Granzyme

Granzymes are a family of serine proteases primarily expressed in the cytotoxic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, where they mediate the induction of apoptosis and other forms of programmed cell death in target cells, including virus-infected cells and malignant tumors.^[1][2] First identified in the 1980s as components of the cytotoxic granules in NK cells and CTLs, granzymes were initially recognized for their role in immune-mediated cell killing, with subsequent research in the 1990s elucidating their mechanisms, such as Granzyme B's activation of caspase-dependent apoptosis pathways.^[1] In humans, the granzyme family consists of five members—Granzyme A (GzmA), Granzyme B (GzmB), Granzyme H (GzmH), Granzyme K (GzmK), and Granzyme M (GzmM)—each exhibiting distinct substrate specificities, such as chymotrypsin-like (e.g., GzmB cleaving after aspartic acid residues) or trypsin-like activity.^[1][2] These enzymes are constitutively expressed in NK cells and γδ T cells but induced in αβ T cells upon antigen stimulation, ensuring targeted release during immune responses.^[2] Granzymes exert their cytotoxic effects primarily through exocytosis of granules, where perforin forms pores in the target cell membrane, allowing granzyme entry via endocytosis or direct translocation.^[1] Once inside, they cleave intracellular substrates to trigger diverse death pathways: GzmB activates Bid and caspases for mitochondrial outer membrane permeabilization, while GzmA induces single-stranded DNA damage and ROS production, leading to caspase-independent apoptosis or pyroptosis.^[1] Beyond cytotoxicity, granzymes contribute to immune regulation by modulating cytokine production, B-cell proliferation, and extracellular matrix remodeling, and they exhibit antimicrobial properties against pathogens.^[2] In health, granzymes are essential for immune surveillance, eliminating infected or cancerous cells to prevent disease progression.^[1] However, dysregulated granzyme activity is implicated in pathology, including autoimmune disorders like rheumatoid arthritis—where elevated GzmB promotes inflammation—and chronic conditions such as chronic obstructive pulmonary disease (COPD) and atherosclerosis, highlighting their dual roles as both protective and potentially harmful mediators in the immune system.^[1] Evolutionarily, granzymes are mammal-specific, clustered in three chromosomal loci in humans (5q11-q12, 14q11, 19p13.3), reflecting their specialized adaptation for adaptive immunity.^[2]

Introduction

Definition and overview

Granzymes are a family of serine proteases that are predominantly expressed and stored within the cytotoxic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, key effectors of the adaptive and innate immune systems, respectively.^[3] These enzymes are synthesized as inactive precursors and packaged into granules alongside other components to enable rapid deployment during immune responses.^[4] The primary function of granzymes is to mediate targeted cell death through the induction of apoptosis in virus-infected cells, tumor cells, or other aberrant targets that pose a threat to the host.^[5] Upon recognition of the target, CTLs or NK cells release their granular contents at the immunological synapse, where granzymes act in concert with perforin, a pore-forming protein that disrupts the target cell's plasma membrane to facilitate granzyme entry into the cytosol.^[6] In humans, the granzyme family consists of five members—granzymes A, B, H, K, and M—each exhibiting distinct substrate specificities that contribute to diverse proteolytic activities within the target cell.^[7] Granzyme levels and activity can be assessed using several established immunological techniques, including Western blotting for protein detection in cell lysates, enzyme-linked immunosorbent assay (ELISA) for quantifying soluble forms in biological fluids, flow cytometry for intracellular staining in cell populations, and enzyme-linked immunospot (ELISPOT) assays for measuring secretion from individual cells.^[8][9][10] These methods provide insights into granzyme expression and function in both research and clinical contexts.

Biological significance

Granzymes serve as key effectors in cell-mediated immunity, primarily produced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to enable the targeted elimination of infected or malignant cells through perforin-dependent granule exocytosis.^[11] This mechanism induces rapid apoptosis in specific threats while minimizing widespread inflammation, as the localized delivery of granzymes confines proteolytic activity to the target cell, preserving surrounding healthy tissue.^[12] By facilitating precise cytotoxicity, granzymes contribute to immune surveillance and homeostasis, ensuring efficient clearance of pathogens and tumors without excessive bystander damage.^[7] Across mammals, granzymes exhibit notable conservation, with humans possessing five members (A, B, H, K, and M) encoded at three chromosomal loci—5q11-q12 (A and K), 14q11.2 (B and H), and 19p13.3 (M)—while mice have ten, reflecting evolutionary adaptations to species-specific pathogens through gene duplication and substrate specificity divergence.^[13][14] This variability allows granzymes to evolve in response to distinct immune pressures, such as varying viral or bacterial challenges, enhancing adaptive immunity over phylogenetic history.^[13] In chronic inflammatory conditions, however, extracellular accumulation of granzymes, particularly granzyme B, occurs due to dysregulated release from activated immune cells, leading to unintended proteolysis of extracellular matrix components and promotion of tissue remodeling that exacerbates disease progression in disorders like rheumatoid arthritis and chronic obstructive pulmonary disease.^[7] Beyond apoptosis, granzymes exert non-apoptotic functions that bolster antimicrobial defenses, including direct cleavage of bacterial and viral proteins to inhibit replication—for instance, granzyme M targets cytomegalovirus phosphoprotein 71 to block viral latency reactivation, while granzyme B disrupts herpes simplex virus ICP4.^[15] These activities, often involving induction of proinflammatory cytokines like IL-6 and TNF-α, enhance innate responses against pathogens such as Mycobacterium tuberculosis and Epstein-Barr virus without necessitating cell death.^[15] Additionally, granzymes support immune tolerance by enabling regulatory T cells to suppress excessive responses through targeted proteolysis, thereby preventing autoimmunity via the elimination of autoreactive lymphocytes while maintaining self-tolerance.^[12]

Structure and classification

Molecular structure

Granzymes belong to the family of serine proteases and exhibit a chymotrypsin-like fold characterized by two β-barrels connected by a loop, with the catalytic triad consisting of histidine, aspartic acid, and serine residues responsible for substrate cleavage.^[3] The catalytic triad, conserved across the family, typically includes His57, Asp102, and Ser195 in the numbering system based on chymotrypsin, enabling nucleophilic attack on the peptide bond during proteolysis.^[16] Granzymes are synthesized as inactive zymogen precursors featuring an N-terminal pro-peptide that maintains latency until proteolytic removal, often by dipeptidyl peptidase I (cathepsin C), activates the mature enzyme.77160-7/fulltext) This activation step exposes the new N-terminus, which inserts into the activation pocket to stabilize the active conformation.^[17] The active site of granzymes includes specificity pockets, particularly the S1 pocket, which dictates substrate preference; for instance, in granzyme A, this pocket accommodates basic residues like arginine or lysine.^[18] These pockets are formed by conserved residues lining the substrate-binding cleft, contributing to the enzymes' selective cleavage activities.^[19] Post-translational modifications in granzymes include N-linked glycosylation at asparagine residues, which aids in protein folding and stability, and typically three conserved disulfide bonds that maintain the structural integrity of the β-barrel domains.^[20] These disulfide bridges, formed between cysteine pairs such as Cys42-Cys58 and Cys188-Cys220, are essential for the protease's resistance to denaturation.^[3] High-resolution crystal structures have elucidated the conserved architecture of granzymes, such as the 2.0 Å structure of human granzyme B in complex with an inhibitor, revealing the β-barrel fold and the spacious S1 pocket adapted for aspartate recognition.^[21] Similar structures for other family members, including granzyme A at 2.5 Å resolution, confirm the shared chymotrypsin-like scaffold with variations in loop regions influencing specificity.^[22]

