Granulysin-Mediated Tumor Rejection in Transgenic Mice

Abstract

Granulysin (GNLY) is a cytolytic molecule expressed by human CTL and NK cells with activity against a variety of tumors and microbes, including Mycobacterium tuberculosis. Although the molecular mechanism of GNLY-induced apoptosis of Jurkat T cells is well defined in vitro, no direct evidence for its in vivo effects has been demonstrated. Because there is no murine homologue of GNLY, we generated mice expressing GNLY using a bacterial artificial chromosome containing the human GNLY gene and its 5′ and 3′ flanking regions. GNLY is expressed in leukocytes from transgenic mice with similar kinetics as in PBMC from humans: GNLY is constitutively expressed in NK cells and, following stimulation through the TCR, appears in T lymphocytes 8–10 days after activation. Both forms of GNLY (9 and 15 kDa) are produced by activated T cells, whereas the 15-kDa form predominates in freshly isolated NK cells from transgenic animals. GNLY mRNA is highest in spleen, with detectable expression in thymus and lungs, and minimal expression in heart, kidney, liver, muscle, intestine, and brain. Allospecific cell lines generated from GNLY transgenic animals showed enhanced killing of target cells. In vivo effects of GNLY were evaluated using the syngeneic T lymphoma tumor C6VL. GNLY transgenic mice survived significantly longer than nontransgenic littermates in response to a lethal tumor challenge. These findings demonstrate for the first time an in vivo effect of GNLY and suggest that GNLY may prove a useful therapeutic modality for the treatment of cancer.

Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites

“…To confirm the role of PFN and GNLY in killer cell-mediated elimination of intracellular parasites, we assessed the killing of anti-CD3-coated T. cruzi-infected RAW 264.7 cells (a mouse macrophage cell line) by interleukin (IL)-15-cultured splenocytes from T. cruziinfected wild-type (WT), GNLY-transgenic (GNLY +/− ) 11 , PFN-deficient (Prf1 −/− ) and Prf1 −/− GNLY +/− mice ( Fig. 1d and Supplementary Fig.…”

“…3b). Transgenic GNLY +/− mice were generated using a large Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites bacterial artificial chromosome that contains the 5 and 3 regulatory domains of the GNLY gene 11,15 . As a consequence, these mice express GNLY protein at levels similar to those in humans, but only in NK cells and CTLs.…”

Granulysin Delivered by Cytotoxic Cells Damages Endoplasmic Reticulum and Activates Caspase-7 in Target Cells

“…Granulysin homologs are found in humans, cows, pigs, and horses, but not in mice or rats (21,22). To test the physiologic role of granulysin, we used a bacterial artificial chromosome to generate GNLY transgenic mice (23). These animals have normal development and phenotype, and their lymphocytes express granulysin in a manner similar to human T and NK cells.…”

Granulysin Production and Anticryptococcal Activity Is Dependent upon a Far Upstream Enhancer That Binds STAT5 in Human Peripheral Blood CD4+ T Cells

“…Studies examining the regulation of granulysin are limited compared with those of perforin and granzymes, in part due to the lack of a murine gene homolog (26). Our results demonstrated that IL-2 stimulated and enhanced the expression of granulysin via transcription factor binding sites upstream of the granulysin promoter.…”

“…The upstream element (219,802 to 21,181 bp) of the human granulysin promoter was digested from a 186-kb human bacterial artificial chromosome clone RP11-439L14 with BglII and BstBI sites (26). The initial reporter gene construct pGL3219,802/+62 was assembled by cloning the digested fragment into the promoterless pGL3 basic reporter vector (Promega, Madison, WI), in which the human granulysin promoter and translation initiation site (21,181/+62) had been fused to the firefly luciferase gene (24).…”