CD8+ T Cells Promote Pathological Angiogenesis in Ocular Neovascular Disease.

BACKGROUND: CD4+ (cluster of differentation) and CD8+ T cells are increased in the ocular fluids of patients with neovascular retinopathy, yet their role in the disease process is unknown. METHODS: We describe how CD8+ T cells migrate into the retina and contribute to pathological angiogenesis by releasing cytokines and cytotoxic factors. RESULTS: In oxygen-induced retinopathy, flow cytometry revealed the numbers of CD4+ and CD8+ T cells were increased in blood, lymphoid organs, and retina throughout the development of neovascular retinopathy. Interestingly, the depletion of CD8+ T cells but not CD4+ T cells reduced retinal neovascularization and vascular leakage. Using reporter mice expressing gfp (green fluorescence protein) in CD8+ T cells, these cells were localized near neovascular tufts in the retina, confirming that CD8+ T cells contribute to the disease. Furthermore, the adoptive transfer of CD8+ T cells deficient in TNF (tumor necrosis factor), IFNγ (interferon gamma), Prf (perforin), or GzmA/B (granzymes A/B) into immunocompetent Rag1-/- mice revealed that CD8+ T cells mediate retinal vascular disease via these factors, with TNF influencing all aspects of vascular pathology. The pathway by which CD8+ T cells migrate into the retina was identified as CXCR3 (C-X-C motif chemokine receptor 3) with the CXCR3 blockade reducing the number of CD8+ T cells within the retina and retinal vascular disease. CONCLUSIONS: We discovered that CXCR3 is central to the migration of CD8+ T cells into the retina as the CXCR3 blockade reduced the number of CD8+ T cells within the retina and vasculopathy. This research identified an unappreciated role for CD8+ T cells in retinal inflammation and vascular disease. Reducing CD8+ T cells via their inflammatory and recruitment pathways is a potential treatment for neovascular retinopathies.

Detection of Granzyme B-associated Binding Targets in Peripheral Blood Samples of Hosts in Sickness and in Health Using a Granzyme B-like Peptide Fluorescent Conjugate (GP1R).

Granzyme B, mostly expressed by cytotoxic T lymphocytes in the fight against cancer and infection, is known to induce cell death based on its active enzymatic activity as a serine protease. Recent studies showed cytotoxicity of a non-enzymatic granzyme B-like peptide (also referred to as granzyme B-associated peptide or GP1 in this report) in tumor cells and presence of binding targets for GP1R (i.e., GP1 conjugated with rhodamine fluorochrome) in tumor cells, bacteria, and circulating platelets/neutrophils of healthy hosts. But there were no data on "sick" hosts to help substantiate any potential GP1 based medical applications. Thus, we adopted similar GP1R binding protocols to further study binding of GP1 in different biological samples (including different blood samples of hosts in sickness and in health, cancer cell lines, and trigeminal ganglia culture of infected hosts treated with and without GP1) and determine if any binding patterns might have any associations with different health conditions. The overall preliminary results appear to show certain GP1R + binding patterns in certain blood components (especially neutrophils) have potential correlations with certain health conditions of hosts at sampling times, indicating potential GP1R applications for diagnostic purposes. Findings of different GP1R binding patterns in different cancer cell lines, whole blood samples and trigeminal ganglia culture of experimental mice infected with HSV-1 virus (might cause neuropathy) within a week post-infection, and blood samples of GP1-treated mouse survivors on day 21 post-infection provided preliminary evidence of potential GP1-led tumor cell-specific cell death and treatment efficacy for greater survival.

Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Noncytotoxic Roles of Granzymes in Health and Disease.

Granzymes are serine proteases previously believed to play exclusive and somewhat redundant roles in lymphocyte-mediated target cell death. However, recent studies have challenged this paradigm. Distinct substrate profiles and functions have since emerged for each granzyme while their dysregulated proteolytic activities have been linked to diverse pathologies.

Relevance of tissue-resident memory CD8 T cells in the onset of Parkinson's disease and examination of its possible etiologies: infectious or autoimmune?

