GZMK Human

What is Granzyme K (GZMK) and how does it differ from other granzymes in humans?

Granzyme K is a cytotoxic effector molecule expressed by both innate and adaptive immune cells. Unlike Granzyme B (GzmB), which has been extensively studied, GZMK has distinct functions and expression patterns. While both are cytotoxic molecules capable of killing virally infected and tumor cells, GZMK is predominantly expressed by innate-like lymphocytes and specific T cell subsets .

GZMK differs from GzmB in several key ways:

• GZMK responds primarily to cytokine stimulation, whereas GzmB is strongly upregulated by TCR stimulation

• GZMK expression is often mutually exclusive with IL-17a production

• GZMK+ cells have reduced IFN-γ production compared to GzmB+ cells

• Recent research has identified GZMK as a major driver of tissue damage and inflammation

Methodologically, researchers should account for these differences when designing functional assays, as the stimulation conditions optimal for studying GZMK may differ from those traditionally used for GzmB studies.

Which human immune cell populations predominantly express GZMK?

GZMK is predominantly expressed by innate-like lymphocytes in humans. Specifically, research has identified several key cell populations that express GZMK :

• Innate-like lymphocytes, including NK cells

• A newly identified population of GzmK+CD8+ non-mucosal-associated invariant T cells with innate-like characteristics

• Tissue-enriched CD8 T cells (termed "T teK CD8 cells")

• Memory T cell subsets with innate-like features

Notably, single-cell RNA sequencing and flow cytometric analyses have shown that GZMK expression defines a transcriptionally distinct T cell subset compared to GzmB-expressing cells . These GZMK+ T cells form a core population of tissue-associated T cells across multiple human tissues and disease states, including rheumatoid arthritis synovium, gut, kidney, and COVID-19 bronchioalveolar lavage fluid .

What are the phenotypic markers of GZMK-expressing T cells in humans?

GZMK+ T cells in humans have a distinctive phenotypic profile that has been characterized through multiple approaches. Flow cytometry and transcriptomic analyses have identified the following characteristic markers :

• Surface marker profile: KLRG1+, IL-7R+, CD62L−

• Transcription factor profile: EOMES+, Tcf7int

• Functional markers: Reduced IFN-γ production compared to GzmB+ cells

These cells show features of central memory T and effector memory T cells. They are not naive T cells but represent an intermediate memory-like or preterminally differentiated population .

For researchers designing flow cytometry panels, it's important to note that GZMK+ cells show downregulation of naive markers like CD62L while maintaining expression of certain memory markers like IL-7R. They also express lower levels of effector markers like perforin compared to GzmB+ cells .

How does GZMK expression change during immune cell development and maturation?

GZMK expression follows a specific developmental pattern in humans. Research indicates that GZMK+ cells are absent or expressed at very low levels in cord blood, suggesting that GZMK expression is upregulated with immune experience rather than being constitutively expressed in naive cells .

The developmental trajectory appears to follow this pattern:

• Naive T cells: Low/absent GZMK expression

• Immune experience/activation: Upregulation of GZMK in specific subsets

• Terminal differentiation: Potential downregulation of GZMK with concomitant upregulation of GzmB

Methodologically, this developmental pattern suggests that researchers should consider using age-matched controls when studying GZMK expression and should interpret results in the context of the subject's immune history .

What is the relationship between GZMK and GZMB expression in human T cells?

GZMK and GZMB exhibit a complex, often inverse relationship in human T cells. Research has revealed several key aspects of this relationship :

• TCR stimulation downregulates GZMK expression while simultaneously upregulating GZMB expression

• GZMK+ cells respond primarily to cytokine stimuli alone, while GZMB expression is more strongly induced by TCR signaling

• Single-cell RNA sequencing has identified distinct transcriptional profiles:

  • A GZMK+ metacluster characterized by high GZMK with intermediate/low GZMB

  • A GZMB+ metacluster expressing GZMB largely without GZMK

  • A naive metacluster lacking both GZMK and GZMB

These patterns suggest that GZMK and GZMB may mark functionally distinct effector cell states, with GZMK potentially representing an intermediate memory-like state before terminal differentiation .

What experimental approaches are most effective for studying GZMK function in human immune cells?

