External expression of CTLA-4 is crucial for CTLA-4–mediated suppression. (A) Resting CTLA-4^TgWT CD4⁺CD25⁻ lymph node T cells are not suppressive. CFSE-labeled CD4⁺CD25⁻ Tconv cells from lymph nodes of normal B6 (CD45.1) mice were either cultured alone (filled curves) or together with purified CD4⁺CD25⁻ T cells from B6 or CTLA-4^TgWT (ie, Ctla4^−/−CTLA-4^TgWT) mice (open curves) and stimulated to proliferate by anti-CD3 (1 μg/mL) and APCs. Proliferation of CD45.1⁺ 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 3 independent experiments. (B-C) Intracellular localization of CTLA-4 proteins in CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ Tconv cells. Resting T cells and T cells preactivated for 2 hours by anti-TCR were stained for TCR, CTLA-4, and GM130. In panel B, T cells were stained for surface TCR (shown in red) followed by fixation and staining for intracellular CTLA-4 (shown in green) and were then visualized by confocal microscopy (see also supplemental Figure 3 and supplemental Videos 1-2). In panel C, T cells were fixed and stained for intracellular CTLA-4 (shown in green) and GM130 (shown in red; see also supplemental Figure 3 and supplemental Videos 3-4) and then visualized by confocal microscopy. (D) Assessment of CTLA-4 externalization in Tregs and Tconv cells. To compare the rate of CTLA-4 recycling and externalization in Tregs and CTLA-4^TgWTTconv cells, resting CD4⁺CD25⁺ Tregs of B6 origin and resting CD4⁺CD25⁻ Tconv cells of either CTLA-4^TgWT or Ctla4^−/− origin were cultured (without APCs) at 37°C for 60 minutes with PE-conjugated anti–CTLA-4 mAb in plates that had been coated either with medium alone or with anti-TCR/CD28 mAbs. The PE-conjugated anti–CTLA-4 mAb was added to capture CTLA-4 proteins cycling to the surface and was detected as surface PE fluorescence. The PE fluorescence acquired in 60 minutes by the indicated T-cell populations during culture in medium alone (blue line) or during culture on plate-bound anti-TCR/CD28 mAbs (red line) is displayed. Data are representative of 2 independent experiments. (E) Preactivation makes CD4⁺CD25⁻ T cells from CTLA-4^TgWT mice suppressive. CFSE-labeled CD4⁺CD25⁻ lymph node Tconv cells from normal B6 mice were cultured alone (filled curves) or with the indicated T-cell populations (open curves) and stimulated to proliferate by anti-CD3 (1 μg/mL) and APCs. Where indicated, preactivated T cells were stimulated overnight with anti-TCR/CD28+IL-2 before addition to the stimulation cultures. The percentage of Tconv cells that failed to divide at least once in cocultures is shown. Data are representative of 3 independent experiments.