CTLA-4 external domain confers Treg suppression. (A) Treg suppression as assessed by CFSE dye dilution. CFSE-labeled CD4⁺CD25⁻ lymph node Tconv cells from normal B6 mice were either cultured alone (filled curves) or cultured together with purified CD4⁺CD25⁺ Tregs of different mice origin and stimulated to proliferate by anti-CD3 (1 μg/mL) and APCs bearing CD80/86–costimulatory ligands. Tregs from Ctla4^−/− and CTLA-4^TgΔ origins were purified from mixed BM chimeras. Proliferation of Tconv cells was assessed by CFSE dye dilution. The percentage of Tconv cells that failed to divide at least once in cocultures is shown. Data are representative of 4 independent experiments. (B) Five-week-old RAG-2–deficient mice were injected with 4 × 10⁵ CD4⁺CD45RB^high (CD45.1⁺) T cells alone or together with 4 × 10⁵ purified CD4⁺CD25⁺ Treg cells (CD45.2) of either B6 or CTLA-4^TgΔ donor origin obtained from B6+CTLA-4^TgΔ→B6 mice. The percentage change from initial body weight of the recipients was monitored over time. The initial weight gain was because of the young age of the recipient mice. Each data point represents 3 or 4 RAG2^−/− recipient mice. (C) RAG-2–deficient mice were injected with 4 × 10⁵ CD4⁺CD45RB^high (CD45.1⁺) T cells alone or with 4 × 10⁵ purified CD4⁺CD25⁺ (CD45.2) Tregs of either Ctla4^−/− or CTLA-4^TgΔ origin obtained from mixed BM chimeras. Tregs of B6 origin were used as a positive control. Five weeks later, mice were killed and their colons stained with H&E. Images are representative of 4 mice from each group. Staining of mesenteric lymph nodes for the relative proportions of CD4⁺CD45RB^high effectors and CD4⁺CD25⁺ Tregs is shown.