The role of tumor-associated macrophages in tumor immune evasion.

BACKGROUND: Tumor growth is closely linked to the activities of various cells in the tumor microenvironment (TME), particularly immune cells. During tumor progression, circulating monocytes and macrophages are recruited, altering the TME and accelerating growth. These macrophages adjust their functions in response to signals from tumor and stromal cells. Tumor-associated macrophages (TAMs), similar to M2 macrophages, are key regulators in the TME. METHODS: We review the origins, characteristics, and functions of TAMs within the TME. This analysis includes the mechanisms through which TAMs facilitate immune evasion and promote tumor metastasis. Additionally, we explore potential therapeutic strategies that target TAMs. RESULTS: TAMs are instrumental in mediating tumor immune evasion and malignant behaviors. They release cytokines that inhibit effector immune cells and attract additional immunosuppressive cells to the TME. TAMs primarily target effector T cells, inducing exhaustion directly, influencing activity indirectly through cellular interactions, or suppressing through immune checkpoints. Additionally, TAMs are directly involved in tumor proliferation, angiogenesis, invasion, and metastasis. Developing innovative tumor-targeted therapies and immunotherapeutic strategies is currently a promising focus in oncology. Given the pivotal role of TAMs in immune evasion, several therapeutic approaches have been devised to target them. These include leveraging epigenetics, metabolic reprogramming, and cellular engineering to repolarize TAMs, inhibiting their recruitment and activity, and using TAMs as drug delivery vehicles. Although some of these strategies remain distant from clinical application, we believe that future therapies targeting TAMs will offer significant benefits to cancer patients.

Role of m6A modifications in immune evasion and immunotherapy.

RNA modification has garnered increasing attention in recent years due to its pivotal role in tumorigenesis and immune surveillance. N6-methyladenosine (m6A) modification is the most prevalent RNA modification, which can affect the expression of RNA by methylating adenylate at the sixth N position to regulate the occurrence and development of tumors. Dysregulation of m6A affects the activation of cancer-promoting pathways, destroys immune cell function, maintains immunosuppressive microenvironment, and promotes tumor cell growth. In this review, we delve into the latest insights into how abnormalities in m6A modification in both tumor and immune cells orchestrate immune evasion through the activation of signaling pathways. Furthermore, we explore how dysregulated m6A modification in tumor cells influences immune cells, thereby regulating tumor immune evasion via interactions within the tumor microenvironment (TME). Lastly, we highlight recent discoveries regarding specific inhibitors of m6A modulators and the encapsulation of m6A-targeting nanomaterials for cancer therapy, discussing their potential applications in immunotherapy.

PGE2 limits effector expansion of tumour-infiltrating stem-like CD8+ T cells.

Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1-4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5-9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2-EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.

Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages.

Signaling through Notch receptors intrinsically regulates tumor cell development and growth. Here, we studied the role of the Notch ligand Jagged2 on immune evasion in non-small cell lung cancer (NSCLC). Higher expression of JAG2 in NSCLC negatively correlated with survival. In NSCLC pre-clinical models, deletion of Jag2, but not Jag1, in cancer cells attenuated tumor growth and activated protective anti-tumor T cell responses. Jag2-/- lung tumors exhibited higher frequencies of macrophages that expressed immunostimulatory mediators and triggered T cell-dependent anti-tumor immunity. Mechanistically, Jag2 ablation promoted Nr4a-mediated induction of Notch ligands DLL1/4 on cancer cells. DLL1/4-initiated Notch1/2 signaling in macrophages induced the expression of transcription factor IRF4 and macrophage immunostimulatory functionality. IRF4 expression was required for the anti-tumor effects of Jag2 deletion in lung tumors. Antibody targeting of Jagged2 inhibited tumor growth and activated IRF4-driven macrophage-mediated anti-tumor immunity. Thus, Jagged2 orchestrates immunosuppressive systems in NSCLC that can be overcome to incite macrophage-mediated anti-tumor immunity.

