New opportunities to overcome T cell dysfunction: the role of transcription factors and how to target them.

Immune checkpoint blockade (ICB) therapies, which block inhibitory receptors on T cells, can be efficacious in reinvigorating dysfunctional T cell responses. However, most cancers do not respond to these therapies and even in those that respond, tumors can acquire resistance. New strategies are needed to rescue and recruit T cell responses across patient populations and disease states. In this review, we define mechanisms of T cell dysfunction, focusing on key transcription factor (TF) networks. We discuss the complex and sometimes contradictory roles of core TFs in both T cell function and dysfunction. Finally, we review strategies to target TFs using small molecule modulators, which represent a challenging but highly promising opportunity to tune the T cell response toward sustained immunity.

Circular RNA-encoded oncogenic PIAS1 variant blocks immunogenic ferroptosis by modulating the balance between SUMOylation and phosphorylation of STAT1.

BACKGROUND: The clinical response rate to immune checkpoint blockade (ICB) therapy in melanoma remains low, despite its widespread use. Circular non-coding RNAs (circRNAs) are known to play a crucial role in cancer progression and may be a key factor limiting the effectiveness of ICB treatment. METHODS: The circRNAs that were downregulated after coadministration compared with single administration of PD-1 inhibitor administration were identified through RNA-seq and Ribo-seq, and thus the circPIAS1 (mmu_circ_0015773 in mouse, has_circ_0008378 in human) with high protein coding potential was revealed. Fluorescence in situ hybridization (FISH) assays were conducted to determine the localization of circPIAS1 in human and mouse melanoma cells, as well as its presence in tumor and adjacent tissues of patients. Validation through dual-luciferase reporter assay and LC-MS/MS confirmed the ability of circPIAS1 to encode a novel 108 amino acid polypeptide (circPIAS1-108aa). Specific antisense oligonucleotides (ASOs) targeting the junction site of circPIAS1 were developed to reduce its intracellular levels. Proliferation changes in melanoma cells were assessed using CCK8, EdU, and colony formation assays. The impact of circPIAS1-108aa on the ferroptosis process of melanoma cells was studied through GSH, MDA, and C11-BODIPY staining assays. Western Blot, Immunoprecipitation (IP), and Immunoprecipitation-Mass Spectrometry (IP-MS) techniques were employed to investigate the impact of circPIAS1-108aa on the P-STAT1/SLC7A11/GPX4 signaling pathway, as well as its influence on the balance between STAT1 SUMOylation and phosphorylation. Additionally, a melanoma subcutaneous transplanted tumor mouse model was utilized to examine the combined effect of reducing circPIAS1 levels alongside PD-1 inhibitor. RESULTS: Compared with the group treated with PD-1 inhibitor alone, circPIAS1 was significantly down-regulated in the coadministration group and demonstrated higher protein coding potential. CircPIAS1, primarily localized in the nucleus, was notably upregulated in tumor tissues compared to adjacent tissues, where it plays a crucial role in promoting cancer cell proliferation. This circRNA can encode a unique polypeptide consisting of 108 amino acids, through which it exerts its cancer-promoting function and impedes the effectiveness of ICB therapy. Mechanistically, circPIAS1-108aa hinders STAT1 phosphorylation by recruiting SUMO E3 ligase Ranbp2 to enhance STAT1 SUMOylation, thereby reactivating the transduction of the SLC7A11/GPX4 signaling pathway and restricting the immunogenic ferroptosis induced by IFNγ. Furthermore, the combination of ASO-circPIAS1 with PD-1 inhibitor effectively inhibits melanoma growth and significantly enhances the efficacy of immune drugs in vivo. CONCLUSIONS: Our study uncovers a novel mechanism regarding immune evasion in melanoma driven by a unique 108aa peptide encoded by circPIAS1 in melanoma that dramatically hinders immunogenic ferroptosis triggered by ICB therapy via modulating the balance between SUMOylation and phosphorylation of STAT1. This work reveals circPIAS1-108aa as a critical factor limiting the immunotherapeutic effects in melanoma and propose a promising strategy for improving ICB treatment outcomes.

Comparing anti-tumor and anti-self immunity in a patient with melanoma receiving immune checkpoint blockade.

