Tsg enhances binding of TGF-β on cell surface receptors. (A) Primed alloreactive CD4⁺ T-cell line was cultured with media or anti-CD3 mAb for 24 hours and subsequently cells were incubated with increasing concentrations of biotinylated TGF-β (1.25 ng/sample, shaded histogram; 2.5 ng/sample, gray line histogram; 12.5 ng/sample black line histogram). A sample of cells from the media-only culture incubated with 12.5 ng biotinylated TGF-β (dotted line histogram) is included as negative control. (The data of this sample were comparable to those obtained when these cells were incubated with 1.25 ng/sample and 2.5 ng/sample of TGF-β [data not shown]). Following incubation with avidin-fluorescein, samples were analyzed by flow cytometry. (B-D) Alloreactive CD4⁺ T cells cultured with anti-CD3 for 24 hours were incubated with increasing concentrations of TGF-β (B, 1.25 ng/sample; C, 2.50 ng/sample; D, 12.5 ng/sample) that had been preincubated overnight with equimolar concentrations of Tsg. Samples were subsequently incubated with avidin-fluorescein and were analyzed by flow cytometry. Results are representative of 2 independent experiments.