Key Immune Checkpoint Research Articles

Molecular Landscape of the Coagulome of Oral Squamous Cell Carcinoma.

Simple SummaryCancer is associated with a wide spectrum of hemostatic complications that range from thrombotic events to hemorrhage. The tumor coagulomes, i.e., the essential actors that locally regulate coagulation and fibrinolysis, play a key role in these complications. They might also play a regulatory role in various cell types of the tumor microenvironment. Here, we explored the coagulome of Oral Squamous Cell Carcinoma (OSCC) across tumor types, between OSCC tumors and within individual tumors. The coagulome of OSCC is characterized by a high expression of antipodal activators of coagulation and fibrinolysis, and subpopulations of pro-coagulant and pro-fibrinolytic cancer cells coexist within individual tumors. Importantly, we noted that dendritic cells within OSCC with a procoagulant profile express high levels of key immune checkpoint molecules. Further studies examining a possible negative modulation of the tumor’s adaptive immune response by the coagulation process are warranted.Background: Hemostatic complications, ranging from thromboembolism to bleeding, are a significant source of morbidity and mortality in cancer patients. The tumor coagulome represents the multiple genes and proteins that locally contribute to the equilibrium between coagulation and fibrinolysis. We aimed to study the coagulome of Oral Squamous Cell Carcinoma (OSCC) and examine its link to the tumor microenvironment (TME). Methods: We used data from bulk tumor DNA/RNA-seq (The Cancer Genome Atlas), single-cell RNA-seq data and OSCC cells in culture. Results: Among all tumor types, OSCC was identified as the tumor with the highest mRNA expression levels of F3 (Tissue Factor, TF) and PLAU (urokinase type-plasminogen activator, uPA). Great inter- and intra-tumor heterogeneity were observed. Single-cell analyses showed the coexistence of subpopulations of pro-coagulant and pro-fibrinolytic cancer cells within individual tumors. Interestingly, OSCC with high F3 expressed higher levels of the key immune checkpoint molecules CD274/PD-L1, PDCD1LG2/PD-L2 and CD80, especially in tumor dendritic cells. In vitro studies confirmed the particularity of the OSCC coagulome and suggested that thrombin exerts indirect effects on OSCC cells. Conclusions: OSCC presents a specific coagulome. Further studies examining a possible negative modulation of the tumor’s adaptive immune response by the coagulation process are warranted.

Integrated Analysis of Ferroptosis-Related Biomarker Signatures to Improve the Diagnosis and Prognosis Prediction of Ovarian Cancer.

Ovarian cancer remains the most lethal gynecological malignancy. Ferroptosis, a specialized form of iron-dependent, nonapoptotic cell death, plays a crucial role in various cancers. However, the contribution of ferroptosis to ovarian cancer is poorly understood. Here, we characterized the diagnostic, prognostic, and therapeutic value of ferroptosis-related genes in ovarian cancer by analyzing transcriptomic data from The Cancer Genome Atlas and Gene Expression Omnibus databases. A reliable 10-gene ferroptosis signature (HIC1, ACSF2, MUC1, etc.) for the diagnosis of ovarian cancer was identified. Notably, we constructed and validated a novel prognostic signature including three FRGs: HIC1, LPCAT3, and DUOX1. We also further developed a risk score model based on these three genes which divided ovarian cancer patients into two risk groups. Functional analysis revealed that immune response and immune-related pathways were enriched in the high-risk group. Meanwhile, the tumor microenvironment was distinct between the two groups, with more M2 Macrophage infiltration and higher expression of key immune checkpoint molecules in the high-risk group than in the other group. Low-risk patients exhibited more favorable immunotherapy and chemotherapy responses. We conclude that crosstalk between ferroptosis and immunity may contribute to the worse prognosis of patients in the high-risk group. In particular, HIC1 showed both diagnostic and prognostic value in ovarian cancer. In vitro experiments demonstrated that inhibition of HIC1 improved drug sensitivity of chemotherapy and immunotherapy agents by inducing ferroptosis. Our findings provide new insights into the potential role of FRGs in the early detection, prognostic prediction, and individualized treatment decision-making for ovarian cancer patients.

LncRNA AC099850.3 promotes hepatocellular carcinoma proliferation and invasion through PRR11/PI3K/AKT axis and is associated with patients prognosis.