For investigating GZMK function in human immune cells, several experimental approaches have proven particularly effective:

• Single-cell RNA sequencing: This approach has been instrumental in identifying distinct GZMK+ cell populations and their transcriptional profiles. Integration of scRNA-seq data from multiple tissue sources enables comprehensive characterization of GZMK+ cells across different contexts .

• Flow cytometry with polychromatic panels: Including markers for GZMK, GZMB, perforin, tissue residency markers (CD69, CD103), and lineage markers enables precise identification of GZMK+ subsets. Example panel:

  • Lineage: CD3, CD4, CD8, CD56

  • Effector molecules: GZMK, GZMB, Perforin

  • Memory/activation: CD45RA, CCR7, CD62L, KLRG1

  • Tissue residency: CD69, CD103, CD49a

• Functional cytokine stimulation assays: Since GZMK+ cells respond to cytokine stimuli rather than TCR stimulation, protocols using IL-12, IL-15, and IL-18 are particularly useful for studying their function .

• T cell receptor repertoire analysis: This approach helps track clonal expansion of GZMK+ cells across tissues and blood, revealing their migratory patterns and enrichment in inflammatory sites .

• Sorting-based transcriptional profiling: Low-input RNA-seq of sorted GZMK+ vs. GZMB+ populations enables detailed comparison of their molecular programs .

How do tissue-resident GZMK+ T cells differ from circulating GZMK+ T cells in terms of function and phenotype?

Tissue-resident and circulating GZMK+ T cells show both similarities and important differences:

Phenotypic differences:

• Tissue-resident GZMK+ cells show downregulation of S1PR1 and SELL (CD62L), genes that must be turned off to allow cells to remain in tissue

• Both subsets downregulate genes associated with memory precursor effector cells (MPECs) and central memory T cells, such as IL7R and TCF7, compared to cells expressing only GZMK

• Tissue-resident cells upregulate genes associated with effector T cells, including KLRG1 and ID2

Functional differences:

• Tissue-resident GZMK+ cells have been found to be major cytokine producers with low cytotoxic potential in inflammatory conditions like rheumatoid arthritis

• They are armed to produce cytokines in response to both antigen-dependent and -independent stimuli, giving them potential to drive inflammation

What are the mechanisms by which GZMK drives inflammatory pathology in human tissues?

Recent research has identified GZMK as a major driver of inflammatory pathology in human tissues. The mechanisms involve:

• Pro-inflammatory cytokine production: GZMK+ cells can produce IFN-γ and nearly as much TNF as CD4 T cells in inflammatory conditions like rheumatoid arthritis .

• Complement system activation: The most recent findings (2025) suggest that GZMK activates the complement system, a group of proteins that patrols the body for signs of infection or injury . This represents a novel mechanism of immune activation.

• Tissue enrichment: GZMK+ cells are enriched in nonlymphoid tissues such as the liver and adipose tissue, as well as in tumors and inflammatory sites .

• Cytokine responsiveness: GZMK+ cells respond to cytokine stimuli alone, which may enable them to participate in "bystander" inflammatory responses even in the absence of specific antigen recognition .

• Clonal expansion: In conditions like rheumatoid arthritis, GZMK+ CD8 T cells show clonal expansion in affected tissues, suggesting antigen-driven accumulation of these potentially pathogenic cells .

The tissue-damaging effects of GZMK appear to be distinct from the classical cytotoxic functions of granzymes like GZMB, representing a specialized inflammatory pathway .

How can researchers distinguish between the cytotoxic and inflammatory functions of GZMK in experimental models?

Distinguishing between the cytotoxic and inflammatory functions of GZMK requires carefully designed experimental approaches:

• Functional assays with readouts for both pathways:

  • Cytotoxicity: Standard 51Cr release assays or flow-based killing assays

  • Inflammation: Multiplex cytokine analysis, NF-κB activation assays, inflammasome activation

• Substrate specificity analysis: Determine whether GZMK is targeting classical cell death substrates versus inflammatory pathway components.

• Inhibitor studies: Use specific inhibitors of cell death pathways (caspase inhibitors) versus inflammatory pathways (NF-κB inhibitors) to dissect the dominant mechanism.

• Co-culture systems with pathway reporters: Design co-culture systems with reporter cells that can distinguish between cell death and inflammatory activation.

