FIGURE 3.

(A) In vitro activation of CD4⁺ T cells induces secretion of sCTLA-4. CD4⁺ T cells of four healthy donors were stimulated in vitro with anti-CD3/CD2 mAb and culture supernatants harvested at the indicated time points. CD4⁺ T cells (3 × 10⁶ cells/ml) from the four donors were cultured on separate days. Isotype controls were negative for all samples (data not shown). (B) Secretion of sCTLA-4 by CD4⁺ T cells (1 × 10⁶ cells/ml) of three independent donors stimulated for 72 h in vitro with either anti-CD3/CD2 mAb or anti-CD3/CD28 microbeads. (C) Detection of sCTLA-4 following immunoprecipitation and Western blot analysis. sCTLA-4 protein in culture supernatants from activated CD4⁺ T cells and from HeLa cells transfected with sCTLA-4 was immunoprecipitated using 4B8 mAb, followed by 4017 polyclonal Ab immunoblotting. No sCTLA-4 was immunoprecipitated with the corresponding IgG2b isotype control (data not shown). Western blot gel is representative of more than three independent experiments.