Granzyme A Expression Research Articles

Decoding the mechanisms of chimeric antigen receptor (CAR) T cell-mediated killing of tumors: insights from granzyme and Fas inhibition

Chimeric antigen receptor (CAR) T cell show promise in cancer treatments, but their mechanism of action is not well understood. Decoding the mechanisms used by individual T cells can help improve the efficacy of T cells while also identifying mechanisms of T cell failure leading to tumor escape. Here, we used a suite of assays including dynamic single-cell imaging of cell-cell interactions, dynamic imaging of fluorescent reporters to directly track cytotoxin activity in tumor cells, and scRNA-seq on patient infusion products to investigate the cytotoxic mechanisms used by individual CAR T cells in killing tumor cells. We show that surprisingly, overexpression of the Granzyme B (GZMB) inhibitor, protease inhibitor-9 (PI9), does not alter the cytotoxicity mediated by CD19-specific CAR T cells against either the leukemic cell line, NALM6; or the ovarian cancer cell line, SkOV3-CD19. We designed and validated reporters to directly assay T cell delivered GZMB activity in tumor cells and confirmed that while PI9 overexpression inhibits GZMB activity at the molecular level, this is not sufficient to impact the kinetics or magnitude of killing mediated by the CAR T cells. Altering cytotoxicity mediated by CAR T cells required combined inhibition of multiple pathways that are tumor cell specific: (a) B-cell lines like NALM6, Raji and Daudi were sensitive to combined GZMB and granzyme A (GZMA) inhibition; whereas (b) solid tumor targets like SkOV3-CD19 and A375-CD19 (melanoma) were sensitive to combined GZMB and Fas ligand inhibition. We realized the translational relevance of these findings by examining the scRNA-seq profiles of Tisa-cel and Axi-cel infusion products and show a significant correlation between GZMB and GZMA expression at the single-cell level in a T cell subset-dependent manner. Our findings highlight the importance of the redundancy in killing mechanisms of CAR T cells and how this redundancy is important for efficacious T cells.

Construction of an enhanced computed tomography radiomics model for non-invasively predicting granzyme A in head and neck squamous cell carcinoma by machine learning.

Classical prognostic indicators of head and neck squamous cell carcinoma (HNSCC) can no longer meet the clinical needs of precision medicine. This study aimed to establish a radiomics model to predict Granzyme A (GZMA) expression in patients with HNSCC. We downloaded transcriptomic data of HNSCC patients from The Cancer Genome Atlas for prognosis analysis and then used corresponding enhanced computed tomography (CT) images from The Cancer Imaging Archive for feature extraction and model construction. We explored the influence of differences in GZMA expression on signaling pathways and analyzed the potential molecular mechanism and its relationship with immune cell infiltration. Subsequently, non-invasive CT radiomics models were established to predict the expression of GZMA mRNA and evaluate the correlation with the radiomics-score (Rad-score), related genes, and prognosis. We found that GZMA was highly expressed in tumor tissues, and high GZMA expression was a protective factor for overall survival. The degree of B and T lymphocyte and natural killer cell infiltration was significantly correlated with GZMA expression. The receiver operating characteristic curve showed that the Relief GBM and RFE_GBM radiomics models had good predictive ability, and there were significant differences in the Rad-score distribution between the high- and low-GZMA-expression groups. GZMA expression can significantly affect the prognosis of patients with HNSCC. Enhanced CT radiomics models can effectively predict the expression of GZMA mRNA.

Identification of GZMA as a Potential Therapeutic Target Involved in Immune Infiltration in Breast Cancer by Integrated Bioinformatical Analysis.

