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Differential Expression of Granzyme B and C in Murine Cytotoxic Lymphocytes¹

Sheng F. Cai^*, Todd A. Fehniger^*, Xuefang Cao^*, Joshua C. Mayer^*, Joel D. Brune^*, Anthony R. French and Timothy J. Ley^2,*

^* Department of Internal Medicine, Division of Oncology and Department of Pediatrics, Division of Pediatric Rheumatology, Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63110

Cytotoxic lymphocytes use the granule exocytosis pathway to kill pathogen-infected cells and tumor cells. Although many genes in this pathway have been extensively characterized (e.g., perforin, granzymes A and B), the role of granzyme C is less clear. We therefore developed a granzyme C-specific mAb and used flow cytometry to examine the expression of granzyme B and C in the lymphocyte compartments of wild-type and mutant GzmB^–/– cre mice, which have a small deletion in the granzyme B gene. We detected granzyme B and C expression in CD4⁺ and CD8⁺ T cells activated with CD3/CD28 beads or MLRs. Stimulation of NK cells in vitro with IL-15 also induced expression of both granzymes. Granzyme C up-regulation was delayed relative to granzyme B in wild-type lymphocytes, whereas GzmB^–/– cre cells expressed granzyme C earlier and more abundantly on a per-cell basis, suggesting that the deleted 350-bp region in the granzyme B gene is important for the regulation of both granzymes B and C. Quantitative RT-PCR revealed that granzyme C protein levels were regulated by mRNA abundance. In vivo, a population of wild-type CD8⁺ intraepithelial lymphocytes constitutively expressed granzyme B and GzmB^–/– cre intraepithelial lymphocytes likewise expressed granzyme C. Using a model of a persistent murine CMV infection, we detected delayed expression of granzyme C in NK cells from infected hosts. Taken together, these findings suggest that granzyme C is activated with persistent antigenic stimulation, providing nonredundant backup protection for the host when granzyme B fails.

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 grants from the National Institutes of Health (DK49786 to T.J.L. and AI059083 to A.R.F.). The granzyme C mAb was developed in part with the Washington University School of Medicine Hybridoma Center (supported by the Department of Pathology and Immunology and National Institutes of Health Grant P30 AR048335).

² Address correspondence and reprint requests to Dr. Timothy J. Ley, Department of Medicine, Division of Oncology, Section of Stem Cell Biology, Washington University School of Medicine and the Siteman Cancer Center, 660 South Euclid Avenue, Campus Box 8007, St. Louis, MO 63110. E-mail address: timley{at}wustl.edu

³ Abbreviations used in this paper: MCMV, murine CMV; WT, wild type; LAK, lymphokine-activated killer; IEL, intraepithelial lymphocyte; qRT-PCR, quantitative RT-PCR; Ct, threshold cycle; MFI, mean fluorescence intensity; DPPI, dipeptidyl peptidase I; LCR, locus control region.

⁴ The online version of this article contains supplemental material.