FIG 2.

tTreg cell differentiation in fetal thymus organ culture requires CD69 expression. (A) Representative density plots of 5-day FTOCs from cd69^+/+ and cd69^−/− embryos in the C57BL/6 background. Embryonic thymuses were removed from 15- to 17-day-old embryos, and the percentages of tTreg development in the lobes were determined by FACS analysis. (B) Cellularity of fetal thymus lobes (left) and total cell numbers of CD4⁺ Foxp3⁺ cells (right) from cd69^+/+ and cd69^−/− embryos. (C) FTOCs from wild-type 17-day-old embryos (E17) were maintained for up to 14 days in culture in the presence of anti-CD69 monoclonal antibody (2.2) or the isotype control antibody (2.8). Density plots shows the percentages of tTreg cells on days 4, 11, and 14 after culture. (D) Cellularity of fetal thymus lobes (left) and total numbers of CD4⁺ Foxp3⁺ cells (right) under each condition. Totals of 31 and 36 embryos from five cd69^+/+ and four cd69^−/− females, respectively, were analyzed. The 2 lobes from each fetal thymus were analyzed separately. Error bars show standard deviations. Values were calculated relative to data for cd69^+/+ control lobes from four independent FTOC assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student's t test).