BACKGROUND: Tumor regression following immune checkpoint blockade (ICB) is often associated with immune-related adverse events (irAEs), marked by inflammation in non-cancerous tissues. This study was undertaken to investigate the functional relationship between anti-tumor and anti-self immunity, to facilitate irAE management while promoting anti-tumor immunity. METHODS: Multiple biopsies from tumor and inflamed tissues were collected from a patient with melanoma experiencing both tumor regression and irAEs on ICB, who underwent rapid autopsy. Immune cells infiltrating melanoma lesions and inflamed normal tissues were subjected to gene expression profiling with multiplex qRT-PCR for 122 candidate genes. Subsequently, immunohistochemistry was conducted to assess the expression of 14 candidate markers of immune cell subsets and checkpoints. TCR-beta sequencing was used to explore T cell clonal repertoires across specimens. RESULTS: While genes involved in MHC I/II antigen presentation, IFN signaling, innate immunity and immunosuppression were abundantly expressed across specimens, irAE tissues over-expressed certain genes associated with immunosuppression (CSF1R, IL10RA, IL27/EBI3, FOXP3, KLRG1, SOCS1, TGFB1), including those in the COX-2/PGE2 pathway (IL1B, PTGER1/EP1 and PTGER4/EP4). Immunohistochemistry revealed similar proportions of immunosuppressive cell subsets and checkpoint molecules across samples. TCRseq did not indicate common TCR repertoires across tumor and inflammation sites, arguing against shared antigen recognition between anti-tumor and anti-self immunity in this patient. CONCLUSIONS: This comprehensive study of a single patient with melanoma experiencing both tumor regression and irAEs on ICB explores the immune landscape across these tissues, revealing similarities between anti-tumor and anti-self immunity. Further, it highlights expression of the COX-2/PGE2 pathway, which is known to be immunosuppressive and potentially mediates ICB resistance. Ongoing clinical trials of COX-2/PGE2 pathway inhibitors targeting the major COX-2 inducer IL-1B, COX-2 itself, or the PGE2 receptors EP2 and EP4 present new opportunities to promote anti-tumor activity, but may also have the potential to enhance the severity of ICB-induced irAEs.

Counting the lifetime cost of obesity: Analysis based on national England data.

AIM: Obesity has a significant impact on all-cause mortality rate and overall health care resource use (HCRU). These outcomes are also strongly linked to age, sex and local deprivation of the population. We aimed to establish the lifetime costs of obesity by demographic group/geographic area using published mortality rates and HCRU use for integrated care boards (ICB) in England in the context of costs of therapeutic intervention. METHODS: Population and expected mortality rates by age, sex and deprivation were obtained from national data. Obesity class prevalence was taken from the health of the nation study. The published impact of obesity by age, group, sex and deprivation on mortality and HCRU were applied to estimate life years lost and lifetime HCRU [by sex, age band and body mass index (BMI) class for each ICB]. The year 2019 was chosen as the study basis data to avoid influences of COVID-19 pandemic on obesity rates with application of 2022/23 HCRU values. Outcomes including prevalence, deaths, life years lost, HCRU and lifetime HCRU were compared by age and sex groups across four BMI classes normal/underweight (BMI <25 kg/m2 ), overweight (25-29.9 kg/m2 ), obese class I and II (30-39.9 kg/m2 ), and obese class III (≥40), with benchmarking being set against all population being BMI <25 kg/m2 overall and by each of the 42 ICBs. We also associated future life with deaths to provide an estimate of 'future life years lost' occurring each year. RESULTS: Total population aged >16 years was 45.4 million (51% female). PREVALENCE: 13.7 million (28% of the total adult population) had a BMI ≥30 mg/m2 and BMI ≥40 kg/m2 were 1.50 million (12%) of these 1.0 million (68%) were female and of these 0.6 million 40% were women aged 16-49 years. In addition, 35% of those with a BMI ≥40 kg/m2 were in the top deprivation quintile (i.e. overall 20%). Mortality was based on expected deaths of 518K/year, and modelling suggested that if a BMI <25 kg/m2 was achieved in all individuals, the death rate would fall by 63K to 455K/year for the English population (12% reduction). For those with a BMI ≥40 kg/m2 the predicted reduction was 12K deaths (54% lower); while in those aged 16-49 years with a BMI ≥40 kg/m2 72% of deaths were linked to obesity. For future life years lost, we estimated 2.5 years were lost in people with BMI 30-39.9 kg/m2 6.7 years when BMI ≥40 kg/m2 . However, for those aged 16-49 years with a BMI ≥40 kg/m2 , 8.3 years were lost. HCRU, for weight reduction, the annual HCRU decrease from BMI ≥40 kg/m2 to BMI 30-39.9 kg/m2 was £342 per person and from BMI 30-39.9 to 25-29.9 kg/m2 the reduction was £316/person. However, lifetime costs were similar because of reduced life expectancy for obese individuals. In quality adjusted life years (QALY), overall, 791 689 future life years were lost (13.1% of all) in people with BMI ≥25 kg/m2 and were related to excess weight. When the NICE £30 000 per QALY value was applied to the estimated total 791 689 future life years lost then the potential QALY value reduction lost was equivalent to £24 billion/year or £522/person in the obese population. For morbidly obese men and women the potential QALY value lost was £2864/person/year. Regarding geography, across the 42 ICBs, we observed significant variation in the prevalence of BMI ≥40 (1.8%-4.3%), excess mortality (11.6%-15.4%) and HCRU linked to higher BMI (7.2%-8.8%). The areas with the greatest impact on HCRU were in the north-west, north-east and Midlands of England, while the south shows less impact. CONCLUSION: The expected increases in annual HCRU because of obesity, when considered over a lifetime, are being mitigated by the increased mortality of obese individuals. Our data suggest that simple short-term HCRU reduction brought about through BMI reduction will be insufficient to fund additional specialist weight reduction interventions. The HRCUs associated with BMI are not in most cases related to short-term health conditions. They are a cumulative result over a number of years, so for age 16-49 years reducing BMI from ≥40 to 30-39.9 kg/m2 might show an annual decrease in HCRU/person by £325/year for women and £80/year for men but this might not have immediately occurred within that year. For those aged >70 years reducing BMI from ≥40 to 30-39.9 kg/m2 might show an annual decrease in HCRU/person by £777/year for women and £796/year for men but also may not be manifest within that year. However, for the morbidly obese men and women, the potential QALY value lost was £2864 per person per year with the potential for these funds to be applied to intensive weight management programmes, including pharmacotherapy.

Drug-Loaded Mesoporous Silica Nanoparticles Enhance Antitumor Immunotherapy by Regulating MDSCs.

Myeloid-derived suppressor cells (MDSCs) are recognized as major immune suppressor cells in the tumor microenvironment that may inhibit immune checkpoint blockade (ICB) therapy. Here, we developed a Stattic-loaded mesoporous silica nanoparticle (PEG-MSN-Stattic) delivery system to tumor sites to reduce the number of MDSCs in tumors. This approach is able to significantly deplete intratumoral MSDCs and thereby increase the infiltration of T lymphocytes in tumors to enhance ICB therapy. Our approach may provide a drug delivery strategy for regulating the tumor microenvironment and enhancing cancer immunotherapy efficacy.

Gamma delta T-cell-based immune checkpoint therapy: attractive candidate for antitumor treatment.

As a nontraditional T-cell subgroup, γδT cells have gained popularity in the field of immunotherapy in recent years. They have extraordinary antitumor potential and prospects for clinical application. Immune checkpoint inhibitors (ICIs), which are efficacious in tumor patients, have become pioneer drugs in the field of tumor immunotherapy since they were incorporated into clinical practice. In addition, γδT cells that have infiltrated into tumor tissues are found to be in a state of exhaustion or anergy, and there is upregulation of many immune checkpoints (ICs) on their surface, suggesting that γδT cells have a similar ability to respond to ICIs as traditional effector T cells. Studies have shown that targeting ICs can reverse the dysfunctional state of γδT cells in the tumor microenvironment (TME) and exert antitumor effects by improving γδT-cell proliferation and activation and enhancing cytotoxicity. Clarification of the functional state of γδT cells in the TME and the mechanisms underlying their interaction with ICs will solidify ICIs combined with γδT cells as a good treatment option.

Endosialin-positive tumor-derived pericytes promote tumor progression through impeding the infiltration of CD8+ T cells in clear cell renal cell carcinoma.

