Kevin Thia

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.

Functional dissociation of ΔΨm and cytochrome c release defines the contribution of mitochondria upstream of caspase activation during granzyme B-induced apoptosis

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage – upstream or downstream of caspases – remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (ΔΨm) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their ΔΨm. Loss of ΔΨm was not required for rapid granzyme B-induced apoptosis and regeneration of ΔΨm following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of ΔΨm demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

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.

Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1.

Met-ase-1 is a 30000Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3− large granular lymphocytes. We screened a mouse genomic library with ratMet-ase-1 cDNA, and obtained bacteriophage clones that contained the mouseMet-ase-1 gene. The mouseMet-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. MouseMet-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3− GM1+ large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1+ cell line 4–16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouseMet-ase-1 mRNA. The 5′ flanking region of the mouseMet-ase-1 gene also shares considerable regions of identity with the 5′ flanking region of the ratMet-ase-1 gene. A 3.3 kb segment of 5′ sequence flanking the mouseMet-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouseMet-ase-1 5′ flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4–16 mouse NK1.1+ cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouseMet-ase-1. The predicted hexapropeptide of mouseMet-ase-1 (Asn−6 to Gln−1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouseMet-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouseMet-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.

Activated Mouse B Cells Lack Expression of Granzyme B

Recently, it has been reported that human B cells express and secrete the cytotoxic protease granzyme B (GrB) after stimulation with IL-21 and BCR cross-linking. To date, there are few clues on the function of GrB in B cell biology. As experimental transgenic murine systems should provide insights into these issues, we assayed for GrB in C57BL/6 B cells using an extensive array of physiologically relevant stimuli but were unable to detect either GrB expression or its proteolytic activity, even when Ag-specific transgenic BCRs were engaged. Similar results were also obtained with B cells from DBA/2, CBA, or BALB/c mice. In vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of GrB in CTLs, but not in B cell populations. We also investigated a possible role of GrB on the humoral immune response to the model Ag 4-hydroxy-3-nitrophenylacetyl–keyhole limpet hemocyanin, but GrB-deficient mice produced normal amounts of Ab with typical affinity maturation and a heightened secondary response, demonstrating conclusively the redundancy of GrB for Ab responses. Our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans. The physiological consequences of GrB expression in human B cells remain unclear, and the current study suggests that experimental mouse models will not be helpful in addressing this issue.

Hypothesis : cytotoxic lymphocyte granule serine proteases activate target cell endonucleases to trigger apoptosis

Upon interaction with target cells, cytotoxic T lymphocytes and natural killer cells vectorially secrete highly specialized cytoplasmic granules containing perforin and a family of serine proteases (granzymes). This granule exocytosis mechanism of cytolysis is of patho‐physiological importance, and usually results in target cell DNA fragmentation. Neither perforin nor granzymes possess inherent nuclease activity, but in combination they can induce target cell apoptosis. Perforin forms transmembrane pores in the target cell, thereby enabling granzymes to access target cell substrates. The target cell substrates of granzymes are unknown, but granzyme A binding and cleavage of the nuclear shuttle protein nucleolin in target cells demonstrates that granzymes may act on nuclear substrates. Furthermore, the presence of granzyme B and other granzyme activities in the nucleus of cytotoxic lymphocytes indicates that granzymes can be transported from the cytoplasm to the nucleus.

Heterozygosity for the common perforin mutation, p.A91V, impairs the cytotoxicity of primary natural killer cells from healthy individuals.

The production and delivery of functional perforin (PRF; PRF1 gene) by cytotoxic lymphocytes maintains immune homeostasis and tumour immune surveillance. In humans, inheritance of the common PRF1 polymorphism, p.A91V, (c.272C>T) found in 8–9% of the Caucasian population, with another mutated allele resulting in reduced PRF function or trafficking, has been shown to result in hyperinflammatory diseases and/or haematological cancers. In this study, we sought to investigate the function of p.A91V on a wild‐type (WT) perforin background. We first developed an assay that distinguishes the relative levels of transcription of individual PRF1 alleles, including p.A91V. The p.A91V allele was seen to be expressed at similar levels as the WT allele in primary human natural killer (NK) cells, ruling out that allelic expression imbalance influenced their function. We then demonstrated that the p.A91V mutation results in protein misfolding and an appreciable reduction in NK‐cell cytotoxicity in healthy carriers of p.A91V. We propose that this level of cytotoxic dysfunction may readily account for the predisposition to immune‐mediated disease in individuals homozygous for p.A91V. Also, the fact that monoallelic mutations of PRF1 decrease NK‐cell cytotoxicity should be considered in individuals presenting with the manifestations of immune deficiency states that impinge on NK‐cell cytotoxicity.

Xenogeneic mouse anti-human NK cytotoxicity is mediated via perforin

Abstract: Natural killer (NK) cells have been identified among the cell populations thought to participate in delayed xenograft rejection. We investigated the relative contribution of perforin‐ and Fas ligand‐pathways in mouse NK‐mediated xenocytotoxicity generated in response to cytokine or xenoantigenic stimulation. Freshly isolated spleen cells exhibited little NK cell‐mediated cytotoxicity against human PHA‐stimulated peripheral blood mononuclear cells (PBMC) or the human NK‐sensitive cell line, K562, despite efficiently lysing mouse NK‐sensitive Yac‐1 target cells. However, after incubation of mouse spleen cells for 7 to 10 days in the presence of mitomycin C‐treated xenogeneic human PBMC or exogenous interleukin‐2 (IL‐2), the NK activity of cultured mouse adherent lymphocytes (A‐NK) against human targets increased dramatically. A‐NK cells generated in immunocompetent mice displayed significant lysis of human targets, as did effector cells generated in gld mice with a mutated Fas ligand. By contrast, the low cytotoxic activity of A‐NK from perforin‐deficient mice responding to K562 target cells suggested that NK xenocytotoxicity is perforin‐mediated. Perforin‐deficient mouse A‐NK cells only lysed Fas‐sensitive human PBMC and K562‐Fas targets to a minor extent. Overall, these data suggest that A‐NK cell xenolysis is predominantly mediated by perforin, irrespective of the stimulus (cytokine or cellular) provided.

Fatal immune dysregulation due to a gain of glycosylation mutation in lymphocyte perforin

Mutations in the perforin gene (PRF1) are a common cause of the fatal immune dysregulation disorder, familial hemophagocytic lymphohistiocytosis (type 2 FHL, FHL2). Here we report a female infant born with biallelic PRF1 mutations: a novel substitution, D49N, and a previously identified in-frame deletion, K285del. We assessed the effects of each mutation on the cytotoxicity of human NK cells in which the expression of endogenous perforin was ablated with miR30-based short hairpin (sh) RNAs. Both mutations were detrimental for function, thereby explaining the clinically severe presentation and rapidly fatal outcome. We demonstrate that D49N exerts its deleterious effect by generating an additional (third) N-linked glycosylation site, resulting in protein misfolding and degradation in the killer cell. Our data provide a rationale for treating some cases of type 2 familial hemophagocytic lymphohistiocytosis, based on the pharmacologic inhibition or modification of glycosylation.

Cloning and characterization of a novel NK cell-specific serine protease gene and its functional 5′-flanking sequences

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30 000 Mr serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5′-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5′-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5′-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5′-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines.