Granzyme A and Granzyme B Signaling Pathways: Cytotoxic Effects and Immune Regulatory Mechanisms

Granzyme A (GZMA) and Granzyme B (GZMB) are serine proteases released by cytotoxic T cells, natural killer cells, and some NKT cells. They are key effector molecules in the perforin–granzyme cytotoxic system. GZMB is mainly associated with caspase-dependent apoptosis, the Bid–mitochondrial pathway, and target cell clearance. GZMA is more commonly involved in non-caspase-dependent injury, disruption of the SET complex, oxidative stress, and inflammatory amplification. Together, they reflect activation of cytotoxic immunity, but expression of a single granzyme cannot directly replace comprehensive interpretation of killing function.

Keywords: Granzyme A; Granzyme B; GZMA; GZMB; perforin; PRF1; cytotoxic T cells; natural killer cells; caspase; mitochondrial apoptosis; SET complex; immune killing

1、Basic Framework of the Granzyme A/B Pathway

1.1 Cytotoxic Granule Release and Cooperation with Perforin

(1) Granule release

GZMA and GZMB are mainly stored in cytoplasmic granules of cytotoxic lymphocytes. After CD8⁺ cytotoxic T cells recognize target cell antigens, or NK cells recognize abnormal cells, effector cells form immune synapses with target cells. Cytotoxic granules polarize toward the synaptic region and are released, creating a locally high-concentration environment for perforin and granzymes.

(2) Cooperation with perforin

Perforin (PRF1) can form pores in the target cell membrane or endosomal membrane, helping GZMA and GZMB enter the cytoplasm and access their substrates. If perforin function is insufficient, increased granzyme expression does not necessarily induce effective target cell killing.

(3) Experimental interpretation

Research on granzyme pathways should simultaneously consider GZMA/GZMB expression, PRF1 level, CD107a degranulation, target cell death, and the source of effector cells. Detecting granzyme expression alone only indicates increased cytotoxic molecule reserves and does not directly prove that killing has been completed.

1.2 Functional Division Between Granzyme A and Granzyme B

(1) Pathway positioning of GZMB

GZMB has strong pro-apoptotic capacity and can cleave caspase family members, Bid, ICAD, and some nuclear and structural proteins. After entering target cells, it often rapidly initiates the caspase cascade and mitochondrial apoptosis pathway, causing PARP cleavage, DNA fragmentation, nuclear condensation, and formation of apoptotic bodies.

(2) Pathway positioning of GZMA

GZMA tends to mediate atypical cytotoxic responses. When it induces cell injury, cleaved caspase-3 may not be obvious. Instead, it more commonly causes SET complex disruption, impaired DNA repair, single-strand DNA damage, increased ROS, and inflammatory mediator release.

(3) Differences in mechanistic interpretation

GZMB is more suitable for explaining apoptotic clearance of target cells and is commonly used in studies of antiviral immunity, antitumor immunity, transplant rejection, and cytotoxic function. GZMA is more suitable for explaining inflammatory cytotoxic responses, non-caspase injury, and chronic tissue inflammation, especially in autoimmune disease, infection-related immunopathology, and chronic inflammation models.

Table 1 Core Differences Between Granzyme A and Granzyme B

Comparison dimension	Granzyme A (GZMA)	Granzyme B (GZMB)
Main mode of action	Non-caspase-dependent death, oxidative damage, inflammatory amplification	Caspase-dependent apoptosis and mitochondrial apoptosis amplification
Key substrate direction	SET complex, nuclear proteins, DNA repair-related proteins	Caspase-3/7, Bid, ICAD, PARP-related pathways
DNA damage characteristics	Single-strand DNA damage and oxidative stress-related injury	DNA fragmentation and CAD-mediated nucleic acid cleavage
Mitochondrial effect	Mainly ROS generation and functional disturbance	Mainly Bid–BAX/BAK-mediated mitochondrial outer membrane permeabilization
Inflammatory attribute	More prominent pro-inflammatory features	Mainly target cell apoptosis, but can also participate in tissue injury
Suitable detection indicators	GZMA, ROS, γH2AX, SET complex, Comet assay	GZMB, cleaved caspase-3, tBid, cleaved PARP, TUNEL
Common research scenarios	Chronic inflammation, autoimmunity, infection-related inflammation, tissue injury	Antiviral immunity, antitumor immunity, CTL/NK cell killing, transplant rejection

2、Granzyme B Signaling Pathway

2.1 Caspase-Dependent Apoptosis Axis

(1) Activation of executioner caspases

After entering target cells, GZMB can directly cleave and activate executioner caspases such as caspase-3 and caspase-7, and may also affect caspase-8-related pathways. Once executioner caspases are activated, target cells enter a typical apoptotic program, showing cell shrinkage, nuclear condensation, cytoskeletal disassembly, and apoptotic body formation.