BACKGROUND: Immune checkpoint blockade (ICB) therapy can be effective against clear cell renal cell carcinoma (ccRCC), but many patients show no benefit. Tumor-derived pericytes (TDPs) may promote tumor progression by influencing T cells and are an immunotherapy target; however, they may comprise functionally distinct subtypes. We aimed to identify markers of tumor-promoting TDPs and develop TDP-targeting strategies to enhance ICB therapy effectiveness against ccRCC. METHODS: We analyzed the relationship between endosialin (EN) expression and cytotoxic T-lymphocyte (CTL) infiltration in ccRCC tumor samples using flow cytometry and in a ccRCC-bearing mice inhibited for EN via knockout or antibody-mediated blockade. The function of ENhigh TDPs in CTL infiltration and tumor progression was analyzed using RNA-sequencing (RNA-seq) data from ccRCC tissue-derived TDPs and single-cell RNA-seq (scRNA-seq) data from an online database. The role of EN in TDP proliferation and migration and in CTL infiltration was examined in vitro. Finally, we examined the anti-tumor effect of combined anti-EN and anti-programmed death 1 (PD-1) antibodies in ccRCC-bearing mice. RESULTS: High EN expression was associated with low CTL infiltration in ccRCC tissues, and inhibition of EN significantly increased CTL infiltration in ccRCC-bearing mice. RNA-seq and scRNA-seq analyses indicated that high EN expression represented the TDP activation state. EN promoted TDP proliferation and migration and impeded CTL infiltration in vitro. Finally, combined treatment with anti-EN and anti-PD-1 antibodies synergistically enhanced anti-tumor efficacy. CONCLUSION: ENhigh TDPs are in an activated state and inhibit CTL infiltration into ccRCC tissues. Combined treatment with anti-EN and anti-PD-1 antibodies may improve ICB therapy effectiveness against ccRCC.

Abnormal generation of IL-17A represses tumor infiltration of stem-like exhausted CD8+ T cells to demote the antitumor immunity.

BACKGROUND: Variated anti-cancer therapies are combined with immune checkpoint blockades (ICBs) for improving ICB therapeutic efficacy. Occurrence of tissue damage is common that triggers multiple inflammatory cytokine generation. Gastrointestinal organs are the commonly affected. We investigated the impact of acute colitis on tumor infiltration of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) for controlling tumor growth and responding to antibody against PD-1 (anti-PD-1). METHODS: Several tumor cell lines were inoculated into syngeneic mice subcutaneously or intra-hepatically. When tumor mass formed, activated CTLs were intravenously transferred into the tumor-bearing mice, that were given the drinking water containing 2% dextran sulfate sodium (DSS) for acute colitis induction. Tumor growth, infiltration of two exhausted CTL subsets, and the CTL interaction with tumor vascular endothelium were examined. RESULTS: Acute colitis dampened CTL-mediated antitumor effects, correlating with IL-17A elevation in the inflamed intestine. In the tumor bed, stem-like exhausted CTLs, which were defined as PD-1+Slamf6+Tim3-, expressed higher IL-17A receptor heterodimers and lower leukocyte function-associated antigen-1 (LFA-1) than terminally exhausted CTLs did, that were defined as PD-1+Slamf6-Tim3+. IL-17A stimulation reduced LFA-1 surface expression on stem-like exhausted CTLs and the counterpart ICAM-1 (intracellular adhesion molecule-1) on tumor vascular endothelium. IL-17A stimulation suppressed the extravasation across tumor vascular endothelium and self-renewal of stem-like, not the terminally exhausted CTLs. Administration of anti-IL-17A neutralizing antibody to the colitis mice restored the CTL tumor infiltration and enhanced anti-PD-1 treatment efficacy against tumors. In 33 hepatocellular carcinoma patients being treated with anti-PD-1 plus antibody against vascular endothelial growth factor, disease progression of 15 patients, that exhibited serum IL-17A increase 24 h post-therapy as compared to pre-therapy level, was poorer than that of 18 patients that exhibited serum IL-17A no-increase. CONCLUSIONS: Abnormal generation of IL-17A mainly repressed tumor infiltration of stem-like exhausted CTLs. ICB-based immunotherapeutic efficacy could be upgraded with administration of anti-IL-17A, when treatment-related IL-17A elevation occurred due to tissue damage, such as acute colitis.

