Fig. 6. Aiolos-deficient CD4⁺ T cells exhibit increased cytotoxic hallmarks in an otherwise wildtype setting.

Naïve CD4⁺ T cells were isolated from OT-II-WT and OT-II-Ikzf3^−/−/ mice. 5 × 10⁵ cells were adoptively transferred into naïve CD45.1 mice which were then infected with 40 PFU OVA_323–339-expressing A/PR8/34 (“PR8-OT-II”). After 8 days, lungs were harvested and viable CD45.2⁺CD4⁺ populations were analyzed via flow cytometry. a, b Analysis of Eomes expression in antigen-specific (CD45.2⁺CD4⁺) cells. Data are representative of 6 independent experiments (n = 20 ± s.e.m; *P < 0.05, two-sided, unpaired Student’s t test). c, d Analysis of T-bet expression in antigen-specific (CD45.2⁺CD4⁺) cells^. Data are representative of five independent experiments (n = 17 ± s.e.m; two-sided, unpaired Student’s t test). e–i Whole-tissue homogenates from the lung were stimulated ex vivo in culture media with OVA_323-339 peptide for 48 h. Suspensions were then incubated in the presence of protein transport inhibitors for 3 h. e, f Flow cytometry analyses of antigen-specific (CD45.2⁺CD4⁺) cells for the expression of NKG2A/C/E and perforin. Data are representative of 4 independent experiments (n = 13 ± s.e.m; **P < 0.01, ****P < 0.0001, two-sided, unpaired Student’s t test). g Analysis of the number of antigen-specific (CD45.2⁺CD4⁺) cells from whole lung tissue. Data are representative of 5 independent experiments (n = 17 ± s.e.m; two-sided, unpaired Student’s t test). h, i Flow cytometry analyses of ex vivo stimulated antigen-specific (CD45.2⁺CD4⁺) cells for the expression of CD25 (IL-2Rα) and CD122 (IL-2Rβ). Data are representative of three independent experiments (n = 10 ± s.e.m; ***P < 0.001, two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.