(2) PARP cleavage and DNA fragmentation

The GZMB–caspase axis can promote PARP cleavage, weaken DNA repair capacity, and release CAD nuclease through ICAD cleavage, thereby inducing DNA fragmentation. TUNEL positivity, DNA laddering, γH2AX changes, and increased cleaved PARP can all serve as supporting evidence of GZMB-related apoptosis.

(3) Result interpretation

If GZMB is increased together with cleaved caspase-3, cleaved PARP, Annexin V-positive cells, and enhanced TUNEL signal, and if these changes are reduced by a GZMB inhibitor or perforin inhibitor, this more strongly supports target cell apoptosis mediated by the perforin–GZMB axis. If only GZMB expression is increased while death endpoints are not obvious, degranulation and target cell sensitivity should be further analyzed.

2.2 Bid–Mitochondrial Apoptosis Amplification

(1) Bid cleavage

GZMB can cleave the BH3-only protein Bid to generate truncated Bid (tBid). After translocating to mitochondria, tBid promotes BAX/BAK-mediated mitochondrial outer membrane permeabilization.

(2) Cytochrome c release

After mitochondrial outer membrane permeabilization, cytochrome c is released into the cytoplasm, promoting apoptosome formation and further activating caspase-9 and caspase-3. This step means that GZMB-induced death does not depend only on direct caspase cleavage but can also amplify apoptotic signaling through the mitochondrial pathway.

(3) Influence of anti-apoptotic proteins

Anti-apoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1 can reduce target cell sensitivity to GZMB. If tumor cells or target cells in a chronic inflammatory context upregulate these proteins, increased GZMB expression may be accompanied by insufficient killing efficiency.

2.3 Extracellular GZMB and Tissue Remodeling

(1) Extracellular substrates

GZMB can also exist in the extracellular microenvironment and cleave some extracellular matrix proteins, junction proteins, and barrier structure-related molecules. This effect can alter tissue structure, cell adhesion, and local permeability.

(2) Pathological scenarios

In chronic inflammation, skin injury, pulmonary inflammation, vascular inflammation, and tumor microenvironments, extracellular GZMB may mainly participate in matrix remodeling, barrier disruption, and inflammation maintenance, rather than directly inducing target cell apoptosis.

(3) Detection focus

If GZMB is increased in tissues but evidence from cleaved caspase-3, TUNEL, or Annexin V is insufficient, extracellular GZMB effects should be considered. In this case, indicators related to matrix degradation, collagen structure, vascular permeability, and inflammatory cell infiltration can be added.

3、Granzyme A Signaling Pathway

3.1 Non-Caspase-Dependent Death

(1) Mode of death

GZMA-induced cell death usually does not rely primarily on the classical caspase cascade. Even with broad-spectrum caspase inhibitors, GZMA may still cause DNA damage, mitochondrial dysfunction, nuclear structural disruption, and cell death.

(2) Difference from GZMB

GZMB-related death is more likely to show increased cleaved caspase-3, PARP cleavage, and DNA fragmentation. GZMA-related injury more often shows increased ROS, single-strand DNA damage, γH2AX changes, nuclear protein complex disruption, and inflammatory mediator release.

(3) Detection boundary

Only detecting cleaved caspase-3 or Annexin V can easily underestimate GZMA-related injury. GZMA studies should include ROS, MitoSOX, γH2AX, Comet assay, SET complex cleavage, and residual death analysis after caspase inhibitor intervention.

3.2 SET Complex and Nuclear Structural Disruption

(1) Composition of the SET complex

The SET complex contains proteins such as SET, Ape1, HMGB2, and pp32, which are associated with chromatin stability, DNA repair, and maintenance of nuclear structure. This complex is one of the important action directions distinguishing GZMA from GZMB.

(2) Nuclear injury mechanism

After entering target cells, GZMA can cleave members of the SET complex, reducing DNA repair capacity and promoting single-strand DNA damage and nuclear structural instability. This type of injury does not fully depend on caspases and does not necessarily form typical apoptotic DNA fragmentation.

