Immune Checkpoint Regulators Research Articles

Reprogramming Cancer Cells to Antigen-presenting Cells.

Cancer cells evade the immune system by downregulating antigen presentation. Although immune checkpoint inhibitors (ICI) and adoptive T-cell therapies revolutionized cancer treatment, their efficacy relies on the intrinsic immunogenicity of tumor cells and antigen presentation by dendritic cells. Here, we describe a protocol to directly reprogram murine and human cancer cells into tumor-antigen-presenting cells (tumor-APCs), using the type 1 conventional dendritic cell (cDC1) transcription factors PU.1, IRF8, and BATF3 delivered by a lentiviral vector. Tumor-APCs acquire a cDC1 cell-like phenotype, transcriptional and epigenetic programs, and function within nine days (Zimmermannova et al., 2023). Tumor-APCs express the hematopoietic marker CD45 and acquire the antigen presentation complexes MHC class I and II as well as co-stimulatory molecules required for antigen presentation to T cells, but do not express high levels of negative immune checkpoint regulators. Enriched tumor-APCs present antigens to Naïve CD8+ and CD4+ T cells, are targeted by activated cytotoxic T lymphocytes, and elicit anti-tumor responses in vivo. The tumor-APC reprogramming protocol described here provides a simple and robust method to revert tumor evasion mechanisms by increasing antigen presentation in cancer cells. This platform has the potential to prime antigen-specific T-cell expansion, which can be leveraged for developing new cancer vaccines, neoantigen discovery, and expansion of tumor-infiltrating lymphocytes. Key features • This protocol describes the generation of antigen-presenting cells from cancer cells by direct reprogramming using lineage-instructive transcription factors of conventional dendritic cells type I. • Verification of reprogramming efficiency by flow cytometry and functional assessment of tumor-APCs by antigen presentation assays.

Immune checkpoints and cancer immunotherapies: insights into newly potential receptors and ligands.

Checkpoint markers and immune checkpoint inhibitors have been increasingly identified and developed as potential immunotherapeutic targets in various human cancers. Despite valuable efforts to discover novel immune checkpoints and their ligands, the precise roles of their therapeutic functions, as well as the broad identification of their counterpart receptors, remain to be addressed. In this context, it has been suggested that various putative checkpoint receptors can be induced upon activation. In the tumor microenvironment, T cells, as crucial immune response against malignant diseases as well as other immune central effector cells, such as natural killer cells, are regulated via co-stimulatory or co-inhibitory signals from immune or tumor cells. Studies have shown that exposure of T cells to tumor antigens upregulates the expression of inhibitory checkpoint receptors, leading to T-cell dysfunction or exhaustion. Although targeting immune checkpoint regulators has shown relative clinical efficacy in some tumor types, most trials in the field of cancer immunotherapies have revealed unsatisfactory results due to de novo or adaptive resistance in cancer patients. To overcome these obstacles, combinational therapies with newly discovered inhibitory molecules or combined blockage of several checkpoints provide a rationale for further research. Moreover, precise identification of their receptors counterparts at crucial checkpoints is likely to promise effective therapies. In this review, we examine the prospects for the application of newly emerging checkpoints, such as T-cell immunoglobulin and mucin domain 3, lymphocyte activation gene-3, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig suppressor of T-cell activation (VISTA), new B7 family proteins, and B- and T-cell lymphocyte attenuator, in association with immunotherapy of malignancies. In addition, their clinical and biological significance is discussed, including their expression in various human cancers, along with their roles in T-cell-mediated immune responses.

Immune checkpoint regulation of cardiac lymphatic remodeling in cardiovascular diseases

Immune checkpoint regulation of cardiac lymphatic remodeling in cardiovascular diseases

TGF-β1 suppresses the T-cell response in teleost fish by initiating Smad3- and Foxp3-mediated transcriptional networks

