Figure 6.

(A and B) The suppressive effect of βGBP is reversible. Cells were either incubated with βGBP for 3 hours, washed and cultured in a fresh medium overnight at 37°C prior to stimulation (pre-incubation) or were treated with βGBP for 3 hours immediately prior to stimulation (co-incubation). Cells were activated with either soluble CD3 Ab (soluble CD3) or plate-bound CD3 Ab (plate CD3). Legend indicates titrated amounts of βGBP (μg/ml). (A) Proportion of proliferated CD8⁺CFSE^low cells was evaluated after 4 days of culture by flow cytometry analysis. Results are presented in percentage of the proliferation rate of untreated CD8⁺ T cells (0).*P<0.05 is for comparisons with untreated cells (0) within the group. (B) CD8⁺ T cells treated with 1 μg/ml βGBP for 3 hours and then were stimulated with either soluble or plate-bound CD3 Ab for 5 min, lysed and were analyzed by western blotting for pERK and ERK levels. *Cells were treated with βGBP for 3 hours one day prior to stimulation. (C) βGBP suppresses proliferation of TCR transgenic CD8⁺ T cells from splenocytes of pmel mice induced by mouse, but not human, gp100 peptide. Pmel mice splenocytes were cultured in the presence or absence (Cnt) of 1 μg/ml βGBP and indicated peptides (both at 1 μg/ml) for 4 days. No peptide was added to control cells (unstimulated). Proliferation was evaluated by BrdU incorporation and ELISA. Data represent mean ± SD of OD values at 450 nm. The peptide stimulation yielded more than 95% CD8⁺ cells at the time of assay (data not shown). *P<0.05 is for comparison with the mgp100 Cnt group.