(3) Experimental validation

If GZMA knockdown, GZMA functional inhibition, or antioxidant treatment reduces SET complex cleavage, γH2AX signal, Comet assay positivity, and inflammatory cytokine release, this supports the involvement of GZMA in atypical cytotoxic injury.

3.3 ROS and Inflammatory Amplification

(1) ROS generation

GZMA can induce increases in mitochondrial ROS and total cellular ROS. ROS can further damage DNA, proteins, and lipids, and form an amplification effect together with nuclear structural disruption.

(2) Inflammatory mediator release

GZMA can promote the production of inflammatory mediators by monocytes/macrophages, epithelial cells, or other target cells, and jointly maintain the inflammatory microenvironment with TNF-α, IL-1β, IL-6, and chemokine networks.

(3) Pathological significance

In autoimmunity, chronic inflammation, and infection-related tissue injury, increased GZMA should usually not be interpreted only as “enhanced cytotoxic killing.” It more likely indicates active inflammatory cytotoxic responses, local tissue injury, and immunopathological processes.

4、Immune Cell Sources and Disease Scenarios

4.1 CD8⁺ T Cells and NK Cells

(1) CD8⁺ T cells

After recognizing peptide–MHC I complexes through TCR, CD8⁺ cytotoxic T cells can release GZMA, GZMB, and PRF1. If GZMB, PRF1, IFN-γ, CD107a, and target cell apoptosis all increase simultaneously, this more strongly supports an effective antigen-specific cytotoxic response.

(2) NK cells

NK cells recognize abnormal cells through a balance between activating and inhibitory receptors, without relying on TCR-specific antigen recognition. GZMB in NK cells is often used to evaluate rapid killing capacity, whereas GZMA can help reflect an inflammatory cytotoxic state.

(3) Source discrimination

GZMA/GZMB positivity in tissue samples does not directly indicate that the source is CD8⁺ T cells. Spatial localization or flow cytometric subgrouping should be performed together with markers such as CD8, CD56, NKp46, NKG2D, CD3, and PRF1.

4.2 Antiviral and Antitumor Immunity

(1) Antiviral immunity

During viral infection, GZMB-mediated target cell apoptosis helps limit viral replication. If GZMB and PRF1 increase while viral load decreases and apoptosis of infected cells increases, this more strongly supports effective cytotoxic clearance.

(2) Tumor immunity

In tumor tissues, increased GZMB⁺CD8⁺ T cells or GZMB⁺NK cells usually suggests enhanced cytotoxic immune infiltration. Whether effective killing occurs still requires analysis of tumor cell apoptosis, spatial proximity, T cell exhaustion markers, and the immunosuppressive microenvironment.

(3) Immunotherapy research

After immunotherapy, if CD8⁺ T cell infiltration, GZMB, PRF1, IFN-γ, and TUNEL signals increase together and tumor burden decreases, this more strongly supports therapy-induced effective cytotoxic responses. If GZMB increases but tumors are not controlled, immune exhaustion, impaired killing access, or enhanced anti-apoptotic capacity of target cells should be considered.

4.3 Autoimmunity, Chronic Inflammation, and Transplant Rejection

(1) Autoimmunity

In autoimmune diseases, increased GZMA/GZMB may be associated with tissue cell injury, inflammatory cytokine release, and immune cell infiltration. GZMA is more suitable for explaining inflammatory amplification and atypical injury, whereas GZMB is more suitable for explaining cell apoptosis and tissue barrier disruption.

(2) Chronic inflammation

In chronic inflammation, persistently increased granzymes do not necessarily represent enhanced protective immunity. Instead, they may indicate long-term activation of cytotoxic responses, matrix remodeling, and sustained tissue injury. In this context, TNF-α, IL-6, IL-1β, MMPs, ECM injury, and histopathological scores should be analyzed together.

(3) Transplant rejection

In transplant rejection, increased GZMB often indicates active cytotoxic rejection, especially when it appears together with CD8⁺ T cell infiltration, PRF1 expression, and target cell apoptosis. Increased GZMA may indicate inflammatory cytotoxic responses and amplification of local tissue injury.

5、Experimental Detection Strategies

5.1 Expression, Release, and Activity Detection

(1) Expression detection

qPCR, Western blot, ELISA, flow cytometry, immunofluorescence, and immunohistochemistry can be used to detect GZMA/GZMB expression. Increased mRNA or protein only indicates increased effector molecule reserves and does not directly prove granule release or target cell death.