Characterization of endoplasmic reticulum stress unveils ZNF703 as a promising target for colorectal cancer immunotherapy.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant tumors globally, with high morbidity and mortality. Endoplasmic reticulum is a major organelle responsible for protein synthesis, processing, and transport. Endoplasmic reticulum stress (ERS) refers to the abnormal accumulation of unfolded and misfolded proteins in the endoplasmic reticulum, which are involved in tumorigenesis and cancer immunity. Nevertheless, the clinical significance of ERS remains largely unexplored in CRC. METHODS: In present study, we performed an unsupervised clustering to identify two types of ERS-related subtypes [ERS clusters, and ERS-related genes (ERSGs) clusters] in multiple large-scale CRC cohorts. Through the utilization of machine learning techniques, we have successfully developed an uncomplicated yet robust gene scoring system (ERSGs signature). Furthermore, a series of analyses, including GO, KEGG, Tumor Immune Dysfunction and Exclusion (TIDE), the Consensus Molecular Subtypes (CMS), were used to explore the underlying biological differences and clinical significance between these groups. And immunohistochemical and bioinformatics analyses were performed to explore ZNF703, a gene of ERSGs scoring system. RESULTS: We observed significant differences in prognosis and tumor immune status between the ERS clusters as well as ERSGs clusters. And the ERSGs scoring system was an independent risk factor for overall survival; and exhibited distinct tumor immune status in multicenter CRC cohorts. Besides, analyses of TNM stages, CMS groups demonstrated that patients in advanced stage and CMS4 had higher ERSGs scores. In addition, the ERSGs scores inversely correlated with positive ICB response predictors (such as, CD8A, CD274 (PD-L1), and TIS), and directly correlated with negative ICB response predictors (such as, TIDE, T cell Exclusion, COX-IS). Notably, immunohistochemical staining and bioinformatics analyses revealed that ZNF70 correlated with CD3 + and CD8 + T cells infiltration. CONCLUSION: Based on large-scale and multicenter transcriptomic data, our study comprehensively revealed the essential role of ERS in CRC; and constructed a novel ERSGs scoring system to predict the prognosis of patients and the efficacy of ICB treatment. Furthermore, we identified ZNF703 as a potentially promising target for ICB therapy in CRC.

Nanoliposome C6-Ceramide in combination with anti-CTLA4 antibody improves anti-tumor immunity in hepatocellular cancer.

Combination therapy represents an effective therapeutic approach to overcome hepatocellular cancer (HCC) resistance to immune checkpoint blockade (ICB). Based upon previous work demonstrating that nanoliposome C6-ceramide (LipC6) not only induces HCC apoptosis but also prevents HCC-induced immune tolerance, we now investigate the potential of LipC6 in combination with ICB in HCC treatment. We generated orthotopic HCC-bearing mice, which have typical features in common with human patients, and then treated them with LipC6 in combination with the antibodies (Abs) for programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte antigen 4 (CTLA4). The tumor growth was monitored by magnetic resonance imaging (MRI) and the intrahepatic immune profiles were checked by flow cytometry in response to the treatments. Realtime PCR (qPCR) was used to detect the expression of target genes. The results show that LipC6 in combination with anti-CTLA4 Ab, but not anti-PD-1 Ab, significantly slowed tumor growth, enhanced tumor-infiltrating CD8+ T cells, and suppressed tumor-resident CD4+ CD25+ FoxP3+ Tregs. Further molecular investigation indicates that the combinational treatment suppressed transcriptional factor Krüppel-like Factor 2 (KLF2), forkhead box protein P3 (FoxP3), and CTLA4. Our studies suggest that LipC6 in combination with anti-CTLA4 Ab represents a novel therapeutic approach with significant potential in activating anti-HCC immune response and suppressing HCC growth.

Current Approaches of Immune Checkpoint Therapy in Chronic Lymphocytic Leukemia.

OPINION STATEMENT: Increasing understanding of the complex interaction between leukemic and immune cells, which is responsible for tumor progression and immune evasion, has paved the way for the development of novel immunotherapy approaches in chronic lymphocytic leukemia (CLL). One of the well-known immune escape mechanisms of tumor cells is the up-regulation of immune checkpoint molecules. In recent years, targeting immune checkpoint receptors is the most clinically effective immunotherapeutic strategy for cancer treatment. In this regard, various immune checkpoint blockade (ICB) drugs are currently been investigating for their potential effects on improving anti-tumor immune response and clinical efficacy in the hematological malignancies; however, their effectiveness in patients with CLL has shown less remarkable success, and ongoing research is focused on identifying strategies to enhance the efficacy of ICB in CLL.

Programmable Bispecific Nano-immunoengager That Captures T Cells and Reprograms Tumor Microenvironment.

