Cytotoxic lymphocytes produce granules armed with a set of 5 serine proteases (granzymes (Gzms)), which, together with the pore-forming protein (perforin), serve as a major defense against viral infections in humans. This granule-exocytosis pathway subsumes a well-established mechanism in which target cell death is induced upon perforin-mediated entry of Gzms and subsequent activation of various (apoptosis) pathways. In the past decade, however, a growing body of evidence demonstrated that Gzms also inhibit viral replication and potential reactivation in cell death-independent manners. For example, Gzms can induce proteolysis of viral or host cell proteins necessary for the viral entry, release, or intracellular trafficking, as well as augment pro-inflammatory antiviral cytokine response. In this review, we summarize current evidence for the noncytotoxic mechanisms and roles by which killer cells can use Gzms to combat viral infections, and we discuss the potential thereof for the development of novel therapies.

Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even "orthologous" granzymes have species-specific functions, having evolved in distinct environments that pose different challenges.

Granzymes are serine proteases that, upon release from cytotoxic cells, induce apoptosis in tumor cells and virally infected cells. In addition, a role of granzymes in inflammation is emerging. Recently, we have demonstrated that extracellular granzyme K (GrK) potentiates lipopolysaccharide (LPS)-induced cytokine response from monocytes. GrK interacts with LPS, disaggregates LPS micelles, and stimulates LPS-CD14 binding and Toll-like receptor signaling. Here we show that human GrA also potentiates cytokine responses in human monocytes initiated by LPS or Gram-negative bacteria. Similar to GrK, this effect is independent of GrA catalytic activity. Unlike GrK, however, GrA does not bind to LPS, has little influence on LPS micelle disaggregation, and does not augment LPS-CD14 complex formation. We conclude that GrA and GrK differentially modulate LPS-Toll-like receptor signaling in monocytes, suggesting functional redundancy among cytotoxic lymphocyte proteases in the anti-bacterial innate immune response.

Aims: Recent in vitro findings suggest that the serine protease Granzyme K (GzmK) may act as a proinflammatory mediator. However, its role in sepsis is unknown. Here we aim to understand the role of GzmK in a mouse model of bacterial sepsis and compare it to the biological relevance of Granzyme A (GzmA). Methods: Sepsis was induced in WT, GzmA-/- and GzmK-/- mice by an intraperitoneal injection of 2x108 CFU from E. coli. Mouse survival was monitored during 5 days. Levels of IL-1α, IL-1β, TNFα and IL-6 in plasma were measured and bacterial load in blood, liver and spleen was analyzed. Finally, profile of cellular expression of GzmA and GzmK was analyzed by FACS. Results: GzmA and GzmK are not involved in the control of bacterial infection. However, GzmA and GzmK deficient mice showed a lower sepsis score in comparison with WT mice, although only GzmA deficient mice exhibited increased survival. GzmA deficient mice also showed reduced expression of some proinflammatory cytokines like IL1-α, IL-β and IL-6. A similar result was found when extracellular GzmA was therapeutically inhibited in WT mice using serpinb6b, which improved survival and reduced IL-6 expression. Mechanistically, active extracellular GzmA induces the production of IL-6 in macrophages by a mechanism dependent on TLR4 and MyD88. Conclusions: These results suggest that although both proteases contribute to the clinical signs of E. coli-induced sepsis, inhibition of GzmA is sufficient to reduce inflammation and improve survival irrespectively of the presence of other inflammatory granzymes, like GzmK.

Escherichia (E.) coli is the most common causative pathogen in peritonitis, the second most common cause of sepsis. Granzymes (gzms) are serine proteases traditionally implicated in cytotoxicity and, more recently, in the inflammatory response. We here sought to investigate the role of gzms in the host response to E. coli-induced peritonitis and sepsis in vivo. For this purpose, we used a murine model of E. coli intraperitoneal infection, resembling the clinical condition commonly associated with septic peritonitis by this bacterium, in wild-type and gzmA-deficient (gzmA-/- ), gzmB-/- , and gzmAxB-/- mice. GzmA and gzmB were predominantly expressed by natural killer cells, and during abdominal sepsis, the percentage of these cells expressing gzms in peritoneal lavage fluid decreased, while the amount of expression in the gzm+ cells increased. Deficiency of gzmA and/or gzmB was associated with increased bacterial loads, especially in the case of gzmB at the primary site of infection at late stage sepsis. While gzm deficiency did not impact neutrophil recruitment into the abdominal cavity, it was accompanied by enhanced nucleosome release at the primary site of infection, earlier hepatic necrosis, and more renal dysfunction. These results suggest that gzms influence bacterial growth and the host inflammatory response during abdominal sepsis caused by E. coli.