(2) Release detection

CD107a externalization is a commonly used indicator of cytotoxic granule release. If high GZMA/GZMB expression is accompanied by increased CD107a, this more strongly supports that effector cells are in a degranulated state.

(3) Activity detection

Granzyme activity detection is closer to functional status than expression detection alone. GZMB can be analyzed using specific substrates, caspase activation, and PARP cleavage. GZMA can be analyzed using substrate cleavage, ROS, SET complex cleavage, and DNA damage.

5.2 Target Cell Death Detection

(1) GZMB direction

For the GZMB pathway, detection should focus on cleaved caspase-3, cleaved caspase-7, cleaved PARP, tBid, mitochondrial membrane potential, Annexin V/PI, and TUNEL. Only when multiple indicators are consistent can GZMB-mediated apoptotic killing be more reliably supported.

(2) GZMA direction

For the GZMA pathway, detection should focus on ROS, MitoSOX, γH2AX, Comet assay, SET complex-related proteins, DNA repair proteins, and residual death after caspase inhibitor intervention. GZMA studies should not rely only on apoptosis indicators.

(3) Co-culture endpoints

When CTLs or NK cells are co-cultured with target cells, the effector-to-target ratio, co-culture time, target cell labeling method, effector cell purity, and death readout should be clearly defined. If a GZMB inhibitor reduces target cell apoptosis, or a perforin inhibitor reduces GZMA/GZMB-related injury, causal evidence is strengthened.

6、Reagents and Detection Tools Related to the Granzyme A/B Signaling Pathway

Table 2 Key Reagents and Detection Tools for the Granzyme A/B Signaling Pathway

Cat. No.	Product Name	Grade/Specification	Product category	Corresponding pathway link	Application positioning
A276505	Ac-IEPD-CHO	≥95%	Granzyme B/caspase-8 inhibitor	GZMB-mediated apoptotic signaling	Used to validate GZMB- and caspase-8-related death pathways; suitable for studies of Granzyme B-induced apoptosis and caspase-dependent mechanisms
A275444	Z-AAD-CH2Cl	≥95%	Granzyme B inhibitor	GZMB functional blockade	Used to inhibit GZMB activity and verify whether target cell apoptosis, PARP cleavage, caspase activation, and related effects depend on Granzyme B
EJ1515114	Human Granzyme A (Gzms-A) ELISA Kit	BioReagent	ELISA kit	GZMA expression/secretion detection	Used for quantitative detection of GZMA in human serum, cell culture supernatants, or tissue extracts; suitable for cytotoxic immunity and inflammatory injury studies
EJ1515115	Human Granzyme B (Gzms-B) ELISA Kit	BioReagent	ELISA kit	GZMB expression/secretion detection	Used for quantitative detection of GZMB in human-derived samples; suitable for evaluating CTL/NK cell killing activation and target cell apoptosis-related immune responses
EJ1513358	Mouse Granzyme A (Gzms-A) ELISA Kit	BioReagent	ELISA kit	GZMA expression/secretion detection	Used to detect GZMA levels in mouse infection, tumor, autoimmune, or inflammation models
EJ1513359	Mouse Granzyme B (Gzms-B) ELISA Kit	BioReagent	ELISA kit	GZMB expression/secretion detection	Used for GZMB quantification in mouse models; suitable for analyzing cytotoxic effects of CD8⁺ T cells and NK cells
Ab106347	Recombinant Granzyme B Antibody	ExactAb™, Validated, Carrier Free, Recombinant, 0.3 mg/mL	Granzyme B antibody	GZMB protein detection	Used for Western blot, immunofluorescence, immunohistochemistry, or flow-related detection to analyze GZMB expression and cytotoxic effector cell infiltration
rp146632	Recombinant Human Granzyme A Protein	Animal Free,Carrier Free,Bioactive,ActiBioPure™,His Tag,≥95%(SDS-PAGE)	Recombinant Granzyme A protein	GZMA functional research	Used for studies of GZMA substrate cleavage, non-caspase-dependent death, ROS, and inflammatory regulation mechanisms
rp181293	Recombinant Human Granzyme B Protein	Animal Free,Carrier Free,Bioactive,ActiBioPure™,His Tag,PBS Only,≥95%(SDS-PAGE)	Recombinant Granzyme B protein	GZMB functional research	Used for mechanistic experiments on GZMB-induced target cell apoptosis, caspase activation, Bid cleavage, and PARP cleavage
rp330176	Recombinant Mouse GZMA Protein	≥90%(SDS-PAGE)	Recombinant Granzyme A protein	Mouse GZMA functional research	Suitable for studying GZMA-related cytotoxic effects, inflammatory injury, and atypical death mechanisms in mouse systems
G1489100	GZMA Human Pre-designed siRNA Set A		siRNA	GZMA gene silencing	Used to knock down GZMA and validate its role in cytotoxic effects, inflammatory amplification, or non-caspase-dependent death
G1486046	GZMB Human Pre-designed siRNA Set A		siRNA	GZMB gene silencing	Used to knock down GZMB and validate GZMB-mediated caspase activation, mitochondrial apoptosis, and target cell killing effects
EJ1514480	Human Perforin 1 (PF1) ELISA Kit	BioReagent	ELISA kit	Perforin–granzyme entry pathway	Used to detect perforin levels in human-derived samples and assist evaluation of the cytotoxic granule release system required for granzyme entry into target cells
EJ1512182	Rat Perforin 1 (PF1) ELISA Kit	BioReagent	ELISA kit	Perforin–granzyme entry pathway	Used to detect perforin levels in rat models; suitable for cytotoxic immunity and tissue injury studies
EJ1512986	Mouse Perforin 1 (PF1) ELISA Kit	BioReagent	ELISA kit	Perforin–granzyme entry pathway	Used for perforin quantification in mouse infection, tumor, and transplant rejection models; helps explain GZMA/GZMB effector release and entry processes
EJ1515199	Monkey Perforin 1 (PF1) ELISA Kit	BioReagent	ELISA kit	Perforin–granzyme entry pathway	Used for perforin detection in monkey-derived samples; suitable for non-human primate immunotoxicology or cytotoxic immunity studies
P1498390	Perforin-IN-2	Moligand™, 10 mM in DMSO	Perforin inhibitor	Granzyme entry into target cells	Used to block perforin function and verify whether GZMA/GZMB-mediated target cell injury depends on perforin-mediated entry
P1455516	Perforin-IN-2	≥99%	Perforin inhibitor	Granzyme entry into target cells	Used for inhibition of perforin-dependent cytotoxic pathways; suitable for functional validation in CTL/NK cell killing assays