• Correlation analyses in clinical samples: Correlate GZMK expression with markers of tissue destruction versus markers of inflammation to identify dominant pathways.

What are the current technical challenges in studying GZMK+ cell populations in human clinical samples?

Researchers face several technical challenges when studying GZMK+ cell populations in human clinical samples:

• Tissue processing effects: Standard tissue processing methods may alter GZMK expression or selectively deplete GZMK+ populations. Optimized protocols for tissue disaggregation that preserve these cells are needed.

• Ex vivo stability: The stability of GZMK expression during ex vivo handling can affect results, particularly since TCR stimulation downregulates GZMK while upregulating GZMB .

• Antibody quality and specificity: The availability of high-quality, specific antibodies for GZMK detection in different applications (flow cytometry, immunohistochemistry, etc.) can be limited.

• Tissue heterogeneity: GZMK+ cells show tissue-specific enrichment patterns, making it difficult to generalize findings across different tissue types or disease states .

• Functional assessment: Since GZMK+ cells respond primarily to cytokine stimulation rather than TCR stimulation, traditional T cell functional assays may not optimally capture their activity .

• Low abundance in circulation: As GZMK+ cells are enriched in tissues, studying them in blood may underrepresent their true biology and requires specialized approaches to identify tissue-derived circulating populations .

Researchers should consider these challenges when designing studies and interpreting results from GZMK analyses in clinical samples.

What flow cytometry panels are recommended for optimal identification of GZMK+ immune cell subsets?

For comprehensive identification and characterization of GZMK+ immune cell subsets, the following flow cytometry panel design is recommended based on current research:

Basic Panel (10-12 colors):

• Lineage markers: CD3, CD4, CD8, CD56 (to identify T cell subsets and NK cells)

• Granzymes: GZMK, GZMB (critical for distinguishing subsets)

• Memory markers: CD45RA, CCR7 (to distinguish naive, memory, and effector populations)

• Innate-like T cell markers: KLRG1, CD161 (enriched on GZMK+ populations)

• Viability dye: To exclude dead cells

Extended Panel (16-20 colors):

• All markers from basic panel plus:

• Additional functional molecules: Perforin, IFN-γ, TNF

• Tissue residency markers: CD69, CD103, CD49a

• Transcription factors: Eomes, T-bet, TCF1

• Additional phenotypic markers: IL-7R, CD62L, PD-1, TIGIT

Gating strategy recommendations:

• Exclude doublets and dead cells

• Identify CD3+ lymphocytes

• Gate CD4+ and CD8+ T cell populations

• Within each subset, create a GZMK vs. GZMB plot to identify:

  • GZMK+GZMB- cells

  • GZMK+GZMB+ cells

  • GZMK-GZMB+ cells

  • GZMK-GZMB- cells

• Further characterize each population using memory, activation, and functional markers

This approach aligns with the methodology used in recent studies that identified GZMK+ cells as having a distinct KLRG1+EOMES+IL-7R+CD62L−Tcf7int phenotype .

How can single-cell RNA sequencing be optimized for studying GZMK expression in heterogeneous immune cell populations?

Optimizing single-cell RNA sequencing (scRNA-seq) for GZMK research requires specific considerations:

Sample preparation:

• Fresh samples are preferred to minimize ex vivo artifacts

• Consider gentle isolation protocols that preserve GZMK expression

• For tissues, optimize digestion protocols to maintain cell viability while ensuring complete dissociation

Sequencing considerations:

• Aim for sufficient sequencing depth (minimum 30,000-50,000 reads per cell) to capture GZMK and related genes that may have moderate expression levels

• Include adequate cell numbers (minimum 5,000-10,000 cells per condition) to capture less abundant GZMK+ populations

Analysis pipeline recommendations:

• Quality control: Apply stringent but appropriate QC to avoid removing GZMK+ populations that might have unique transcriptional features

• Dimensionality reduction: Use UMAP or t-SNE visualization to identify cell clusters

• Feature selection: Ensure GZMK and related genes (GZMB, PRF1, IFNG, etc.) are included in variable gene selection

• Metaclustering approach: Follow methods from recent studies that used hierarchical metaclustering of graph-based clusters to identify GZMK+ populations

• Integration methods: When working with multiple samples, use appropriate batch correction and integration methods (e.g., Harmony, Seurat integration)

Validation strategies:

• Confirm key findings with protein-level assays (flow cytometry, CyTOF)

• Use RNA velocity analysis to infer developmental trajectories between GZMK+ and other populations

• Consider spatial transcriptomics to validate tissue localization of GZMK+ cells

These approaches were successfully implemented in studies that identified distinct metaclusters corresponding to differential expression of GZMK and GZMB .

