Cell death induced by recombinant GzmH. (A) K562 or HL60 cells exposed to GzmH and sublytic concentrations of SLO (i) or PFN (ii) resulted in near-complete cell death. Viability of K562 and HL60 cells was examined by annexin V–FITC (AV) and propidium iodide (PI) staining 10 to 12 (SLO) and 24 (PFN) hours after the indicated treatments. Depending on the translocator of choice, GzmB or GzmH was used at final concentrations of 5 or 10 μg/mL and 5 or 20 μg/mL, respectively, with the lower concentration used in combination with perforin. GzmH^S195A and mast cell chymase were used at 25 μg/mL. Presented for both perforin- and SLO-mediated experiments is the sum of apoptotic (AV⁺ positive and PI^- negative) and necrotic (AV⁺ and PI⁻ cells from 3 pooled independent experiments (n = 3; ± standard deviation [SD]). Statistical significance is shown as *P > .005 (significant) and **P > .001 (very significant). In contrast to GzmH, mast cell chymase did not induce cell death. (B) Effective range of GzmH concentrations that induced cell death. K562 or HL60 cells were treated for 24 hours with sublytic SLO and GzmH or GzmB. The experiment shows the mean of triplicates ± SD and is representative of 2 independent experiments. (C) Time course of GzmH killing (shown is the mean of triplicates, representative of 2 independent experiments). GzmH induced cell death within 10 hours. Sublytic SLO induces 10% cell death on top of the natural background values. (D) Representative FACS data for K562 cells 12 hours after treatment with the indicated proteases, GzmB (7.5 μg/mL), GzmH and GzmH^S195A (15 μg/mL each), and sublytic SLO. Apoptotic and dead cells were identified by annexin V and PI staining (ii). Dead cells are characterized by low FCS and high SSC signals (i). (E) Morphology of GzmH-treated K562 cells (magnification × 40). Ten hours after GzmH treatment, K562 cells displayed a characteristic morphology with increased granularity, condensation of nuclei, and membrane irregularities. Importantly, inactive GzmH^S195A together with SLO did not trigger similar changes. (F) GzmH-induced cell death leads to the quick loss of membrane integrity. GzmH/SLO-treated K562 cells were stained with trypan blue at various time points (n = 3; ± SD). Contrary to GzmB, GzmH-induced cell death resulted in a much more pronounced trypan staining, with nearly 50% of cells positive after 4 hours. At this time point, GzmB-treated cells, in late-phase apoptosis, accounted for only 30%. Med. indicates medium; Chy., Chymase; n.d, not determined.