Tissue-resident memory CD8 T cells are responsible for local immune surveillance in different tissues, including the brain. They constitute the first line of defense against pathogens and cancer cells and play a role in autoimmunity. A recently published study demonstrated that CD8 T cells with markers of residency containing distinct granzymes and interferon-γ infiltrate the parenchyma of the substantia nigra and contact dopaminergic neurons in an early premotor stage of Parkinson's disease. This infiltration precedes α-synuclein aggregation and neuronal loss in the substantia nigra, suggesting a relevant role for CD8 T cells in the onset of the disease. To date, the nature of the antigen that initiates the adaptive immune response remains unknown. This review will discuss the role of tissue-resident memory CD8 T cells in brain immune homeostasis and in the onset of Parkinson's disease and other neurological diseases. We also discuss how aging and genetic factors can affect the CD8 T cell immune response and how animal models can be misleading when studying human-related immune response. Finally, we speculate about a possible infectious or autoimmune origin of Parkinson's disease.

Super-killer CTLs are generated by single gene deletion of Bach2.

Bach2 codes for a transcriptional regulator exerting major influences on T cell-mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8+ T cells showed increased proliferative and cytolytic capacity in the absence of BACH2. Before activation, BACH2-deficient splenic CD8+ T cells had a higher abundance of memory and reduced abundance of naïve cells compared to wild-type. CTLs derived from central memory T cells were more potently cytotoxic than those derived from naïve T cells, but even within separated subsets, BACH2-deficiency conferred a cytotoxic advantage. Immunofluorescence and electron microscopy revealed larger granules in BACH2-deficient compared to wild-type CTLs, and proteomic analysis showed an increase in granule content, including perforin and granzymes. Thus, the enhanced cytotoxicity observed in effector CTLs lacking BACH2 arises not only from differences in their initial differentiation state but also inherent production of enlarged cytolytic granules. These results demonstrate how a single gene deletion can produce a CTL super-killer.

Granzymes-Their Role in Colorectal Cancer.

Colorectal cancer (CRC) is among the most common malignancies worldwide. CRC is considered a heterogeneous disease due to various clinical symptoms, biological behaviours, and a variety of mutations. A number of studies demonstrate that as many as 50% of CRC patients have distant metastases at the time of diagnosis. However, despite the fact that social and medical awareness of CRC has increased in recent years and screening programmes have expanded, there is still an urgent need to find new diagnostic tools for early detection of CRC. The effectiveness of the currently used classical tumour markers in CRC diagnostics is very limited. Therefore, new proteins that play an important role in the formation and progression of CRC are being sought. A number of recent studies show the potential significance of granzymes (GZMs) in carcinogenesis. These proteins are released by cytotoxic lymphocytes, which protect the body against viral infection as well specific signalling pathways that ultimately lead to cell death. Some studies suggest a link between GZMs, particularly the expression of Granzyme A, and inflammation. This paper summarises the role of GZMs in CRC pathogenesis through their involvement in the inflammatory process. Therefore, it seems that GZMs could become the focus of research into new CRC biomarkers.

Effective Natural Killer Cell Degranulation Is an Essential Key in COVID-19 Evolution.

NK degranulation plays an important role in the cytotoxic activity of innate immunity in the clearance of intracellular infections and is an important factor in the outcome of the disease. This work has studied NK degranulation and innate immunological profiles and functionalities in COVID-19 patients and its association with the severity of the disease. A prospective observational study with 99 COVID-19 patients was conducted. Patients were grouped according to hospital requirements and severity. Innate immune cell subpopulations and functionalities were analyzed. The profile and functionality of innate immune cells differ between healthy controls and severe patients; CD56dim NK cells increased and MAIT cells and NK degranulation rates decreased in the COVID-19 subjects. Higher degranulation rates were observed in the non-severe patients and in the healthy controls compared to the severe patients. Benign forms of the disease had a higher granzymeA/granzymeB ratio than complex forms. In a multivariate analysis, the degranulation capacity resulted in a protective factor against severe forms of the disease (OR: 0.86), whereas the permanent expression of NKG2D in NKT cells was an independent risk factor (OR: 3.81; AUC: 0.84). In conclusion, a prompt and efficient degranulation functionality in the early stages of infection could be used as a tool to identify patients who will have a better evolution.

Intracellular expression of granzymes A, B, K and M in blood lymphocyte subsets of critically ill patients with or without sepsis.

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

Granzyme family acts as a predict biomarker in cutaneous melanoma and indicates more benefit from anti-PD-1 immunotherapy.

