Expression and identity of a non-CD28 receptor for B7-1 on the cell surface of P1CTL.

(A) CD28-independent expression of CTLA4 on activated T cells. Naive (i-ii), in vitro–activated (iii-iv), and ex vivo–activated (v-vi) P1CTL were stained with either PE-conjugated anti-CTLA4 mAb (solid lines) or isotype control (dotted lines). Data shown were gated CD8 T cells, marked by FITC-conjugated anti-CD8 mAb. (B) Blocking of B7-1 immunoglobulin binding to activated CD28^−/− P1CTL by anti-CTLA4, but not anti-CD8 mAbs. CD28^−/− P1CTL were stimulated for 3 days in vitro with P1A peptide and were stained with biotinylated B7-1 immunoglobulin or control HSA immunoglobulin followed by PE-conjugated streptavidin. To verify the involvement of CTLA4, half the cells were pretreated with either anti-CD8 or anti-CTLA4 mAb (100 μg/mL) for 30 minutes before the addition of biotinylated fusion proteins. Data shown are normalized histograms using Flowjo software (version 3.4). Essentially identical numbers of cells were analyzed to produce the overlaid histograms. The number of gated CD8 T cells analyzed were naive, 10 000 events; in vitro activated, 5000 events; ex vivo activated CD28^−/− T cells, 4000 events; ex vivo– activated WT CD8 T cells, 5000 events. These experiments were repeated twice with similar results.