What are the best approaches for functional validation of GZMK activity in human tissues?

Functional validation of GZMK activity in human tissues requires multifaceted approaches:

Ex vivo tissue explant cultures:

• Culture fresh tissue explants with GZMK inhibitors versus controls

• Measure inflammatory mediators and tissue damage markers

• Utilize multiplex cytokine assays to capture the range of inflammatory responses

In situ detection methods:

• Multiplex immunofluorescence to co-localize GZMK with its substrates and downstream signaling molecules

• RNAscope to visualize GZMK transcripts alongside activity markers

• Proximity ligation assays to detect GZMK-substrate interactions in tissue sections

Functional assays with sorted cells:

• Isolate GZMK+ cells from tissues and assess:

  • Cytokine production capacity with/without stimulation

  • Cytotoxic potential against relevant target cells

  • Response to tissue-specific factors

Recombinant protein studies:

• Use purified recombinant GZMK to test direct effects on:

  • Primary tissue-derived cells

  • Extracellular matrix components

  • Complement system components

In vitro modeling:

• Develop 3D organoid systems with GZMK+ cells to model tissue interactions

• Use live cell imaging to track GZMK delivery and downstream effects

These approaches help distinguish direct GZMK-mediated effects from indirect consequences of GZMK+ cell activation, providing a comprehensive view of GZMK function in the tissue microenvironment.

What in vitro models best recapitulate GZMK-mediated inflammation for experimental studies?

Several in vitro models can effectively recapitulate GZMK-mediated inflammation:

Primary cell co-culture systems:

• Co-culture of sorted GZMK+ cells with tissue-specific cells (e.g., synoviocytes, epithelial cells)

• Include relevant cytokine milieu to maintain GZMK expression

• Monitor inflammatory readouts (cytokine production, tissue damage markers)

3D tissue models:

• Organoid systems incorporating both immune and tissue-specific cells

• Synovial organoids for studying rheumatoid arthritis

• Tumor spheroids with infiltrating immune cells for cancer studies

Microfluidic "organ-on-chip" platforms:

• Systems that incorporate flow and tissue architecture

• Allow for spatial organization of immune and tissue cells

• Enable real-time monitoring of cellular interactions

Ex vivo tissue explant cultures:

• Short-term culture of tissue biopsies preserving the native microenvironment

• Can be manipulated with GZMK inhibitors or depleted of GZMK+ cells

• Particularly useful for studying rheumatoid arthritis synovium, where GZMK+ cells form a core population

Specialized stimulation protocols:

• Use cytokine stimulation rather than TCR stimulation to activate GZMK+ cells

• Combinations of IL-12, IL-15, and IL-18 have been shown to be effective

The optimal model depends on the specific research question, with consideration given to maintaining physiological GZMK expression levels and the relevant tissue microenvironment.

How should researchers account for tissue-specific differences in GZMK expression when designing experiments?

Accounting for tissue-specific differences in GZMK expression is crucial for robust experimental design:

Tissue-specific sampling strategies:

• Sample multiple regions within tissues to account for spatial heterogeneity

• Include paired blood samples to compare tissue-enriched versus circulating GZMK+ populations

• Consider time-of-day effects on GZMK expression, especially in tissues with circadian regulation

Control selection:

• Use tissue-matched controls rather than peripheral blood when studying tissue GZMK+ cells

• In disease states, include both healthy tissue controls and disease-affected tissues from different sites

Analytical approaches:

• Perform hierarchical analyses that first identify tissue-specific signatures before focusing on GZMK+ populations

• Use tissue-specific reference transcriptomes when analyzing RNA-seq data

• Apply tissue-specific normalization strategies

Experimental conditions:

• Optimize tissue processing protocols for each tissue type to preserve GZMK expression

