Secretion Of Granzyme Research Articles

DNMT1 silencing induces KIR2DL1/2/3 expression via methylation to alleviate graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Natural killer (NK) cells are the promoters in graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), while demethylation can regulate NK cell function. We explored the mechanism of demethylation regulating NK cell function to affect GVHD after allo-HSCT. BALB/c mice were transfused with C57BL/6 mouse-derived NK and bone marrow cells to establish GVHD models, followed by isolation and in-vitro expansion of NK cells. NK cell purity, cytokine levels, proliferation, and cytokine-producing NK cell levels were measured via flow cytometry. KIR2DL1/2/3 methylation was tested by Methylation-specific polymerase chain reaction (MSP), with determination of mouse survival and GVHD scores. KIR2DL1/2/3 and DNMT1 expression was detected through qRT-PCR and/or western blot. Methylation levels were upregulated and KIR2DL1/2/3 expression was downregulated in GVHD mouse model-derived NK cells following IL-2 stimulation. DNMT1 silencing promoted KIR2DL1/2/3 expression, proliferation, and the secretion of Granzyme, Perforin, and Interferon-γ (IFN-γ) in C57BL/6 mouse-derived NK cells. DNMT1 silencing also enhanced mouse survival, reduced GVHD scores, promoted KIR2DL1/2/3 expression on the NK cell surface, and increased the secretion of Granzyme, Perforin, IFN-γ, and the number of cytokine-producing NK cells in the spleen, liver, and lung tissues of the models. Collectively, DNMT1 silencing induced KIR2DL1/2/3 expression in NK cells through reducing methylation to alleviate GVHD after allo-HSCT.

Circulating CD8 + LGALS9 + T Cell Population Exhibiting Low Cytotoxic Characteristics are Decreased in Patients with Systemic Lupus Erythematosus.

LGALS9, also known as Galectin-9 and a member of the β-galactosidase family, plays a crucial role in immune regulation. However, its expression and function in CD8 T cells, as well as its association with cytotoxic T lymphocytes (CTL), remain unclear. This study aims to investigate LGALS9 expression patterns in human circulating CD8 T lymphocytes and elucidate its clinical significance in Systemic Lupus Erythematosus (SLE). Blood samples from 56 healthy controls and 50 new-onset SLE patients were collected. Flow cytometry was utilized to analyze LGALS9 expression in circulating CD8 T lymphocytes via intracellular staining. Compared to LGALS9 + CD8 + T cells, LGALS9-CD8 + T cells showed increased secretion of Granzyme B (GZMB) and Perforin, along with elevated expression levels of GPR56, CX3CR1, KLRD1, KLRF1, PD1, and CD29. A higher proportion of Tn (naive T cells) and TCM (central memory T cells) showed LGALS9 positivity, compared to TEM (effector memory T cells) and TEMRA (terminally differentiated effector memory T cells re-expressing CD45RA). Clinically, the downregulation of LGALS9 expression was significant in SLE patients. LGALS9 + CD8 + T cells exhibited an Area Under the Curve (AUC) of 0.6916, while CX3CR1 + in LGALS9 + CD8 + T cells had an AUC of 0.6478, and KLRF1 + had an AUC of 0.6419, for distinguishing SLE from healthy individuals. In conclusion, CD8 + LGALS9 + T cells display characteristics of low cytotoxicity, and their reduction is evident in SLE patients, potentially implicating them in SLE pathogenesis and providing diagnostic assistance.

PD-L2 drives resistance to EGFR-TKIs: dynamic changes of the tumor immune environment and targeted therapy.

There is a lack of effective treatments to overcome resistance to EGFR-TKIs in EGFR mutant tumors. A deeper understanding of resistance mechanisms can provide insights into reducing or eliminating resistance, and can potentially deliver targeted treatment measures to overcome resistance. Here, we identified that the dynamic changes of the tumor immune environment were important extrinsic factors driving tumor resistance to EGFR-TKIs in EGFR mutant cell lines and syngeneic tumor-bearing mice. Our results demonstrate that the acquired resistance to EGFR-TKIs is accompanied by aberrant expression of PD-L2, leading a dynamic shift from an initially favorable tumor immune environment to an immunosuppressive phenotype. PD-L2 expression significantly affected EGFR mutant cell apoptosis that depended on the proportion and function of CD8+ T cells in the tumor immune environment. Combined with single-cell sequencing and experimental results, we demonstrated that PD-L2 specifically inhibited the proliferation of CD8+ T cells and the secretion of granzyme B and perforin, leading to reduced apoptosis mediated by CD8+ T cells and enhanced immune escape of tumor cells, which drives EGFR-TKIs resistance. Importantly, we have identified a potent natural small-molecule inhibitor of PD-L2, zinc undecylenate. In vitro, it selectively and potently blocks the PD-L2/PD-1 interaction. In vivo, it abolishes the suppressive effect of the PD-L2-overexpressing tumor immune microenvironment by blocking PD-L2/PD-1 signaling. Moreover, the combination of zinc undecylenate and EGFR-TKIs can synergistically reverse tumor resistance, which is dependent on CD8+ T cells mediating apoptosis. Our study uncovers the PD-L2/PD-1 signaling pathway as a driving factor to mediate EGFR-TKIs resistance, and identifies a new naturally-derived agent to reverse EGFR-TKIs resistance.