Transforming growth factor-β1 (TGF-β1) can suppress the activation, proliferation, and function of many T-cell subsets, protecting organisms from inflammatory and autoimmune disease caused by an overexuberant immune response. However, whether and how TGF-β1 regulates T-cell immunity in early vertebrates remain unknown. Here, using a Nile tilapia (Oreochromis niloticus) model, we investigated suppression of the T-cell response by TGF-β1 in teleost species. Tilapia encodes an evolutionarily conserved TGF-β1, the expression of which in lymphocytes is significantly induced during the immune response following Edwardsiella piscicida infection. Once activated, tilapia T cells increase TGF-β1 production, which in turn suppresses proinflammatory cytokine expression and inhibits T-cell activation. Notably, we found administration of TGF-β1 cripples the proliferation of tilapia T cells, reduces the potential capacity of Th1/2 differentiation, and impairs the cytotoxic function, rendering the fish more vulnerable to bacterial infection. Mechanistically, TGF-β1 initiates the TGF-βR/Smad signaling pathway and triggers the phosphorylation and nuclear translocation of Smad2/3. Smad3 subsequently interacts with several transcriptional partners to repress transcription of cytokines IL-2 and IFN-γ but promote transcription of immune checkpoint regulator CTLA4 and transcription factor Foxp3. Furthermore, TGF-β1/Smad signaling further utilizes Foxp3 to achieve the cascade regulation of these T-cell genes. Taken together, our findings reveal a detailed mechanism by which TGF-β1 suppresses the T cell-based immunity in Nile tilapia and support the notion that TGF-β1 had already been employed to inhibit the T-cell response early in vertebrate evolution, thus providing novel insights into the evolution of the adaptive immune system.

Disruption of CISH promotes the antitumor activity of human T cells and decreases PD-1 expression levels

Tumor cells and the immunosuppressive tumor microenvironment suppress the antitumor activity of Tcells through immune checkpoints, including the PD-L1/PD-1 axis. Cytokine-inducible SH2-containing protein (CISH), a member of the suppressor of cytokine signaling (SOCS) family, inhibits JAK-STAT and Tcell receptor (TCR) signaling in T and natural killer (NK) cells. However, its role in the regulation of immune checkpoints in Tcells remains unclear. In this study, we ablated CISH in Tcells with CRISPR-Cas9 and found that the sensitivity of Tcells to TCR and cytokine stimulation was increased. In addition, chimeric antigen receptor Tcells with CISH deficiency exhibited longer survival and higher cytokine secretion and antitumor activity. Notably, PD-1 expression was decreased in activated CISH-deficient Tcells invitro and invivo. The level of FBXO38, a ubiquitination-regulating protein that reduces PD-1 expression, was elevated in activated Tcells after CISH ablation. Hence, this study reveals a mechanism by which CISH promotes PD-1 expression by suppressing the expression of FBXO38 and proposes a new strategy for augmenting the therapeutic effect of CAR-T cells by inhibiting CISH.

Digital Spatial Profiling of Glomerular Gene Expression in Pauci-Immune Focal Necrotizing Glomerulonephritis.

Pauci-immune focal necrotizing glomerulonephritis (piFNGN) involves asynchronous onset and progression of injurious lesions in biopsies. Pathologists can describe this heterogeneity within a biopsy, but translating the information into prognostic or expression analyses is challenging. Understanding the underlying molecular processes could improve treatment; however, bulk or single-cell transcriptomic analyses of dissociated tissue disregard the heterogeneity of glomerular injury. We characterize protein and mRNA expression of individual glomeruli in 20 biopsies from 18 patients with antineutrophil cytoplasmic antibody-associated piFNGN using the NanoString digital spatial profiling (DSP) platform. For this purpose, circular annotations of glomeruli were analyzed using protein, immuno-oncology RNA, and Cancer Transcriptome Atlas panels (n=120, 72, and 48 glomeruli, respectively). Histologic evaluation of glomerular patterns of injury was performed in adjacent serial sections. Expression data were processed by log2 transformation, quantile normalization, and batch adjustment. DSP revealed distinct but overlapping gene expression profiles relating to the morphological evolution of injurious lesions, including dynamic expression of various immune checkpoint regulators. Enrichment analysis indicated deregulated pathways that underline known and highlight novel potential mechanisms of disease. Moreover, by capturing individual glomeruli, DSP describes heterogeneity between and within biopsies. We demonstrate the benefit of spatial profiling for characterization of heterogeneous glomerular injury, indicating novel molecular correlates of glomerular injury in piFNGN.

Digital spatial profiling of glomerular gene expression in pauci-immune focal necrotizing glomerulonephritis

