Extended Data Fig. 9. Gating strategy to detect antigen-reactive CD4⁺ T cells.

PBMC were stimulated with Candida albicans antigen (0.05 Units/mL) for 6 days. Antigen-reactive T cells were detected by flow cytometry. (a-c). Cells of interest were identified by forward versus side scatter (FSC vs SSC) based on size and granularity (a). Alive cells were identified as 7-AAD^negative (b). CD4⁺ T cells were identified as CD3⁺ CD4⁺ cells (c).

(d). Antigen-reactive CD3⁺CD4⁺ T cells were identified as CD69⁺ CD40L⁺. Frequencies in FMO, in non-stimulated cells and in antigen-stimulated cells.

(e). T cells were primed with antigen as in a-d and restimulated with Mϕ that were loaded with vehicle or antigen. CD3⁺CD4⁺ CD69⁺ CD40L⁺ T cells were measured after 6 hours.