Background: LncRNA is a key factor influencing tumor development. The present study aimed to investigate the effect of a novel lncRNA on the progression of hepatocellular carcinoma (HCC).Methods: A candidate lncRNA in The Cancer Genome Atlas database was identified using limma and survival R packages. The effect of lncRNA AC099850.3 on cell proliferation, apoptosis, migration, and invasion, as well as its association with immune cells in HCC were investigated. Furthermore, the functional mechanisms of lncRNA AC099850.3 in HCC were elucidated.Results: The aberrant expression of lncRNA AC099850.3 was identified in tumor tissues and its prognostic relevance in HCC was determined. The results revealed that AC099850.3 was highly expressed in HCC tissues and cell lines, and it predicted poor prognosis in patients with HCC. Furthermore, knockdown of AC099850.3 significantly suppressed the proliferation and metastatic potential of HCC cells, and promoted cell apoptosis in HCC cells. The results of gene set enrichment analysis revealed that the PI3K/AKT pathway was associated with the biological function of AC099850.3, which was further validated by western blotting. PRR11 was identified as the target gene of AC099850.3 and we established that AC099850.3 acted as an oncogene in the PRR11/PI3K/AKT axis. Immune cell infiltration analyses results revealed that AC099850.3 was positively correlated with T follicular helper cells, M0 macrophages, CD4+ memory T cells, and memory B cells. Conversely, AC099850.3 was negatively correlated with M2 macrophages, monocytes, natural killer cells, and CD8+ T cells, which could be responsible for its oncogenic effect. Of note, a significantly positive correlation was observed between AC099850.3 and key immune checkpoint molecules (PD-1, PD-L1, PD-L2, and CTLA4) in the present study, making AC099850.3 a potential immune therapeutic target for HCC.Conclusion: AC099850.3 can promote malignant biological behavior of HCC cells, and could be a potential biomarker and therapeutic target for HCC.

Construction and verification of a prognostic risk model based on immunogenomic landscape analysis of bladder caner

This study was designed to construct a prognostic risk model to predict prognosis and immunotherapy response of bladder cancer (BCa) patinets. 350 differential expressed immune-related genes (DEIRGs) were obtained according to the transcriptome profiling and immune-related genes from the Cancer Genome Atlas (TCGA) database and ImmPort database, respectively. A prognostic risk model was constructed based on 15 hub genes through univariate, multivariate, and LASSO Cox regression analyses. The area under the receiver operating characteristic (ROC) curve was 0.743, indicating the superiority of the model. The scatter plot showed that as the risk score increased, the overall survival decreased significantly. In addition, all results were internally verified by the TCGA cohort. The model showed that the higher the grade, clinical stage, and TNM stage of BCa, the higher the risk score of patients. The tumor mutation burden of the low-risk group was generally higher than that of the high-risk group. Immune cell infiltration analysis showed that CD8 T cells, naive CD4 T cells, follicular helper T cells and M0 Macrophage were significantly different between the two groups. Several key immune checkpoint genes were found to be significantly different between the two groups, such as CTLA4, PD-L1, CD47, CD276, CXCL8, and HAVCR2/TIM3. Finally, the analysis of immunotherapy revealed that the efficacy of CTLA4 or PD1 blockers alone was better in the low-risk group than in the high-risk group. Taken together, we developed and validated a prognostic risk model based on 15 hub genes, which performed well in predicting prognosis and immunotherapy response of BCa patients.

Comprehensive Analysis of Innate Immunophenotyping Based on Immune Score Predicting Immune Alterations and Prognosis in Breast Cancer Patients.

Breast cancer is the most common cancer, with the highest mortality rate and the most diagnosed cancer type in women worldwide. To identify the effect innate immune checkpoint for breast cancer immunotherapy, the innate immune prognostic biomarkers were selected through the ICI score model and the risk model in breast cancer patients. Moreover, the reliability and accuracy of the ICI score model and the risk model were further examined through the analysis of breast cancer prognosis and immune cell infiltration. The pan cancer analysis further confirmed and selected CXCL9 as the key innate immune checkpoint for breast cancer immunotherapy and identified three small molecular drugs for target CXCL9 through molecular docking analysis. In summary, CXCL9 significantly correlated with the prognostic of breast cancer and immune cell infiltration and could be innate immune checkpoint for breast cancer immunotherapy.