Granzyme subtypes

The human granzyme family comprises five serine proteases—granzyme A (GZMA), granzyme B (GZMB), granzyme H (GZMH), granzyme K (GZMK), and granzyme M (GZMM)—classified primarily by their substrate specificities at the P1 position of peptide bonds. These enzymes are expressed predominantly in cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, with variations across species such as mice, where orthologs exhibit functional divergences like altered substrate preferences in GZMB.^[3][5][4] Granzyme A (GZMA) is a tryptase-like protease with specificity for cleaving after basic residues, particularly arginine and lysine, and exists as a homodimer with a monomeric molecular weight of approximately 27-30 kDa. It induces single-stranded DNA nicks and promotes cytokine release, such as IL-6 and IL-8, contributing to inflammatory responses. GZMA is abundantly expressed in CTLs, NK cells, and γδ T cells in humans and mice, with similar patterns in both species.^[3][5][4] Granzyme B (GZMB), the most studied subtype, functions as a caspase-like protease that preferentially cleaves after aspartic acid (and to a lesser extent glutamic acid), with a molecular weight of about 32 kDa. It is the most potent inducer of apoptosis among granzymes through direct cleavage of key substrates. GZMB is widely expressed in activated CTLs, NK cells, and thymocytes in humans and mice, though its extended substrate specificity differs between species, with mouse GZMB showing chymase-like activity.^[3][5][4] Granzyme H (GZMH) is a chymase-like enzyme that targets aromatic residues, primarily phenylalanine and tyrosine, and has a molecular weight of approximately 27-32 kDa. It is less extensively characterized but shows potential roles in inflammatory processes, including cleavage of viral proteins. GZMH expression is restricted to CTLs and NK cells in humans, with limited ortholog expression in mice.^[3][5][4] Granzyme K (GZMK), another tryptase-like protease similar to GZMA, cleaves after basic residues like arginine and lysine, with a molecular weight around 28 kDa. It supports neutrophil recruitment and proinflammatory cytokine production via PAR-1 activation. GZMK is expressed in CTLs, NK cells, and epithelial cells in humans and mice, often co-expressed with GZMA.^{[3][5][23][4]} Granzyme M (GZMM) is a metase-like protease that cleaves after methionine (and leucine), possessing a molecular weight of about 25-30 kDa, and exhibits antimicrobial activity alongside extracellular matrix (ECM) degradation. It is uniquely expressed almost exclusively in NK cells in humans, contrasting with broader CTL expression in mice.^{[3][5][24][25][4]}

Biosynthesis and expression

Gene organization

The human granzyme genes are distributed across three chromosomal clusters, reflecting their evolutionary divergence within the serine protease superfamily. The genes encoding granzymes A (GZMA) and K (GZMK), both tryptase-like enzymes, are located on chromosome 5q11-12 and span approximately 86 kb.^[26][27] In contrast, the genes for granzymes B (GZMB) and H (GZMH), which are chymase-like, form a cluster on chromosome 14q11.2 alongside the cathepsin G gene (CTSG), encompassing roughly 140 kb.^[28][29][30] The granzyme M gene (GZMM), a metase, resides separately on chromosome 19p13.3.^[31] Each functional granzyme gene exhibits a conserved structure consisting of five exons separated by introns at homologous boundaries, a feature shared with other S1 family serine proteases.^[14] This organization includes an initial exon encoding the signal peptide, followed by exons that code for the propeptide and mature protease domains. Granzyme B (GZMB) exemplifies this architecture, with its ~3.2 kb gene featuring well-defined promoter regions, including TATA-like elements and binding sites for transcription factors such as Ikaros, which contribute to lineage-specific expression.^[14] The granzyme family arose and expanded through tandem gene duplication events predating the divergence of rodents and primates. In humans, five functional genes (GZMA, GZMB, GZMH, GZMK, GZMM) represent a streamlined set compared to mice, which possess ten granzymes due to additional duplications, particularly of the ancestral GZMB/GZMH lineage after rodent-primate separation.^[32] These duplications likely facilitated adaptation to species-specific immune challenges, with conserved synteny across the clusters underscoring their ancient origins. Pseudogenes, indicative of historical duplication without selective pressure, are interspersed within or near these clusters. For instance, a granzyme O-like pseudogene, containing premature stop codons and lacking full functionality, lies adjacent to the GZMB/GZMH cluster on chromosome 14q11.2.^[33] This highlights ongoing genomic instability in the family. Among human granzymes, amino acid sequence identity varies from 40% to 60%, with the greatest conservation (often exceeding 70%) in the catalytic domains harboring the canonical His-Asp-Ser triad essential for serine protease activity. This homology supports shared structural folds akin to trypsin, while divergences in substrate-binding pockets underlie functional specialization.

Cellular expression and regulation

Granzymes are primarily expressed in cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, where they play a central role in immune-mediated cytotoxicity. In human NK cells, granzyme B is constitutively expressed at both the transcript and protein levels, enabling these cells to exert cytotoxic activity even in a resting state.^[34] Upon activation, expression of granzymes, particularly granzyme B, is upregulated in both CTLs and NK cells through stimulation by cytokines such as interleukin-2 (IL-2) or IL-15, or by antigen recognition in the case of CTLs.^[35] For instance, high concentrations of IL-2 or IL-15 induce granzyme B in up to 80% of CD8⁺ T cells and 25-30% of CD4⁺ T cells within three days, mediated via CD122/CD132 receptors, without requiring antigen-specific triggers.^[35] Beyond immune effector cells, granzymes can be expressed in various non-immune cell types under conditions of stress or inflammation. Dendritic cells and mast cells produce granzyme B during inflammatory responses, contributing to local tissue modulation.^[36] Keratinocytes express granzyme B following ultraviolet B irradiation, acquiring cytotoxic properties that may influence skin homeostasis.^[36] Similarly, type II pneumocytes in the lungs of patients with chronic obstructive pulmonary disease (COPD) show granzyme B expression, associated with stress-induced apoptosis and airway remodeling.^[36] The regulation of granzyme expression involves key transcription factors and epigenetic mechanisms. In cytotoxic lymphocytes, the T-box transcription factors T-bet and Eomesodermin (Eomes) drive differentiation and effector function, with Eomes being essential for activating granzyme B (GZMB) and perforin (PRF1) transcription in NK cells.^[37] Eomes promotes histone acetylation at the GZMB locus, facilitating chromatin accessibility and enhanced expression.^[37] Subtype-specific patterns exist, such as granzyme B dominance in NK cells, though details vary by cell type.^[37] Granzymes are stored in specialized cytotoxic granules within CTLs and NK cells, which are secretory lysosomes containing serglycin proteoglycan that binds and inactivates the proteases until release.^[12] Upon target cell recognition, these granules polarize toward the immunological synapse, where they undergo directed exocytosis, releasing granzymes and perforin into the synaptic cleft for targeted delivery to the target cell.^[12] Expression patterns differ across species, with mice exhibiting broader granzyme distribution in non-lymphoid cells compared to humans. For example, murine granzyme C is dynamically expressed in skin-resident antiviral T cells, including dendritic epidermal T cells (15-35% at steady state, upregulated to ~36% during vaccinia virus infection) and CD8⁺ tissue-resident memory T cells (~54% at steady state, with increased expression post-infection), often localized to cellular extensions interacting with keratinocytes.^[38] This expression is enhanced by IL-15, suggesting adaptive roles in tissue immunity.^[38]

Mechanism of action

Role in granule-mediated cytotoxicity

Granule exocytosis represents the primary mechanism by which granzymes are deployed during cytotoxicity by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Upon recognition of target cells, engagement of activating receptors induces calcium influx in the effector cell, polarizing the microtubule-organizing center toward the immunological synapse formed at the contact site. This triggers the rapid migration and docking of lytic granules to the plasma membrane, followed by their fusion and polarized release of contents, including perforin and granzymes, directly into the synaptic cleft to minimize bystander damage.^[39] The synergy between perforin and granzymes is essential for effective cytotoxicity. Perforin monomers oligomerize in a calcium-dependent manner to form transient pores, approximately 10-20 nm in diameter, in the target cell plasma membrane. These pores enable granzymes to enter the cytosol primarily through passive diffusion or clathrin-mediated endocytosis, where perforin may further disrupt endosomal membranes to release the proteases intracellularly. This delivery targets a range of abnormal cells, including virus-infected cells, tumor cells, and allografts, allowing precise elimination of threats without widespread tissue damage.^[40][39][41] The efficiency of this pathway is remarkable, as only a small number of granzyme molecules—estimated at 3-5 for granzyme B—are required to initiate target cell death, facilitated by high local concentration gradients within the confined space of the immunological synapse. This low threshold ensures rapid and potent killing, with effector cells releasing sufficient granules (each containing thousands of perforin and granzyme molecules) to overwhelm repair mechanisms in susceptible targets. In certain contexts, granzymes can also enter cells via alternative, perforin-independent routes, such as receptor-mediated endocytosis involving the mannose-6-phosphate/insulin-like growth factor II receptor, which binds granzyme B and internalizes it into endosomes.^[42][43][44]

