A natural genetic variant of granzyme B confers lethality to a common viral infection.

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining 'Asp-ase' activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV-infected targets in vitro. Our results suggest that known virally-encoded inhibitors of the intrinsic (mitochondrial) apoptotic pathway account for the increased susceptibility of GzmBW mice to MCMV. We conclude that different natural variants of GzmB have a profound impact on the immune response to a common and authentic viral pathogen.

A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

Human perforin mutations and susceptibility to multiple primary cancers.

Loss-of-function mutations in the gene coding for perforin (PRF1) markedly reduce the ability of cytotoxic T lymphocytes and natural killer cells to kill target cells, causing immunosuppression and impairing immune regulation. In humans, nearly half of the cases of type 2 familial hemophagocytic lymphohistiocytosis are due to bi-allelic PRF1 mutations. The partial inactivation of PRF1 due to mutations that promote protein misfolding or the common hypomorphic allele coding for the A91V substitution have been associated with lymphoid malignancies in childhood and adolescence. To investigate whether PRF1 mutations also predispose adults to cancer, we genotyped 566 individuals diagnosed with melanoma (101), lymphoma (65), colorectal carcinoma (30) or ovarian cancer (370). The frequency of PRF1 genotypes was similar in all disease groups and 424 matched controls, indicating that the PRF1 status is not associated with an increased susceptibility to these malignancies. However, four out of 15 additional individuals diagnosed with melanoma and B-cell lymphoma during their lifetime expressed either PRF1A91V or the rare pathogenic PRF1R28C variant (p = 0.04), and developed melanoma relatively early in life. Both PRF1A91V- and PRF1R28C-expressing lymphocytes exhibited severely impaired but measurable cytotoxic function. Our results suggest that defects in human PRF1 predispose individuals to develop both melanoma and lymphoma. However, these findings require validation in larger patient cohorts.

The granzyme B gene is highly polymorphic in wild mice but essentially invariant in common inbred laboratory strains.

Granzyme B is a 247 amino acid pro-apoptotic protease secreted by effector lymphocytes for the purpose of killing virus-infected cells. While the capacity of granzyme B to potently induce caspase-dependent apoptosis has long been recognized, it has only recently been found that human and mouse granzyme B activate overlapping but distinct apoptotic pathways. To investigate a possible evolutionary basis for this observation, we sequenced the exons and flanking intronic sequences of the mouse Gzmb gene from a variety of inbred laboratory strains and wild mice. The sequences of 12/13 inbred strains encoded identical proteins, the exception being DBA/2, whose sequence varied at two amino acids. By contrast with the laboratory strains, there was extensive polymorphism in the Gzmb gene of 54 wild mice and 28 wild-derived inbred mice examined, resulting in 2-18 amino acid differences in the predicted proteins, a discrepancy rate of up to 7.3%. Many of these amino acid variations were found in rat and/or human granzyme B. The granzyme B allotype of inbred laboratory strains could be identified in only one of three geographically dispersed clans of wild mice and was absent from all 28 wild-derived inbred strains. The Gzmb gene of Mus musculus castaneus, a close relative of laboratory mice, encoded six amino acid differences compared with the laboratory strains, all of which were also found in corresponding positions in the granzyme B molecules of wild mice. Unlike the protease, the extended granzyme B recognition and cleavage site in Bid, a key pro-apoptotic substrate, was invariant.