Introduction: Pauci-immune focal necrotizing glomerulonephritis (piFNGN) involves asynchronous onset and progression of injurious lesions in biopsies. Pathologists describe this heterogeneity within a biopsy, but translating the information into prognostic or expression analyses is challenging. Understanding the underlying molecular processes could improve treatment; however, bulk or single cell transcriptomic analyses of dissociated tissue disregard the heterogeneity of glomerular injury. Methods: We characterize protein and mRNA expression of individual glomeruli in 20 biopsies from 18 patients with anti-neutrophil cytoplasmic antibody-associated piFNGN using the Nanostring digital spatial profiling (DSP) platform. For this purpose, circular annotations of glomeruli were analyzed using protein, immuno-oncology RNA, and cancer transcriptome atlas panels (n=120, 72, and 48 glomeruli, respectively). Histological evaluation of glomerular patterns of injury was performed in adjacent serial sections. Expression data was processed by log2 transformation, quantile normalization and batch adjustment. Results: DSP revealed distinct but overlapping gene expression profiles relating to the morphological evolution of injurious lesions, including dynamic expression of various immune checkpoint regulators. Enrichment analysis indicated deregulated pathways that underline known and highlight novel potential mechanisms of disease. Moreover, by capturing individual glomeruli, DSP describes heterogeneity between and within biopsies. Conclusions: We demonstrate the benefit of spatial profiling for characterization of heterogeneous glomerular injury, indicating novel molecular correlates of glomerular injury in piFNGN.

Comprehensive Transcriptomic Analysis of VISTA in Acute Myeloid Leukemia: Insights into Its Prognostic Value.

The V-domain Ig suppressor of T-cell activation (VISTA) has been recognized as a critical negative regulator of antitumor immune response and is gaining growing interest as a potential pharmacological target in immunotherapy. This molecule is highly expressed in hematopoietic stem cells and myeloid compartment, and it has been found upmodulated in acute myeloid leukemia (AML). However, VISTA-associated immune features are relatively unexplored in myeloid malignancies. Herein, we aimed to explore whether this immune checkpoint regulator could play a role in the generation of an immune escape environment in AML patients. We characterized VISTA mRNA expression levels in leukemia cell lines and in large publicly available cohorts of specimens from bone marrow of healthy individuals and AML patients at diagnosis by deploying bulk and single-cell RNA sequencing. We also defined the correlations with leukemia-associated burden using results of whole-exome sequencing of AML samples at disease onset. We showed that VISTA expression linearly increased across the myeloid differentiation tree in normal hematopoiesis. Accordingly, its transcript was highly enriched in AML cell lines as well as in AML patients at diagnosis presenting with myelomonocytic and monocytic differentiation. A strong correlation was seen with NPM1 mutations regardless of the presence of FLT3 lesions. Furthermore, VISTA expression levels at baseline correlated with disease recurrence in patients with normal karyotype and NPM1 mutations, a subgroup traditionally considered as favorable according to current diagnostic schemes. Indeed, when compared to patients with long-term remission (>5 years after standard chemotherapy regimens), cases relapsing within 2 years from diagnosis had increased VISTA expression in both leukemia and T cells. Our results suggest a rationale for developing VISTA-targeted therapeutic strategies to treat molecularly defined subgroups of AML patients to prevent disease recurrence and treatment resistance.

Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation

Cases of the spontaneous regression of multiple pulmonary metastases, after radiofrequency ablation (RFA), of a single lung metastasis, have been documented to be mediated by the immune system. The interaction of immune checkpoints, e.g., PD-1/PD-L1 and CTLA-4/CD80, may explain this phenomenon. The purpose of this study is to identify and quantify immune mechanisms triggered by RFA of pulmonary metastases originating from colorectal cancer. We used two-site time-resolved Förster resonance energy transfer as determined by frequency-domain FLIM (iFRET) for the quantification of receptor-ligand interactions. iFRET provides a method by which immune checkpoint interaction states can be quantified in a spatiotemporal manner. The same patient sections were used for assessment of ligand-receptor interaction and intratumoral T-cell labeling. The checkpoint interaction states quantified by iFRET did not correlate with ligand expression. We show that immune checkpoint ligand expression as a predictive biomarker may be unsuitable as it does not confirm checkpoint interactions. In pre-RFA-treated metastases, there was a significant and negative correlation between PD-1/PD-L1 interaction state and intratumoral CD3+ and CD8+ density. The negative correlation of CD8+ and interactive states of PD-1/PD-L1 can be used to assess the state of immune suppression in RFA-treated patients.

The role of YAP1 in survival prediction, immune modulation, and drug response: A pan-cancer perspective.

Dysregulation of the Hippo signaling pathway has been implicated in multiple pathologies, including cancer, and YAP1 is the major effector of the pathway. In this study, we assessed the role of YAP1 in prognostic value, immunomodulation, and drug response from a pan-cancer perspective. We compared YAP1 expression between normal and cancerous tissues and among different pathologic stages survival analysis and gene set enrichment analysis were performed. Additionally, we performed correlation analyses of YAP1 expression with RNA modification-related gene expression, tumor mutation burden (TMB), microsatellite instability (MSI), immune checkpoint regulator expression, and infiltration of immune cells. Correlations between YAP1 expression and IC50s (half-maximal inhibitory concentrations) of drugs in the CellMiner database were calculated. We found that YAP1 was aberrantly expressed in various cancer types and regulated by its DNA methylation and post-transcriptional modifications, particularly m6A methylation. High expression of YAP1 was associated with poor survival outcomes in ACC, BLCA, LGG, LUAD, and PAAD. YAP1 expression was negatively correlated with the infiltration of CD8+ T lymphocytes, CD4+ Th1 cells, T follicular helper cells, NKT cells, and activated NK cells, and positively correlated with the infiltration of myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs) in pan-cancer. Higher YAP1 expression showed upregulation of TGF-β signaling, Hedgehog signaling, and KRAS signaling. IC50s of FDA-approved chemotherapeutic drugs capable of inhibiting DNA synthesis, including teniposide, dacarbazine, and doxorubicin, as well as inhibitors of hypoxia-inducible factor, MCL-1, ribonucleotide reductase, and FASN in clinical trials were negatively correlated with YAP1 expression. In conclusion, YAP1 is aberrantly expressed in various cancer types and regulated by its DNA methylation and post-transcriptional modifications. High expression of YAP1 is associated with poor survival outcomes in certain cancer types. YAP1 may promote tumor progression through immunosuppression, particularly by suppressing the infiltration of CD8+ T lymphocytes, CD4+ Th1 cells, T follicular helper cells, NKT cells, and activated NK cells, as well as recruiting MDSCs and CAFs in pan-cancer. The tumor-promoting activity of YAP1 is attributed to the activation of TGF-β, Hedgehog, and KRAS signaling pathways. AZD2858 and varlitinib might be effective in cancer patients with high YAP1 expression.

High Dimensional Mapping of Bone Marrow Immune Characteristics in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an acquired autoimmune disease that is characterized by increased antibody-mediated platelet destruction, decreased megakaryopoiesis and impaired thrombopoiesis. An abnormal bone marrow microenvironment has been proposed to play an essential role in the progression of ITP [Stem Cells Dev, 2017]. Cytometry by Time of Flight (CyTOF) is a new analytic strategy that provides single-cell proteomics for enhanced cellular analysis, and describes the components of immune cell populations and the characteristics of new biomarkers. Here, we aim to provide a high-dimensional overview and summary of the changes in cell subsets and functions in the bone marrow of patients with ITP using CyTOF. Methods A total of 40 patients diagnosed with ITP (21 men and 19 women; 23-71 years of age, median 47 years) were enrolled between July and October 2021. Based on the onset and progression of the disease, the ITP patients were classified into 3 subgroups. Group ITP-N1 included 15 newly diagnosed treatment-naïve patients ≤3 months after diagnosis, Group ITP-N2 included 10 patients with an ITP diagnosis of > 3 months who had not received any standard treatment (>3-12 months after diagnosis), and Group ITP-R included 15 patients who had received standard treatment but relapsed following remission. The healthy control (HC) group consisted of 10 donors who underwent hematopoietic stem cell transplantation. CyTOF panels were designed using 42 parameters that were measured at the single-cell level in the bone marrow of the ITP patients and HCs. The plasma levels of immune checkpoint regulators, such as PD-1, PD-L1, CTLA-4, TIM-3, LAG-3 and Galectin-9, were measured using a multianalyte flow assay kit (BioLegend). Results The platelet counts of enrolled ITP patients ranged from 1 to 96×109/L, with a median count of 25×109/L. Using CyTOF, the immune cells in the bone marrow were classified into 7 major subsets, including B cells, γδT cells, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, dendritic cells (DCs), and monocytes. The proportions of DCs and monocytes were significantly different between the ITP patients and HCs. The proportion of monocytes was observed to increase significantly in the bone marrow of the ITP patients. The proportion of the pDC subset of DCs showed a trend of being reduced in the ITP patients, while the proportion of mDCs was increased significantly in the ITP patients. NK cells can be divided into 2 subsets according to the expression of CD56, and these subsets exhibit different cytokine production patterns and cytotoxic functions. The proportion of CD56low NK cells was elevated and the proportion of CD56bright NK cells decreased significantly in ITP-R patients compared to HCs; similar results were not observed in the ITP-N1 or ITP-N2 groups. NK cells in bone marrow may play an important role in the relapse of ITP. We further investigated the functional status of T cells in the ITP patients using a CyTOF panel. The results showed that the proportions of both CD4+ naïve and CD8+ naïve T cells were decreased in the bone marrow of the ITP patients. In particular, the proportion of CD8+ effector T cells was increased significantly in the ITP patients. Next, we analyzed coinhibitory and costimulatory receptor expression in each T-cell subpopulation. CTLA4 expression was observed to be reduced in CD8+ effector T cells and CD4+ Tfh cells. In addition to the changes in coinhibitory receptor expression, costimulatory molecules such as CD28 and ICOS were found to be expressed at higher levels in CD4+ central memory T cells. Combined with the soluble regulators in plasma, we found that the B7.2 levels in plasma was positively correlated with the cell surface expression of CD28 and negatively correlated with the expression of CTLA4 in T cells. Conclusions This study provides a comprehensive overview of the human bone marrow immune characteristics of patients with ITP. Using CyTOF to conduct single-cell analyses, we mapped myeloid and T-cell subset phenotypes in particular and focused on the expression of coinhibitory molecules such as CTLA4. The different proportions of bone marrow immune cells and patterns of immune checkpoint molecule expression in different stages of ITP may provide novel insights into disease etiology and offer promising therapeutic targets for patients with ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Construction of an Iodine-Labeled CS1001 Antibody for Targeting PD-L1 Detection and Comparison with Low-Molecular-Peptide Micro-PET Imaging.

Programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), the research focus in immune checkpoint regulation, play an important role in tumor immunotherapy. Inhibitors of this pathway are also the focus of tumor immunotherapy research. The PD-1/PD-L1 pathway can be blocked by selective binding to PD-L1. Clinical trials have been conducted in a variety of patients with advanced solid tumors. CS1001 is a high-affinity humanized full-length anti-PD-L1 monoclonal antibody with great clinical significance. We constructed a PD-L1-targeted radioactive molecular probe, 124/125I-labeled full-length antibody CS1001, and evaluated its binding specificity and targeting ability to PD-L1 in tumor cells and tumor models. Additionally, a comparison study with 68Ga-WL12, a PD-L1 targeting peptide, was conducted. The binding potency of 125I-CS1001 to human PD-L1 was evaluated by enzyme-linked immunosorbent assay (ELISA), and the Kd value was 52.1 ± 19.3 nM. The cellular uptake of 125I-CS1001 was examined in Chinese hamster ovary cells (CHO) and CHO expressing human PD-L1 (CHO-hPD-L1). At 2 h, the uptake values of 125I-CS1001 in CHO-hPD-L1 without blocking and in the presence of 0.1 mg non-radiolabeled CS1001 were 3.60 ± 0.08 and 0.09 ± 0.005 (%AD/2 × 105 cells, p < 0.001). Micro-PET imaging was performed between 8 to 192 h after injection of 124I-CS1001 into normal KM mice and CHO-hPD-L1 and HeLa tumor models. The standard uptake value (SUV) of relevant organs in PET images was calculated by drawing regions of interest (ROI). SUVmean of CHO-hPD-L1 tumors was significantly higher than that of HeLa tumors at 48 h (1.98 ± 0.04 vs 0.73 ± 0.14, p = 0.005). The SUVmean of 124I-CS1001 in CHO-hPD-L1 tumors at 48 h was higher than that of 68Ga-WL12 in CHO-hPD-L1 tumors at 0.5 h (1.98 ± 0.04 vs 1.09 ± 0.1 SUVmean, p = 0.007). In conclusion, this work provides a new method for monitoring and evaluating the in vivo expression of PD-L1 in tumors.

Plasma-Based microRNA Expression Analysis in Advanced Stage NSCLC Patients Treated with Nivolumab.

Simple SummaryNivolumab (anti-PD-1 inhibitor) is the first monoclonal antibody approved for the treatment of NSCLC, with research results showing that patients who had received previous lines of therapy had a better response to this treatment and better overall survival. Tissue-level analyses fail to capture the dynamic tumor-host relationship, in contrast to circulating biomarkers, which can reflect the systemic response of the tumor, allowing for repeated sampling and monitoring. In the context of liquid biopsy, microRNAs are studied as biomarkers of immunotherapy efficacy based on their role in regulating antitumor immunity. The present study suggests that miR-200c and miR-34a plasma expression levels have a prognostic role in patients with advanced NSCLC receiving Nivolumab. It further supports that the expression profile of circulating immunomodulatory microRNAs provides information on the survival of patients with advanced NSCLC receiving Nivolumab and could represent promising circulating biomarkers that may provide information about patients’ responses to immunotherapy.Since circulating microRNAs (miRNAs) are involved in the modulation of the immune response, they are tested as liquid biopsy-based biomarkers in patients with NSCLC treated with immunotherapy. We analyzed the expression levels and examined the clinical significance of immunoregulatory miRNAs involved in immune checkpoint regulation (miR-34a, miR-200b, miR-200c), T-cell activity (miR-155), and the function of myeloid-derived suppressive cells (MDSCs) (miR-223) or regulatory T lymphocytes (Tregs) (miR-146a), in patients with advanced NSCLC (N = 69) treated with anti-PD-1 (Nivolumab) immunotherapy as 2nd or 3rd line of treatment therapy. Plasma levels of circulating miRNAs were analyzed by RT-qPCR before the initiation of immunotherapy. Expression of miR-34a, miR-146a, mir-200c, and miR-223 was found to be associated with response to immunotherapy. High miR-200c expression emerged as an independent prognostic factor for inferior overall survival in all patients with NSCLC (OS, HR: 2.243, 95% CI: 1.208–4.163; p = 0.010) and in patients with non-Squamous (non-SqCC) subtype (N = 38) (HR: 2.809, 95% CI: 1.116–7.074; p = 0.028). Low miR-34a expression independently predicted for shorter OS (HR: 3.189, 95% CI: 1.193–8.527; p = 0.021) in the non-SqCC subgroup. Our findings suggest that alterations in circulating miR-200c and miR-34a expression levels are associated with the response and outcome in patients with advanced NSCLC treated with anti-PD1 immunotherapy.

CMTM3 as a Potential New Immune Checkpoint Regulator.

Objectives To evaluate the role of CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) in tumor microenvironment and cancer immunotherapy and explore its potential mechanism. Method The cancer genome map was obtained from the UCSC Xena database. RNAseq data from the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases were utilized for evaluating the expression and prognostic value of CMTM3 through survival data of clinical trials. The enrichment analysis of CMTM3 was performed using the R package “clusterProfiler.” The scores of immune cell infiltration in TCGA samples were downloaded from the ImmuCellAI database and TIMER2 database, and the relationship between both immune cell invasion and CMTM3 expression was investigated. Immunological activation and suppression genes, immune checkpoints, chemokines, and their receptors were all investigated in relation to CMTM3. Results Most tumor types had varied levels of CMTM3 expression and predicted poor survival status. The CMTM3 expression is closely associated with cancer-associated fibroblasts, macrophages, myeloid dendritic cells, endothelial cells, immune activation genes, immune suppressor genes, immune checkpoints, chemokines, and related receptors. Conclusion Our data reveal that CMTM3 might be used as a cancer biomarker. CMTM3 may work in conjunction with other immunological checkpoints to alter the immune milieu, which could lead to the establishment of new immunotherapy medicines.

Expression of immune checkpoint regulators, programmed death-ligand 1 (PD-L1/PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), and indolaimine-2, 3-deoxygenase (IDO) in uterine mesenchymal tumors

BackgroundImmune checkpoints including programmed death-ligand 1/programmed death-1/ (PD-L1/PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), and indolaimine-2, 3-deoxygenase (IDO) have recently emerged as effective candidates for treatment against a range of human malignancies. We have investigated their expression in the uterine mesenchymal tumors.MethodsSixty-eight mesenchymal tumors were categorized into 6 diagnostic groups. We assessed PD-L1, PD-1, CTLA-4, and IDO expression on paraffin embedded tissue blocks of the uterine tumors using the respective antibodies. Immunohistochemical (IHC) stains were classified as positive when the reactions were present in at least 1% of the cell membranes for PD-L1/PD-1 or in cytoplasm for CTLA-4 and IDO, regardless of intensity. Student’s t-test and McNemar’s chi-square tests were carried out to analyze the results.ResultsThe mesenchymal neoplasms had expressed the immune checkpoints in the tumor and/or the lymphoid cells at the rate of 49% and 54% respectively. The tumor cells were positive in 10 (18%, PD-L1), 0 (0%, PD-1), 18 (32%, CTLA-4), and 13 (23%, IDO) cases while the infiltrating lymphoid cells were positive in 10 (18%, PD-L1), 23 (40%, PD-1), 18 (32%, CTLA-4), and 13 (23%, IDO) cases. Overall, comparison of paired tumor vs lymphoid cells resulted in p-values of ≤ 0.04.ConclusionsNearly 50% of the uterine tumors express at least one of the immune checkpoints in tumor and/or the infiltrating lymphoid cells. However, expression of the proteins in the two cellular components are mutually exclusive. Namely, when tumor cells express an immune checkpoint, the infiltrating lymphoid cells do not, and vice versa. Since the leiomyosarcomas are reportedly resistant to the immunotherapy when PD-L1 is expressed in the tumor cells, it can be posited that presence of the IHC positive lymphoid cells may be a better indicator of response to the treatment.