Specific apoptosis pathways

Granzymes induce apoptosis through distinct intracellular pathways following their delivery into target cells via perforin pores.^[45] The granzyme B pathway encompasses both caspase-dependent and mitochondrial mechanisms. In the caspase-dependent route, granzyme B directly cleaves and activates initiator caspases such as caspase-8 and -10, as well as effector caspases-3 and -7, thereby initiating the proteolytic cascade that dismantles cellular structures.^[46] Concurrently, the mitochondrial pathway involves granzyme B-mediated cleavage of Bid into truncated Bid (tBid), which translocates to the mitochondria and promotes Bax/Bak oligomerization, leading to outer membrane permeabilization and cytochrome c release; this forms the apoptosome with Apaf-1 and procaspase-9, amplifying caspase activation.^[46] Additionally, granzyme B can enter mitochondria via the Sam50-Tim22 complex, cleaving respiratory chain complex I subunits to generate reactive oxygen species (ROS), which further destabilizes mitochondrial integrity and enhances cytochrome c efflux independently of Bid in certain contexts.^[46] In contrast, the granzyme A pathway operates through a caspase-independent mechanism focused on nuclear and mitochondrial disruption. Granzyme A targets the SET complex in the endoplasmic reticulum, cleaving the SET protein (also known as TAF-Iβ or inhibitor of NM23-H1) to release and activate the NM23-H1 DNase, which introduces single-stranded nicks in DNA; these lesions are extended by the Trex1 exonuclease, culminating in genomic fragmentation.^[47] Granzyme A also accesses mitochondria to cleave complex I subunits, producing superoxide ROS that drives SET nuclear translocation and exacerbates DNA damage while inducing endoplasmic reticulum stress.^[47] Other granzyme subtypes engage alternative death routes. Granzyme K mirrors granzyme A by inducing caspase-independent cell death through SET cleavage, NM23-H1 activation for DNA nicking, and mitochondrial ROS production, resulting in a pyroptosis-like inflammatory response with rapid membrane disruption.^[47] Granzyme H promotes caspase-independent apoptosis primarily via a Bcl-2-sensitive mitochondrial pathway involving DNA damage through cleavage of DFF45 and disruption of viral proteins, though its substrate specificity remains less defined.^[47][48] Granzyme M induces a perforin-dependent, caspase-dependent form of cell death distinct from other granzymes. It targets topoisomerase IIα, leading to cell cycle arrest at the G2/M phase and subsequent apoptosis, and also processes FADD to potentiate death receptor signaling.^[49][50] These pathways converge on common apoptotic hallmarks, including extensive DNA fragmentation, cytoplasmic condensation, membrane blebbing, and formation of apoptotic bodies that facilitate non-inflammatory phagocytosis by macrophages.^[51] Notably, granzyme A and K pathways bypass Bcl-2-mediated resistance, as they do not rely on mitochondrial outer membrane permeabilization, providing a safeguard against cells overexpressing anti-apoptotic Bcl-2 family members that inhibit granzyme B's mitochondrial effects.^[47][51]

Physiological functions

Immune defense against pathogens

Granzymes, primarily released by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), are essential effectors in the innate and adaptive immune responses against viral pathogens, enabling the targeted elimination of infected cells through granule-mediated cytotoxicity. This process involves perforin forming pores in the target cell membrane, allowing granzymes to enter and activate intracellular apoptosis pathways, thereby restricting viral replication and spread. For instance, granzyme B (GZMB) directly inhibits viral protein function by cleaving key viral components, such as the immediate-early protein ICP27 of herpes simplex virus 1 (HSV-1), which disrupts viral gene expression and transcription. Similarly, granzyme A (GZMA) induces rapid DNA damage in virally infected cells via single-stranded nicks and activation of the SET complex, leading to caspase-independent cell death and limiting pathogen propagation. Beyond cytotoxicity, granzymes exhibit noncytotoxic antiviral activities by degrading host and viral factors essential for replication. In influenza A virus infections, granzyme K (GZMK) cleaves the nuclear import complex importin α1/β, blocking viral nucleoprotein transport into the nucleus and thereby suppressing viral genome replication. During HIV-1 infection, granzyme M (GZMM) targets the viral Gag polyprotein, impairing virion assembly and release from infected cells. These mechanisms complement direct cell killing, providing a multifaceted defense that enhances viral clearance without solely relying on apoptosis. Granzymes also contribute to antibacterial immunity, both extracellularly by degrading bacterial virulence factors and intracellularly by inducing death in infected host cells. Extracellular GZMB attenuates bacterial pathogenicity by proteolytically targeting secreted toxins and adhesins, as observed in Listeria monocytogenes infections where it cleaves listeriolysin O, reducing bacterial invasion and survival. Intracellularly, granzymes delivered via granulysin pores into bacteria like Listeria and Mycobacterium tuberculosis disrupt essential metabolic pathways, such as electron transport chains, leading to bacterial lysis and preventing intracellular persistence. This dual action supports effective control of bacterial infections in collaboration with other antimicrobial peptides. The synergy between innate and adaptive immunity amplifies granzyme-mediated defense: NK cells provide rapid, early responses to pathogens through constitutive granzyme expression, while antigen-specific CTLs expand during the adaptive phase for sustained eradication. However, pathogens can evade these mechanisms; many viruses, including HIV and HSV-1, produce inhibitors that block perforin pore formation or sequester granzymes, thereby promoting immune escape and chronic infection.

