Functional characterization of distinct populations of Th1 responses using multiparameter flow cytometry. A, The staining panel and gating strategy used to identify Ag-specific IFN-γ–, TNF-α–, and IL-2–producing CD4+ T cells in the splenocytes from a representative mouse with intranasal live EB immunization. B, Boolean combinations of the three total cytokine gates (IFN-γ, TNF-α, and IL-2) to uniquely discriminate responding cells based on their functionality or quality with respect to cytokine production.