Alloactivated T_reg cells express granzyme B during in vitro MLRs. The 129/SvJ WT (H-2K^b) splenocytes were cultured with irradiated (2000 cGy) Balb/c WT (H-2K^d) splenocytes in complete medium supplemented with 50 U/mL IL-2. At various time points, splenocytes were harvested and analyzed by flow cytometry for granzyme B expression. (A) Representative flow plots of granzyme B expression, gated on naive and day 6 MLR-stimulated CD8⁺ T cells. (B) Representative flow plots of granzyme B expression, gated on naive and day 6 MLR-stimulated CD4⁺Foxp3⁺ T_reg cells. (C) Summary graph of percentage granzyme B–expressing T_reg cells (n = 3 independent MLR cultures per time point).