Role in antitumor immunity

Granzymes, particularly granzyme B (GZMB), play a pivotal role in antitumor immunity by enabling cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to eliminate tumor cells, including those that downregulate MHC class I to evade recognition. Upon forming an immunological synapse with target cells, CTLs and NK cells release granules containing perforin and granzymes, which perforin facilitates entry into the tumor cell cytoplasm, where GZMB cleaves key substrates to activate multiple apoptosis pathways, such as caspase-dependent and mitochondrial routes, leading to rapid tumor cell death.^[52][53][54] This mechanism is crucial for surveilling and destroying nascent tumors, with GZMB being the dominant effector in granule-mediated cytotoxicity against MHC-I low or negative cancers.^[55] GZMB expression serves as a biomarker for T cell activation and inversely correlates with exhaustion in the tumor microenvironment, where PD-1 blockade reinvigorates exhausted CD8+ T cells to restore granzyme-mediated killing. In non-small cell lung cancer, low baseline serum GZMB levels predict poor response to PD-1 inhibitors, highlighting its utility in assessing therapeutic efficacy and T cell functional status.^[56][57] Enhanced GZMB production post-checkpoint inhibition promotes infiltration and cytotoxicity of tumor-attacking lymphocytes, amplifying antitumor responses.^[58] In immunotherapy, chimeric antigen receptor (CAR) T cells leverage granzyme release for potent cytotoxicity, mimicking natural CTL function to induce perforin/granzyme-dependent tumor lysis in solid tumors.^[59] Similarly, bispecific antibodies redirect T cells to tumor antigens, facilitating immunological synapse formation that triggers granzyme influx and apoptosis in target cells, as demonstrated in preclinical models of colorectal cancer.^[60][61] While primarily tumoricidal, granzymes can exhibit context-dependent suppressive effects in certain tumor microenvironments, though their protective role dominates in immune surveillance (detailed in pathological roles).^[62] Clinically, elevated GZMB expression in tumor-infiltrating lymphocytes correlates with improved prognosis; in melanoma, CD8+ GZMB+ TILs are associated with longer overall and progression-free survival, reflecting robust antitumor activity.^[63] In colorectal cancer, high granzyme B-positive TIL density serves as a positive prognostic factor for overall survival, underscoring its value in predicting favorable outcomes.^[64]

Pathological roles

Involvement in cancer

Granzymes, particularly granzyme B (GZMB), exhibit complex roles in the tumor microenvironment (TME), where they can paradoxically contribute to cancer progression despite their primary cytotoxic functions. Extracellular GZMB, secreted by immune cells or tumor-associated sources, degrades extracellular matrix (ECM) components such as decorin and fibronectin, thereby releasing sequestered vascular endothelial growth factor (VEGF) and promoting tumor angiogenesis and vascular permeability.^[65] This ECM remodeling facilitates nutrient supply and metastatic dissemination, as observed in models of choroidal neovascularization and solid tumors.^[66] Additionally, GZMB expressed by regulatory T cells (Tregs) enhances their immunosuppressive activity, suppressing antitumor T cell responses and promoting tumor growth in murine models.^[67] In mechanisms of tumor escape, chronic exposure to GZMB can induce resistance in cancer cells through upregulation of inhibitors like proteinase inhibitor-9 (PI-9) and activation of autophagy, which degrades intracellular GZMB and reduces susceptibility to cytotoxic lymphocyte-mediated lysis.^[68] Similarly, granzyme K (GZMK) secreted by CD8+ T effector memory cells in the TME recruits CD15high suppressive neutrophils, fostering an immunosuppressive niche that correlates with poor clinical outcomes in non-metastatic colorectal cancer (CRC).^[69] Within the TME, elevated granzyme levels, including extracellular GZMA and GZMB, are linked to enhanced CRC progression and metastasis by promoting inflammation and epithelial-mesenchymal transition (EMT).^[70] Granzymes play a dual role in immunotherapy responses; while intracellular GZMB drives tumor cell apoptosis in responders, extracellular forms can exacerbate immune evasion, with GZMB activity serving as a biomarker for both efficacy and resistance in checkpoint blockade therapies.^[62] As biomarkers, low serum GZMB levels predict worse progression-free and overall survival in non-small cell lung cancer (NSCLC) patients undergoing PD-1 blockade, reflecting impaired cytotoxic potential.^[71] Genetic variants in GZMB, such as the triple-mutated allele (Q55R/P88A/Y247H), reduce enzymatic activity and are associated with increased cancer risk and poorer outcomes by diminishing effective antitumor immunity.^[72] Therapeutic strategies targeting granzymes' pro-tumor functions include inhibitors to block extracellular GZMB-mediated ECM degradation and angiogenesis, potentially sensitizing tumors to immune attack.^[73] Conversely, enhancers like perforin-GZMB fusion constructs or melittin-augmented GZMB delivery systems overcome resistance by improving tumor penetration and cytotoxicity, showing promise in preclinical models of head and neck cancers.^[74]