Granzyme A (GZMA) is a potential prognostic target for various cancer types. However, its therapeutic significance in breast cancer with immune infiltration remains controversial. We analyzed GZMA expression and its prognostic value in breast cancer with immune cell infiltration. Data was obtained from patients with breast cancer registered at The Cancer Genome Atlas. A correlation was performed between GZMA expression and patient's clinicopathological features such as age, pathologic stage, metastasis stage, overall survival (OS), disease-specific survival (DSS), and progress free interval (PFI). Kaplan-Meier analyses and Cox proportional hazard regression model were used to examine the predictive significance of GZMA expression for breast cancer. The co-expression pattern of GZMA was assessed by the LinkedOmics web portal. The relationship between GZMA expression and immune cells was analyzed using the TIMER database. The correlation between GZMA and lymphocytes and immunomodulators was established with the TISIDB database. There was a lower GZMA expression in breast cancer tissue than in normal tissue. Interestingly, GZMA expression was associated with age, pathologic stage, and the Tumour, Node, and Metastasis stage. Overexpression of GZMA was also associated with better OS, DSS, and PFI. Based on the Cox regression analysis, GZMA was identified as an independent favorable prognostic factor for breast cancer. Our findings demonstrated a strong association between GZMA and T-cell checkpoints (PD-1, PD-L1, and cytotoxic T lymphocyte-associated antigen (CTLA-4)) in breast cancer. Moreover, we evaluated the interactions between GZMA expression and markers of dendritic and CD8+ T cells using quantitative immunofluorescence. We discovered that increased infiltration of dendritic and CD8+ T cells was associated with GZMA expression in breast cancer. GZMA expression is associated with a favorable prognosis in breast cancer and is significantly correlated with immune cell infiltration. GZMA may be considered a promising therapeutic target for patients with breast cancer.

Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies.

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA's physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA's bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

Abstract 6490: PD-L1 and granzyme A mRNA expression associated with prognosis and response to immune checkpoint inhibitors in non-small cell lung cancer

Abstract Background Lung cancer is one of the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer. The discovery of immune checkpoints and the development of immune checkpoint inhibitors (ICIs) is revolutionary in cancer immunology. However, only about 20-30% of cancer patients could benefit from ICIs. Recently, secreted and 5' UTR truncated PD-L1 isoforms were shown to mediate resistance to ICI therapies. Immunohistochemistry (IHC) is the current standard PD-L1 detection method to triage patients for ICI therapies but has limitations, including various antibodies with various detection thresholds, challenge to distinguish PD-L1 isoforms, and inability in multiplex analysis of other immune related factors and genetic alterations. Here, we developed a new method to detect the mRNA expression of CD274 (encoding PD-L1), its various splicing variants and multiplex with T-cell activity factors and other genetic drivers by next-generation sequencing (NGS). Patients and Methods We recruited two patient cohorts. Cohort I included 182 NSCLC (number of stage I, II, III and IV was 73, 48, 59, and 2, respectively) patients with surgery resections. Cohort II included 33 patients who failed first-line therapies and further received anti-PD-1 therapy, prior to which needle biopsies were obtained. Tumor tissues were used for total nucleic acids (DNA and RNA) extraction and subjected to an anchored multiplex PCR NGS panel for one-tube enrichment of 63 genes relevant in NSCLC, including EGFR, KRAS, BRAF, ALK, ROS1, RET, MET, NTRK1/2/3, NRG1 and immune related genes CD274, GZMA (encoding granzyme A) and PRF1 (encoding perforin). We calculated exon-level mRNA expression per copy genomic DNA of the same target, expressed as RNA-to-DNA ratio, without relying on the conventional housekeeping gene normalization. Gene fusions and isoforms were analyzed using our bioinformatics pipeline designed for a clinically established Anchored Multiplex PCR NGS platform (Zheng, et. al, Nat Med, 2014). Kaplan-Meier curve and Cox regression were used for analyses of overall survival (OS) in Cohort I and progression free survival (PSF) in Cohort II. Results In Cohort I, the mRNA expression of CD274 was correlated with its protein expression assayed by a clinical IHC assay (antibody 22C3; R2 = 0.51, P value < 0.01). ALK mRNA expression were highest among those with ALK fusions. Interestingly, patients with high expression of CD274 exon3 and low expression of exon5 (presumably resembling those secreted and 5-UTR-trancated PD-L1 isoforms) had worst overall survival when compared to patients with other CD274 splicing isoforms and patients with low CD274 expression (P value < 0.01). In Cohort II, the analysis of pre-ICI treatment needle biopsies showed that a high expression of CD274 was associated with response to ICIs and with a better PFS compared to patients with low CD274 expression (P value < 0.01). Further, among patients with high CD274 expression, a high GZMA expression was associated with a trend towards better PFS (P value = 0.06), whereas a high PRF1 expression level was not. Conclusion The data suggests that PD-L1and GZMA mRNA expression may predict the prognosis and response to immune checkpoint blockade therapies in NSCLC patients, and larger studies are warranted. Citation Format: Zhengbo Song, Chenyu Lu, Hongxiang Wang, Chunwei Xu, Baifeng Zhang, Wenxian Wang, Juxiang Chen, Zongli Zheng. PD-L1 and granzyme A mRNA expression associated with prognosis and response to immune checkpoint inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6490.

