sGARP enhances Foxp3 expression and represses proliferation and cytokine production in CD4⁺ effector T cells. (A) sGARP increases Foxp3 expression. CFSE-labeled T cells were stimulated with anti-CD3 (0.5 µg/mL) and anti-CD28 mAb (1 µg/mL) in the presence of sGARP (1 µg/mL). Foxp3 expression was analyzed on day 3 by flow cytometry. Dot blots show one representative result for each T-cell subset (upper part). Diagrams (lower part) display summarized data of 7 independent experiments. Data represent the mean ± SEM of experiments (n = 7, **P < .01, ***P < .001; n.s., not significant). (B) sGARP inhibits proliferation. Each T-cell subset was stimulated under the same conditions described in (A) in the presence or absence of sGARP (1 µg/mL). Proliferation was analyzed on day 12 by ³H-TdR incorporation. Bar diagrams show pooled data of 3 independent experiments (percentage of proliferation) in the presence of sGARP normalized to proliferation of cells without sGARP (n = 3, mean ± SEM, **P < .01; n.s., not significant). (C) Each T-cell subset was analyzed for CD62L and CCR7 expression by flow cytometry as described. Peripheral CD4⁺CD45RA⁺ T cells displayed a mixed population of CD62L^high and CD62L^low cells (gray represents isotype control). Dot blot shows one representative out of 8 experiments (n = 8). (D) TGF-βR and membrane-bound TGF-β (TGF-β) expression on resting and activated (16 hour) naïve CD4⁺ T cells and CD4⁺CD45RO⁺ T cells. Histograms show one representative experiment of 4 independent experiments. Numbers indicate mean fluorescence intensities (MFI) of TGF-βR (naïve resting: 51.9 ± 19, activated: 34.5 ± 6; CD4⁺CD45RO⁺ resting: 22.1 ± 5, activated: 27.8 ± 14) or TGF-β (naïve resting: 15.2 ± 9, activated: 27.0 ± 4; CD4⁺CD45RO⁺ resting: 5.13 ± 0.5, activated: 13.3 ± 7) expression are indicated. (E) CFSE-labeled naïve CD4⁺ T cells and CD4⁺CD45RO⁺ T cells were stimulated with anti-CD3 and anti-CD28 mAb in the presence or absence of sGARP (1 µg/mL). Cytokines were analyzed on day 10 upon restimulation. Dot blots show one representative result of 5. Diagrams show summarized data of 5 independent experiments.