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Granzyme C Supports Efficient CTL-Mediated Killing Late in Primary Alloimmune Responses¹

Yonas Getachew^*, Heather Stout-Delgado^*,, Bonnie C. Miller^*, and Dwain L. Thiele^2,*,

^* Division of Digestive and Liver Diseases, Department of Internal Medicine, and Immunology Graduate Program, University of Texas Southwestern Medical Center, Dallas, TX 75390

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants R01 DK53933 and AI24639 (to D.L.T.) and 3T32 DK007745 (to Y.G.).

² Address correspondence and reprint requests to Dr. Dwain L. Thiele, Department of Internal Medicine, 5323 Harry Hines Boulevard, Dallas, TX 75390-9151. E-mail address: dwain.thiele{at}utsouthwestern.edu

³ Abbreviations used in this paper: FasL, Fas ligand; Gzm, granzyme; 7-AAD, 7-aminoactinomycin D; siRNA, small interfering RNA; qRT-PCR, quantitative RT-PCR; WT, wild type.

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