The understanding of GzmB expression, from transcriptional activation to post-translational modifications, remains a dynamic area of research. In a study conducted in 2005, the direct impact of IL-2 on Perforin and GzmB expression in lymphocytes was revealed, enhancing cytotoxic activity without affecting survival or division of the lymphocyte itself (13). Aside from cytotoxic lymphocytes, a study in 2009 observed that activated human pDCs secrete GzmB, triggered by IL-3 and IL-10, causing suppression of T cell proliferation (9). Additionally, B cell receptor activation along with IL-21 treatment induced proliferation of human B cells with high levels of GzmB expression (10). Furthermore, Pardo et al. presented evidence that skin-, but not lung-associated primary mast cells and in vitro-differentiated bone marrow-derived mast cells (BMMC) express GzmB (but not GzmA or Perforin), which is associated with cytoplasmic granules in BMMC and is secreted after Fcϵ-receptor-mediated activation (7). In the meantime, post-translational modifications, such as phosphorylation and proteolytic cleavage, are involved in converting inactive GzmB into its functional form, and may also influence apoptosis induction and inflammatory response. There are ongoing investigations to elucidate the diverse signals that drive GzmB production by different cell types, from gene expression to functional activation, and the respective contributions to immune response and inflammatory condition.

Activated lymphocytes including CTLs and NK cells are known to express Perforin and Granzymes, and package these molecules into the cytotoxic granules that are derived from lysosome compartments (14). When a CTL recognizes and binds a target cell, the formation of immunological synapse following MHC-TCR interaction activates linker for activation of T cells (LAT) signalosome, which triggers calcium influx through phospholipase C (PLC) pathway (15, 16). Subsequently, the elevated calcium mobilizes cytotoxic granules to polarize towards the immunological synapse, then Granzymes and Perforin are released through degranulation (17). Interestingly, recent studies have shown that other types of immune cells including B cells, pDCs and mast cells may express and secrete GzmB in the absence of Perforin. However, the molecular machinery governing GzmB release from these conventionally known non-cytotoxic immune cells is not well studied and demands further investigation.

After release, the dual mechanisms of GzmB delivery—receptor-mediated and Perforin-mediated pathways—underlie its versatile functions ( Figure 1 ). The receptor-mediated pathway engages cell surface receptors and involves receptor-mediated endocytosis, impacting protein cleavage and caspase interplay. Meanwhile, the Perforin-dependent route involves extracellular release via the immunological synapse, translocating into target cells to induce apoptosis via caspase cleavage. A recent study exemplified the separation and collaboration between GzmB and Perforin in this process, by examining intracellular damage in intestinal epithelial cells induced by anti-CD3 mAb-activated intra-epithelial lymphocytes (18). It revealed GzmB-dependent yet Perforin-independent DNA fragmentation in intestinal epithelial cells, while GzmB can still concurrently engage Perforin-dependent pathways. This challenges the binary perception of GzmB delivery, emphasizing its nuanced interplay with both mechanisms. Such adaptability adds complexity to immune surveillance, requiring comprehensive investigation into its diverse actions. The relationship between GzmB, Perforin, heparan sulfate, and mannose-6-phosphate receptor (M6PR) underscores their joint role in efficient GzmB delivery. While receptors significantly aid entry, alternative routes highlight GzmB’s versatility. Granule-mediated killing is augmented by heparan sulfate and M6PR, broadening the scope of GzmB’s action in immune responses (19).

