Altered thymic selections of Tconv and Treg cells in ZAP-70 mutant mice. (A and B) CD4SP and CD8SP thymocytes (top row), and Foxp3⁺ Treg cells among CD4SP thymocytes (bottom row) in 8-wk-old BALB/c or ZAP-70 mutant mice. Frequency (A) and cell numbers of CD4SP and Foxp3⁺ CD4SP Treg cells (B; n = 11 each). (C) TCRβ expression levels of DP thymocytes after TCR stimulation in vitro. Data are representative of seven independent experiments. (D) TCR Vβ usage of CD4SP thymocytes in BALB/c and ZAP-70 mutant mice (n = 5 each). (E and F) Thymic development of CD4SP (top row) and Treg cells (bottom row, gated on CD4SP) in DO11.10⁺ RAG2^−/− or Ld-nOVA⁺ DO11.10⁺ RAG2^−/− mice on the ZAC or WT background. Representative plots (E) and summary (F; n = 5 each). (G and H) Proliferation of splenic Foxp3⁻CD4⁺ Tconv cells from indicated mice cultured with irradiated autologous APCs and anti-CD3 antibody (G) or without anti-CD3 antibody (H; n = 5 each). (I) Number of splenic CD4⁺ T cells in RAG2^−/− mice 8 wk after cell transfer of 5 × 10⁵ CD4⁺ T cells from the spleen of ZAP-70 mutant mice (n = 5 each). Two-tailed unpaired Student’s t test and mean ± SD in B, D, and F–I. *P < 0.05; **P < 0.005; ***P < 0.0005.