Fig. 1.

Br treatment reduces CD25 expression on CD4⁺ T cells. A) CD4⁺CD25⁻ T cells (1×10⁶) were stimulated in vitro with anti-CD3 (10 µg/ml) for 48 h. Activated T cells were removed from stimulation, washed thrice, and re-plated with Br (25, 50, 100 µg/ml) for 8 h. Post culture, cells were analyzed for cell surface expression of CD25. As compared to baseline (0.5%), activated control CD4⁺ T cells up regulated CD25 surface expression (78%). Br treatment reduced the expression of CD25 to 52% (Br 25 µg/ml), 40% (Br 50 µg/ml) and 18% (Br 100 µg/ml). Inactivation of the cysteine protease activity of Br (100 µg/ml) with E64 prevents the loss of CD25 surface expression (72%). Experiments were carried out in triplicate. CD4 T cells (x-Axis) are compared to CD25 expression (y-Axis). B) The mean fluorescent intensity (MFI) of CD25 was compared between groups from A) above. C) CD4⁺CD25⁺ Endogenous Tregs (1×10⁵) were treated with Br (100 µg/ml) or media for 8 h and CD25 expression was compared between groups and to CD4⁺CD25⁻ (Negative) controls. The * represents a significant difference between the control anti-CD3 stimulated cells and all other groups, while the ** denotes significant differences between the E64 Inactivated Br and all other treatment groups. The ‡ represents p=0.05 when comparing the Br 50 µg/ml and Br 100 µg /ml treatment groups. Experiments were carried out in triplicate.