Blockage of IL-4 and IL-15 inhibits the induction of the innate phenotype in DP thymocytes.

A bulk population of CD45.2+ thymocytes either from WT control, WT T. cruzi-infected (Tulahuen) or IL-4KO T. cruzi-infected (Tulahuen) mice were obtained at day 14 post-infection and cultured for 2h at 37°C in the presence of PMA/ionomycin. Cells were washed twice and co-cultured with sorted DP cells from OT-I (Vβ5+ OVA-tetramer+) control mice at a 1:1 ratio in the presence or absence of a neutralizing anti-IL-15 Ab. After 48h, thymocytes were obtained and CD44, CD122, CD49d, Eomes and Tbet expression were analyzed by Flow cytometry only in DP CD8 OVA-specific OT-I thymocytes. Eomes or Tbet were measured by intranuclear staining using Flow cytometry analysis and were expressed as the difference of the mean fluorescence intensity (MFI) of Eomes or Tbet vs the MFI of the correspondent isotype control (IC). Histograms are representative of two independent experiments with 3–6 mice/experiment. The statistical test applied was a One-way ANOVA. T. cruzi vs the rest of the groups, *p.05, **p.01 and ***p.001. Tc = T. cruzi; Tc+α15 = T. cruzi + anti-IL-15 neutralizing Ab; Tc4KO = IL-4 KO T. cruzi; Tc4KO+α15 = IL-4 KO T. cruzi + anti-IL-15 neutralizing Ab.