The incidence of cutaneous melanoma (CM) increased since the 1970s, and also along with an unfavorable prognosis. CM patients have been verified benefits from immunotherapy, and granzymes (GZMs) comprise more than 90% of the cytolytic granules secreted by cytotoxic T lymphocytes and nature killer cell. Therefore, it is essential to evaluate the prognostic value of GZMs in CM. A total of 633 CM patients was enrolled to access the prognostic value of GZMs. The integrated prognostic value of five GZMs was validated in TCGA-SKCM, GSE65904, GSE53118, GSE19234 and GSE22153 cohorts. GZMscore, age, Breslow's depth and tumor stage are the independent risk factors for CM patients, risk score based on these factors was calculated in TCGA-SKCM and GSE65906 cohorts, which could polarize the CM patients to high- and low-risk groups with diverse prognosis. Patients in low-risk group obtained the activated immune signaling pathways and response, especially for the activated CD8+ T cells, and could benefit more from anti-PD-1 therapy. A higher tumor mutation burden was observed in low-risk group, especially for the mutation of BRAF. The protect function of GZMK was confirmed by CM cell lines, overexpression of GZMK in A375 and G361 cells suppresses cell proliferation, migration, but not cell apoptosis. All in all, we revealed the prognostic value of GZMs in CM patients, which could also act as a predicted value for the selection of responders of anti-PD-1 immunotherapy.

Human CD4+ T-Cell Clone Expansion Leads to the Expression of the Cysteine Peptidase Inhibitor Cystatin F.

The existence of CD4+ cytotoxic T cells (CTLs) at relatively high levels under different pathological conditions in vivo suggests their role in protective and/or pathogenic immune functions. CD4+ CTLs utilize the fundamental cytotoxic effector mechanisms also utilized by CD8+ CTLs and natural killer cells. During long-term cultivation, CD4+ T cells were also shown to acquire cytotoxic functions. In this study, CD4+ human T-cell clones derived from activated peripheral blood lymphocytes of healthy young adults were examined for the expression of cytotoxic machinery components. Cystatin F is a protein inhibitor of cysteine cathepsins, synthesized by CD8+ CTLs and natural killer cells. Cystatin F affects the cytotoxic efficacy of these cells by inhibiting the major progranzyme convertases cathepsins C and H as well as cathepsin L, which is involved in perforin activation. Here, we show that human CD4+ T-cell clones express the cysteine cathepsins that are involved in the activation of granzymes and perforin. CD4+ T-cell clones contained both the inactive, dimeric form as well as the active, monomeric form of cystatin F. As in CD8+ CTLs, cysteine cathepsins C and H were the major targets of cystatin F in CD4+ T-cell clones. Furthermore, CD4+ T-cell clones expressed the active forms of perforin and granzymes A and B. The levels of the cystatin F decreased with time in culture concomitantly with an increase in the activities of granzymes A and B. Therefore, our results suggest that cystatin F plays a role in regulating CD4+ T cell cytotoxicity. Since cystatin F can be secreted and taken up by bystander cells, our results suggest that CD4+ CTLs may also be involved in regulating immune responses through cystatin F secretion.

Mechanisms of Apoptosis Resistance to NK Cell-Mediated Cytotoxicity in Cancer.

Natural killer (NK) cells are major contributors to immunosurveillance and control of tumor development by inducing apoptosis of malignant cells. Among the main mechanisms involved in NK cell-mediated cytotoxicity, the death receptor pathway and the release of granules containing perforin/granzymes stand out due to their efficacy in eliminating tumor cells. However, accumulated evidence suggest a profound immune suppression in the context of tumor progression affecting effector cells, such as NK cells, leading to decreased cytotoxicity. This diminished capability, together with the development of resistance to apoptosis by cancer cells, favor the loss of immunogenicity and promote immunosuppression, thus partially inducing NK cell-mediated killing resistance. Altered expression patterns of pro- and anti-apoptotic proteins along with genetic background comprise the main mechanisms of resistance to NK cell-related apoptosis. Herein, we summarize the main effector cytotoxic mechanisms against tumor cells, as well as the major resistance strategies acquired by tumor cells that hamper the extrinsic and intrinsic apoptotic pathways related to NK cell-mediated killing.

