Induction of CD95 in T cells by EBV and purified gp350. (A) Affinity-purified T cells were incubated at 1 × 106 T cells/200 μL for 2 hours on ice with 1/10 vol of mock virus, 1/10 vol of purified Akata virus, or 1/10 vol of purified Akata virus that was previously incubated on ice for 1 hour with either 1 μg/mL 72A1 or 858-3 antibody. Cells were washed 2 times with cold PBS and cultured for 24 hours. Cells were stained with FITC-conjugated CD95 MoAb and PE-conjugated antihuman CD4 MoAb (□) or antihuman CD8 MoAb (▪). Purified T cells (2 × 105/200 μL) were cultured for 18 hours with either plate-bound anti-gp350/220 MoAbs 72A1, 2L10, control MoAb 858-3, antibody-gp350 complexes, or PHA. Cells were stained with FITC-conjugated anti-CD95 MoAb and PE-conjugated antihuman CD4 MoAb or antihuman CD8 MoAb. CD95 expression in medium-treated CD4+ and CD8+ T cells for the 3 separate donors was subtracted during analysis and the mean CD95 percentage positive cells ± SD was plotted. Statistically significant values after analysis by the Student’s t-test are designated by an asterisk. (B) Purified Akata virus used in the EBV stimulation experiments is shown (original magnification × 86,500). (C) The purified gp350 used in the formation of the antibody-gp350 complexes was resolved in a 6.5% gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver stained.
