Differential Regulation of Granzyme B and C Expression in Murine Cytotoxic T and NK Cells.
Authors: Cai Sheng F. , Fehniger Todd A. , Dai Xuezhi , Cao Xuefang , Ley Timothy J. 
Published: April 2020

Abstract
Cytotoxic lymphocytes, which include CD4+ and CD8+ T cells as well as NK cells, use the granule exocytosis pathway to kill virus-infected and tumor cells. Previous studies have characterized the expression of many genes in this pathway (e.g. perforin, granzyme A, granzyme B, etc.), although no reagent has been developed to measure granzyme C protein at the single-cell level. The murine granzyme C gene, orthologous to human granzyme H, lies 24.2Kb directly downstream from granzyme B in the granzyme B gene cluster. Recombinant granzyme C rapidly induces target cell death in a manner distinct from granzyme A- or B-induced death. To further understand the regulation and function of murine granzyme C, we developed a granzyme C-specific monoclonal antibody and used flow cytometry to examine the expression of granzyme C in resting and activated murine cytotoxic lymphocyte compartments. Naive CD4+ and CD8+ T cells express little or no granzyme C (0.1 + 0.1% of cells are positive). After activation of splenocytes with plate-bound CD3 and CD28 agonistic antibodies for 4 days, a small percentage of CD4+ (2.3 + 1.0% positive) and CD8+ (6.6 + 0.3% positive) T cells express granzyme C. However, only 24 hours later, almost all CD4+ (96.7 + 2.7%) and CD8+ (98.7 + 1.4%) T cells express granzyme C. Granzyme B co-staining revealed that granzyme B is detectable in both CD4+ and CD8+ T cells at least 48 hours before granzyme C is expressed. Furthermore, we employed a fully mismatched GVHD mouse model in order to examine T cell expression of granzymes B and C in vivo; similarly, granzyme B expression preceded granzyme C by at least 48 hours. In addition, CD4+Foxp3+ regulatory T cells also expressed granzyme C in this model. Murine NK cell granzyme C mRNA expression was assessed using Affymetrix microarrays (MOE430v2) at rest and following IL-15 activation. Resting NK cells express minimal granzyme C mRNA (mean gzmC probe set signal intensity + SD [n=3]): 356 + 202, which increased after IL-15 activation as follows: 4,180 + 1,106 (day 1), 60,788 + 8,455 (day 2), 167,448 + 26,398 (day 4), and 178,451+14,037 (day 6). Utilizing our granzyme C-specific mAb, we showed that very few resting murine NK cells express granzyme C protein (2.3 + 0.4% positive). Consistent with the mRNA data, the percentage of granzyme C-expressing NK cells was substantially increased after 3 days of IL-15 activation (88.8 + 5.2% positive). In contrast, granzyme B mRNA levels are high in resting NK cells (41,208 + 2,534), and increase only incrementally with IL-15 treatment (Fehniger et al., Immunity 2007 Jun;26(6):798–811). Granzyme B protein expression is controlled at a post-transcriptional level in NK cells, whereas granzyme C expression is transcriptionally regulated. Taken together, our findings show that the mouse granzyme B and C genes, despite being only 24.2Kb apart, are activated in cytotoxic lymphocytes with different kinetics; moreover, granzyme B and C protein abundance is controlled by completely different mechanisms in NK cells. These data suggest that the granzyme genes are differentially regulated in lymphocyte compartments, and that this regulation may be relevant for how cytotoxic lymphocytes function.