Roles in inflammatory and autoimmune diseases

Granzymes, particularly granzyme B (GZMB), play a significant role in the pathogenesis of autoimmune diseases by contributing to tissue damage through cytotoxic mechanisms. In rheumatoid arthritis (RA), elevated levels of GZMB are detected in the synovial fluid and tissue of affected joints, where it promotes inflammation and drives joint destruction by inducing apoptosis in chondrocytes and fibroblasts, as well as degrading extracellular matrix components.^[75][76] Similarly, in multiple sclerosis (MS), granzyme B is present in cerebrospinal fluid and within demyelinating plaques, where it mediates oligodendrocyte and neuronal damage via perforin-dependent cytotoxicity from CD8+ T cells and natural killer (NK) cells, exacerbating neuroinflammation and lesion progression.^[5][77] Granzyme A (GZMA) has also been implicated in MS pathology, with altered plasma levels potentially contributing to immune dysregulation in the central nervous system. In chronic inflammatory conditions, granzymes facilitate extracellular matrix remodeling and tissue remodeling processes that perpetuate disease. In atherosclerosis, extracellular GZMB degrades matrix proteins such as decorin and fibrillin-1, promoting plaque instability and vascular remodeling by activating smooth muscle cells and inflammatory cascades.^[78][79] Additionally, GZMB contributes to skin aging by inducing apoptosis in keratinocytes exposed to ultraviolet radiation, leading to collagen disorganization, epidermal thinning, and impaired wound healing in aged tissues.^[80][81] Dysregulation of granzyme expression underlies aberrant immune responses in various inflammatory and autoimmune contexts. In systemic lupus erythematosus (SLE), NK cells exhibit heightened granzyme B production, correlating with increased cytotoxicity against autoreactive lymphocytes but also contributing to overall immune imbalance and tissue injury.^[82] Granzyme K (GZMK), expressed by CD8+ T cells, promotes fibrosis by interacting with fibroblasts via CXCR4-CXCL12 signaling, inducing neutrophil chemoattractants and exacerbating fibrotic remodeling in affected tissues.^[23][83] Despite their pathological roles, granzymes can exert protective effects by suppressing excessive inflammation. In sepsis models, GZMB produced by regulatory T cells and NK cells aids in resolving hyperinflammation by inducing apoptosis in overactivated immune cells, thereby mitigating multiorgan damage and promoting recovery.^[84]00480-X) Therapeutically, targeting granzyme B holds promise for mitigating inflammatory damage in autoimmune and transplant-related conditions. Preclinical studies demonstrate that GZMB inhibitors, such as serpins, reduce T cell-mediated cytotoxicity and alleviate symptoms in graft-versus-host disease (GVHD) models by limiting donor T cell activation and tissue infiltration, with ongoing efforts to translate these findings into clinical trials.^[85][86]