Abstract 1223: KDM6A orchestrates NK cell response via CD38/48 regulation in multiple myeloma

Abstract Daratumumab (Dara), a first-in-class humanized monoclonal antibody targeting CD38, demonstrates notable efficacy in both newly diagnosed and relapsed/refractory multiple myeloma (MM) when administrated alone or in combination with other agents. However, the development of Dara resistance and subsequent relapse is common.To identify the tumor-intrinsic genes involved in resistance to NK-mediated cytotoxicity in the presence of Dara in MM cells, we conducted an in vitro genome-wide CRISPR-Cas9 knockout (KO) screen and found 433 genes exhibiting a positive correlation with MM cytotoxicity elicited by Dara and primary NK cells. Remarkably, 16 of these genes demonstrated a consistent correlation with cytolytic activity across the majority of the 36 cancer types in the TCGA database.The efficacy of Dara is closely linked to the expression of CD38, with nonresponders exhibiting low CD38 expression. Therefore, we conducted another CRISPR screen to identify the genes whose KO resulted in diminished CD38 expression. The genes that overlapped in these two screens are implicated in regulating both Dara-mediated ADCC and CD38 expression. Our focus turned to KDM6A among the overlapping genes. We validated that KDM6A KO significantly decreased CD38 expression and that reintroducing KDM6A into KDM6A KO cells restored the expression of CD38 and Dara-induced cytotoxicity.KDM6A demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. Our ChIP-seq data unveiled that KDM6A-KO cells exhibited an increased H3K27me3 level at the CD38 promoter area than control cells. Remarkably, this pattern was consistent with Dara-resistant cell lines, which displayed reduced CD38 expression alongside elevated levels of H3K27me3 at the CD38 promoter. Interestingly, we found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment, implying the existence of an additional mechanism. We performed RNA-seq in KDM6A KO cells and found CD48, an NK-activating ligand, was significantly repressed. Overexpression of CD48 in KDM6A KO cells resulted in a substantial increase in the secretion of Granzyme B and Perforin by NK cells and restored the activity of NK cells and re-sensitized KDM6A KO cells to Dara treatment. These data suggest that KDM6A mediates Dara-mediated ADCC sensitivity through the dual regulation of CD38 and CD48. KDM6A acts to oppose the EZH2/PRC2 complex by demethylating H3K27me3, and we found that Tazemetostat (Taze), an FDA-approved EZH2 inhibitor, increased the expression of CD38 and CD48, especially in KDM6A-KO cells. Importantly, Taze also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity. In summary, our findings illuminate the dual role of KDM6A in enhancing the efficacy of Dara in MM. Imitating KDM6A function using an FDA-approved inhibitor holds promise in overcoming Dara resistance and improving patient outcomes in MM. Citation Format: Jiye Liu, Lijie Xing, Jiang Li, Kenneth Wen, Ning Liu, Yuntong Liu, Annamaria Gulla, Nikhil Munshi, Teru Hideshima, Kenneth Carl Anderson. KDM6A orchestrates NK cell response via CD38/48 regulation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1223.

A Programmable Peptidic Hydrogel Adjuvant for Personalized Immunotherapy in Resected Stage Tumors.

