Effect of RELT-hFc fusion protein in T-cell responses.
(A) RELT-hFc binds PMA and ionomycin-activated T cells. Nylon wool–purified T cells were activated for 18 hours in 200 ng/mL ionomycin and 20 ng/mL PMA and then double stained with RELT-hFc biotin and anti-CD3 phycoerythrin. Histogram shows CD3 gated cells. Filled histogram is hIgG biotin control, and open histogram is RELT-hFc biotin. (B) RELT-hFc costimulates T-cell proliferation. For proliferation assays, 96-well plates were coated with the indicated concentration of anti-CD3, washed with PBS, and coated with 10 μg/mL of the indicated protein. After coating with fusion protein, wells were washed again with PBS, and T cells were added. T cells were incubated for 72 hours and pulsed with 1 μCi 3H thymidine for the last 18 hours before being harvested. All CPM values are from triplicate wells. This experiment is representative of 3 experiments. (C) Addition of soluble RELT-hFc selectively inhibits RELT-hFc costimulation of T-cell proliferation. Proliferation assays were performed as in (A). Soluble RELT-hFc was added at a dose of 10 μg/mL, and the dose of anti-CD3 is 6.25 ng/mL. (D) RELT-hFc does not inhibit the one-way mixed lymphocyte reaction. Nylon wool–purified T cells and stimulator PBMCs that had been irradiated for 3000 rads were incubated at various responder-to-stimulator ratios with 10 μg/mL hIgG or RELT-hFc. The mixed cells were incubated for 5 days and pulsed with 1 μCi 3H thymidine for the last 18 hours. All CPM values are from triplicate wells. This graph is representative of 3 experiments.