MiR-4759 suppresses breast cancer through immune checkpoint blockade

Programmed cell death protein 1 (PD-1)/ programmed cell death protein ligand 1 (PD-L1) is the key immune checkpoint governing evasion of advanced cancer from immune surveillance. Immuno-oncology (IO) therapy targeting PD-1/PD-L1 with traditional antibodies is a promising approach to multiple cancer types but to which the response rate remains moderate in breast cancer, calling for the need of exploring alternative IO targeting approaches. A miRNA-gene network was integrated by a bioinformatics approach and corroborated with The Cancer Genome Atlas (TCGA) to screen miRNAs regulating immune checkpoint genes and associated with patient survival. Here we show the identification of a novel microRNA miR-4759 which repressed RNA expression of the PD-L1 gene. miR-4759 targeted the PD-L1 gene through two binding motifs in the 3′ untranslated region (3′-UTR) of PD-L1. Reconstitution of miR-4759 inhibited PD-L1 expression and sensitized breast cancer cells to killing by immune cells. Treatment with miR-4759 suppressed tumor growth of orthotopic xenografts and promoted tumor infiltration of CD8+ T lymphocytes in immunocompetent mice. In contrast, miR-4759 had no effect to tumor growth in immunodeficient mice. In patients with breast cancer, expression of miR-4759 was preferentially downregulated in tumors compared to normal tissues and was associated with poor overall survival. Together, our results demonstrated miR-4759 as a novel non-coding RNA which promotes anti-tumor immunity of breast cancer.

Identification of a novel signature based on unfolded protein response-related gene for predicting prognosis in bladder cancer

BackgroundThe unfolded protein response (UPR) served as a vital role in the progression of tumors, but the molecule mechanisms of UPR in bladder cancer (BLCA) have been not fully investigated.MethodsWe identified differentially expressed unfolded protein response-related genes (UPRRGs) between BLCA samples and normal bladder samples in the Cancer Genome Atlas (TCGA) database. Univariate Cox analysis and the least absolute shrinkage and selection operator penalized Cox regression analysis were used to construct a prognostic signature in the TCGA set. We implemented the validation of the prognostic signature in GSE13507 from the Gene Expression Omnibus database. The ESTIMATE, CIBERSORT, and ssGSEA algorithms were used to explore the correlation between the prognostic signature and immune cells infiltration as well as key immune checkpoints (PD-1, PD-L1, CTLA-4, and HAVCR2). GDSC database analyses were conducted to investigate the chemotherapy sensitivity among different groups. GSEA analysis was used to explore the potential mechanisms of UPR-based signature.ResultsA prognostic signature comprising of seven genes (CALR, CRYAB, DNAJB4, KDELR3, CREB3L3, HSPB6, and FBXO6) was constructed to predict the outcome of BLCA. Based on the UPRRGs signature, the patients with BLCA could be classified into low-risk groups and high-risk groups. Patients with BLCA in the low-risk groups showed the more favorable outcomes than those in the high-risk groups, which was verified in GSE13507 set. This signature could serve as an autocephalous prognostic factor in BLCA. A nomogram based on risk score and clinical characteristics was established to predict the over survival of BLCA patients. Furthermore, the signature was closely related to immune checkpoints (PD-L1, CTLA-4, and HAVCR2) and immune cells infiltration including CD8+ T cells, follicular helper T cells, activated dendritic cells, and M2 macrophages. GSEA analysis indicated that immune and carcinogenic pathways were enriched in high-risk group.ConclusionsWe identified a novel unfolded protein response-related gene signature which could predict the over survival, immune microenvironment, and chemotherapy response of patients with bladder cancer.

Development and validation of a novel lipid metabolism-related gene prognostic signature and candidate drugs for patients with bladder cancer

BackgroundBladder cancer (BLCA) is a common cancer associated with an unfavorable prognosis. Increasing numbers of studies have demonstrated that lipid metabolism affects the progression and treatment of tumors. Therefore, this study aimed to explore the function and prognostic value of lipid metabolism-related genes in patients with bladder cancer.MethodsLipid metabolism-related genes (LRGs) were acquired from the Molecular Signature Database (MSigDB). LRG mRNA expression and patient clinical data were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Cox regression analysis and least absolute shrinkage and selection operator (LASSO) regression analysis was used to construct a signature for predicting overall survival of patients with BLCA. Kaplan-Meier analysis was performed to assess prognosis. The connectivity Map (CMAP) database was used to identify small molecule drugs for treatment. A nomogram was constructed and assessed by combining the signature and other clinical factors. The CIBERSORT, MCPcounter, QUANTISEQ, XCELL, CIBERSORT-ABS, TIMER and EPIC algorithms were used to analyze the immunological characteristics.ResultsAn 11-LRG signature was successfully constructed and validated to predict the prognosis of BLCA patients. Furthermore, we also found that the 11-gene signature was an independent hazardous factor. Functional analysis suggested that the LRGs were closely related to the PPAR signaling pathway, fatty acid metabolism and AMPK signaling pathway. The prognostic model was closely related to immune cell infiltration. Moreover, the expression of key immune checkpoint genes (PD1, CTLA4, PD-L1, LAG3, and HAVCR2) was higher in patients in the high-risk group than in those in the low-risk group. The prognostic signature based on 11-LRGs exhibited better performance in predicting overall survival than conventional clinical characteristics. Five small molecule drugs could be candidate drug treatments for BLCA patients based on the CMAP dataset.ConclusionsIn conclusion, the current study identified a reliable signature based on 11-LRGs for predicting the prognosis and response to immunotherapy in patients with BLCA. Five small molecule drugs were identified for the treatments of BLCA patients.