The transcription factor Tcf1 is essential for the development of natural killer (NK) cells. However, its precise role has not been clarified. Our combined analysis of Tcf1-deficient and transgenic mice indicated that Tcf1 guides NK cells through three stages of development. Tcf1 expression directed bone marrow progenitors toward the NK cell lineage and ensured the survival of NK-committed cells, and its downregulation was needed for terminal maturation. Impaired survival of NK-committed cells was due to excessive expression of granzyme B (GzmB) and other granzyme family members, which induced NK cell self-destruction during maturation and following activation with cytokines or target cells. Mechanistically, Tcf1 binding reduced the activity of a Gzmb-associated regulatory element, and this accounted for the reduced Gzmb expression in Tcf1-expressing NK cells. These data identify an unexpected requirement to limit the expression of cytotoxic effector molecules for the normal expansion and function of NK cells.

Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.

Sepsis is a complex syndrome related to an infection-induced exaggerated inflammatory response, which is associated with a high mortality. Granzymes (Gzm) are proteases mainly found in cytotoxic lymphocytes that not only have a role in target cell death, but also as mediators of infection and inflammation. In this study we sought to analyse the intracellular expression of GzmA, B, M and K by flow cytometry in diverse blood lymphocyte populations from 22 sepsis patients, 12 non-infected intensive care unit (ICU) patients and 32 healthy controls. Additionally, we measured GzmA and B plasma levels. Both groups of patients presented decreased percentage of natural killer (NK) cells expressing GzmA, B and M relative to healthy controls, while sepsis patients showed an increased proportion of CD8+ T cells expressing GzmB compared to controls. Expression of GzmK remained relatively unaltered between groups. Extracellular levels of GzmB were increased in non-infected ICU patients relative to sepsis patients and healthy controls. Our results show differential alterations in intracellular expression of Gzm in sepsis patients and non-infected critically ill patients compared to healthy individuals depending on the lymphocyte population and on the Gzm.

Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB).

Human milk has been previously found to contain various types of leukocytes however specific characteristics of these cells, such as whether they contain cytolytic antimicrobial proteins that may induce pathogen directed cell death, are unknown. This project aims to examine the presence and localization of immune proteins such as perforin, granulysin and granzymes in human milk cells at the protein and mRNA level. Genes encoding these proteins were confirmed in human milk cell samples, which were particularly enriched in early milk and in the case of maternal infection. Fluorescence activated cell sorting (FACS) was used to investigate the co-expression of these proteins with pan-immune cell marker CD45 and epithelial marker EPCAM. Co-expression of antimicrobial proteins was found predominantly in CD45 positive cells, also increasing in the case of maternal infection. Our study suggests that human milk contains cells that carry hallmarks of activated or memory T-cells which are enriched early in lactation and in the case of maternal infection. Presence and prevalence of these cells in human milk may indicate a role in the protection of the maternal breast or for delivery to the vulnerable infant.

NK cells kill target cells mainly via exocytosis of granules containing perforin (perf) and granzymes (gzm). In vitro, gzm delivery into the target cell cytosol results in apoptosis, and induction of apoptosis is severely impaired in the absence of gzm A and B. However, their importance for in vivo cytotoxicity by cytotoxic T cells has been questioned. We used an in vivo NK cytotoxicity assay, in which splenocytes from wild-type and β(2)microglobulin-deficient (MHC-I(neg)) mice are co-injected into recipients whose NK cells were activated by virus infection or synthetic Toll-like receptor ligands. Elimination of adoptively transferred MHC-I(neg) splenocytes was unimpaired in the absence of gzmA and gzmB, but dependent on perforin. This target cell rejection was NK cell dependent, since NK cell depletion abrogated it. Furthermore, target cell elimination in vivo was equally rapid in both wild-type and gzmAxB-deficient recipients, with the majority of specific target cells lost from lymphoid tissue within less than one to two hours after transfer. Thus, similar to T cell cytotoxicity, the contribution of gzmA and B to in vivo target cell elimination remains unresolved.

Cytotoxic T lymphocyte (CTL)-induced death triggered by the granule exocytosis pathway involves the perforin-dependent delivery of granzymes to the target cell. Gene targeting has shown that perforin is essential for this process; however, CTL deficient in the key granzymes A and B maintain the ability to kill their targets by granule exocytosis. It is not clear how granzyme AB(-/-) CTLs kill their targets, although it has been proposed that this occurs through perforin-induced lysis. We found that purified granzyme B or CTLs from wild-type mice induced classic apoptotic cell death. Perforin-induced lysis was far more rapid and involved the formation of large plasma membrane protrusions. Cell death induced by granzyme AB(-/-) CTLs shared similar kinetics and morphological characteristics to apoptosis but followed a distinct series of molecular events. Therefore, CTLs from granzyme AB(-/-) mice induce target cell death by a unique mechanism that is distinct from both perforin lysis and apoptosis.

How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

As many SARS-CoV-2 infections are asymptomatic, it could be useful to be able to determine how much time has passed since infection. We explored the changes in the temporal levels of T cell-related proteins (including perforin and granzymes) in the sera of patients with SARS-CoV-2 infection using a commercially available assay.

