MHC-dependent inhibition of alloreactive CD8⁺ T cells in MLR: perforin-mediated killing is associated with granzyme A activity. To determine granule location in imDCs, the cells were stained with LysoTracker Red, anti-perforin (green), anti-granzyme A (red) or granzyme B (red), and Hoechst 33342 (blue). Images were taken using a Zeiss ApoTome system. (B) Granzyme B (36 kDa) and granzyme A (28 kDa) expression determined by Western blotting in CB6/F1 or PKO imDCs. Expression of granzyme A and B in CB6/F1 CTLs was used as a positive control. (Ci) Total numbers of live calcein AM green–stained CD8⁺ 2C cells after incubation for 5 hours in the absence (blue) or presence of imDCs from CB6/F1 (yellow), FVB (red), or PKO (green) at the indicated ratios was determined by FACS. (Cii) At the end of the short-term MLR shown in panel C1, the percentage of specific BLT esterase release was calculated as described in the “Methods.” The bars represent the average ± SD (n ≥ 3).