A signature based on 11 autophagy genes for prognosis prediction of colorectal cancer.

AimTo develop an autophagy-gene-based signature that could help to anticipate the therapeutic effects of Colorectal Cancer (CRC).MethodsWe downloaded the gene expression profiles of CRC samples from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) datasets. Genes with significant prognostic value in CRC were screened through univariate Cox regression analysis, while the LASSO Cox regression method was applied to screen optimal genes to construct the autophagy‐related prognostic signature.Results11 autophagy genes were identified and selected for the establishment of prognosis prediction model for CRC patients. The CRC patients were classified into the low- and high-risk groups according to the optimal cutoff value. The time-dependent ROC curves indicated the good performance of this model in prognosis prediction, with AUC values of 0.66, 0.66, and 0.67 at 1, 3 and 5 years for TCGA samples, as well as AUC values of 0.63, 0.65 and 0.64 for GEO samples, respectively. The multivariate Cox regression analysis results confirmed risk score as the independent marker for prognosis prediction in CRC. Besides, the constructed nomogram also had high predictive value. The results analysis on the tumor infiltrating immune cells (TIICs) relative ratios and mRNA levels of key immune checkpoint receptors indicated the signature was closely related to immune microenvironment of CRC in the context of TIICs and immune checkpoint receptors’ mRNA level. The proportion of MSI-L + MSI-H in the high-risk group was higher than that in the low-risk group. Moreover, the tumor purity was evaluated by estimate function package suggested that lower tumor purity in CRC might lead to a poorer prognosis.ConclusionThe autophagy-related features obtained in this study were able to divide the CRC patients into low- and high-risk groups, which should be contribute to the decision-making of CRC treatment.

Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

The PD-1/PD-L1 pathway is a key immune checkpoint that regulates T cell activation. There is strong rationale to develop PD-1 agonists as therapeutics against autoimmunity, but progress in this area has been limited. Here, we generated T cell receptor (TCR) targeting, PD-1 agonist bispecifics called ImmTAAI molecules that mimic the ability of PD-L1 to facilitate the colocalization of PD-1 with the TCR complex at the target cell–T cell interface. PD-1 agonist ImmTAAI molecules specifically bound to target cells and were highly effective in activating the PD-1 receptor on interacting T cells to achieve immune suppression. Potent PD-1 antibody ImmTAAI molecules closely mimicked the mechanism of action of endogenously expressed PD-L1 in their localization to the target cell–T cell interface, inhibition of proximal TCR signaling events, and suppression of T cell function. At picomolar concentrations, these bispecifics suppressed cytokine production and inhibited CD8+ T cell–mediated cytotoxicity in vitro. Crucially, in soluble form, the PD-1 ImmTAAI molecules were inactive and, hence, could avoid systemic immunosuppression. This study outlines a promising new route to generate more effective, potent, tissue-targeted PD-1 agonists that can inhibit T cell function locally with the potential to treat autoimmune and chronic inflammatory diseases of high unmet need.

Immune suppressive checkpoint interactions in the tumour microenvironment of primary liver cancers.

Liver cancer is one of the most prevalent cancers, and the third most common cause of cancer-related mortality worldwide. The therapeutic options for the main types of primary liver cancer-hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA)-are very limited. HCC and CCA are immunogenic cancers, but effective immune-mediated tumour control is prevented by their immunosuppressive tumour microenvironment. Despite the critical involvement of key co-inhibitory immune checkpoint interactions in immunosuppression in liver cancer, only a minority of patients with HCC respond to monotherapy using approved checkpoint inhibitor antibodies. To develop effective (combinatorial) therapeutic immune checkpoint strategies for liver cancer, in-depth knowledge of the different mechanisms that contribute to intratumoral immunosuppression is needed. Here, we review the co-inhibitory pathways that are known to suppress intratumoral T cells in HCC and CCA. We provide a detailed description of insights from preclinical studies in cellular crosstalk within the tumour microenvironment that results in interactions between co-inhibitory receptors on different T-cell subsets and their ligands on other cell types, including tumour cells. We suggest alternative immune checkpoints as promising targets, and draw attention to the possibility of combined targeting of co-inhibitory and co-stimulatory pathways to abrogate immunosuppression.