CD8+ T cell-based cancer immunotherapy.

The immune system in humans is a defense department against both exogenous and endogenous hazards, where CD8+ T cells play a crucial role in opposing pathological threats. Various immunotherapies based on CD8+ T cells have emerged in recent decades, showing their promising results in treating intractable diseases. However, in the fight against the constantly changing and evolving cancers, the formation and function of CD8+ T cells can be challenged by tumors that might train a group of accomplices to resist the T cell killing. As cancer therapy stepped into the era of immunotherapy, understanding the physiological role of CD8+ T cells, studying the machinery of tumor immune escape, and thereby formulating different therapeutic strategies become the imperative missions for clinical and translational researchers to fulfill. After brief basics of CD8+ T cell-based biology is covered, this review delineates the mechanisms of tumor immune escape and discusses different cancer immunotherapy regimens with their own advantages and setbacks, embracing challenges and perspectives in near future.

Aldehyde Dehydrogenese-1 High Cancer Stem-like Cells/Cancer-initiating Cells Escape from Cytotoxic T Lymphocytes due to Lower Expression of Human Leukocyte Antigen Class 1.

BACKGROUND/AIM: Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDHhigh) cells. Cancer immunotherapy employing immune checkpoint blockade has been approved for advanced gastric cancer cases. However, the effectiveness of cancer immunotherapy against gastric CSCs/CICs remains unclear. This study aimed to investigate the susceptibility of gastric CSCs/CICs to immunotherapy. MATERIALS AND METHODS: Gastric CSCs/CICs were isolated as ALDHhigh cells using the human gastric cancer cell line, MKN-45. ALDHhigh clone cells and ALDHlow clone cells were isolated using the ALDEFLUOR assay. ALDH1A1 expression was assessed via qRT-PCR. Sphere-forming ability was evaluated to confirm the presence of CSCs/CICs. A model neoantigen, AP2S1, was over-expressed in ALDHhigh clone cells and ALDHlow clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay. RESULTS: Three ALDHhigh clone cells were isolated from MKN-45 cells. ALDHhigh clone cells exhibited a stable phenotype in in vitro culture for more than 2 months. The High-36 clone cells demonstrated the highest sphere-forming ability, whereas the Low-8 cells showed the lowest sphere-forming ability. High-36 cells exhibited lower expression of HLA-A24 compared to Low-8 cells. TCR-T cells specific for AP2S1 showed lower reactivity to High-36 cells compared to Low-8 cells. CONCLUSION: High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDHhigh CSCs/CICs evade T cells due to lower expression of HLA class 1.

Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells.

Gastric cancer has a high rate of recurrence, and as such, immunotherapy strategies are being investigated as a potential therapeutic strategy. Although the involvement of immune checkpoints in immunotherapy is well studied, biomechanical cues, such as target cell stiffness, have not yet been subject to the same level of investigation. Changes in the cholesterol content of the cell membrane directly influence tumor cell stiffness. Here, we investigated the effect of cholesterol on NK cell-mediated killing of gastric cancer stem-like cells. We report that surviving tumor cells with stem-like properties elevated cholesterol metabolism to evade NK cell cytotoxicity. Inhibition of cholesterol metabolism enhances NK cell-mediated killing of gastric cancer stem-like cells, highlighting a potential avenue for improving immunotherapy efficacy. This study suggests a possible effect of cancer cell stiffness on immune evasion and offers insights into enhancing immunotherapeutic strategies against tumors.

Regulatory T cell-mediated immunosuppression orchestrated by cancer: towards an immuno-genomic paradigm for precision medicine.

Accumulating evidence indicates that aberrant signalling stemming from genetic abnormalities in cancer cells has a fundamental role in their evasion of antitumour immunity. Immune escape mechanisms include enhanced expression of immunosuppressive molecules, such as immune-checkpoint proteins, and the accumulation of immunosuppressive cells, including regulatory T (Treg) cells, in the tumour microenvironment. Therefore, Treg cells are key targets for cancer immunotherapy. Given that therapies targeting molecules predominantly expressed by Treg cells, such as CD25 or GITR, have thus far had limited antitumour efficacy, elucidating how certain characteristics of cancer, particularly genetic abnormalities, influence Treg cells is necessary to develop novel immunotherapeutic strategies. Hence, Treg cell-targeted strategies based on the particular characteristics of cancer in each patient, such as the combination of immune-checkpoint inhibitors with molecularly targeted agents that disrupt the immunosuppressive networks mediating Treg cell recruitment and/or activation, could become a new paradigm of cancer therapy. In this Review, we discuss new insights on the mechanisms by which cancers generate immunosuppressive networks that attenuate antitumour immunity and how these networks confer resistance to cancer immunotherapy, with a focus on Treg cells. These insights lead us to propose the concept of 'immuno-genomic precision medicine' based on specific characteristics of cancer, especially genetic profiles, that correlate with particular mechanisms of tumour immune escape and might, therefore, inform the optimal choice of immunotherapy for individual patients.

Phagocytosis-initiated tumor hybrid cells acquire a c-Myc-mediated quasi-polarization state for immunoevasion and distant dissemination.

While macrophage phagocytosis is an immune defense mechanism against invading cellular organisms, cancer cells expressing the CD47 ligand send forward signals to repel this engulfment. Here we report that the reverse signaling using CD47 as a receptor additionally enhances a pro-survival function of prostate cancer cells under phagocytic attack. Although low CD47-expressing cancer cells still allow phagocytosis, the reverse signaling delays the process, leading to incomplete digestion of the entrapped cells and subsequent tumor hybrid cell (THC) formation. Viable THCs acquire c-Myc from parental cancer cells to upregulate both M1- and M2-like macrophage polarization genes. Consequently, THCs imitating dual macrophage features can confound immunosurveillance, gaining survival advantage in the host. Furthermore, these cells intrinsically express low levels of androgen receptor and its targets, resembling an adenocarcinoma-immune subtype of metastatic castration-resistant prostate cancer. Therefore, phagocytosis-generated THCs may represent a potential target for treating the disease.

Y chromosome loss in cancer drives growth by evasion of adaptive immunity.

Loss of the Y chromosome (LOY) is observed in multiple cancer types, including 10-40% of bladder cancers1-6, but its clinical and biological significance is unknown. Here, using genomic and transcriptomic studies, we report that LOY correlates with poor prognoses in patients with bladder cancer. We performed in-depth studies of naturally occurring LOY mutant bladder cancer cells as well as those with targeted deletion of Y chromosome by CRISPR-Cas9. Y-positive (Y+) and Y-negative (Y-) tumours grew similarly in vitro, whereas Y- tumours were more aggressive than Y+ tumours in immune-competent hosts in a T cell-dependent manner. High-dimensional flow cytometric analyses demonstrated that Y- tumours promote striking dysfunction or exhaustion of CD8+ T cells in the tumour microenvironment. These findings were validated using single-nuclei RNA sequencing and spatial proteomic evaluation of human bladder cancers. Of note, compared with Y+ tumours, Y- tumours exhibited an increased response to anti-PD-1 immune checkpoint blockade therapy in both mice and patients with cancer. Together, these results demonstrate that cancer cells with LOY mutations alter T cell function, promoting T cell exhaustion and sensitizing them to PD-1-targeted immunotherapy. This work provides insights into the basic biology of LOY mutation and potential biomarkers for improving cancer immunotherapy.

Towards a consensus definition of immune exclusion in cancer.

