sGARP protein induces TGF-β production in naïve CD4⁺ T cells and signals through the TGF-β receptor. (A) Induction of TGF-β in naïve CD4⁺ T cells by sGARP. Cells were stimulated with anti-CD3 and anti-CD28 mAb with and without sGARP (1 µg/mL) or control protein. After 16 hours, supernatants were analyzed for TGF-β by enzyme-linked immunosorbent assay. TGF-β mRNA expression was analyzed 16 hours after stimulation normalized to EF1α. Relative expression of TGF-β mRNA is shown in percent normalized to untreated cells. Bar diagrams represent pooled data of 4 independent experiments (mean ± SEM, *P < .05). (B-C) sGARP binds to the TGF-β receptor. (B) CFSE-labeled naïve T cells were stimulated with anti-CD3 and anti-CD28 mAb in the presence of sGARP (1 µg/mL) or TGF-β (1 ng/mL) with and without blocking anti–TGF-βRII mAb (10 µg/mL) or were left untreated. Numbers indicate percentage of Foxp3⁺ cells 24 hours after stimulation. Dot blots of one representative experiment of 8 are shown (left). Bar diagrams (right) show pooled data (mean ± SEM, *P < .05, **P < .01). (C) GARP-mediated cytokine repression. Naïve T cells were stimulated as indicated, and intracellular cytokine staining was performed 10 days after primary stimulation. Numbers indicate percentage of cytokine-producing cells. Dot blots of one representative experiment are shown in the upper part. The bar diagrams (lower part) show pooled data (n = 8, mean ± SEM, *P < .05). (D) Mononuclear cord blood cells were stimulated with 0.5 µg/mL anti-CD3 mAb with and without sGARP in the presence or absence of blocking anti–TGF-βRII mAb. Smad2/3 phosphorylation in gated CD4⁺ T cells is shown 30 minutes after stimulation. A histogram of one representative experiment of 3 is shown. The bar diagram shows pooled data of 3 independent experiments (n = 3, mean ± SEM). (E) sGARP-mediated Treg induction. Naïve CD4⁺ T cells (Donor 1) were stimulated with anti-CD3 and anti-CD28 mAb and cultured in the presence and absence of sGARP (1 µg/mL) for 7 days before being used as suppressor cells in a coculture experiment. Allogeneic CD4⁺ T cells (Donor 2) simultaneously stimulated for 7 days with anti-CD3 and anti-CD28 mAb served as responder cells. Cocultures (10⁵ responder cells, Donor 2) plus titrated suppressor cells (Donor 1) were stimulated with 0.5 µg/mL anti-CD3 mAb in the presence of 3 × 10⁵ irradiated T cell–depleted PBMC feeder cells (Donor 3). As a control, CD4⁺ T cells from Donor 1 cultured without sGARP were cocultured at a ratio of 1:1 with CD4⁺ T cells of Donor 2. Proliferation was determined on day 4 by a 16-hour [³H] TdR pulse. Results show the pooled data of 3 independent experiments (mean ± SEM).