Abstract LB208: Obesity is associated with lower tumor oxidative phosphorylation (OXPhos) in metastatic melanoma (MM)

Abstract Immune checkpoint inhibitors (ICI) and BRAF targeted therapies improve overall survival (OS) for patients (pts) with MM, but treatment responses are heterogeneous. Recent data strongly implicates host factors in response and resistance to these agents. We and others have shown that overweight/obese BMI [OW/OB: body mass index (BMI≥25)] is associated with improved OS with both ICI and BRAF targeted therapies in MM pts. The biologic basis for this association is unknown. Thus, we examined the molecular and immune correlates of BMI in MM. In melanoma TCGA regionally metastatic specimens with available BMI (n=202), BMI was not associated with DNA mutations or copy number variations, nor with protein expression. To assess for associations with gene expression, we performed a gene set enrichment analysis (GSEA) on pooled data for cohorts of MM patients with RNAseq and BMI data (TCGA, n=202; MDACC, n=61; MIA, n=68; UCLA, n=26). Following batch correction, GSEA identified downregulation of OXPHOS and adipogenesis in tumors from OW/OB pts compared to normal (NL) BMI (BMI<25). Mass spectrometry of 36 MM specimens from the TCGA cohort confirmed downregulation of the TCA cycle intermediates citrate (p=0.01) and succinate (p<0.05) in OW/OB MM. Immunohistochemistry for key immune cell populations and checkpoints in the MIA and MDACC cohorts did not identify significant differences by BMI, consistent with the lack of difference in inflammatory pathways by gene expression. Finally, we evaluated associations of BMI with gut microbiome features in MM pts (n=272). OW/OB was associated with lower microbial alpha diversity vs. NL BMI (p=0.02), but no significant differences were observed in the composition of individual species. As increased microbiome diversity has been associated with improved outcomes with ICI in MM, this finding supports that changes in the microbiome are unlikely to explain the improved outcomes seen with ICI in OW/OB pts. In sum, our findings suggest that host energy balance influences tumor metabolism in MM, with downregulation of OXPHOS in MM from OB/OW pts, which may influence outcomes with targeted and immune therapies. These hypothesis generating findings suggest one possible mechanism underlying the obesity paradox observed in MM pts. Citation Format: Andrew W. Hahn, Mingchu Xu, Jun Li, Lauren E. Haydu, M.A. Wadud Khan, Tuba N. Gide, Alexander M. Menzies, Courtney W. Hudgens, Theodore S. Nowicki, Jeffrey E. Gershenwald, Antoni Ribas, Michael T. Tetzlaff, Alexander J. Lazar, James Willmott, Jennifer Wargo, Andrew Futreal, Jianhua Zhang, Michael A. Davies, Jennifer L. McQuade. Obesity is associated with lower tumor oxidative phosphorylation (OXPhos) in metastatic melanoma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB208.

58-LB: ß-Cell Targeted Immune Suppressive PD-1 Bispecific Agonists: A Novel Approach to Treat Type 1 Diabetes

The success of immunosuppressive therapies to preserve pancreatic islets and treat Type 1 Diabetes (T1D) has, so far, been limited either due to safety concerns or lack of persistent efficacy. Restricting T cell suppression to the pancreas, thereby avoiding systemic immunosuppression, may overcome these issues and is thus an attractive option for T1D. One possible approach to inhibit the destruction of pancreatic β-cells in T1D is to activate the PD-1 receptor on autoreactive T cells only when they enter in the pancreas. The PD-1 pathway is a key immune checkpoint that limits T cell responses and helps to maintain peripheral tolerance. Blocking this pathway in cancer patients has been shown to cause diabetes-like symptoms. Therefore, stimulating PD-1 to inhibit autoreactive T cells and treat this disease is an appealing concept. To create β-cell-restricted inhibitors of autoreactive T cells, we designed targeted PD-1 agonist bispecific molecules that contain a high affinity TCR specific for a pHLA complex expressed on the surface of pancreatic β-cells. We show that these bispecific molecules bind specifically to immortalized β-cell lines and primary β-cells from disaggregated pancreatic islets. Once bound to target pHLA, these bispecifics engage PD-1 on attacking T cells and inhibit TCR signaling resulting in potent inhibition of T cell effector functions. At picomolar concentrations, these bispecifics suppress secretion of inflammatory cytokines from cytotoxic T cells and inhibit β-cell killing by autoreactive CD8+ T cells. Importantly, these PD-1 agonists are unable to inhibit T cells when free in solution and not bound to target cells. Hence, these immune modulating bispecifics have the potential to limit autoimmunity in pancreatic islets while avoiding systemic immunosuppression. These features make them an attractive and novel platform to treat T1D. Disclosure G. Bossi: Employee; Self; Immunocore Limited. V. Gonzalez: None. C. M. Lucato: None. K. Page: None. N. Smith: Employee; Self; Immunocore Limited. L. Whaley: Employee; Self; Immunocore Limited. H. Ghadbane: Employee; Self; Immunocore Ltd. D. Knight: Employee; Self; Immunocore Limited. E. Henderson: Employee; Self; Immunocore Ltd. S. Hearty: Employee; Self; Immunocore Limited. T. Mahon: None. A. P. Curnock: Employee; Self; Immunocore Ltd. P. Weber: Employee; Self; Immunocore Ltd., Stock/Shareholder; Self; Vertex Pharmaceuticals Incorporated. P. Weber: Employee; Self; Immunocore Ltd., Stock/Shareholder; Self; Vertex Pharmaceuticals Incorporated. J. Kumaran: None. L. J. Bawden: Employee; Self; Immunocore Ltd. R. Figueiredo: None. K. E. Wiseman: None. R. O’dwyer: Employee; Self; Immunocore ltd. D. Overton: None.