Background: The immune cell topography of solid tumors has been increasingly recognized as an important predictive factor for progression of disease and response to immunotherapy. The distribution pattern of immune cells in solid tumors is commonly classified into three categories - namely, "Immune inflamed", "Immune desert" and "Immune excluded" - which, to some degree, connect immune cell presence and positioning within the tumor microenvironment to anti-tumor activity. Materials and methods: In this review, we look at the ways immune exclusion has been defined in published literature and identify opportunities to develop consistent, quantifiable definitions, which in turn, will allow better determination of the underlying mechanisms that span cancer types and, ultimately, aid in the development of treatments to target these mechanisms. Results: The definitions of tumor immune phenotypes, especially immune exclusion, have largely been conceptual. The existing literature lacks in consistency when it comes to practically defining immune exclusion, and there is no consensus on a definition. Majority of the definitions use somewhat arbitrary cut-offs in an attempt to place each tumor into a distinct phenotypic category. Tumor heterogeneity is often not accounted for, which limits the practical application of a definition. Conclusions: We have identified two key issues in existing definitions of immune exclusion, establishing clinically relevant cut-offs within the spectrum of immune cell infiltration as well as tumor heterogeneity. We propose an approach to overcome these limitations, by reporting the degree of immune cell infiltration, tying cut-offs to clinically meaningful outcome measures, maximizing the number of regions of a tumor that are analyzed and reporting the degree of heterogeneity. This will allow for a consensus practical definition for operationalizing this categorization into clinical trial and signal-seeking endpoints.

EHBP1L1 Drives Immune Evasion in Renal Cell Carcinoma through Binding and Stabilizing JAK1.

High lymphocyte infiltration and immunosuppression characterize the tumor microenvironment (TME) in renal cell carcinoma (RCC). There is an urgent need to elucidate how tumor cells escape the immune attack and to develop novel therapeutic targets to enhance the efficacy of immune checkpoint blockade (ICB) in RCC. Overactivated IFN-γ-induced JAK/STAT signaling involves in such TME, but the underlying mechanisms remain elusive. Here, EH domain-binding protein 1-like protein 1 (EHBP1L1) is identified as a crucial mediator of IFN-γ/JAK1/STAT1/PD-L1 signaling in RCC. EHBP1L1 is highly expressed in RCC, and high EHBP1L1 expression levels are correlated with poor prognosis and resistance to ICB. EHBP1L1 depletion significantly inhibits tumor growth, which is attributed to enhanced CD8+ T cell-mediated antitumor immunity. Mechanistically, EHBP1L1 interacts with and stabilizes JAK1. By competing with SOCS1, EHBP1L1 protects JAK1 from proteasomal degradation, which leads to elevated JAK1 protein levels and JAK1/STAT1/PD-L1 signaling activity, thereby forming an immunosuppressive TME. Furthermore, the combination of EHBP1L1 inhibition and ICB reprograms the immunosuppressive TME and prevents tumor immune evasion, thus significantly reinforcing the therapeutic efficacy of ICB in RCC patient-derived xenograft (PDX) models. These findings reveal the vital role of EHBP1L1 in immune evasion in RCC, which may be a potential complement for ICB therapy.

NKG2D engagement on human NK cells leads to DNAM-1 hypo-responsiveness through different converging mechanisms.

Natural killer (NK) cell activation is regulated by activating and inhibitory receptors that facilitate diseased cell recognition. Among activating receptors, NKG2D and DNAM-1 play a pivotal role in anticancer immune responses since they bind ligands upregulated on transformed cells. During tumor progression, however, these receptors are frequently downmodulated and rendered functionally inactive. Of note, NKG2D internalization has been associated with the acquisition of a dysfunctional phenotype characterized by the cross-tolerization of unrelated activating receptors. However, our knowledge of the consequences of NKG2D engagement is still incomplete. Here, by cytotoxicity assays combined with confocal microscopy, we demonstrate that NKG2D engagement on human NK cells impairs DNAM-1-mediated killing through two different converging mechanisms: by the upregulation of the checkpoint inhibitory receptor TIGIT, that in turn suppresses DNAM-1-mediated cytotoxic function, and by direct inhibition of DNAM-1-promoted signaling. Our results highlight a novel interplay between NKG2D and DNAM-1/TIGIT receptors that may facilitate neoplastic cell evasion from NK cell-mediated clearance.