Adjuvant treatment after surgical resection usually plays an important role in delaying disease recurrence. Immunotherapy displays encouraging results in increasing patients' chances of staying cancer-free after surgery, as reported by recent clinical trials. However, the clinical outcomes of current immunotherapy need to be improved due to the limited responses, patient heterogeneity, nontargeted distribution, and immune-related adverse effects. This work describes a programmable hydrogel adjuvant for personalized immunotherapy after surgical resection. By filling the hydrogel in the cavity, this system aims to address the limited secretion of granzyme B (GrB) during immunotherapy and improve the low immunotherapy responses typically observed, while minimizing immune-related side effects. The TLR7/8 agonist imidazoquinoline (IMDQ) is linked to the self-assembling peptide backbone through a GrB-responsive linkage. Its release could enhance the activation and function of immune cells, which will lead to increased secretion of GrB and enhance the effectiveness of immunotherapy together. The hydrogel adjuvant recruits immune cells, initiates dendritic cell maturation, and induces M1 polarized macrophages to reverse the immunosuppressive tumor microenvironment in situ. In multiple murine tumor models, the hydrogel adjuvant suppresses tumor growth, increases animal survival and long-term immunological memory, and protects mice against tumor rechallenge, leading to effective prophylactic and therapeutic responses. This work provides a potential chemical strategy to overcome the limitations associated with immunotherapy.

Enhanced NK cell activation via eEF2K-mediated potentiation of the cGAS–STING pathway in hepatocellular carcinoma

BackgroundLiver cancer, particularly hepatocellular carcinoma (HCC), is characterized by a high mortality rate, attributed primarily to the establishment of an immunosuppressive microenvironment. Within this context, we aimed to elucidate the pivotal role of eukaryotic elongation factor 2 kinase (eEF2K) in orchestrating the infiltration and activation of natural killer (NK) cells within the HCC tumor microenvironment. By shedding light on the immunomodulatory mechanisms at play, our findings should clarify HCC pathogenesis and help identify potential therapeutic intervention venues. MethodsWe performed a comprehensive bioinformatics analysis to determine the functions of eEF2K in the context of HCC. We initially used paired tumor and adjacent normal tissue samples from patients with HCC to measure eEF2K expression and its correlation with prognosis. Subsequently, we enrolled a cohort of patients with HCC undergoing immunotherapy to examine the ability of eEF2K to predict treatment efficacy. To delve deeper into the mechanistic aspects, we established an eEF2K-knockout cell line using CRISPR/Cas9 gene editing. This step was crucial for verifying activation of the cGAS–STING pathway and the subsequent secretion of cytokines. To further elucidate the role of eEF2K in NK cell function, we applied siRNA-based techniques to effectively suppress eEF2K expression in vitro. For in vivo validation, we developed a tumor-bearing mouse model that enabled us to compare the infiltration and activation of NK cells within the tumor microenvironment following various treatment strategies. ResultsWe detected elevated eEF2K expression within HCC tissues, and this was correlated with an unfavorable prognosis (30.84 vs. 20.99 months, P = 0.033). In addition, co-culturing eEF2K-knockout HepG2 cells with dendritic cells led to activation of the cGAS–STING pathway and a subsequent increase in the secretion of IL-2 and CXCL9. Moreover, inhibiting eEF2K resulted in notable NK cell proliferation along with apoptosis reduction. Remarkably, after combining NH125 and PD-1 treatments, we found a significant increase in NK cell infiltration within HCC tumors in our murine model. Our flow cytometry analysis revealed reduced NKG2A expression and elevated NKG2D expression and secretion of granzyme B, TNF-α, and IFN-γ in NK cells. Immunohistochemical examination confirmed no evidence of damage to vital organs in the mice treated with the combination therapy. Additionally, we noted higher levels of glutathione peroxidase and lipid peroxidation in the peripheral blood serum of the treated mice. ConclusionTargeted eEF2K blockade may result in cGAS–STING pathway activation, leading to enhanced infiltration and activity of NK cells within HCC tumors. The synergistic effect achieved by combining an eEF2K inhibitor with PD-1 antibody therapy represents a novel and promising approach for the treatment of HCC.

High HPK1+PD-1+TIM-3+CD8+ T cells infiltration predicts poor prognosis to immunotherapy in NSCLC patients

