The induction and release of granzyme B in CD4⁺CD25⁺ T cells. (A) Induction of granzyme B mRNA after activation. CD4⁺CD25^– (⋄), CD4⁺CD25⁺ (♦), and CD8⁺ (▴) T cells that were either freshly isolated or activated for indicated periods were collected, total RNA isolated, and reverse transcribed. Granzyme B mRNA was then analyzed by quantitative PCR. (B) Induction of granzyme B protein after activation. CD4⁺CD25^–, CD4⁺CD25⁺, and CD8⁺ T cells were isolated by cell sorting and activated as indicated. Granzyme B expression was determined by intracellular staining. (C) Concomitant cell-surface expression of CD107a and release of granzyme B on restimulation. The 5-day preactivated CD4⁺CD25^–, CD4⁺CD25⁺, and CD8⁺ T cells were restimulated with soluble anti-CD3 (1 μg/mL) and irradiated T-depleted splenocytes for the indicated periods. The cells were stained for cell-surface CD4, CD8, and CD107a, fixed, permeabilized, and stained for intracellular granzyme B. Representative results from at least 3 independent experiments are shown.