gating on antigen-experienced (non-CD45RA⁺CCR7⁺) CD4⁺ T cells from HC and CoV2⁺ V2 PBMCs following incubation with SARS-CoV-2 spike for 20 h. Gating on CD69⁺ CCR6^+/− T effector cells (Teff, CXCR5^-) and CCR6^+/− circulating T follicular helper cells (cTfh, CXCR5⁺) for IL-2, IFN-γ and IL-17A effector cytokines expression.

(D) Number of IL-4-producing, antigen-experienced CD69⁺CD4⁺ T cells per 1x10⁶ CD4⁺ T cells after incubation with vehicle (Veh.) or SARS-CoV2 spike (S) (left) and calculated number of spike-responsive, cytokine-producing CD4⁺ T cells (number after incubation with spike minus number after incubation with vehicle)(right).

Statistics determined by two-tailed Mann-Whitney tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value < 0.05. Error bars represent mean and SD. Data from two experiments per visit.