Development of TβRII^-/- thymocytes in FTOC. (A) (1) Thymocytes were harvested from bone marrow recipients at 3 weeks after transplantation and enriched for CD4^-CD8^- thymocytes. (2) Simultaneously, thymic lobes from day-14.5 embryos were dissected. (3) Thymocytes and lobes were put together to aggregate in hanging drop culture for 2 days and (4) subsequently plated into FTOC cultures for 7 or 14 days until flow cytometric analysis. (B,D) Representative dot plots showing flow cytometric analysis of thymocytes at days 7 and 14 of FTOC. CD25/CD44 plots were gated for CD3⁺ in addition to CD4^-CD8^- cells to ensure immaturity. Percentage values for each subset are indicated. (C,E) Mean percentage values of T-cell subsets from a number of independent FTOC experiments. (▪) TβRII^-/- FTOC thymocytes; (▦) control FTOC thymocytes (TβRII^+/- or TβRII^flox/flox). Number of experiments: CD4/CD8 analysis: TβRII^-/- (n = 5), control (n = 4); CD25/CD44 analysis: TβRII^-/- (n = 5), control (n = 5). The scale represents mean percentage of the Ly5.2⁺ gated populations indicated. Results are expressed as mean ± SDs.