Immune checkpoint blockade (ICB) therapy has revolutionized clinical oncology. However, the efficacy of ICB therapy is limited by the ineffective infiltration of T effector (Teff) cells to tumors and the immunosuppressive tumor microenvironment (TME). Here, we report a programmable tumor cells/Teff cells bispecific nano-immunoengager (NIE) that can circumvent these limitations to improve ICB therapy. The peptidic nanoparticles (NIE-NPs) bind tumor cell surface α3ß1 integrin and undergo in situ transformation into nanofibrillar network nanofibers (NIE-NFs). The prolonged retained nanofibrillar network at the TME captures Teff cells via the activatable α4ß1 integrin ligand and allows sustained release of resiquimod for immunomodulation. This bispecific NIE eliminates syngeneic 4T1 breast cancer and Lewis lung cancer models in mice, when given together with anti-PD-1 antibody. The in vivo structural transformation-based supramolecular bispecific NIE represents an innovative class of programmable receptor-mediated targeted immunotherapeutics to greatly enhance ICB therapy against cancers.

Mechanisms underlying immune-related adverse events during checkpoint immunotherapy.

Immune checkpoint (IC) proteins are some of the most important factors that tumor cells hijack to escape immune surveillance, and inhibiting ICs to enhance or relieve antitumor immunity has been proven efficient in tumor treatment. Immune checkpoint blockade (ICB) agents such as antibodies blocking programmed death (PD) 1, PD-1 ligand (PD-L) 1, and cytotoxic T lymphocyte-associated antigen (CTLA)-4 have been approved by the U.S. Food and Drug Administration (FDA) to treat several types of cancers. Although ICB agents have shown outstanding clinical success, and their application has continued to expand to additional tumor types in the past decade, immune-related adverse events (irAEs) have been observed in a wide range of patients who receive ICB treatment. Numerous studies have focused on the clinical manifestations and pathology of ICB-related irAEs, but the detailed mechanisms underlying irAEs remain largely unknown. Owing to the wide expression of IC molecules on distinct immune cell subpopulations and the fact that ICB agents generally affect IC-expressing cells, the influences of ICB agents on immune cells in irAEs need to be determined. Here, we discuss the expression and functions of IC proteins on distinct immune cells and the potential mechanism(s) related to ICB-targeted immune cell subsets in irAEs.

Identification of Neoantigens in Cancer Cells as Targets for Immunotherapy.

The clinical benefits of immune checkpoint blockage (ICB) therapy have been widely reported. In patients with cancer, researchers have demonstrated the clinical potential of antitumor cytotoxic T cells that can be reinvigorated or enhanced by ICB. Compared to self-antigens, neoantigens derived from tumor somatic mutations are believed to be ideal immune targets in tumors. Candidate tumor neoantigens can be identified through immunogenomic or immunopeptidomic approaches. Identification of neoantigens has revealed several points of the clinical relevance. For instance, tumor mutation burden (TMB) may be an indicator of immunotherapy. In various cancers, mutation rates accompanying neoantigen loads may be indicative of immunotherapy. Furthermore, mismatch repair-deficient tumors can be eradicated by T cells in ICB treatment. Hence, immunotherapies using vaccines or adoptive T-cell transfer targeting neoantigens are potential innovative strategies. However, significant efforts are required to identify the optimal epitopes. In this review, we summarize the recent progress in the identification of neoantigens and discussed preclinical and clinical studies based on neoantigens. We also discuss the issues remaining to be addressed before clinical applications of these new therapeutic strategies can be materialized.

Dynamic serum biomarkers to predict the efficacy of PD-1 in patients with nasopharyngeal carcinoma.

BACKGROUND: There is a lack of effective treatments for recurrent or metastatic nasopharyngeal carcinoma (RM-NPC). Furthermore, the response rate of NPC patients to programmed death 1 (PD-1) inhibitors is approximately 20% to 30%. Thus, we aimed to explore reliable and minimally invasive prognostic indicators to predict the efficacy of PD-1 inhibitors combination therapy in RM-NPC. METHODS: The serum markers of 160 RM-NPC patients were measured before and three weeks after the first anti-PD-1 treatment. The least absolute shrinkage and selection operator (LASSO) logistic regression was carried out to select dynamic serum indicators and construct a prediction model. Furthermore, we carried out univariate, multivariate, nomogram and survival analyses to identify independent prognostic factors that were associated with 1-year progression-free survival (PFS). RESULTS: Based on two markers that were screened by Lasso logistic regression, we constructed a risk score prediction model for the prediction of anti-PD-1 efficacy at 8-12 weeks with an AUC of 0.737 in the training cohort and 0.723 in the validation cohort. Risk score and metastases were included in the nomogram, and the Kaplan-Meier survival curves demonstrated that the high-risk group has shorter PFS compared to the low-risk group. The concordance index (C-index) of the nomogram for PFS is higher than that of the TNM stage in the training and validation cohort. CONCLUSION: We proposed a strategy to monitor dynamic changes in the biochemistry markers and emphasized their importance as potential prognostic biomarkers for the treatment of advanced NPC treated with PD-1 inhibitors. Our risk score prediction model was based on the dynamic change of LDH and AST/ALT, which has predictive and prognostic value for NPC patients who were treated with PD-1 inhibitors.