At present the efficacy of immune checkpoint inhibitors (ICIs) remains limited. The lack of responsiveness in certain patients may be attributed to CD8+ T cell exhaustion within the tumor microenvironment (TME). Hematopoietic progenitor kinase 1 (HPK1) has been identified as a mediator of T cell dysfunction, leading to our hypothesis that HPK1 positive exhausted CD8+ T cells could serve as a predictor for ICIs' efficacy in NSCLC patients, and potentially indicate key cellular subset causing ICIs resistance. Here, we retrospectively collected tumor tissue samples from 36 NSCLC patients who underwent first-line immunotherapy. Using multiplex immunohistochemistry, we visualized various PD-1+CD8+ T cell subsets and explore biomarkers for response. The analysis endpoints included overall response rate (ORR), progression free survival (PFS), and overall survival (OS), correlating them with levels of cell infiltration or effective density. We found that the proportion of PD-1+CD8+ T cell subsets did not align with predictions for ORR, PFS, and OS. Conversely, a high infiltration of HPK1+PD-1+TIM-3+CD8+ T cells was identified as an independent risk factor for both PFS (P = 0.019) and OS (P = 0.03). These cells were found to express the highest levels of Granzyme B, and the secretion of Granzyme B in CD8+ T cell subsets was related to TCF-1. In conclusion, these data suggest that a high infiltration of HPK1+PD-1+TIM-3+CD8+ T cells correlates with poor clinical outcomes in NSCLC patients receiving immunotherapy. These cells may represent terminally exhausted T cells that fail to respond to ICIs, thereby laying the groundwork for the potential integration of HPK1 inhibitors with immunotherapy to enhance treatment strategy.

Mn-Anti-CTLA4-CREKA-Sericin Nanotheragnostics for Enhanced Magnetic Resonance Imaging and Tumor Immunotherapy.

The integration of magnetic resonance imaging (MRI), cGAS-STING, and anti-CTLA-4 (aCTLA-4) based immunotherapy offers new opportunities for tumor precision therapy. However, the precise delivery of aCTLA-4 and manganese (Mn), an activator of cGAS, to tumors remains a major challenge for enhanced MRI and active immunotherapy. Herein, a theragnostic nanosphere Mn-CREKA-aCTLA-4-SS (MCCS) is prepared by covalently assembling Mn2+, silk sericin (SS), pentapeptide CREKA, and aCTLA-4. MCCS are stable with an average size of 160nm and is almost negatively charged or neutral at pH 5.5/7.4. T1-weighted images showed MCCS actively targeted tumors to improve the relaxation rate r1 and contrast time of MRI. This studies demonstrated MCCS raises reactive oxygen species levels, activates the cGAS-STING pathway, stimulates effectors CD8+ and CD80+ T cells, reduces regulatory T cell numbers, and increases IFN-γ and granzyme secretion, thereby inducing tumor cells autophagy and apoptosis in vitro and in vivo. Also, MCCS are biocompatible and biosafe. These studies show the great potential of Mn-/SS-based integrative material MCCS for precision and personalized tumor nanotheragnostics.

Anti-tumor Effect of Activated Canine B Cells With Interleukin-21 and Anti-B Cell Receptor.

Recently, novel studies on the pivotal role of B cells in the tumor-microenvironment and anti-tumor immunity have been conducted. Additionally, Interleukin-21 (IL-21) and anti-B cell receptor (BCR) have been reported to stimulate B cells to secrete granzyme B, which exhibits cytotoxic effects on tumor cells. However, the direct anti-tumor effect of B cells is not yet fully understood in the veterinary field. This study is the first attempt in veterinary medicine to identify the immediate effect of B cells on tumor suppression and the underlying mechanisms involved. Canine B cells were isolated from peripheral blood and activated with IL-21 and anti-B cell receptor (BCR). The canine leukemia cell line GL-1 was co-cultured with B cells, and the anti-tumor effect was confirmed by assessing the changes in cell viability and apoptotic ratio. When B cells were activated with IL-21 and anti-BCR, the secretion of granzyme B and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increased. Simultaneously, the viability of GL-1 cells decreased, and the apoptotic ratio increased, particularly when co-cultured with activated B cells. The results demonstrated the direct anti-tumor effect of granzyme B-and TRAIL and its enhanced potential of B cells to inhibit tumor cell growth after activation with IL-21 and anti-BCR. This study is the first study dealing with immunomodulation in the canine tumor micro-environment.

The mRNA expression and secretion of granzyme B are up-regulated via the histamine H2 receptor in human CD4+ T cells