• Adjust ex vivo culture conditions to maintain tissue-specific GZMK expression patterns

• Consider tissue-specific cytokine environments when stimulating cells

Validation across tissues:

• Validate findings in multiple tissue types before generalizing

• Identify tissue-invariant versus tissue-specific aspects of GZMK biology

• Use multimodal approaches (protein, RNA, functional) to confirm findings

Research has shown that GZMK+ cells are enriched in nonlymphoid tissues such as the liver and adipose tissue, as well as in tumors and sites of inflammation . They form a core population of tissue-associated T cells across diseases and human tissues, suggesting both common features and tissue-specific adaptations .

How does GZMK expression in tumors correlate with patient prognosis across different cancer types?

GZMK expression in tumors has emerged as a significant prognostic indicator in several cancer types:

Colorectal cancer:

• GZMK+ cells are enriched in colorectal tumors and can produce IFN-γ

• GZMK expression is mutually exclusive with IL-17a production

• Recent studies have associated GZMK expression with improved prognosis in solid tumors

General observations across tumor types:

• GZMK+ cells represent a distinct population from GzmB+ cells in the tumor microenvironment

• They have characteristics of tissue-resident memory-like cells that respond to cytokine stimulation

• Their prognostic significance may vary by cancer type, potentially reflecting different immune contextures

Methodological considerations for clinical studies:

• Stratify analyses by tumor type and stage

• Account for treatment history when assessing GZMK's prognostic value

• Consider spatial distribution within the tumor (invasive margin vs. tumor core)

• Evaluate GZMK in context with other immune markers (CD8, PD-1, etc.)

Potential mechanisms underlying prognostic associations:

• GZMK+ cells may represent effective anti-tumor responses due to their cytokine production capabilities

• They might indicate a favorable immunogenic tumor environment

• Their presence could reflect specific antigen-driven responses against tumor-associated antigens

These findings suggest that GZMK+ cells may be important effector cells in the tumor microenvironment, with potential implications for immunotherapy response and patient outcomes .

What is the role of GZMK+ cells in rheumatoid arthritis and other inflammatory conditions?

GZMK+ cells play a significant role in rheumatoid arthritis (RA) and potentially other inflammatory conditions:

In rheumatoid arthritis:

• CD8 T cells expressing GZMK are strikingly abundant in synovium

• They produce substantial amounts of IFN-γ and nearly as much TNF as CD4 T cells

• The vast majority of synovial tissue and synovial fluid CD8 T cells belong to a GZMK+GzmB− population

• These cells are functionally characterized as major cytokine producers with low cytotoxic potential

• They are clonally expanded in synovial tissues, suggesting antigen-driven responses

Functional characteristics in inflammatory settings:

• GZMK+ cells respond to both antigen-dependent and -independent stimuli

• They have the potential to drive inflammation through cytokine production

• They form a core population of tissue-associated T cells across different inflammatory diseases

Presence in other inflammatory conditions:

• GZMK-expressing CD8 T cells are a major CD8 T cell population in the gut and kidney

• They are also abundant in COVID-19 bronchioalveolar lavage fluid

• Recent research identifies GZMK as a major driver of inflammatory pathology through activation of the complement system

This evidence suggests that GZMK+ cells represent a specialized subset of effector T cells that contribute to tissue inflammation across multiple disease states, with particularly well-characterized roles in rheumatoid arthritis.

How can GZMK expression be used as a biomarker for immune responses in clinical trials?

GZMK expression holds promise as a biomarker for immune responses in clinical trials:

Potential applications as a biomarker:

• Treatment response prediction: Baseline GZMK levels might predict response to immunotherapies or anti-inflammatory treatments

• Pharmacodynamic marker: Changes in GZMK+ cell frequency or function could indicate biological activity of immune-modulating drugs

• Tissue inflammation indicator: GZMK expression in tissue biopsies may serve as a marker of ongoing inflammatory responses

• Monitoring immune competence: GZMK responses to stimulation could assess functional immune capacity

Methodological recommendations for clinical trials:

• Standardized assessment protocols:

  • Flow cytometry panels including GZMK, GZMB, and relevant functional markers

  • Standardized stimulation conditions (cytokine vs. TCR stimulation)