Association of increased eomesodermin, BCL6, and granzyme B expression with major clinical manifestations of Hashimoto's thyroiditis - an observational study.

PURPOSE: Studies of cytotoxic T cells and their respective lineage master regulators have been limited in Hashimoto's thyroiditis (HT). It is unclear whether their transcriptomes are changed in HT patients and how these changes are associated with the thyroid damage, major clinical manifestations, and disease progression. METHODS: We explored the gene expression patterns of selected transcription factors [eomesodermin (EOMES), BACH2, BCL6, TCF1] and cytolytic molecules [granzyme B (GZMB)] in peripheral blood (PB) T cells of 10 healthy controls and 30 HT patients of various subtypes (hypothyroid, untreated HT; L-thyroxine (T4)-treated HT, and spontaneously euthyroid HT) using real-time quantitative PCR. RESULTS: EOMES (Mann-Whitney P = 0.044), GZMB (P = 0.028), and BCL6 mRNA (P = 0.001) were overrepresented in PB T cells from HT and showed levels varying by age, thyroid volume and disease severity. BCL6 transcripts were predominantly enriched in severely affected, hypothyroid cases, both on and off LT4. Increased EOMES RNA expression was associated with advancing age, lower thyroid volumes and higher peak adjusted TSH levels over the course of the disease. The body mass-adjusted, steady-state maintenance dose of LT4 increased with GZMB and BCL6 levels in PB T cells of hypothyroid cases, mostly postmenopausal women having long-standing, non-goitrous and atrophic disease form. CONCLUSIONS: Our exploratory results suggest a role for GZMB, EOMES, and BCL6 in the context of HT, thyroid injury, and aggressive/advanced disease forms. Functions enriched within differentially expressed transcripts could be an important new target in understanding the pathogenesis of HT.

Mass-Spectrometric Analysis of Proteome of Microvesicles Produced by NK-92 Natural Killer Cells.

Membrane extracellular microvesicles serve as carriers of a wide range of molecules, the most important among these are proteins, lipids, and nucleic acids. Cytotoxic proteins of natural killer cells play a key role in the realization of their cytolytic functions. An important stage in understanding of the distant communication of cells and mechanisms of its regulation is analysis of the proteome composition of microvesicles. We studied the proteomic composition of microvesicles produced by NK-92 natural killer cells. Granzyme A, a specific protein of cytotoxic cells, has been identified in the microvesicles by QTOF-mass spectrometry. It was shown that heat shock proteins, components of the ubiquitin-proteasome system, enzymes of protein biosynthesis and energy metabolism, nuclear and serum proteins, as well as cytoskeleton proteins are associated with the microvesicles.

CD4+ natural killer T cells potently augment aortic root atherosclerosis by perforin- and granzyme B-dependent cytotoxicity.

RATIONALE: CD4(+) natural killer T (NKT) cells augment atherosclerosis in apolipoprotein E-deficient (ApoE)(-/-) mice but their mechanisms of action are unknown. OBJECTIVES: We investigated the roles of bystander T, B, and NK cells; NKT cell-derived interferon-γ, interleukin (IL)-4, and IL-21 cytokines; and NKT cell-derived perforin and granzyme B cytotoxins in promoting CD4(+) NKT cell atherogenicity. METHODS AND RESULTS: Transfer of CD4(+) NKT cells into T- and B-cell-deficient ApoE(-/-)Rag2(-/-) mice augmented aortic root atherosclerosis by ≈75% that was ≈30% of lesions in ApoE(-/-) mice; macrophage accumulation similarly increased. Transferred NKT cells were identified in the liver and atherosclerotic lesions of recipient mice. Transfer of CD4(+) NKT cells into T-, B-cell-deficient, and NK cell-deficient ApoE(-/-)Rag2(-/-)γC(-/-) mice also augmented atherosclerosis. These data indicate that CD4(+) NKT cells can exert proatherogenic effects independent of other lymphocytes. To investigate the role of NKT cell-derived interferon-γ, IL-4, and IL-21 cytokines and perforin and granzyme B cytotoxins, CD4(+) NKT cells from mice deficient in these molecules were transferred into NKT cell-deficient ApoE(-/-)Jα18(-/-) mice. CD4(+) NKT cells deficient in IL-4, interferon-γ, or IL-21 augmented atherosclerosis in ApoE(-/-)Jα18(-/-) mice by ≈95%, ≈80%, and ≈70%, respectively. Transfer of CD4(+) NKT cells deficient in perforin or granzyme B failed to augment atherosclerosis. Apoptotic cells, necrotic cores, and proinflammatory VCAM-1 (vascular cell adhesion molecule) and MCP-1 (monocyte chemotactic protein) were reduced in mice receiving perforin-deficient NKT cells. CD4(+) NKT cells are twice as potent as CD4(+) T cells in promoting atherosclerosis. CONCLUSIONS: CD4(+) NKT cells potently promote atherosclerosis by perforin and granzyme B-dependent apoptosis that increases postapoptotic necrosis and inflammation.