In the classical model, intracellular GzmB is detected within cytotoxic lymphocytes or their corresponding target cells following Perforin-mediated delivery (26). Once delivered into target cells, GzmB activates a precise apoptotic cascade via caspase cleavage, notably pro-caspase-8 and pro-caspase-10. This orchestrated process involves a pivotal interaction with Bid, generating tBid and triggering mitochondrial release of cytochrome c. Cytochrome c, along with other factors, activates caspase-9, initiating downstream effector caspases, ultimately causing DNA fragmentation, a defining feature of programmed cell death (27). In contrast, a recent study identified a previously unexpected role of GzmB in triggering sublethal DNA damage response in cancer cells independent of Perforin, where GzmB performs proteolytic function without causing cell death by cleaving intracellular proteins, including ICAD, caspase-7, caspase-8, DDX21, topoisomerase-IIα, and NuMA, indicative of GzmB’s protease activity within cells (28). Interestingly, these intracellular enzymatic activities lead to a non-cytotoxic DNA damage response. Consequently, interferon regulatory transcription factor 3 (IRF3) was activated, which may regulate the expression of a set of interferon-stimulated genes involved in inflammatory response (28).

A number of studies have extended Perforin-independent GzmB activity to the membrane surface of target cells. First, NK cell-derived GzmB was reported to specifically bind cell surface-bound heat shock protein 70 (Hsp70) in a process that mediates Perforin-independent GzmB uptake (29). This interaction increases the sensitivity of tumor cells to NK cell-mediated killing. The study identified a 14 amino acid sequence from Hsp70, termed TKD, sensitizing tumor cells to NK cytotoxic activity, orchestrating GzmB release by activated NK cells and inducing apoptosis in Hsp70 membrane-positive tumor cells. These findings support an immunotherapeutic strategy that uses GzmB as a lever to target membrane Hsp70-expressing tumors (30). In addition, in a model of T cell-mediated neuroinflammatory disorder, GzmB was found to mediate neurotoxicity through proteolytic activity on a G-protein-coupled receptor (11). GzmB cleaves and activates the protease-activated receptor-1 (PAR-1) on neuronal cell membrane, leading to increased expression and translocation of the voltage gated potassium channel, Kv1.3 to the neuronal cell membrane, followed by activation of Notch-1 resulting in neurotoxicity (12). These observations suggest that GzmB released from T cells induced neuronal injury and neurite atrophy by interacting with the membrane bound Gi-coupled PAR-1 receptor and subsequently activated Kv1.3 and Notch-1, which was required for GzmB-induced neurotoxicity (12). Furthermore, GzmB displayed Perforin-independent function by cleaving the extracellular domain of Notch-1, yet GzmB cleavage of Notch-1 can occur in all subcellular compartments, during maturation of the Notch-1 receptor, at the membrane, and in the nucleus (31). Cleavage of Notch-1 by GzmB resulted in a loss of transcriptional activity, independent of Notch-1 activation, which may influence target cell proliferation and survival (31).

Understanding GzmB’s extracellular functions remains an active research area despite significant progress in deciphering its intracellular mechanisms. Recent studies accentuate the diverse extracellular roles of GzmB, expanding beyond apoptosis, demonstrating its involvement in extracellular matrix (ECM) remodeling and immune regulation (8, 18). Released independently of perforin or GzmA, mast cell-derived GzmB plays a role in ECM modulation, impacting cellular adhesion and migration (27). Also, immunological synapse leakage of GzmB may influence cellular behavior. Anoikis induction in smooth muscle and endothelial cells reveals GzmB’s role in cellular motility and its potential anti-carcinogenic impact. Several ECM substrates for GzmB have been identified, including aggrecan, fibronectin, vitronectin, and laminin. GzmB cleaves vitronectin at the RGD domain, crucial for integrin binding, disrupting cellular-vitronectin interactions and affecting cell adhesion and migration (27). Notably, a recent study using 2-photon microscopy shows that extracellular GzmB activity is important for the migration of primed CD8+ CTLs (32). Upon engagement with postcapillary venules, CTLs secrete a small amount of GzmB that works like scissors to cut through basement membrane constituents to facilitate their recruitment into the inflammatory site (32).