Emerging immune checkpoints in the tumor microenvironment: Implications for cancer immunotherapy.

Immune checkpoints within the tumor microenvironment (TME) play important roles in modulating host antitumor immunity. Checkpoint-based immunotherapies (e.g. immune checkpoint inhibitors) have revolutionized cancer therapy. However, there are still many drawbacks with current checkpoint immunotherapies in clinical practice, such as unresponsiveness, resistance, tumor hyperprogression, autoimmune-related adverse events, and limited efficacy with some solid malignances. These drawbacks highlight the need to further investigate the mechanisms underlying the therapeutic effects, as well as the need to identify new targets for cancer immunotherapy. With the discovery of emerging immune checkpoints in the TME, the development of strategies targeting the pivotal immunomodulators for cancer treatment has been significantly advanced in the past decade. In this review, we summarize and classify the novel emerging immune checkpoints beyond the extensively studied ones (e.g. PD-1, PD-L1, CTLA-4, LAG-3 and TIM-3) in the TME, and provide an update on the clinical trials targeting these key immune molecules.

Hedgehog-induced PD-L1 on tumor-associated macrophages is critical for suppression of tumor-infiltrating CD8+ T cell function.

The programmed death-1 (PD-1) and the PD ligand 1 (PD-L1) interaction represents a key immune checkpoint within the tumor microenvironment (TME), and PD-1 blockade has led to exciting therapeutic advances in clinical oncology. Although IFN-γ–dependent PD-L1 induction on tumor cells was initially thought to mediate the suppression on effector cells, recent studies have shown that PD-L1 is also expressed at high level on tumor-associated macrophages (TAMs) in certain types of tumors. However, the precise role of PD-L1 expression on TAMs in suppressing antitumor immunity within the TME remains to be defined. Using a myeloid-specific Pdl1-knockout mouse model, here we showed definitive evidence that PD-L1 expression on TAMs is critical for suppression of intratumor CD8+ T cell function. We further demonstrated that tumor-derived Sonic hedgehog (Shh) drives PD-L1 expression in TAMs to suppress tumor-infiltrating CD8+ T cell function, leading to tumor progression. Mechanistically, Shh-dependent upregulation of PD-L1 in TAMs is mediated by signal transducer and activator of transcription 3, a cascade that has not been previously reported to our knowledge. Last, single-cell RNA sequencing analysis of human hepatocellular carcinoma revealed that PD-L1 is mainly expressed on M2 TAMs, supporting the clinical relevance of our findings. Collectively, our data revealed an essential role for Shh-dependent PD-L1 upregulation in TAMs in suppressing antitumor immunity within the TME, which could lead to the development of new immunotherapeutic strategies for treating cancer.

Unraveling the Role of Immune Checkpoints in Leishmaniasis

Leishmaniasis are Neglected Tropical Diseases affecting millions of people every year in at least 98 countries and is one of the major unsolved world health issues. Leishmania is a parasitic protozoa which are transmitted by infected sandflies and in the host they mainly infect macrophages. Immunity elicited against those parasites is complex and immune checkpoints play a key role regulating its function. T cell receptors and their respective ligands, such as PD-1, CTLA-4, CD200, CD40, OX40, HVEM, LIGHT, 2B4 and TIM-3 have been characterized for their role in regulating adaptive immunity against different pathogens. However, the exact role those receptors perform during Leishmania infections remains to be better determined. This article addresses the key role immune checkpoints play during Leishmania infections, the limiting factors and translational implications.

