Altered development and self-skewed TCR repertoire selection in the thymus of ZAP-70 mutant mice. (A) Cell number of total thymocytes, CD8 SP thymocytes, and frequency of Foxp3⁺ cells in CD4SP thymocytes of 8-wk-old BALB/c, SKG, ZAC, and W163A mice (n = 11 each). (B) TCRβ, CD5, CD69, and HSA (Heat-Stable Antigen; CD24) expressions by DP and CD4SP thymocytes from BALB/c and ZAP-70 mutant mice. MFI in parentheses (n = 3 each). (C) TCR Vβ usage of splenic CD4⁺ T cells from ZAC, SKG, W163A, and WT BALB/c mice by FACS analysis (n = 5 each). (D) TCR Vβ usage of splenic Foxp3⁺ CD4⁺ Treg cells from BALB/c or ZAC mice (n = 7 each). (E) Kinetics of ERK phosphorylation upon TCR stimulation in CD4⁺ T cells from ZAC, SKG, W163A, and BALB/c mice (n = 7). (F and G) Proliferation of total CD4⁺ T cells from indicated mice cultured in the presence of irradiated autologous APCs with anti-CD3 antibody (F) or without anti-CD3 antibody (G; n = 5 each). (H) Lck protein levels in Foxp3⁻CD4⁺ T and Foxp3⁺ Treg cells in ZAC or WT mice. Numbers indicate MFI (n = 5). (I) Phosphorylated-tyrosine 394 of Lck (pY394-Lck) in Foxp3⁺ Treg and Foxp3⁻CD4⁺T cells from ZAC or WT mice (n = 3). Two-tailed unpaired Student’s t test in A, C, D, F, and G. Mean ± SD in A and C–G. Source data are available for this figure: SourceData FS4.