Dynamic regulation of permissive histone modifications and GATA3 binding underpin acquisition of granzyme A expression by virus-specific CD8(+) T cells.

Numerous studies have focused on the molecular regulation of perforin (PFP) and granzyme B (GZMB) expression by activated cytotoxic T lymphocytes (CTLs), but little is known about the molecular factors that underpin granzyme A (GZMA) expression. In vitro activation of naïve CD8(+) T cells, in the presence of IL-4, enhanced STAT6-dependent GZMA expression and was associated with GATA3 binding and enrichment of transcriptionally permissive histone posttranslational modifications (PTMs) across the Gzma gene locus. While GZMA expression by effector influenza A virus specific CTLs was also associated with a similar permissive epigenetic signature, memory CTL lacked enrichment of permissive histone PTMs at the Gzma locus, although this was restored within recalled secondary effector CTLs. Importantly, GZMA expression byvirus-specific CTLswas associated with GATA3 binding at the Gzma locus, and independent of STAT6-mediated signaling. This suggests regulation of GZMA expression is underpinned by differentiation-dependentregulation of chromatin composition at the Gzma locus and that, given GATA3 is key for CTL differentiation in response to infection, GATA3 expression is regulated by a distinct, IL-4 independent, signaling pathway. Overall, this study provides insights into the molecular mechanisms that control transcription of Gzma during virus-induced CD8(+) T-cell differentiation.

Pars planitis is associated with an increased frequency of effector-memory CD57+ T cells

Aim:To evaluate the frequency, phenotype and the potential function of CD57+ T cell subsets in patients with pars planitis.Methods:CD4+CD57+ and CD8+CD57+ T cells were quantitated in peripheral blood from 15...

Glucocorticoid-induced granzyme A expression can be used as a marker of glucocorticoid sensitivity for acute lymphoblastic leukemia therapy

The ability of glucocorticoids (GC) to efficiently kill lymphoid cells has led to their inclusion in essentially all chemotherapy procedures used to treat acute lymphoblastic leukemia (ALL). GC sensitivity is an important prognostic factor in ALL treatment, and it is used to classify patients into risk groups. Clinical assessment for GC sensitivity is very time-consuming, however. We have recently found that granzyme A (GZMA) mediates GC-induced apoptosis in ALL-derived cell line 697. In this study we examined the correlation between GC sensitivity and GC-induced GZMA expression by using seven established cell lines derived from ALL patients. The apoptosis assay showed four cell lines were GC-sensitive and three were GC-resistant. GC treatment markedly enhanced GZMA expression in GC-sensitive cell lines only, and not in GC-resistant cell lines. GC-induced GZMA expression also correlated well with the amount of GC-induced apoptosis. GC-induced GZMA expression could thus be a useful early biomarker for "personalized" ALL therapy.