P14.20 Multi-Spectral Imaging of Lung Adenocarcinoma Reveals Importance of Cancer Specific HLA-DR on the TME and Clinical Outcome

Within the tumor microenvironment (TME), the interactions between cancer cells and immune cells are of increasing importance as targeting key immune checkpoints have redefined clinical care for patients with lung cancer. While immune checkpoint inhibitors have improved outcomes for patients with NSCLC, only a subset of patients respond. Tremendous efforts have been made to define factors that mediate tumor progression and response to immunotherapy. Regulation of antigen-presentation has been shown to play a critical role in this response. MHCII (HLA-DR) is normally expressed on antigen presenting cells and leads to activation of CD4+ T cells. Notably, HLA-DR expression is observed on cancer cells and appears to shape local anti-tumor immunity through potential direct presentation of tumor neoantigens to CD4+ helper T cells. While several studies have demonstrated that HLA-DR on cancer cells leads to more T cell infiltration, its effect on the TME and outcomes for patients with NSCLC have not been systemically examined. Advances in multispectral immunohistochemistry techniques allow for quantitative and spatial identification of multiple immune cells. Imaging with multiple markers can reveal the diversity of the local immune infiltrate, cell-cell interactions, and correlations of spatial distribution and location of immune cells in NSCLC with clinical outcome of patients with cancer cells. Slides from 153 patients with resected lung adenocarcinoma were stained using Opal multiplex with a panel of markers: DAPI, HLA-DR, CK, CD3, CD14, CD19 and CD8. Multispectral imaging was performed using wide field microscopy on the Vectra3.0. For each patient, 3-5 regions of interest (ROIs) were chosen and spectral unmixing was utilized for the detection and separation of seven different fluorescent signals in each NSCLC tissue samples. Cells were segmented based on nuclear and membrane markers and classified in phenotypes using inForm machine learning algorithms. The immunophenotypes were quantified and nearest neighbor analysis was performed. The presence of HLA-DR on cancer cells and immune cell infiltration were correlated with clinical outcome. Co-expression of HLA-DR and CK+ defined cancer cells that express MHCII. HLA-DR high and HLA-DR low patients were stratified using a cutoff of >5% of cancer cells expressing HLA-DR. Patients with HLA-DR high tumors had significantly increased immune cell infiltration of CD4+, CD8+, and CD14+ cells. Nearest neighbor analysis demonstrated CK+ tumor cells in HLA-DR high patients were in closer proximity to CD14+, CD19+, CD4+, and CD8+ T cells. In HLA-DR high patients, more CK+ cancer cells were directly interfaced with CD4+ and CD8+ T cells. Patients with high levels of cancer cell HLA-DR experienced improved overall survival at 5 years (p=0.046). HLA-DR high tumors are significantly associated with increased T cell and myeloid cell infiltration, resulting in closer proximity of all immune cells and more cell-cell contact. Imaging analysis of HLA-DR high tumors demonstrated more direct contact with CD4+ and CD8+ T cells compared to HLA-DR low tumors. Patients with HLA-DR high tumors experienced an improved overall survival compared to patients with HLA-DR low tumors. These data suggest cancer cell HLA-DR may represent a new biomarker for TME inflammation and patient outcomes in NSCLC.

Polymorphisms and Circulating Plasma Protein Levels of Immune Checkpoints (CTLA-4 and PD-1) Are Associated With Posner-Schlossman Syndrome in Southern Chinese.

Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) are well-known key immune checkpoints that play a crucial dampening effect on regulating T-cell homeostasis and self-tolerance. In this study, we aimed to evaluate the association between immune checkpoints (CTLA-4 and PD-1) and Posner-Schlossman syndrome (PSS) in a southern Chinese population. A total of 137 patients with PSS and 139 healthy controls from a southern Chinese population were recruited. Five single nucleotide polymorphisms (SNPs) of CTLA-4 (rs733618, rs4553808, rs5742909, rs231775, and rs3087243) and five SNPs of PD-1 (rs10204525, rs2227981, rs2227982, rs41386349, and rs36084323) were genotyped by SNaPshot technique. Soluble CTLA-4 (sCTLA-4) and soluble PD-1 (sPD-1) were determined by ELISA and antibody array assay, respectively. The frequencies of T allele at rs733618 and A allele at rs231775 of CTLA-4 were significantly higher in PSS patients than in healthy controls (corrected p (Pc) = 0.037; Pc = 0.044, respectively). The haplotype frequencies of CACGG haplotype (rs733618-rs4553808-rs5742909-rs231775-rs3087243) of CTLA-4 and TGAGC haplotype (rs10204525-rs2227981-rs2227982-rs41386349-rs36084323) of PD-1 in the PSS group was significantly lower than those in the control group (Pc = 0.015, p = 0.034, respectively). Circulating plasma levels of sCTLA-4 and sPD-1 in PSS patients were significantly higher than those in controls (all p < 0.001). The present study suggests that CTLA-4 and PD-1 genetic polymorphisms are associated with the susceptibility to PSS in a southern Chinese population. The upregulated circulating plasma protein levels of sCTLA-4 and sPD-1 might provide some hints regarding the dysfunction of immune checkpoints in PSS during the active status.