[Quantities and function of NK cells in patients with immune thrombocytopenia].

Objective: To investigate natural killer (NK) cell quantities and function in patients with immune thrombocytopenia (ITP) . Methods: A total of 66 ITP patients (34 newly diagnosed and 32 in complete remission) were collected from September 2015 to May 2016 in Tianjin Medical University General Hospital, and 30 healthy volunteers were recruited as controls. The percentages of NK cells and their subsets in peripheral blood, the expression of activating receptor (NKp44), inhibitory receptor (NKG2A) and CD16, perforin and granzyme ß were detected by flow cytometry. The correlation between the above parameters and patients' immune status and platelet level were evaluated. Results: (1)The percentage of CD3(-)CD56(+) NK cells in newly diagnosed patients (10.99%±4.89%)and patients in complete remission (9.73%±6.75%) were significantly lower than that in healthy controls (14.67%±7.24%)(P=0.023, 0.003). The percentage of NK cells Bright subset was significantly lower in the newly diagnosed patients(0.48%±0.23%)and those in complete remission (0.41%±0.33%) than in healthy controls(0.64%±0.32%)(P=0.037, 0.002); the percentage of Dim subset was also significantly lower in the newly diagnosed (10.16%±5.02%) and patients in complete remission (8.07%±5.74%) than in healthy controls(14.16%±7.19%) (P=0.009, 0.007). (2)The proportion of Bright subset in total NK cells in new diagnosed ITP patients (6.48%±4.33%) was significantly higher than that in healthy controls (4.21%±2.70%)(P=0.020); the proportion of Dim NK cells subset in new diagnosed ITP patients (93.51%±4.33%) was significantly lower than that in healthy controls(95.79%±2.70%) (P=0.020). (3)The expression of activating receptor NKp44 in new diagnosed ITP patients was significantly lower than that in complete remission group and healthy controls[0.28%(0.95%)vs 0.61%(2.05%), 0.92%(0.90%); P=0.047, 0.048]; the expression of inhibitory receptor NKG2A in new diagnosed ITP patients was significantly higher than that in healthy controls(42.34%±23.86% vs 29.25%±12.83%, P=0.009). The proportion of CD16 was significantly lower in the newly diagnosed patients than in healthy controls(93.51%±4.33%95.79%±2.70%, P=0.020). (4)The expression of perforin in the newly diagnosed ITP patients was significantly lower than that in healthy controls [87.52%(25.29%)vs 91.55%(8.29%), P=0.025]; the expression of granzyme ß in ITP patients and controls showed no statistically significant difference. (5)The level of NK cells in ITP patients was negatively correlated with CD3(+) CD8(+) T cells (r=-0.387, P=0.012) and CD5(+) CD19(+) B cells in peripheral blood (r=-0.273, P=0.028), positively correlated with the ratio of CD3(+) CD4(+) /CD3(+) CD8(+) (r=0.358, P=0.028) and peripheral platelet count (r=0.314, P=0.011). Conclusion: Deceased quantities and impaired total NK function, insufficient suppression of autoreactive T and B cells might play a role in the pathogenesis of ITP.

Detection of Cancer Cell Death Mediated by a Synthetic Granzyme B-like Peptide Fluorescent Conjugate and the same Peptide Binding in Bacteria.

