Figure 4

SRA-ECD protein inhibits T cell proliferation and cytokine production. (A, B) T cells purified from human PBMCs were stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies in the presence of the indicated concentration of SRA-ECD protein for 72 hr. T cell proliferation was assessed by FACS analysis of CD3⁺ T cells based on the dilution of CFSE intensity. Histograms are representative of two independent experiments (A). Supernatants were collected after 48 h and assayed for IL-2, IFN-γ and IL-4 using ELISA (B). (C) Naïve T cells stimulated with anti-CD3 and anti-CD28 antibodies for 72 hr. Then the cells were re-stimulated with anti-CD3 and anti-CD28 antibodies in the absence (medium) or presence of different concentration of SRA-ECD protein for 6 hours and subjected to intracellular cytokine staining assays. The percentages of IFN-γ-producing CD3⁺ T cells were calculated. Representative dot plots from two independent experiments are shown. (D) CFSE-labeled naïve T cells were stimulated in plates coated with anti-CD3 anxd anti-CD28 antibodies in the absence or presence of 10 μg/mL of SRA-ECD protein. After 72 h, T cells were stained for CD3, CD4 and CD8 respectively. T-cell proliferation was assessed by FACS analysis based on the dilution of CFSE intensity. Histograms are representative of three independent experiments. (E) Human PBMCs were activated with anti-CD3 and anti-CD28 antibodies co-stimulation for 48 h, and cells with and without stimulation were analyzed for SRA-ECD binding together with antibodies for CD3. Data are representative of three independent experiments.