Oxidation of Innate Immune Checkpoint CD47 on Cancer Cells with Non-Thermal Plasma.

Simple SummaryNon-thermal plasma is being developed for cancer immunotherapy. The aim of our study was to determine the effect of non-thermal plasma on immunosuppressive immune checkpoint, CD47. The direct effect of non-thermal plasma on CD47 was measured in vitro and in vivo, and the mechanism of action was studied in silico. Non-thermal plasma immediately oxidized CD47, suggesting a dual role of non-thermal plasma therapy to simultaneously increase immunogenic signals and reduce immunosuppressive ones.Non-thermal plasma (NTP) therapy has been emerging as a promising cancer treatment strategy, and recently, its ability to locally induce immunogenic cancer cell death is being unraveled. We hypothesized that the chemical species produced by NTP reduce immunosuppressive surface proteins and checkpoints that are overexpressed on cancerous cells. Here, 3D in vitro tumor models, an in vivo mouse model, and molecular dynamics simulations are used to investigate the effect of NTP on CD47, a key innate immune checkpoint. CD47 is immediately modulated after NTP treatment and simulations reveal the potential oxidized salt-bridges responsible for conformational changes. Umbrella sampling simulations of CD47 with its receptor, signal-regulatory protein alpha (SIRPα), demonstrate that the induced-conformational changes reduce its binding affinity. Taken together, this work provides new insight into fundamental, chemical NTP-cancer cell interaction mechanisms and a previously overlooked advantage of present NTP cancer therapy: reducing immunosuppressive signals on the surface of cancer cells.

Hexokinase 3 Dysfunction Promotes Tumorigenesis and Immune Escape by Regulating Monocyte/Macrophage Infiltration of Clear Cell Renal Cell Carcinoma Microenvironment

Purpose: This study aims to identify potential prognostic role of ccRCC and provide clues for glycolysis and the benefits of immune checkpoint therapies (ICTs) of ccRCC. Methods: Based on TCGA (n=533), GEO (n=118), real-world (n=377) ccRCC cohorts, and approximately 15,000 cancer samples, prognostic value of HK3 was identified using survival, Cox regression and ROC analysis. Afterwards, functional effects of HK3 in ccRCC were analyzed in silico and in vivo. Results: The large-scale findings suggested significantly higher HK3 expression in ccRCC tissues and the predictive efficacy of HK3 for tumor progression and poor prognosis. Next, the subgroup survival and Cox regression analysis found that HK3 serves as a promising and independent marker predicting prognosis and survival of ccRCC patients from bioinformatics and real-world cohorts. Subsequently, we found that HK3 could modulate malignant behaviors of ccRCC cells. Additionally, the comprehensive results suggested that HK3 highly correlated with abundance of immune cells, and specifically stimulates the infiltration of monocyte/macrophage surface markers, regulates the immune checkpoint molecules PD-1 and CTLA-4 of exhaustive T cells, affects the immune escape process of cancers and predicts outcomes for ccRCC patients receiving ICT. Conclusion: In conclusion, the large-scale data first revealed that HK3 could predict aggressive progression and poor prognosis of ccRCC, and improve predictive outcomes for ccRCC patients receiving ICT. HK3 activated ccRCC microenvironment stimulates the abundance of monocyte/macrophage surface markers infiltration, and may regulate the key molecular subgroups immune checkpoint molecules of exhaustive T cells, promoting the formation of tumor immune escape in cancers. Funding Statement: This work is supported by Grants from National Key Research and Development Project (No.2019YFC1316000), the Natural Science Foundation of Shanghai (No.20ZR1413100) and Shanghai Municipal Health Commission Project (2020CXJQ03) Declaration of Interests: The author reports no conflicts of interest in this work. Ethics Approval Statement: The Ethics approval and participation consent of this study was approved and agreed by the ethics committee of Fudan University Shanghai Cancer Center (Shanghai, China).