IntroductionGranzyme B (GZMB), a serine protease with cytotoxic and immunomodulatory functions, shows elevated levels in blood plasma of patients with atopic dermatitis (AD). It has been observed that GZMB expression in CD4+ and CD8+ T cells is higher in lesional skin in AD than in healthy skin. Since histamine is present in high concentration in the skin of AD patients, we investigated the regulation of GZMB in human CD4+ T cells by histamine.MethodsNaïve CD4+ T cells polarized into Th2 cells, total CD4+ T cells treated with IL-4 for 72 h and CD4+ T cells isolated from healthy donors and AD patients were investigated. The cells were stimulated with histamine or with different histamine-receptor agonists. Gene expression was evaluated by RNA-Seq. GZMB mRNA expression was detected by quantitative real time PCR, whereas GZMB secretion was measured by ELISpot and ELISA. T cell degranulation was evaluated by flow cytometry using CD107a surface expression as a degranulation marker.ResultsBy RNA-Seq, we identified the up-regulation of various genes of the cytotoxic pathway, in particular of GZMB, by histamine in Th2-polarized CD4+ T cells. In Th2-polarized CD4+ T cells and in CD4+ T cells activated by IL-4 the mRNA expression of GZMB was significantly up-regulated by histamine and by histamine H2 receptor (H2R) agonists. The induction of GZMB secretion by histamine was significantly higher in CD4+ T cells from AD patients than in those from healthy donors. CD107a surface expression was up-regulated by trend in response to histamine in Th2-polarized CD4+ T cells.ConclusionOur findings may help to elucidate novel mechanisms of the H2R and to achieve a better understanding of the role of GZMB in the pathogenesis of AD.

Single-cell RNA sequencing reveals enhanced antitumor immunity after combined application of PD-1 inhibitor and Shenmai injection in non-small cell lung cancer

BackgroundImmune checkpoint inhibitors (ICIs) have altered the clinical management of non-small cell lung cancer (NSCLC). However, the low response rate, severe immune-related adverse events (irAEs), and hyperprogressive disease following ICIs monotherapy require attention. Combination therapy may overcome these limitations and traditional Chinese medicine with immunomodulatory effects provides a promising approach. Shenmai injection (SMI) is a clinically effective adjuvant treatment for cancer with chemotherapy and radiotherapy. Therefore, the combined effects and mechanisms of SMI and programmed death-1 (PD-1) inhibitor against NSCLC was focused on this study.MethodsA Lewis lung carcinoma mouse model and a lung squamous cell carcinoma humanized mouse model were used to investigate the combined efficacy and safety of SMI and PD-1 inhibitor. The synergistic mechanisms of the combination therapy against NSCLC were explored using single-cell RNA sequencing. Validation experiments were performed using immunofluorescence analysis, in vitro experiment, and bulk transcriptomic datasets.ResultsIn both models, combination therapy alleviated tumor growth and prolonged survival without increasing irAEs. The GZMAhigh and XCL1high natural killer (NK) cell subclusters with cytotoxic and chemokine signatures increased in the combination therapy, while malignant cells from combination therapy were mainly in the apoptotic state, suggesting that mediating tumor cell apoptosis through NK cells is the main synergistic mechanisms of combination therapy. In vitro experiment confirmed that combination therapy increased secretion of Granzyme A by NK cells. Moreover, we discovered that PD-1 inhibitor and SMI combination blocked inhibitory receptors on NK and T cells and restores their antitumoral activity in NSCLC better than PD-1 inhibitor monotherapy, and immune and stromal cells exhibited a decrease of angiogenic features and attenuated cancer metabolism reprogramming in microenvironment of combination therapy.ConclusionsThis study demonstrated that SMI reprograms tumor immune microenvironment mainly by inducing NK cells infiltration and synergizes with PD-1 inhibitor against NSCLC, suggested that targeting NK cells may be an important strategy for combining with ICIs.4EoQVoCxPUSr8SKnFMutzeVideo

Single-cell RNA sequencing highlights the role of PVR/PVRL2 in the immunosuppressive tumour microenvironment in hepatocellular carcinoma.

The conflict between cancer cells and the host immune system shapes the immune tumour microenvironment (TME) in hepatocellular carcinoma (HCC). A deep understanding of the heterogeneity and intercellular communication network in the TME of HCC will provide promising strategies to orchestrate the immune system to target and eradicate cancers. Here, we performed single-cell RNA sequencing (scRNA-seq) and computational analysis of 35786 unselected single cells from 3 human HCC tumour and 3 matched adjacent samples to elucidate the heterogeneity and intercellular communication network of the TME. The specific lysis of HCC cell lines was examined in vitro using cytotoxicity assays. Granzyme B concentration in supernatants of cytotoxicity assays was measured by ELISA. We found that VCAN+ tumour-associated macrophages (TAMs) might undergo M2-like polarization and differentiate in the tumour region. Regulatory dendritic cells (DCs) exhibited immune regulatory and tolerogenic phenotypes in the TME. Furthermore, we observed intensive potential intercellular crosstalk among C1QC+ TAMs, regulatory DCs, regulator T (Treg) cells, and exhausted CD8+ T cells that fostered an immunosuppressive niche in the HCC TME. Moreover, we identified that the TIGIT-PVR/PVRL2 axis provides a prominent coinhibitory signal in the immunosuppressive TME. In vitro, antibody blockade of PVR or PVRL2 on HCC cell lines or TIGIT blockade on immune cells increased immune cell-mediated lysis of tumour cell. This enhanced immune response is paralleled by the increased secretion of Granzyme B by immune cells. Collectively, our study revealed the functional state, clinical significance, and intercellular communication of immunosuppressive cells in HCC at single-cell resolution. Moreover, PVR/PVRL2, interact with TIGIT act as prominent coinhibitory signals and might represent a promising, efficacious immunotherapy strategy in HCC.

