Figure 3.

iCD8SP cells generated from CD4SP thymocytes in the presence of IL-4 are stable and diverse. (A) Proliferation of CD4SP thymocytes, sorted as CD3^highCD4⁺CD8^negCD25^neg TCRγδ^neg cells, in response to IL-2, IL-4 and IL-7, as measured by Cell Trace Violet (CTV) dilution. Frequency of CTV^neg cells is presented in each population, and data are representative of 3 experiments. (B) Proliferation of tCD4SP, iDP and iCD8SP thymocytes in response to IL-4, as measured by CTV dilution or Ki67 frequency (graph: n=5). (C) Stability of IL-4-induced CD8 expression, as assessed by sorting iDP and iCD8SP populations on day 7 of IL-4 culture and either maintaining cells in IL-4 or switching them to IL-2 for 7 more days. (D) CD8 induction in CD4⁺CD8^neg (tCD4SP) cells sorted 7 days after IL-4 culture of CD4SP thymocytes and cultured in the presence of IL-4 for 7 additional days. (E) Bcl-2 median fluorescence intensity (MFI) of different populations before and after exposure to IL-4 or IL-7. (F) TCR Vβ repertoire of CD8SP, CD4SP and IL-4-induced iCD8SP thymocytes (n=3). Results in graphs are presented as mean ± SD.