Prognostic impacts of soluble immune checkpoint regulators and cytokines in patients with SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) has been a pandemic for the past two years. Predicting patient prognosis is critical. Although immune checkpoints (ICs) were shown to be involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, quantitative studies of ICs in clinical practice are limited. In this study, various soluble ICs (sICs) and cytokine levels in patients with SARS-CoV-2 infection at different time points were compared between survivors and deaths; we also examined whether sICs are useful for predicting prognosis. sICs and cytokines were measured in serum samples from 38 patients diagnosed with COVID-19 in the first and second week post-diagnosis. All assays were performed by bead-based multiplexed immunoassay system using Luminex Bio-Plex 200 system. The correlation of sICs and cytokines with laboratory markers was evaluated, and the levels of sICs in survivors were compared with those in deaths. Among the sICs, the second-week levels of soluble cluster of differentiation (sCD27, p = 0.012), sCD40 (p< 0.001), cytotoxic T-lymphocyte-associated protein 4 (sCTLA-4, p< 0.001), herpes virus entry mediator (sHVEM, p = 0.026), and T-cell immunoglobulin and mucin-domain containing-3 (sTIM-3, p = 0.002) were significantly higher in deaths than in survivors. The levels of nine cytokines assessed in the second week of deaths were significantly higher than those in survivors. The sICs sCD27, sCD40, sCTLA-4, and sTIM-3 and cytokines chemokine CC motif ligand 2 (CCL2), GM-CSF, IL-10, and IL-8 showed significant positive correlations with the levels of C-reactive protein (CRP) and procalcitonin and were negatively correlated with the absolute lymphocyte count and platelet values. Increased levels of sICs including sCD27, sCD40, sCTLA-4, and sTIM-3 and cytokines were significant factors for poor prognosis. sICs, together with cytokines and inflammatory markers, may be useful as prognostic stratification markers in SARS-CoV-2-infected patients.

CTLA4 , PD-1 , PD-L1 , PD-L2 , TIM-3 , TIGIT , and LAG3 DNA Methylation Is Associated With BAP1 -Aberrancy, Transcriptional Activity, and Overall Survival in Uveal Melanoma.

Uveal melanoma (UM) is an aggressive disease with poor response to oncological treatment, including immunotherapy. Loss of the epigenetic modifier BRCA1-associated protein 1 (BAP1) function drives UM oncogenesis and is associated with an immune-suppressive tumor microenvironment, poor prognosis, and a distinct DNA methylation and gene expression profile. Our study aimed to analyze comprehensively the DNA methylation status of the immune checkpoint genes PD-1 , PD-L1 , PD-L2 , CTLA4, TIM-3 ( HAVCR2 ), TIGIT , and LAG3 and its association with mRNA expression, BAP1 -aberrancy, and patients' survival. We analyzed the DNA methylation landscape of immune checkpoint genes at single CpG resolution in N=80 UM samples provided by The Cancer Genome Atlas. We analyzed CpG methylation levels of the immune checkpoints with regard to their transcriptional signatures and patient outcomes.Methylation of specific CpG sites within the immune checkpoint genes PD-1 , PD-L1 , PD-L2 , CTLA4 , TIM-3 , TIGIT , and LAG3 correlated strongly with mRNA expression levels, indicating a strong regulation of gene expression through DNA methylation. Moreover, immune checkpoint gene methylation was strongly associated with BAP1 -mutation status and associated with overall survival in UM. Our data indicate an epigenetic regulation of immune checkpoints through DNA methylation in UM. Further, our data highlight the prognostic significance of DNA methylation of immune checkpoint genes in UM thereby providing a rationale for methylation testing as predictive biomarkers for immunotherapy response.

626 Targeting a novel carbohydrate that serves as an immune checkpoint and angiogenic regulator for treatment of melanoma

We discovered DC-HIL/Gpnmb receptor expressed by melanoma and myeloid-derived suppressor cells (MDSC), with 2 functions, immune checkpoint and angiogenesis, both mediated by its binding to syndecan-4 (SD4) on activated T cells and select endothelial cells (EC), respectively. Using microarray technology, we now report that DC-HIL’s precise ligand on SD4 is a rare heparan sulfate (rHS) saccharide sulfated highly at positions 2-O/6-O. Consistently with this discovery, HS sulfotransferases required for rHS synthesis rises markedly during T-cell activation, and knocking-down gene expression by specific shRNA abrogated DC-HIL binding to T cells/EC.

