ESAT-6–specific CD4 T cells produce IFN-γ in a TCR signaling–dependent manner. Mice were infected as described in Fig. 1. (A) Intracellular IFN-γ was assessed by flow cytometry directly ex vivo using lung cells (150 d after infection) processed in the presence of Brefeldin A. Gating strategy and representative flow cytometry plots for naive CD44low (gray), nontetramer-binding CD44hi (blue), and ESAT-6 tetramer-binding CD44hi (red) CD4 T cells are shown. (B) Flow cytometry plots show intracellular IFN-γ, or staining with isotype matched control, in ESAT-6–specific CD4 T cells (70 d after infection). Graph shows the percentage of ESAT-6 tetramer-binding or naive CD44low T cells producing IFN-γ at the indicated time points. (C) Mice infected with Mtb 35 d prior were treated intraperitoneally with either cyclosporine-A (CsA) or vehicle, and ESAT-6–specific cells in the lung were assessed for intracellular IFN-γ as in A and B. Numbers in parentheses represent the percentage of ESAT-6 tetramer-binding CD4 T cells producing IFN-γ and the graph shows this value for each mouse and the mean ± SEM for each group. Significance was determined by two-tailed Student's t test (**, P < 0.01). Data are representative of two independent experiments with four to five mice per group.
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