The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT.

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

Genome-Wide CRISPR-Cas9 Screen Identifies KDM6A As a Modulator of Daratumumab Sensitivity in Multiple Myeloma

Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38, which triggers both direct and immune-mediated cytotoxicity of multiple myeloma (MM) cells in the bone marrow (BM) milieu. Importantly, Dara combination therapies have a high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse is commonly observed due to the development of Dara resistance. As an immune-targeting drug, the anti-tumor activity of Dara is closely associated with the expression of CD38, with nonresponders having a lower expression of CD38. To identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells, we performed a genome-wide CRISPR-Cas9 knockout (KO) screen in vitro. We identified 46 different sgRNAs that were positively correlated with MM cytotoxicity triggered by Dara and primary NK cells. Using the cells with the lowest expression of CD38 (bottom 5%), we performed a second round of screening and found that KDM6A was the top-ranked gene out of the set of overlapping genes from these two screenings. We confirmed that CD38 expression was significantly decreased (>2 times, p<0.01) by KDM6A KO and that re-introducing KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity. KDM6A encodes lysine-specific demethylase 6A, which demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. To confirm that CD38 expression was altered by the methylation status of H3K27, we compared the CD38 promoter area between KDM6A KO and control cells using ChIP-seq and ChIP qPCR and found that the H3K27me3 level was higher in KDM6A KO cells. H3K27 is methylated by EZH2, and we found that EZH2 inhibitor treatment significantly decreased the H3K27me3 level of the CD38 promoter area. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein expression in KDM6A KO cells. Importantly, EZH2 inhibitor also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity in vitro and in xenograft mouse model. We next re-introduced CD38 into KDM6A KO cells and found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment. This suggested that besides CD38 downregulation, there are other mechanisms underlying Dara resistance induced by KDM6A KO. We performed RNA-seq in KDM6A KO cells and found that CD48, an NK-activating ligand, was downregulated. Overexpression of CD48 in KDM6A KO cells significantly increased secretion of Granzyme B and Perforin, restoring the activity of NK cells and re-sensitizing KDM6A KO cells to Dara treatment. These data suggest that KDM6A not only regulates CD38 expression, but also NK cell activity by CD48 regulation, thereby modulating the efficacy of Dara. Taken together, our studies demonstrate that KDM6A regulates CD38 and CD48 expression in MM. Moreover, we validate that combination treatment with FDA-approved EZH2 inhibitor and Dara can overcome Dara resistance in preclinical MM models, providing the rationale for combination clinical trials to overcome Dara resistance and improve patient outcome.

Dysfunction of Cytotoxic T Lymphocyte Induced by Hepatoma Cells through the Gln-GLS2-Endoplasmic Reticulum Stress Pathway.

Metabolic activities of tumor cells lead to a depletion of nutrients within the tumor microenvironment, which results in the dysfunction of infiltrating T cells. Here, we explored how glutamine (gln) metabolism, which is essential for biosynthesis and cellular function, can affect the functions of cytotoxic T lymphocytes (CTLs). Activated CTLs were co-cultured with hepatoma cells. Western blot was used to analyze changes of proteins and ELISA was used to analyze changes of effector. RNA-sequencing was used to detect differentially expressed genes in CTLs. The status of the endoplasmic reticulum (ER) was investigated using transmission electron microscopy experiments. Co-culturing CTLs and hepatoma cells revealed that CTLL-2 cells in the co-culture group expressed high levels of PD-1 (Programmed cell death protein 1), TIM-3 (T cell immunoglobulin and mucin domain-containing protein-3), GRP78 (Glucose regulated protein 78), and P-PERK (phosphorylated protein kinase RNA-activated-like endoplasmic reticulum kinase) and secreted low levels of Granzyme B and perforin. Additionally, the substructure of the ER was severely damaged. When CTLs were treated with an inhibitor of ER stress, their functions were restored. Next, complete medium without Gln was used to culture cells, causing CTLs to display dysfunction and ER stress. WB results revealed decreased expression levels of GLS2 and SLC1A5 (Solute carrier family 1 member 5) in CTLs in the co-culture group. Subsequently, glutaminase (GLS) inhibitors were added to the cultures. As expected, CTLs treated with a GLS2 inhibitor had increased protein content of PD-1 and TIM-3, decreased secretion of Granzyme B and perforin, and an enhanced ER stress response. In summary, CTLs are functionally downregulated induced by hepatoma cells through the Gln-GLS2-ERS pathway.