ATP and Adenosine Metabolism in Cancer: Exploitation for Therapeutic Gain.

Adenosine is an evolutionary ancient metabolic regulator linking energy state to physiologic processes, including immunomodulation and cell proliferation. Tumors create an adenosine-rich immunosuppressive microenvironment through the increased release of ATP from dying and stressed cells and its ectoenzymatic conversion into adenosine. Therefore, the adenosine pathway becomes an important therapeutic target to improve the effectiveness of immune therapies. Prior research has focused largely on the two major ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase 1/cluster of differentiation (CD)39 and ecto-5'-nucleotidase/CD73, which catalyze the breakdown of extracellular ATP into adenosine, and on the subsequent activation of different subtypes of adenosine receptors with mixed findings of antitumor and protumor effects. New findings, needed for more effective therapeutic approaches, require consideration of redundant pathways controlling intratumoral adenosine levels, including the alternative NAD-inactivating pathway through the CD38-ectonucleotide pyrophosphatase phosphodiesterase (ENPP)1-CD73 axis, the counteracting ATP-regenerating ectoenzymatic pathway, and cellular adenosine uptake and its phosphorylation by adenosine kinase. This review provides a holistic view of extracellular and intracellular adenosine metabolism as an integrated complex network and summarizes recent data on the underlying mechanisms through which adenosine and its precursors ATP and ADP control cancer immunosurveillance, tumor angiogenesis, lymphangiogenesis, cancer-associated thrombosis, blood flow, and tumor perfusion. Special attention is given to differences and commonalities in the purinome of different cancers, heterogeneity of the tumor microenvironment, subcellular compartmentalization of the adenosine system, and novel roles of purine-converting enzymes as targets for cancer therapy. SIGNIFICANCE STATEMENT: The discovery of the role of adenosine as immune checkpoint regulator in cancer has led to the development of novel therapeutic strategies targeting extracellular adenosine metabolism and signaling in multiple clinical trials and preclinical models. Here we identify major gaps in knowledge that need to be filled to improve the therapeutic gain from agents targeting key components of the adenosine metabolic network and, on this basis, provide a holistic view of the cancer purinome as a complex and integrated network.

The relationship of 3'UTR HLA-G14-bp insertion/deletion and +3142 C/G polymorphisms and soluble HLA-G expression with gynecological cancers: An updated meta-analysis.

ObjectivesHuman leukocyte antigen‐G (HLA‐G) is implicated in several cancers and is considered to be an immune checkpoint regulator. We determined the association between polymorphisms in the 3′ untranslated region of HLA‐G and soluble HLA‐G (sHLA‐G) expression with gynecological cancers (GCs).MethodsA meta‐analysis was conducted to examine the association between HLA‐G14‐bp insertion (I)/deletion (D) and +3142C/G polymorphism in GC and to evaluate sHLA‐G expressionResultsWe revealed a significant association between the +3142C/G polymorphism and invasive cervical cancer (ICC) based on the allelic model G versus C (odds ratio [OR] = 0.738, 95% confidence interval [CI] = 0.563–0.966, p = 0.027), dominant GG+GC versus CC (OR = 0.584, 95% CI = 0.395–0.862, p = 0.007), and codominant GG versus CC (OR = 0.527, 95% CI = 0.312–0.891, p = 0.017) models, suggesting that the G allele and GG genotype are protective against ICC. In gynecological precancerous patients with human papillomavirus (HPV) infection, we found that the 14‐bp I/D under the codominant DD versus DI model (OR = 0.492, 95% CI = 0.241–1.004, p = 0.051) was of borderline significance. Soluble HLA‐G levels were significantly higher in patients compared with healthy controls (standardized mean differences [SMD] = 1.434, 95% CI = 0.442–2.526, p = 0.005). Stratification by cancer type revealed that the sHLA‐G levels were significantly increased in cervical cancer (SMD = 4.889, 95% CI = 0.468–9.310, p = 0.030) and in subjects of Asian ethnicity (SMD = 4.889, 95% CI = 0.467–9.309, p = 0.030).Conclusions HLA‐G14‐bp I/D and +3142 C/G polymorphisms are associated with GC and HPV‐associated cervical cancer. In addition, we found significantly increased sHLA‐G levels in cancer patients. These results provide a basis for further studies in diagnostics and immunotherapy of GC.