(a-e) Flow cytometric assessment of thymocyte development showing proportion of single positive (SP) CD4 and SP CD8 thymocytes (a), total number of SP CD4 thymocytes (b), proportion and count of FOXP3+CD25+ thymic regulatory T cells (tTreg) from SP CD4 thymocytes (c), surface expression TCRβ on SP CD4 thymocytes (d), and cell number of peripheral CD4+ T cell output in the spleen (e). (f,g) d8 post-sheep red blood cell (SRBC) immunization of mice showing the proportion of IFNγ+ Th1, IL4+ Th2, and IL-17+ Th17 cells (f), and the proportion of FOXP3+CD25+ Tregs (g) from splenic total CD4+ T cells. Data are representative of four independent experiments. (h-i) In vitro CD4+ T cell differentiation assay on naive T cells cultured under Th1, Th2, Th17, or iTreg-polarizing conditions and analyzed d3 by flow cytometry (h) to measure expression of Th cell transcription factors in CD25+CD69+ activated T cells and on FOXP3+ activated T cells for iTreg cultures (i). Statistics were calculated by Student’s t-test, n.s., not significant; Dot symbols, individual mice; columns, median.