Fig. 7: Impact of IFN-γ and TNF-α on specific signaling cascades in Tregs.

a–e Splenic Tregs, non-Treg CD4⁺ T cells, and CD8⁺ Teff were cultured for 8 days in the presence of anti-CD3/anti-CD28 beads and IL-2 with or without IFN-γ and TNF-α. Culture supernatant was replaced biweekly with media containing fresh cytokines. a Expression of Ki67 and b expression of annexin V and viability dye aqua on Tregs. c Expression of Ki67 on CD4⁺ Foxp3⁻ Teff (left panel) and CD8⁺ Teff (right panel) is shown, analyzed on day 9. Data (mean ± SEM) is representative of 3–6 independent experiments (**p < 0.01, ****P < 0.0001; unpaired t test). d, e Reverse-phase protein array (RPPA) profiling of cultured Tregs on day 4. Enriched functional pathway and molecular pathway analysis on differentially expressed proteins (P value < 0.05 and fold change >1.25 or <1/1.25). Activation z score depicting up- or downregulated d functions and e signaling pathways in IL-2 and IFN-γ plus TNF-α-treated Tregs relative to Tregs treated with IL-2 alone. Data are from three experimental and three technical replicates.