DIgR2 binds with its unknown ligand expressed by T cells and inhibits DC-induced T-cell proliferation in vitro. The binding of DIgR2-Ig fusion protein to CD3⁺ T cells (A), CD4⁺ T cells, CD8⁺ T cells, B cells, NK cells, monocytes, and DCs (B) was analyzed by fluorescence-activated cell sorting (FACS). CD3⁺, CD4⁺, and CD8⁺ T cells, B cells, and NK cells were freshly isolated from mouse splenocytes with CD3, CD4, CD8a, CD19, and NK1.1 MicroBeads, respectively. Macrophages were isolated from peritoneal exudates, and bone marrow-derived DCs were prepared conventionally. Purity of each cell subset exceeds more than 90%. Cells were stained with 40 ng/mL DIgR2-Ig or hIg (CD3⁺ T cells were also stained with DIgR2-Ig mixed with 400 ng/mL GST-DIgR2) and then stained with FITC-conjugated sheep anti-human IgG (Sigma). Data shown are representative of 3 independent experiments. (C) Mixed lymphocyte reaction (MLR) of DCs from C57BL/6 mice incubated with splenocytes from Balb/c mice, with 50 μg/mL DIgR2-Ig or hIg added into the coculture. Data represent the mean (+SE) of [³H] thymidine uptake in 3 independent experiments. (D) Analysis of DIgR2 levels in siRNA-transfected DCs. Day 6 BM-DCs were transfected with synthetic DIgR2 siRNA, siRNA mutant duplexes, or mock. Expression of DIgR2 was determined in triplicate after stimulation with LPS (100 ng/mL) by Western blotting 48 hours after transfection (bottom panel), and DIgR2 mRNA was assessed by RT-PCR on different days after transfection (top panel). Data shown are representative of 3 independent experiments. (E) MLR was conducted as described. DCs were transfected with DIgR2-siRNA, DIgR2-mut-siRNA, or mock before coculturing with splenocytes. Data represent the mean (+SE) of [³H] thymidine uptake in 3 independent experiments.