Effect of CTLA-4 on TCR signaling and anergy. (A) Impairment of TCR signaling in Tregs from normal B6 mice. In the left panel, whole-cell lysates from CD4⁺CD25⁻ and CD4⁺CD25⁺ B6 lymph node T cells that were either unstimulated or stimulated by anti–TCR/CD4 cross-linking (in the absence of APCs) were blotted for p23 phospho-ζ (p23ζ) with a phospho-tyrosine (p-Y)–specific Ab. The p23ζ band intensity from anti-TCR/CD4–stimulated CD4⁺CD25⁺ T cells was set to 100. As an intrinsic loading control, membranes were also blotted for total ζ with a ζ-specific Ab. Data are representative of 3 independent experiments. In the right panel, calcium flux was induced in Indo-1–loaded CD4⁺CD25⁻ and CD4⁺CD25⁺ B6 lymph node T cells by avidin-induced cross-linking of biotinylated anti-TCR/CD4 mAbs. Data are representative of 6 independent experiments. (B) TCR-induced ITAM phosphorylation in Tregs of different donor origin. Whole-cell lysates from CD4⁺CD25⁺ lymph node T cells of different origin from B6+Ctla4^−/−→B6 mice that were either unstimulated or stimulated by anti–TCR/CD4 cross-linking (in the absence of APCs) were blotted for p23ζ. The p23ζ band intensity from anti–TCR/CD4–stimulated CD4⁺CD25⁺ T cells of B6 origin was set to 100. As an intrinsic loading control, membranes were also blotted for total ζ. Data are representative of 2 independent experiments from 20 mixed BM chimeras. (C) The CTLA-4 internal domain is responsible for TCR hyposignaling. TCR-induced calcium mobilization in peripheral T cells was induced in Indo-1–loaded CD4⁺CD25⁻ and CD4⁺CD25⁺ lymph node T cells by avidin-induced cross-linking of biotinylated anti–TCR/CD4 mAbs or by ionomycin (in the absence of APC). In mixed-radiation BM chimeras, B6 donor origin T cells were identified as CD45.2⁻ T cells, whereas Ctla4^−/−, CTLA-4^TgWT or CTLA-4^TgΔ origin T cells were identified as CD45.2⁺. Data for each experimental group are representative of 4 independent experiments. (D) The CTLA-4 external domain induces Treg anergy. Tregs from the indicated populations of CD4⁺CD25⁺ lymph node T cells were sorted and stimulated by anti-CD3 (1 μg/mL) with APC (left panel) or by APC with rIL-2 (200 U/mL) with or without anti-CD3 (right panel). Tregs from Ctla4^−/− and CTLA-4^TgΔ origins were purified from mixed BM chimeras. Proliferation was measured by ³H-thymidine incorporation and mean cpm ± SD of triplicate wells are shown. Data are representative of 3 independent experiments. (E) B6 lymph node CD4⁺CD25⁻ Tconv cells and Ctla4^−/− origin Tregs from B6+Ctla4^−/−→B6 mice were stimulated by anti-CD3 (1 μg/mL) and APCs in the presence or absence of CTLA-4Ig (10 μg/mL). Proliferation was measured by ³H-thymidine incorporation and mean cpm ± SD of triplicate wells are shown. Data are representative of 2 independent experiments.