Gene expression and immune infiltration in melanoma patients with different mutation burden.

BACKGROUND: Immunotherapy is a vital component in cancer treatment. However, due to the complex genetic bases of cancer, a clear prediction index for efficacy has not been established. Tumor mutation burden (TMB) is one of the essential factors that affect immunotherapeutic efficacies, but it has not been determined whether the mutation is associated with the survival of Skin Cutaneous Melanoma (SKCM) patients. This study aimed at evaluating the correlation between TMB and immune infiltration. METHODS: Somatic mutation profiles (n = 467), transcriptome data (n = 471), and their clinical information (n = 447) of all SKCM samples were downloaded from The Cancer Genome Atlas (TCGA) database. For each sample, TMB was calculated as the number of variants per megabase. Based on K-M survival analysis, they were allocated into the high-TMB and low-TMB groups (the optimal cutoff was determined by the 'surv_cutpoint' algorithm of survival R package). Then, Gene ontology (GO) and Gene Set Enrichment Analyses (GSEA) were performed, with immune-associated biological pathways found to be significantly enriched in the low-TMB group. Therefore, immune genes that were differentially expressed between the two groups were evaluated in Cox regression to determine their prognostic values, and a four-gene TMB immune prognostic model (TMB-IP) was constructed. RESULTS: Elevated TMB levels were associated with better survival outcomes in SKCM patients. Based on the cutoff value in OS analysis, they were divided into high-TMB and low-TMB groups. GSEA revealed that the low-TMB group was associated with immunity while intersection analysis revealed that there were 38 differentially expressed immune-related genes between the two groups. Four TMB-associated immune genes were used to construct a TMB-IP model. The AUC of the ROC curve of this model reached a maximum of 0.75 (95%CI, 0.66-0.85) for OS outcomes. Validation in each clinical subgroup confirmed the efficacy of the model to distinguish between high and low TMB-IP score patients. CONCLUSIONS: In SKCM patients, low TMB was associated with worse survival outcomes and enriched immune-associated pathways. The four TMB-associated immune genes model can effectively distinguish between high and low-risk patients.

Accurate detection of Zika virus IgG using a novel immune complex binding ELISA.

OBJECTIVES: Accurate serological assays are urgently needed to support public health responses to Zika virus (ZIKV) infection with its potential to cause foetal damage during pregnancy. Current flavivirus serology for ZIKV infections lacks specificity due to cross-reacting antibodies from closely related other flaviviruses. In this study, we evaluated novel serological tests for accurate ZIKV IgG detection. METHODS: Our ELISAs are based on immune complex binding. The high specificity is achieved by the simultaneous incubation of labelled ZIKV antigen and unlabelled flavivirus homolog protein competitors. Two assays were validated with a panel of 406 human samples from PCR-confirmed ZIKV patients collected in Brazil (n = 154), healthy blood donors and other infections from Brazil, Europe, Canada and Colombia (n = 252). RESULTS: The highest specificity (100% [252/252, 95% confidence interval (CI) 98.5-100.0]) was shown by the ZIKV ED3 ICB ELISA using the ED3 antigen of the ZIKV envelope. A similar test using the NS1 antigen (ZIKV NS1 ICB ELISA) was slightly less specific (92.1% [232/252, 95% CI 88.0-95.1]). The commercial Euroimmun ZIKV ELISA had a specificity of only 82.1% (207/252, 95% CI 76.8-86.7). Sensitivity was high (93-100%) from day 12 after onset of symptoms in all three tests. Seroprevalence of ZIKV IgG was analysed in 87 samples from Laos (Asia) confirming that the ED3 ELISA showed specific reactions in other populations. CONCLUSIONS: The novel ED3 ICB ELISA will be useful for ZIKV-specific IgG detection for seroepidemiological studies and serological diagnosis for case management in travellers and in countries where other flavivirus infections are co-circulating.

Manipulating the Wnt/ß-catenin signaling pathway to promote anti-tumor immune infiltration into the TME to sensitize ovarian cancer to ICB therapy.