7、Research Design Recommendations

7.1 Cell-Level Studies

(1) Model selection

Cell-level studies can use co-culture models of CTLs or NK cells with target cells. The effector-to-target ratio, co-culture time, target cell labeling method, effector cell purity, and stimulation conditions should be clearly defined.

(2) GZMB direction

For GZMB-oriented studies, the focus should be on caspase activation, Bid cleavage, PARP cleavage, decreased mitochondrial membrane potential, Annexin V positivity, and TUNEL signal. If these indicators decrease after GZMB inhibition, apoptotic killing is more likely to depend on GZMB.

(3) GZMA direction

For GZMA-oriented studies, the focus should be on ROS generation, SET complex disruption, DNA damage, γH2AX changes, and caspase-independent death. If GZMA knockdown reduces inflammatory cytokines, ROS, or DNA damage, it has mechanistic interpretive value even when cleaved caspase-3 changes are not obvious.

7.2 Tissue Samples and Disease Model Studies

(1) Spatial localization

In tissue sample studies, GZMA/GZMB should be spatially correlated with CD8, CD56, NKp46, PRF1, TUNEL, or cleaved caspase-3 to distinguish cytotoxic cell infiltration, granule release, and target cell death.

(2) Disease endpoints

Antiviral and antitumor studies should combine viral load, tumor burden, cytotoxic cell infiltration, and target cell apoptosis. Autoimmune and chronic inflammation studies should combine tissue injury scores, inflammatory cytokines, barrier disruption, and oxidative stress indicators.

(3) Comprehensive interpretation

Increased granzymes cannot be directly equated with effective immune killing. Conclusions about the Granzyme A/B pathway are more reliable only when a consistent evidence chain is established among effector cell source, granule release, perforin cooperation, granzyme activity, target cell death, and disease endpoints.

Granzyme A and Granzyme B together form an important effector axis of the perforin–granzyme system, but the cytotoxic mechanisms represented by the two are different. GZMB focuses on caspase activation, the Bid–mitochondrial pathway, and apoptotic clearance. GZMA focuses on SET complex disruption, ROS generation, non-caspase injury, and inflammatory amplification.

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