Tsg selectively inhibits proliferation of primed alloreactive T cells and enhances TGF-β–mediated inhibition. Primary CD4⁺ T cells (A) or primed alloreactive CD4⁺ T cell lines (B) were cultured with anti-CD3 mAb in the presence of either BMP2, BMP4 (100 ng/mL), or TGF-β (2 ng/mL) in the absence or in the presence of Tsg (2 μg/mL). Proliferation was determined by ³H-thymidine incorporation for the last 16 hours of a 72-hour total period of culture. A wide range of BMP2/4 concentrations (5-200 ng/mL) and Tsg concentrations (1-10 μg/mL) showed a similar pattern of results (data not shown). Results are representative of 6 independent experiments using cells from different donors. Similar effects on proliferation and cytokine production by alloreactive primed CD4⁺ T cells line were observed when commercial recombinant Tsg (R&D Systems) was used (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). (C) sTGF-β RII reverses the inhibitory effect of Tsg on proliferation of primed alloreactive T lymphocytes. Primed alloreactive CD4⁺ T-cell lines were cultured with the indicated stimuli in the presence of Tsg alone or with TGF-β. Various concentrations (100-500 ng/mL) of TFG-β RII were added (results shown represent TFG-β RII concentrations 250 ng/mL). Proliferation was determined by ³H-thymidine incorporation for the last 16 hours of a 72-hour total period of culture. (D) Tsg inhibits proliferation of primary T lymphocytes that receive optimal activation by anti-CD3 plus anti-CD28 mAbs. Primary CD4⁺ T cells were cultured as in panels A-B but with anti-CD3 and anti-CD28 mAbs instead of anti-CD3 mAb alone. Proliferation was determined by ³H-thymidine incorporation for the last 16 hours of a 72-hour total period of culture. Error bars indicate variation of cpm values.