Neoantigen quality predicts immunoediting in survivors of pancreatic cancer.

Cancer immunoediting1 is a hallmark of cancer2 that predicts that lymphocytes kill more immunogenic cancer cells to cause less immunogenic clones to dominate a population. Although proven in mice1,3, whether immunoediting occurs naturally in human cancers remains unclear. Here, to address this, we investigate how 70 human pancreatic cancers evolved over 10 years. We find that, despite having more time to accumulate mutations, rare long-term survivors of pancreatic cancer who have stronger T cell activity in primary tumours develop genetically less heterogeneous recurrent tumours with fewer immunogenic mutations (neoantigens). To quantify whether immunoediting underlies these observations, we infer that a neoantigen is immunogenic (high-quality) by two features-'non-selfness'  based on neoantigen similarity to known antigens4,5, and 'selfness'  based on the antigenic distance required for a neoantigen to differentially bind to the MHC or activate a T cell compared with its wild-type peptide. Using these features, we estimate cancer clone fitness as the aggregate cost of T cells recognizing high-quality neoantigens offset by gains from oncogenic mutations. With this model, we predict the clonal evolution of tumours to reveal that long-term survivors of pancreatic cancer develop recurrent tumours with fewer high-quality neoantigens. Thus, we submit evidence that that the human immune system naturally edits neoantigens. Furthermore, we present a model to predict how immune pressure induces cancer cell populations to evolve over time. More broadly, our results argue that the immune system fundamentally surveils host genetic changes to suppress cancer.

The Effect of Hypoxia and Hypoxia-Associated Pathways in the Regulation of Antitumor Response: Friends or Foes?

Hypoxia is an environmental stressor that is instigated by low oxygen availability. It fuels the progression of solid tumors by driving tumor plasticity, heterogeneity, stemness and genomic instability. Hypoxia metabolically reprograms the tumor microenvironment (TME), adding insult to injury to the acidic, nutrient deprived and poorly vascularized conditions that act to dampen immune cell function. Through its impact on key cancer hallmarks and by creating a physical barrier conducive to tumor survival, hypoxia modulates tumor cell escape from the mounted immune response. The tumor cell-immune cell crosstalk in the context of a hypoxic TME tips the balance towards a cold and immunosuppressed microenvironment that is resistant to immune checkpoint inhibitors (ICI). Nonetheless, evidence is emerging that could make hypoxia an asset for improving response to ICI. Tackling the tumor immune contexture has taken on an in silico, digitalized approach with an increasing number of studies applying bioinformatics to deconvolute the cellular and non-cellular elements of the TME. Such approaches have additionally been combined with signature-based proxies of hypoxia to further dissect the turbulent hypoxia-immune relationship. In this review we will be highlighting the mechanisms by which hypoxia impacts immune cell functions and how that could translate to predicting response to immunotherapy in an era of machine learning and computational biology.

Overexpression of DAPK1-mediated inhibition of IKKß/CSN5/PD-L1 axis enhances natural killer cell killing ability and inhibits tumor immune evasion in gastric cancer.