Granzyme-mediated apoptosis, supported by pore-forming perforin, plays an important role in CD8+ T lymphocytes (CTL)-dependent cellular immunity protection against both cancer and viral infection. Quantitative and qualitative problems with CTL are potential contributing factors to disease progression. The feasibility of developing CTL-independent cellular immunity is desired but must first overcome the barrier of CTL-independent target cell recognition. Granzyme B with its strong pro-apoptotic activity in many different target cells is investigated for use in the CTL-independent cellular immunity approach, and granzyme B or its bioactive peptides without the enzymatic activity are more desirable for use. Native granzyme B with enzymatic activity is usually investigated in cancer cells for its mediation of apoptosis by detection of DNA fragmentation. Detection of cell death mediated by such peptides in cancer cells is needed to demonstrate the potential therapeutic purposes. We show with never-before-seen microscopic images using fluorescence microscopy that a synthetic granzyme B-like peptide fluorescent conjugate (GP1R) can: 1) mediate cell death of different cancer cells via membrane extrusion, 2) bind to constitutively expressed binding targets in different cancer cells and bacteria, and 3) promote bacterial phagocytosis. The putative binding targets may serve as a universal pathologic biomarker detectable by GP1R. Our data taken together demonstrate the potential applications of GP1R for use in CTL-independent target cell recognition and target cell death induction. It may lead to development of rapid targeted detection and new treatment of cancer, viral and bacterial infections. The new treatment may show mutual benefits for two or more diseases.

Granzyme B in aging and age-related pathologies.

Aging is a major risk factor for pathologies that manifest later in life. Much attention is devoted towards elucidating how prolonged environmental exposures and inflammation promote biological (accelerated) tissue aging. Granzymes, a family of serine proteases, are increasingly recognized for their emerging roles in biological aging and disease. Widely recognized as intracellular mediators of cell death, granzymes, particularly granzyme B (GzmB), also accumulate in the extracellular milieu of tissues with age, contributing to chronic tissue injury, inflammation, and impaired healing. Consequently, this has prompted the field to reconsider how GzmB regulation, accumulation, and proteolysis impact health and disease with age. While GzmB is observed in numerous age-related conditions, the current review focuses on mechanistic studies where proof-of-concept has been forwarded.

Distinct cell death pathways induced by granzymes collectively protect against intestinal Salmonella infection.

Intestinal intraepithelial T lymphocytes (IEL) constitutively express high amounts of the cytotoxic proteases Granzymes (Gzm) A and B and are therefore thought to protect the intestinal epithelium against infection by killing infected epithelial cells. However, the role of IEL granzymes in a protective immune response has yet to be demonstrated. We show that GzmA and GzmB are required to protect mice against oral, but not intravenous, infection with Salmonella enterica serovar Typhimurium, consistent with an intestine-specific role. IEL-intrinsic granzymes mediate the protective effects by controlling intracellular bacterial growth and aiding in cell-intrinsic pyroptotic cell death of epithelial cells. Surprisingly, we found that both granzymes play non-redundant roles. GzmB-/- mice carried significantly lower burdens of Salmonella, as predominant GzmA-mediated cell death effectively reduced bacterial translocation across the intestinal barrier. Conversely, in GzmA-/- mice, GzmB-driven apoptosis favored luminal Salmonella growth by providing nutrients, while still reducing translocation across the epithelial barrier. Together, the concerted actions of both GzmA and GzmB balance cell death mechanisms at the intestinal epithelium to provide optimal control that Salmonella cannot subvert.

Granzyme K mediates IL-23-dependent inflammation and keratinocyte proliferation in psoriasis.

Psoriasis is an inflammatory disease with systemic manifestations that most commonly presents as itchy, erythematous, scaly plaques on extensor surfaces. Activation of the IL-23/IL-17 pro-inflammatory signaling pathway is a hallmark of psoriasis and its inhibition is key to clinical management. Granzyme K (GzmK) is an immune cell-secreted serine protease elevated in inflammatory and proliferative skin conditions. In the present study, human psoriasis lesions exhibited elevated GzmK levels compared to non-lesional psoriasis and healthy control skin. In an established murine model of imiquimod (IMQ)-induced psoriasis, genetic loss of GzmK significantly reduced disease severity, as determined by delayed plaque formation, decreased erythema and desquamation, reduced epidermal thickness, and inflammatory infiltrate. Molecular characterization in vitro revealed that GzmK contributed to macrophage secretion of IL-23 as well as PAR-1-dependent keratinocyte proliferation. These findings demonstrate that GzmK enhances IL-23-driven inflammation as well as keratinocyte proliferation to exacerbate psoriasis severity.