Cytotoxic T cells and natural killer cells can kill target cells based on their expression and release of perforin, granulysin, and granzymes. Genes encoding these molecules have been only poorly annotated in camelids. Based on bioinformatic analyses of genomic resources, sequences corresponding to perforin, granulysin, and granzymes were identified in genomes of camelids and related ungulate species, and annotation of the corresponding genes was performed. A phylogenetic tree was constructed to study evolutionary relationships between the species analyzed. Re-sequencing of all genes in a panel of 10 dromedaries and 10 domestic Bactrian camels allowed analyzing their individual genetic polymorphisms. The data showed that all extant Old World camelids possess functional genes for two pore-forming proteins (PRF1, GNLY) and six granzymes (GZMA, GZMB, GZMH, GZMK, GZMM, and GZMO). All these genes were represented as single copies in the genome except the GZMH gene exhibiting interspecific differences in the number of loci. High protein sequence similarities with other camelid and ungulate species were observed for GZMK and GZMM. The protein variability in dromedaries and Bactrian camels was rather low, except for GNLY and chymotrypsin-like granzymes (GZMB, GZMH).

The mechanism underlying perforin (PFN)-dependent delivery of apoptotic granzymes during cytotoxic cell granule-mediated death remains speculative. Granzyme B (GrB) and perforin were found to coexist as multimeric complexes with the proteoglycan serglycin (SG) in cytotoxic granules, and cytotoxic cells were observed to secrete exclusively macromolecular GrB-SG. Contrary to the view that PFN acts as a gateway for granzymes through the plasma membrane, monomeric PFN and, strikingly, PFN-SG complexes were shown to mediate cytosolic delivery of macromolecular GrB-SG without producing detectable plasma membrane pores. These results indicate that granule-mediated apoptosis represents a phenomenon whereby the target cell perceives granule contents as a multimeric complex consisting of SG, PFN, and granzymes, which are, respectively, the scaffold, translocator, and targeting/informational components of this modular delivery system.

Type 1 innate lymphoid cells (ILC1s) represent the predominant population of liver ILCs and function as important effectors and regulators of immune responses, but the cellular heterogeneity of ILC1s is not fully understood. Here, single-cell RNA sequencing and flow cytometric analysis demonstrated that liver ILC1s could be dissected into Ly49E+ and Ly49E- populations with unique transcriptional and phenotypic features. Genetic fate-mapping analysis revealed that liver Ly49E+ ILC1s with strong cytotoxicity originated from embryonic non-bone marrow hematopoietic progenitor cells (HPCs), persisted locally during postnatal life, and mediated protective immunity against cytomegalovirus infection in newborn mice. However, Ly49E- ILC1s developed from BM and extramedullary HPCs after birth, gradually replaced Ly49E+ ILC1s in the livers with age, and contained the memory subset in recall response to hapten challenge. Thus, our study shows that Ly49E dissects liver ILC1s into two unique subpopulations, with distinct origins and a bias toward neonatal innate or adult immune memory responses.

MS is thought to be mediated by CD4(+) T-helper cells. To investigate the importance of CD8(+) cytotoxic T-cells in MS we analyzed peripheral blood T-cells by DNA microarray, and plasma and CSF levels of granzymes from MS patients and controls. Cytotoxic gene expression was decreased in peripheral T-cells from RRMS patients whereas plasma levels of granzymes were unchanged. However, granzyme levels were elevated in the CSF of RRMS patients at relapse compared with controls and remission. Thus, CD8+ T-cell-mediated cytotoxicity is confined to the CSF/CNS compartment in RRMS patients and may be involved in the immunopathogenesis of clinical relapses.

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.

Granzymes are a family of serine proteases found in the lytic granules of cytotoxic T lymphocytes and natural killer (NK) cells, which are involved in killing of susceptible target cells. Most information on granzymes and their enzymatic specificities derive from studies in humans and mice. Although granzymes shared by both species show a high level of conservation, the complement of granzyme genes differs between the species. The aim of this study was to identify granzyme genes expressed in cattle, determine their genomic locations and analyse their sequences to predict likely functional specificities. Orthologues of the five granzyme genes found in humans (A, B, H, K and M) were identified, as well a novel gene designated granzyme O, most closely related to granzyme A. An orthologue of granzyme O was found in pigs and a non-function version was detected in the human genome. Use of specific PCRs demonstrated that all of these genes, including granzyme O, are expressed in activated subsets of bovine lymphocytes, with particularly high levels in CD8 T cells. Consistent with findings in humans and mice, the granzyme-encoding genes were located on three distinct genomic loci, which correspond to different proteolytic enzymatic activities, namely trypsin-like, chymotrypsin-like and metase-like. Analysis of amino acid sequences indicated that the granzyme proteins have broadly similar enzymatic specificities to their human and murine counterparts but indicated that granzyme B has a different secondary specificity. These findings provide the basis for further work to examine their role in the cytotoxic activity of bovine CD8 T cells.