  • Assay validation across multiple clinical sites

• Sampling considerations:

  • Include paired blood and tissue samples when possible

  • Consider longitudinal sampling to capture dynamic changes

  • Account for tissue heterogeneity in biopsy-based analyses

• Analysis approaches:

  • Assess both frequency of GZMK+ cells and per-cell expression levels

  • Evaluate GZMK in conjunction with other inflammatory markers

  • Consider GZMK:GZMB ratio as a potential indicator of inflammatory vs. cytotoxic balance

• Context-specific interpretation:

  • Interpret GZMK changes in the context of the specific disease and intervention

  • Account for baseline variation in GZMK expression across individuals

  • Consider tissue-specific reference ranges

The unique characteristics of GZMK+ cells—their tissue enrichment, responsiveness to cytokines, and association with inflammatory conditions—make GZMK expression a potentially valuable biomarker, particularly in diseases where tissue inflammation is a key component .

What are the implications of GZMK expression for immunotherapy response in cancer patients?

GZMK expression may have significant implications for immunotherapy response in cancer patients:

Potential impact on checkpoint inhibitor therapy:

• GZMK+ cells represent a distinct effector population that may respond differently to checkpoint blockade than GzmB+ cytotoxic cells

• Their cytokine-producing capacity could contribute to inflammatory responses after checkpoint inhibition

• Pre-existing GZMK+ populations in tumors might indicate an immune-responsive tumor microenvironment

Considerations for cell-based therapies:

• GZMK expression patterns could inform optimal manufacturing conditions for adoptive cell therapies

• Engineering GZMK expression in therapeutic T cells might confer specific functional advantages

• GZMK+ CAR-T cells might have distinct persistence and activity profiles compared to conventional designs

Monitoring considerations:

• Changes in circulating and tumor-infiltrating GZMK+ cells could serve as pharmacodynamic markers of immunotherapy effect

• The ratio of GZMK to GZMB expression might indicate shifts between inflammatory and cytotoxic immune responses

• Spatial distribution of GZMK+ cells in relation to tumor cells and other immune populations could predict response patterns

Research directions to establish clinical relevance:

• Correlative studies of baseline GZMK expression with clinical outcomes in immunotherapy trials

• Investigation of changes in GZMK+ populations during successful versus failed immunotherapy

• Mechanistic studies of how GZMK+ cells interact with other immune populations in the tumor microenvironment

Given that GZMK has been associated with improved prognosis in solid tumors and that GZMK+ cells are enriched in colorectal tumors , understanding their role in immunotherapy response represents an important research direction with potential clinical implications.

How do therapeutic interventions targeting inflammatory pathways affect GZMK+ cell populations?

The impact of anti-inflammatory therapies on GZMK+ cell populations remains an area of active investigation:

Potential effects of current therapies:

• TNF inhibitors: May indirectly affect GZMK+ cells by altering the inflammatory microenvironment that supports their maintenance

• JAK inhibitors: Could directly impact GZMK+ cells by interfering with cytokine signaling pathways that drive their activation

• Corticosteroids: Likely to suppress GZMK+ cell function along with other inflammatory cell types

• Targeted biologics: Effects may vary depending on the specific pathway targeted

Monitoring considerations for clinical studies:

• Track both frequency and functional capacity of GZMK+ cells during treatment

• Assess tissue-resident versus circulating GZMK+ populations, as they may respond differently

• Evaluate changes in GZMK:GZMB ratio as a potential indicator of shifting immune profiles

Research questions for therapeutic development:

• Could selective targeting of GZMK+ cells provide therapeutic benefit in inflammatory diseases?

• Might GZMK itself represent a therapeutic target, given its recently discovered role in driving inflammatory pathology ?

• How do GZMK+ cells contribute to treatment resistance or disease recurrence?

Experimental approaches to address these questions:

• Ex vivo treatment of patient samples with candidate therapeutics to assess effects on GZMK+ cells

• Animal models with human immune system components to evaluate in vivo responses

• Clinical trials with detailed immune monitoring focused on GZMK+ populations

As GZMK is now recognized as a major driver of inflammatory pathology , understanding how therapeutic interventions affect GZMK+ cell populations may lead to more targeted and effective treatment strategies for inflammatory conditions.