CTLA-4 externalization confers suppressor function to preactivated Tconv cells. (A) CTLA-4 expression in preactivated Tconv cells from B6 mice. CTLA-4 expression in resting B6 CD4⁺CD25⁺, resting B6 CD4⁺CD25⁻, and preactivated B6 CD4⁺CD25⁻ cells are displayed (shown in green). Preactivated B6 CD4⁺CD25⁻ T cells were purified B6 CD4⁺CD25⁻ T cells that had been stimulated overnight by plate-bound anti–TCR/CD28+IL-2 (in the absence of APCs). (B) Preactivation makes Tconv cells from B6 mice suppressive. CFSE-labeled CD4⁺CD25⁻ lymph node Tconv cells from normal B6 mice (CD45.1⁺) were either cultured alone (filled curves) or with resting B6 CD4⁺CD25⁺ Tregs and resting or preactivated B6 CD4⁺CD25⁻ lymph node T cells (open curves), and were stimulated to proliferate by anti-CD3 (1 μg/mL) and APCs. Preactivated B6 CD4⁺CD25⁻ T cells were obtained as in panel A. The percentage of Tconv cells that failed to divide at least once in cocultures is shown. (C) Suppression by preactivated Tconv cells requires CTLA-4 externalization and expression. Preactivated or resting CD4⁺CD25⁻ lymph node T cells of B6, Ctla4^−/−, CTLA-4^TgWT, or CTLA-4^TgΔ origin were cocultured with CFSE-labeled B6 CD4⁺CD25⁻ lymph node Tconv cells and stimulated to proliferate by anti-CD3 (1 μg/mL) and APCs. Lymph node T cells of Ctla4^−/− and CTLA-4^TgΔ origin were purified from mixed BM chimeras. In the left panel, Tconv cell proliferation was assessed by CFSE dilution. The percentage of suppression = 100*[(% of Tconv cells cultured alone that had divided at least once) − (% of Tconv cells from Treg cocultures that had divided at least once)] / (% of Tconv cells alone that had divided at least once). Means ± SE of 3 independent experiments and statistically significant differences as determined by the 2-tailed Student t test are indicated. In the right panel, CD25 surface staining on preactivated or resting CD4⁺CD25⁻ T cells was quantified into mean fluorescence intensity. Data are representative of 3 independent experiments.