Granzyme C Supports Efficient CTL-Mediated Killing Late in Primary Alloimmune Responses

It is well established that granzymes A and B play a role in CTL killing of target cells by the perforin-dependent granule exocytosis pathway. The functions of multiple additional granzymes expressed in CTL are less well defined. In the present studies, CTL generated from mice deficient in dipeptidyl peptidase 1 (DPP1) were used to investigate the contribution of granzyme C to CTL killing of allogeneic target cells. DPP1 is required for activation of granzymes A and B by proteolytic removal of their N-terminal dipeptide prodomains while a significant portion of granzyme C is processed normally in the absence of DPP1. Cytotoxicity of DPP1(-/-) CTL generated in early (5-day) MLC in vitro and in peritoneal exudate cells 5 days after initial allogeneic sensitization in vivo was significantly impaired compared with wild-type CTL. Following 3 days of restimulation with fresh allogeneic stimulators however, cytotoxicity of these DPP1(-/-) effector cells was comparable to that of wild-type CTL. Killing mediated by DPP1(-/-) CTL following restimulation was rapid, perforin dependent, Fas independent and associated with early mitochondrial injury, phosphatidyl serine externalization, and DNA degradation, implicating a granzyme-dependent apoptotic pathway. The increased cytotoxicity of DPP1(-/-) CTL following restimulation coincided with increased expression of granzyme C. Moreover, small interfering RNA inhibition of granzyme C expression during restimulation significantly decreased cytotoxicity of DPP1(-/-) but not wild-type CTL. These results indicate that during late primary alloimmune responses, granzyme C can support CTL-mediated killing by the granule exocytosis pathway in the absence of functional granzymes A or B.