History and research

Discovery and early characterization

Granzymes were first identified in the mid-1980s as a family of neutral serine proteases stored within the cytotoxic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. In 1986, Jürg Tschopp and colleagues purified granzyme A from rat CTL granules, characterizing it as a trypsin-like enzyme with specificity for basic amino acid residues, and coined the term "granzyme" to describe these granule-associated serine proteases. This discovery built on prior observations of serine esterase activity in lytic granules, highlighting their potential role in cell-mediated cytotoxicity. Concurrently, perforin, the pore-forming protein that facilitates granzyme delivery into target cells, was isolated from the same granular fractions, establishing the cooperative mechanism of granule exocytosis in immune killing.^[87] Early studies revealed a diverse family of these proteases, with initial nomenclature reflecting their biochemical properties and functions. In rats, eight distinct granzymes (A through G and another variant) were noted based on cDNA cloning and enzymatic assays from cytotoxic lymphocyte sources. Terms like "fragmentin" emerged for certain members, such as fragmentin-2 (later granzyme B), due to their ability to induce rapid DNA fragmentation in susceptible target cells, as demonstrated in 1988 experiments using purified granule extracts. Others were designated as T-cell serine proteases (TSP), with granzyme A termed TSP-1 for its expression in activated T cells. These names underscored the proteases' localization and proteolytic nature before standardization. Subsequent refinement identified only five functional granzymes in humans (A, B, H, K, and M), reflecting species-specific gene clusters.^[87] Functional characterization in the late 1980s linked granzymes to apoptosis induction, particularly through DNA damage. In 1988, cloning of the human granzyme B (GZMB) gene from cytotoxic lymphocytes enabled recombinant expression and assays showing its chymase-like activity, distinct from granzyme A's trypsin-like profile. Studies that year using isolated human granzymes A and B with perforin demonstrated their capacity to trigger DNA fragmentation in target cells, mimicking the effects of intact CTL granules. This perforin-dependent delivery was critical, as granzymes alone lacked cytolytic potency without the pore-forming partner. These findings positioned granzymes as key effectors in granule-mediated cytotoxicity.^[87] A seminal 2001 review in Genome Biology solidified the granzyme family as lymphocyte-specific serine proteases, synthesizing early cloning data and functional studies to define their genomic organization and conserved roles in immunity. This work emphasized their exclusive expression in cytotoxic lineages and highlighted the evolutionary divergence between rodent (multiple members) and human (fewer, specialized) orthologs, providing a foundational framework for subsequent research.

Key developments and recent advances

In the 2000s, research expanded the understanding of granzymes beyond their canonical role in apoptosis, revealing non-apoptotic functions such as extracellular proteolytic activities that modulate inflammation and extracellular matrix remodeling. A key study highlighted how granzyme B can cleave extracellular substrates like fibronectin in the absence of perforin, contributing to tissue remodeling in vascular diseases. This period also emphasized granzymes' involvement in immune regulation, including cytokine processing and antimicrobial effects independent of cell death.^[88][89] During the 2010s, structural biology advanced significantly with the determination of crystal structures for multiple granzyme subtypes, enabling insights into their distinct substrate specificities and activation mechanisms. For instance, the 2.2 Å crystal structure of pro-granzyme K revealed unique zymogen features that differ from other granzymes, informing subtype-specific pathways in cytotoxicity and inflammation. Concurrently, studies delineated the dual roles of granzymes in cancer, where they promote antitumor immunity through target cell killing but can also foster tumor progression via extracellular matrix degradation and pro-inflammatory signaling.^[90][55] In the 2020s, comprehensive reviews have synthesized granzymes' multifaceted roles, framing them as "the good, the bad, and the ugly" in health and disease—beneficial in pathogen clearance and antitumor responses, detrimental in chronic inflammation and autoimmunity, and paradoxical in cancer contexts. A notable advance identified granzyme K (GZMK)-expressing CD8+ T cells as drivers of neutrophilic inflammation in nasal polyps, where they interact with fibroblasts to induce neutrophil chemoattractants via CXCR4-CXCL12 signaling, linking GZMK to tissue-specific immune pathology. Single-cell RNA sequencing has further uncovered expression diversity, showing granzymes like GZMA, GZMB, and GZMK variably distributed across immune subsets such as NK cells and tissue-resident T cells, beyond traditional cytotoxic lymphocytes.^[5][23][91] In 2025, research identified GZMK-expressing CD8+ T cells as key drivers of recurrent airway inflammation, promoting tissue damage through interactions with fibroblasts in conditions like chronic rhinosinusitis.^[92] Therapeutic developments have progressed with granzyme B (GZMB)-based immunotoxins in preclinical optimization as of 2024, designed to enhance tumor targeting while overcoming inhibitors like serpin B9.^[93] Recent studies employing AI and machine learning models, such as graph neural networks for protease-substrate prediction, have aided the design of engineered variants for precision therapies. Additionally, recognition of five human granzyme members (A, B, H, K, M) has been solidified through genomic and transcriptomic analyses, addressing prior gaps in subfamily classification.