OBJECTIVE: Immune checkpoint blockade (ICB) therapy shows limited efficacy in ovarian cancers due to the "cold" immune phenotype surrounding these tumors. Previous studies have shown that in ovarian cancer Wnt/ß-catenin pathway activation contributes to this immune phenotype. Here, we evaluated the anti-tumor and immune-enhancing properties of the Wnt inhibitor, CGX-1321, used alone or in combination with either DKN-01 or anti-PD-1 therapy, in pre-clinical ovarian cancer models. METHODS: The parental ID8 murine ovarian cancer model harboring a knock-out of p53 (ID8p53-/-) and MISIIR-Tag spontaneous ovarian cancer models were used to test the effects of CGX-1321 alone or in combination therapies on tumor burden and immune cell landscape in the tumor microenvironment (TME). Flow cytometry and NanoString analyses were used to characterize the changes in tumor-intrinsic signaling and immune-related profiles in the TME of ovarian cancer in response to treatments. RESULTS: CGX-1321 significantly reduced tumor burden and constrained tumor progression in the ID8p53-/- and MISIIR-Tag models. Furthermore, CGX-1321 increased infiltrating CD8+ T cells in the TME. Combining CGX-1321 with either DKN-01 or anti-PD-1 therapy also decreased tumor burden and increased CD8+ T cell infiltration in the omentum TME but did not do so to a greater extent that CGX-1321 monotherapy. CONCLUSIONS: CGX-1321 significantly reduced tumor burden and enhanced CD8+ T cell levels in ovarian cancer, nevertheless the addition of DKN-01 or anti-PD-1 therapies did not enhance these effects of CGX-1321. Further investigation is needed to determine if CGX-1321 + DKN-01 combination treatment sensitizes pre-clinical ovarian cancer to ICB therapy.

Immuno-oncology gene expression profiling of formalin-fixed and paraffin-embedded clear cell renal cell carcinoma: Performance comparison of the NanoString nCounter technology with targeted RNA sequencing.

Inflammatory gene signatures are currently being explored as predictive biomarkers for immune checkpoint blockade, and particularly for the treatment of renal cell cancers. From a diagnostic point of view, the nCounter analysis platform and targeted RNA sequencing are emerging alternatives to microarrays and comprehensive transcriptome sequencing in assessing formalin-fixed and paraffin-embedded (FFPE) cancer samples. So far, no systematic study has analyzed and compared the technical performance metrics of these two approaches. Filling this gap, we performed a head-to-head comparison of two commercially available immune gene expression assays, using clear cell renal cell cancer FFPE specimens. We compared the nCounter system that utilizes a direct hybridization technology without amplification with an NGS assay that is based on targeted RNA-sequencing with preamplification. We found that both platforms displayed high technical reproducibility and accuracy (Pearson coefficient: ≥0.96, concordance correlation coefficient [CCC]: ≥0.93). A density plot for normalized expression of shared genes on both platforms showed a comparable bi-modal distribution and dynamic range. RNA-Seq demonstrated relatively larger signaling intensity whereas the nCounter system displayed higher inter-sample variability. Estimated fold changes for all shared genes showed high correlation (Spearman coefficient: 0.73). This agreement is even better when only significantly differentially expressed genes were compared. Composite gene expression profiles, such as an interferon gamma (IFNg) signature, can be reliably inferred by both assays. In summary, our study demonstrates that focused transcript read-outs can reliably be achieved by both technologies and that both approaches achieve comparable results despite their intrinsic technical differences.

Tissue-resident memory CD8+ T cells in cancer immunology and immunotherapy.

Memory T cells can be generated and remain long-term in different tissues following infection or immunization. Tissue-resident memory T (TRM) cells are a unique group of memory T cells that form and persist mainly in peripheral non-lymphoid organs. Unlike effector or central memory T (TEM or TCM) cells, TRM cells do not circulate to the blood but can provide a rapid and robust local response to re-infection. Recently, a large body of clinical studies has shown that CD103+ CD8+ TRM-like cells also exist intratumorally and strongly correlate with favorable prognosis in cancer patients. Cancer vaccine-induced CD103+ CD8+ TRM cells have been reported to suppress tumor growth in mouse models. This suggests that CD8+ TRM-like cells play a crucial role in cancer immunosurveillance and immunotherapy. In this review, we focus on the features and cytotoxic mechanisms of CD8+ TRM-like cells in multiple solid tumors and discuss their potential implications for cancer immunotherapy. We believe a better understanding of the generation, function, and longevity of CD8+ TRM-like cells in the tumor microenvironment will provide new insights for cancer immunotherapies.