Cd80 higg1 fc

To determine whether αCTLA-4 clone 4F10 blocks CTLA-4 binding to CD80 and CD86, we permeabilized splenocytes and treated them with graded doses of 4F10 or an isotype control mAb for 60 min at 4°C. All subsequent incubations lasted 30 min at 4°C. Cells were washed, and the amount of residual functionally available CTLA-4 was determined by incubation with 2 μg/ml of recombinant CD80-hIgG1 Fc or CD80-hIgG1 Fc (Biolegend), revealed by subsequent incubation first with 1.25 μg/ml of PE-conjugated goat αhu-man IgG (Southern Biotech) and then with 5 μg/ml PE-conjugated αgoat IgG Abs (Invitrogen). To prevent binding of CD80-hIgG1 Fc or CD80-hIgG1 Fc to CD28 on splenocytes, we blocked CD28 with 10 μg/ml of αCD28 clone 37.51 (Biolegend) during incubation with titrated doses of 4F10. Alternatively to CD80- and CD86-Fc, residual non-blocked CTLA-4 was revealed by incubation with 4 μg/ml APC-conjugated 4F10 mAb (Millipore) under conditions at which displacement of the originally applied blocking antibody is negligible (data not shown). Further staining for CD4 and Foxp3 through conventional protocols identified Treg and Th cells.