In an autoimmune condition such as rheumatoid arthritis, characterized by immune system attacking normal self-tissues, especially joints, GzmB may play a significant extracellular role, contributing to tissue damage (9). Its significance even extends to cardiovascular pathogenesis. Atherosclerosis, a major contributor to heart attacks and strokes, involves lipid-driven inflammation where T lymphocytes, macrophages, and other immune cells contribute to plaque formation. Mast cell-derived GzmB may induce detachment of endothelial and smooth muscle cells, implicating in atherosclerotic lesions (7). Elevated GzmB levels correlate with unstable plaques and post-infarct ventricular remodeling, signifying its potential role in disease progression. In arterial walls, inflammatory responses attract immune cells that secrete GzmB, which may contribute to arterial wall damage and susceptibility to aortic dilation and aneurysms. Two reports highlighted GzmB’s role in aneurysm formation independent of Perforin, positioning it as a potential therapeutic target (33, 34). Studies in murine models show that GzmB deficiency reduces aneurysm incidence, emphasizing its involvement in aneurysm progression. Elevated GzmB levels and the cleavage of fibrillin-1 signify its crucial role in vessel wall destabilization, a hallmark of aneurysm pathology. Further exploration of SA3N’s effects on GzmB and decorin degradation presents a promising avenue targeting GzmB for therapeutic intervention in aortic aneurysms. Furthermore, a few studies have implicated GzmB in T cell-mediated neurological disorders. GzmB released from CD4+ and CD8+ T cells can induce considerable neuronal injury. Recombinant GzmB, even at low concentrations in the absence of Perforin, induced significant toxicity in neurons through Giα-coupled receptors (11). GzmB’s interaction with neuronal receptors, particularly PAR-1 and Kv1.3, contributed to progression of neuronal damage (12). These findings not only shed light on the intricacies of GzmB’s role in neurotoxicity but also open promising therapeutic avenues for addressing T cell-mediated neurological diseases.

Conventional treatments struggle against tumor progression, but emerging therapies including cancer vaccines and adoptive T cell therapy face challenges in generating effective immune responses due to immunosuppression. Innovative strategies in identifying tumor antigens, counteracting immune checkpoint and tumor-induced immune suppression have been fueling the growth of cancer immunotherapy. In this context, GzmB has exhibited complex roles in antitumor cytotoxic lymphocytes as well as immunosuppressive pDCs and Treg cells (9, 35, 36). Recent trials combining chemotherapy with immunotherapy show promise but still lack clarity in mechanisms. A study in 2010 highlighted chemotherapy’s role in enhancing tumor susceptibility to CTL-mediated killing. Chemotherapy, TAX, DOX, and CIS, made tumor cells more susceptible to GzmB-induced cell death independently of Perforin, facilitating non-antigen-specific CTL killing. This effect was mediated via upregulation of M6PR on the surface of tumor cells (37). These models suggest that combining radiation/chemotherapy with cancer vaccines enhances T cell responses associated with antitumor effects. Further studies are warranted to delineate the relevant mechanisms and design GzmB-targeting strategies that improve combinatory cancer treatments.

Many publications have provided strong evidence showing the significance of Perforin and GzmB in allogeneic hematopoietic cell transplantation (38). Several studies suggest that GzmB plays differential roles in graft-versus-host disease (GVHD) and graft-versus-tumor effect mediated by different types of donor T cells. While GzmB is required for CD8+ T cells to cause GVHD, GzmB-mediated damage of CD8+ T cells impairs graft-versus-tumor effect (4). On the other hand, GzmB-mediated activation-induced death of CD4+ T cells inhibits murine acute GVHD (39), while GzmB contributes to the optimal graft-versus-tumor effect mediated by CD4+ T cells (40). Furthermore, the endogenous inhibitor of GzmB, serine protease inhibitor 6, protects alloreactive T cells from GzmB-mediated mitochondrial damage without affecting graft-versus-tumor effect (41). Notably, host-derived serine protease inhibitor 6 also provides GzmB-independent protection of intestinal epithelial cells in acute GVHD (42), suggesting that it may interact with other proteases. These studies have underscored its complex roles in adverse GVHD and the desired graft-versus-tumor effect. There are ongoing studies investigating the roles of Perforin-independent GzmB activity in both acute and chronic GVHD models. Understanding the nuanced regulation and function of GzmB in allogeneic immune response may offer a promising avenue for managing GVHD severity.