FIG. 8.

Adoptively transferred wild-type, IFN-γ^−/− or IL-17AF^−/− CD4⁺ T cells improve survival of neonatal CD4^−/− mice following infection with Y. enterocolitica, and MLN cells from these mice produce inflammatory cytokines upon in vitro stimulation with Y. enterocolitica antigens. Lymph node CD4⁺ T cells (6 × 10⁶) from adult C57BL/6, IFN-γ^−/−, or IL-17AF^−/− mice were injected intravenously into 1-day-old CD4^−/− mice. (A) Seven-day-old wild-type C57BL/6, CD4^−/−, CD4^−/− mice injected with wild type CD4⁺ cells, CD4^−/− mice injected with IFN-γ^−/− CD4⁺ cells, and CD4^−/− mice injected with IL-17AF^−/− CD4⁺ cells were infected orogastrically with 1 × 10⁷ CFU of Y. enterocolitica. The percent survival values from two to three individual experiments are connected with a line. (B) The data from the experiments in panel A were pooled to generate survival curves and analyzed by the Mantel-Haenszel log rank test. n.s., not significant; *, P ≤ 0.03; **, P = 0.0025 (for CD4^−/− versus CD4^−/− mice injected with CD4⁺ cells). (C) The small intestines were collected at 25 days p.i. from infected wild-type, CD4^−/−, CD4^−/− mice injected with IFN-γ^−/− CD4⁺ cells, or CD4^−/− mice injected with IL-17AF^−/− CD4⁺ cells, and tissues analyzed for Yersinia titers. The dashed line is at the limit of detection. The data from two experiments were pooled (n = 7 to 9 mice per group) and analyzed by the Mann-Whitney test. n.s., not significant; *, P = 0.01; **, P = 0.0012. (D) MLN from mice that survived the infection at 21 to 25 days p.i. were collected, and cell suspensions were stained with anti-CD4 and anti-CD44 antibodies. Representative flow cytometry profiles show the percentages of CD4⁺ (top) and CD44⁺ cells within CD4⁺ cells (bottom), with the range of cells from three to four experiments (n = 5 to 17 mice). (E) MLN cells from individual mice described in panel D were cultured for 72 h with 10 μg/well of HKY. MLN cells from uninfected C57BL/6 (n = 8), CD4^−/− (n = 25), and CD4^−/− mice injected with wild-type CD4⁺ cells (n = 5) were stimulated along with cells from infected CD4^−/− (n = 15), CD4^−/− mice injected with wild-type CD4⁺ cells (n = 8), CD4^−/− mice injected with IFN-γ^−/− CD4⁺ cells (n = 17), and CD4^−/− mice injected with IL-17AF^−/− CD4⁺ cells (n = 9). Supernatants were tested for IFN-γ and IL-17A by ELISA. Differences were analyzed by the Mann-Whitney test. n.s., not significant; For IL-17A, significance is indicated as follows: *, P ≤ 0.013 between infected CD4^−/− plus IL-17AF^−/− CD4⁺ cells versus infected wild-type or infected CD4^−/− mice; **, P ≤ 0.0049, ***P ≤ 0.0004 (between selected groups). For IFN-γ, significance is indicated as follows: **, P ≤ 0.0025 between infected CD4 ^−/− plus IFN-γ^−/− CD4⁺ cells versus infected wild-type or infected CD4^−/− mice; ***, P ≤ 0.0004 (betwen selected groups).