T-bet haploinsufficiency prevents the development of KLRG1⁺ CD4⁺ T cells and promotes improved control of Mtb. (A) FACS analysis of T-bet expression by I-A^b ESAT-6_4–17–specific CD4⁺ (left) and H-2K^b TB10.4_4–11–specific CD8⁺ (right) T cells in the lungs of B6 or Il27ra^−/− mice over time. CD4 data are representative of a total of five independent experiments with four to five mice per group. CD8 data are representative of two independent experiments with four mice per group. ***, P < 0.0001. (B) Representative T-bet expression in I-A^b ESAT-6_4–17–specific CD4⁺ T cells from the lungs of B6, Il27ra^−/−, T-bet–haploinsufficient (Tbx21^+/−), and T-bet gene–deleted (Tbx21^−/−) mice at day 60 after aerosol Mtb infection was determined by flow cytometry. (C) Representative FACS analysis of PD-1 and KLRG1 expression in I-A^b ESAT-6_4–17–specific CD4⁺ T cells from Mtb-infected B6, Il27ra^−/−, Tbx21^−/+, and Tbx21^−/− KLRG1^hi CD4⁺ T cells. (D) The frequency of I-A^b ESAT-6_4–17 KLRG1^hi CD4⁺ T cells in the lungs of Mtb-infected B6, Il27ra^−/−, or Tbx21^+/− mice over time. One representative experiment of two total experiments is shown, each with three to five mice per group. **, P < 0.005; ***, P < 0.0001 by ANOVA followed by Dunnett’s comparison to control (B6). (E) Tbx21^+/− and B6 mice were infected via the aerosol route, and the number of bacteria in the lung was determined over time. Data are pooled from four independent experiments with 8–16 total mice per group. *, P < 0.05; **, P < 0.005. (A, D, and E) Error bars represent the mean ± SD.