Gastric cancer (GC) originates from the stomach and is a prevalent human malignancy. Dysfunction of death associated protein kinase 1 (DAPK1) has been identified as a major regulator involved in the development and progression of GC. However, there's limited data regarding the regulatory mechanism of GC. Herein, we investigated role of DAPK1 in natural killer (NK) cell killing ability and immune evasion of GC cells and mediated pathway. Samples from GC-related gene expression profile and clinical samples from 67 patients with GC were collected to determine the expression of DAPK1, IκB kinase ß (IKKß), programmed death receptor-ligand 1 (PD-L1), and photomorphogenesis 9 (COP9) signalosome 5 (CSN5). The binding affinity among DAPK1, IKKß, CSN5, and PD-L1 was characterized to verify the underlying mechanism. GC lines were transfected with overexpressed plasmid or siRNA to determine the effect of DAPK1/IKKß/CSN5/PD-L1 axis on NK cell killing ability and immune evasion of GC cells. GC cells and tissues presented low expression of DAPK1 and high expression of IKKß, CSN5 and PD-L1. IKKß, negatively regulated by DAPK1, was capable of activating CSN5 and upregulating PD-L1 expression. Overexpression of DAPK1 promoted NK cell killing ability and reduced immune evasion, coupled with reduction of NK cell apoptosis and increases in levels of TNF-α, IFN-γ, CD107a, and Granzyme B cytokines. The tumor-suppressing properties of DAPK1 through downregulation of IKKß/CSN5/PD-L1 axis in GC were further confirmed in vivo. In summary, overexpression of DAPK1 promoted the NK cell killing ability and restrained immune evasion of GC cells, providing a potential therapeutic strategy for GC treatment by modulating immune evasion.

EBV miRNAs BART11 and BART17-3p promote immune escape through the enhancer-mediated transcription of PD-L1.

Epstein-Barr virus (EBV) is reportedly the first identified human tumor virus, and is closely related to the occurrence and development of nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and several lymphomas. PD-L1 expression is elevated in EBV-positive NPC and GC tissues; however, the specific mechanisms underlying the EBV-dependent promotion of PD-L1 expression to induce immune escape warrant clarification. EBV encodes 44 mature miRNAs. In this study, we find that EBV-miR-BART11 and EBV-miR-BART17-3p upregulate the expression of PD-L1 in EBV-associated NPC and GC. Furthermore, EBV-miR-BART11 targets FOXP1, EBV-miR-BART17-3p targets PBRM1, and FOXP1 and PBRM1 bind to the enhancer region of PD-L1 to inhibit its expression. Therefore, EBV-miR-BART11 and EBV-miR-BART17-3p inhibit FOXP1 and PBRM1, respectively, and enhance the transcription of PD-L1 (CD274, http://www.ncbi.nlm.nih.gov/gene/29126 ), resulting in the promotion of tumor immune escape, which provides insights into potential targets for EBV-related tumor immunotherapy.

Knockout of Factor-Inhibiting HIF (Hif1an) in Colon Epithelium Attenuates Chronic Colitis but Does Not Reduce Colorectal Cancer in Mice.

Inflammatory bowel disease such as chronic colitis promotes colorectal cancer, which is a common cause of cancer mortality worldwide. Hypoxia is a characteristic of inflammation as well as of solid tumors and enforces a gene expression response controlled by hypoxia-inducible factors (HIFs). Once established, solid tumors are immunosuppressive to escape their abatement through immune cells. Although HIF activity is known to 1) promote cancer development and 2) drive tumor immune suppression through the secretion of adenosine, both prolyl hydroxylases and an asparaginyl hydroxylase termed factor-inhibiting HIF (FIH) negatively regulate HIF. Thus, FIH may act as a tumor suppressor in colorectal cancer development. In this study, we examined the role of colon epithelial FIH in a mouse model of colitis-induced colorectal cancer. We recapitulated colitis-associated colorectal cancer development in mice using the azoxymethane/dextran sodium sulfate model in Vil1-Cre/FIH+f/+f and wild-type siblings. Colon samples were analyzed regarding RNA and protein expression and histology. Vil1-Cre/FIH+f/+f mice showed a less severe colitis progress compared with FIH+f/+f animals and a lower number of infiltrating macrophages in the inflamed tissue. RNA sequencing analyses of colon tissue revealed a lower expression of genes associated with the immune response in Vil1-Cre/FIH+f/+f mice. However, tumor occurrence did not significantly differ between Vil1-Cre/FIH+f/+f and wild-type mice. Thus, FIH knockout in colon epithelial cells did not modulate colorectal cancer development but reduced the inflammatory response in chronic colitis.

G6PD functions as a metabolic checkpoint to regulate granzyme B expression in tumor-specific cytotoxic T lymphocytes.