The pharmacology of blinatumomab: state of the art on pharmacodynamics, pharmacokinetics, adverse drug reactions and evaluation in clinical trials.

Bispecific drugs (BDs) belong to the family of immunotherapies along with checkpoint inhibitors and CAR-T cells. In the field of oncology, BDs are designed to simultaneously bind a tumour antigen on the one side and an antigen present on the surface of effector cells on the other. This review summarizes the information available to date on the first marketed BiTE-format bispecific antibody, blinatumomab BLINCYTO® in acute lymphoblastic leukaemia. A literature search was conducted in the PubMed database by including studies published in English using the term blinatumomab. Furthermore, bibliographies of selected references were also evaluated for relevant articles. Clinical trial (CT) data were retrieved from clinicaltrials.gov (ongoing trials, adverse events [AEs]) and global pharmacovigilance data were retrieved from VigiBase®. Blinatumomab is a fusion protein which consists of two single-chain variable fragments arranged in tandem: the first binds the CD19 surface antigen of all B cells and the second targets the CD3 antigen of T cells. Binding of blinatumomab to B and T cells induces apoptosis of B cells after secretion of granzymes and perforins by T cells. T-cell activation results in secretion of pro-inflammatory cytokines and upregulation of activation markers and adhesion molecules on the surface of T cells. The major CTs that led to an indication show increased overall survival with blinatumomab with better efficacy in patients in haematological remission with minimal residual disease ≥10-3 . The major AEs are cytokine release syndrome, neurotoxicity and hypogammaglobulinemia. The three most frequent system organ classes in CTs are haematological, gastrointestinal and general disorders. These results are also found in VigiBase® but neurological disorders and infections appear more frequently in real life. This review summarizes the current knowledge of blinatumomab in the literature. The subject of many CTs is to improve the route of administration and expand the indications for treatment.

Enhanced half-life and antitumor activity of interleukin-15 through genetic fusion of a serum albumin-specific protein binder

Human interleukin-15 (hIL-15) has attracted a considerable attention as a promising cancer immunotherapeutic due to its function to directly stimulate the proliferation and cytotoxic activity of NK and T cells. Nevertheless, a relatively short half-life of hIL-15 requires repeated administration and higher doses, causing serious side effects. Here, we demonstrate an enhanced blood half-life and biological activity of hIL-15 through genetic fusion of a human serum albumin-specific protein binder (rHSA). The fusion construct (rHSA-IL15) was observed to maintain respective binding activities for both hIL-15 receptor α and human serum albumin. The rHSA-IL15 led to a significant increase in the secretion of Granzyme B and INF-γ by immune cells compare to free hIL-15, expanding the population of activated T cell subset such as CD4 + T and CD8+ T cells. The terminal half-life of the rHSA-IL15 was prolonged by around a 40-fold in transgenic mice expressing human serum albumin, compared to free hIL-15. The rHSA-IL15 resulted in distinct anti-tumor activities in xenograft SCC (squamous cell carcinoma) mouse and allograft melanoma mouse models through activation of NK and CD8+ T cells. The rHSA-IL15 is expected to be used in cancer immunotherapy, assisting in the development of other cytokines as immunotherapeutic agents with greater efficacy.