BACKGROUND: Granzyme B is a key effector of cytotoxic T lymphocytes (CTLs), and its expression level positively correlates with the response of patients with mesothelioma to immune checkpoint inhibitor immunotherapy. Whether metabolic pathways regulate Gzmb expression in CTLs is incompletely understood. METHODS: A tumor-specific CTL and tumor coculture model and a tumor-bearing mouse model were used to determine the role of glucose-6-phosphate dehydrogenase (G6PD) in CTL function and tumor immune evasion. A link between granzyme B expression and patient survival was analyzed in human patients with epithelioid mesothelioma. RESULTS: Mesothelioma cells alone are sufficient to activate tumor-specific CTLs and to enhance aerobic glycolysis to induce a PD-1hi Gzmblo CTL phenotype. However, inhibition of lactate dehydrogenase A, the key enzyme of the aerobic glycolysis pathway, has no significant effect on tumor-induced CTL activation. Tumor cells induce H3K9me3 deposition at the promoter of G6pd, the gene that encodes the rate-limiting enzyme G6PD in the pentose phosphate pathway, to downregulate G6pd expression in tumor-specific CTLs. G6PD activation increases acetyl-coenzyme A (CoA) production to increase H3K9ac deposition at the Gzmb promoter and to increase Gzmb expression in tumor-specific CTLs converting them from a Gzmblo to a Gzmbhi phenotype, thus increasing CTL tumor lytic activity. Activation of G6PD increases Gzmb+ tumor-specific CTLs and suppresses tumor growth in tumor-bearing mice. Consistent with these findings, GZMB expression level was found to correlate with increased survival in patients with epithelioid mesothelioma. CONCLUSION: G6PD is a metabolic checkpoint in tumor-activated CTLs. The H3K9me3/G6PD/acetyl-CoA/H3K9ac/Gzmb pathway is particularly important in CTL activation and immune evasion in epithelioid mesothelioma.

Digital gene expression analysis of NSCLC-patients reveals strong immune pressure, resulting in an immune escape under immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are currently one of the most promising therapy options in the field of oncology. Although the first pivotal ICI trial results were published in 2011, few biomarkers exist to predict their therapy outcome. PD-L1 expression and tumor mutational burden (TMB) were proven to be sometimes-unreliable biomarkers. We have previously suggested the analysis of processing escapes, a qualitative measurement of epitope structure alterations under immune system pressure, to provide predictive information on ICI response. Here, we sought to further validate this approach and characterize interactions with different forms of immune pressure. METHODS: We identified a cohort consisting of 48 patients with advanced non-small cell lung cancer (NSCLC) treated with nivolumab as ICI monotherapy. Tumor samples were subjected to targeted amplicon-based sequencing using a panel of 22 cancer-associated genes covering 98 mutational hotspots. Altered antigen processing was predicted by NetChop, and MHC binding verified by NetMHC. The NanoString nCounter® platform was utilized to provide gene expression data of 770 immune-related genes. Patient data from 408 patients with NSCLC were retrieved from The Cancer Genome Atlas (TCGA) as a validation cohort. RESULTS: The two immune escape mechanisms of PD-L1 expression (TPS score) (n = 18) and presence of altered antigen processing (n = 10) are mutually non-exclusive and can occur in the same patient (n = 6). Both mechanisms have exclusive influence on different genes and pathways, according to differential gene expression analysis and gene set enrichment analysis, respectively. Interestingly, gene expression patterns associated with altered processing were enriched in T cell and NK cell immune activity. Though both mechanisms influence different genes, they are similarly linked to increased immune activity. CONCLUSION: Pressure from the immune system will lay the foundations for escape mechanisms, leading to acquisition of resistance under therapy. Both PD-L1 expression and altered antigen processing are induced similarly by pronounced immunoactivity but in different context. The present data help to deepen our understanding of the underlying mechanisms behind those immune escapes.