Abstract 287: Engineered 3D tissues facilitate preclinical immunotherapy studies in fully human platforms

Abstract Cancer treatment has evolved with the advent of immunotherapies. This drug class now plays a key role in supplementing or even replacing chemotherapies as first line treatments. Immunotherapies such as immune checkpoint inhibitors, bispecific antibodies, and chimeric antigen receptor T-cells (CAR-T) are just a few of the therapeutic strategies currently utilized to promote an anti-cancer response. Preclinical testing of current and novel immune modulators is necessary to expand our knowledge of these drugs and better predict their efficacy. Animal models have numerous deficiencies and complications that make them insufficient for accurate representation of human response, including an insufficient human immune system and a lack of genomic heterogeneity. Thus, there is an urgent need to develop preclinical models that incorporate human tumors and autologous immune cells together to create an accurate microenvironment for effective monitoring of tumor and immune cell interactions. We have developed a tumor-type agnostic platform in which 3D cell cultures are engineered with cells from resected human tumors. This ex vivo model not only recapitulates the tumor immune microenvironment (TIME), but it includes multiple downstream read-outs to address a single question such as whether an agent or combination of agents stimulates an immune response resulting in an anti-tumor effect. Our model can incorporate tumor and immune cells directly from the TIME or tumor cells from patient-derived xenografts or organoids which are then supplemented with allogenic or autologous immune cells or even CAR-T cells. Immune response can be detected via T-cell activation, degranulation assays, and immune cell-mediated tumor cell killing which can be multiplexed with anti-tumor activity assays. We can measure a reduction in viable tumor cells via flow cytometry following treatment or at increasing effector to target cell ratios, detect fluctuations in production and secretion of granzyme B indicating T-cell cytotoxic activity, and evaluate reduced cell viability for synergy assessments of drug combinations. We have detected increased secretion of IFNγ with combination drug treatment compared to single agent treatment. Macrophage polarization can be monitored with co-culture of CD14+ cells with tumor cells in our system inducing an M2 phenotype from an M1 phenotype. Finally, we can detect dendritic cell maturation following stimulation by measuring increased expression of MHC class II and CD103. Taken together this model system supports complex cell-cell interactions necessary to detect immunotherapy response and represents an ideal preclinical testing platform. Citation Format: Kathryn M. Appleton, Katy A. Lassahn, Ashley K. Elrod, Teresa M. DesRochers. Engineered 3D tissues facilitate preclinical immunotherapy studies in fully human platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 287.

Dominant negative TGF-β receptor type II in T lymphocytes promotes anti-tumor immunity by modulating T cell subsets and enhancing CTL responses

Transforming growth factor-β (TGF-β) is a multifunctional regulatory cytokine that maintains tolerance in the immune system by regulating the proliferation, differentiation and survival of lymphocytes. TGF-β blockade therapy for cancer has achieved some results but shows limited efficacy and side effects because these drugs are not selective and act on various types of cells throughout the body. We demonstrate here that dominant negative TGF-β receptor type II specifically targeting T cells decreases tumor load in tumor-bearing mice. In addition, the dominant negative TGF-β receptor type II promotes the proliferation and differentiation of T cells and increases the expression of T-bet, which in turn promotes the secretion of granzyme A, granzyme B, perforin and IFN-γ secreted by T cells, and enhances the cytotoxicity and anti-tumor effects of T cells. Moreover, we also found that dominant negative TGF-β receptor type II reduces the proportion of regulatory T cells (Tregs) in tumor tissue and spleen of tumor-bearing mice. Co-culture experiments with T cells and tumor cells revealed that dominant negative TGF-β receptor type II inhibited tumor cell proliferation and increased apoptosis. Our results indicate that specifically inhibiting TGF-β receptor type II in T cells increases anti-tumor immunity and has a strong therapeutic potential.

Orchestration of Immune Cells Contributes to Fibrosis in IgG4-Related Disease

This review summarizes recent progress in understanding the pathogenesis of IgG4-related disease (IgG4-RD), with a focus on fibrosis. Several studies reported that CD4+ T cells with cytotoxic activity promoted by the secretion of granzyme and perforin, cytotoxic CD4+ T cells (CD4+CTLs), and disease-specific activated B cells, infiltrated inflamed tissues and cooperated to induce tissue fibrosis in autoimmune fibrotic diseases such as IgG4-RD, systemic sclerosis, and fibrosing mediastinitis. An accumulation of cells undergoing apoptotic cell death induced by CD4+CTLs and CD8+CTLs followed by macrophage-mediated clearing and finally tissue remodeling driven by cytokines released by CD4+CTLs, activated B cells, and M2 macrophages may contribute to the activation of fibroblasts and collagen production. In IgG4-RD, this process likely involves the apoptosis of non-immune, non-endothelial cells of mesenchymal origin and subsequent tissue remodeling. In summary, CD4+CTLs infiltrate affected tissues where they may cooperate with activated B cells, CD8+CTLs, and M2 macrophages, to induce apoptosis by secreting cytotoxic cytokines. These immune cells also drive fibrosis by secreting pro-fibrotic molecules in IgG4-RD.