Background: T cell activation is associated with increase in glycolysis and glutaminolysis. T cell immunoglobulin and mucin domain containing protein-3 (TIM-3), a T cell surface molecule, downregulates T cell activation and leads to insufficient immunity in cancer and chronic infection. TIM-3 regulates T cell activation possibly through alterations in metabolism; however, the relationship between TIM-3 expression and T cell metabolic changes has not been well studied.

Results: We investigated the association between TIM-3 expression and metabolic changes by analyzing glucose metabolism, glutamine metabolism, and mitochondrial function in TIM-3 overexpressing or knockout Jurkat T cell lines relative to their control cell lines. Glucose uptake and consumption, and lactate release were downregulated by TIM-3 expression but upregulated by TIM-3 knockout. Concomitantly, the expression of the glucose transporter, Glut1, but not Glut2, 3, or 4 was altered by TIM-3 expression. However, TIM-3 expression alone could not account for the change in glutamine consumption, glutamate release, and mitochondrial mass, ROS production or membrane potential in these cell lines.

Conclusion: Our results show the association of TIM-3 expression with T cell glucose metabolism. These results are significant in chronic infections and cancers where it is necessary to control TIM-3 expressing T cells.

Keywords: CD4+ T cell; Glucose transporter; Glutaminolysis; Glycolysis; HAVCR2.

Patterns of DNA methylation are significantly altered in cancers. Interpreting the functional consequences of DNA methylation requires the integration of multiple forms of data. The recent advancement in the next-generation sequencing can help to decode this relationship and in biomarker discovery. In this study, we investigated the methylation patterns of papillary renal cell carcinoma (PRCC) and its relationship with the gene expression using The Cancer Genome Atlas (TCGA) multi-omics data. We found that the promoter and body of tumor suppressor genes, microRNAs and gene clusters and families, including cadherins, protocadherins, claudins and collagens, are hypermethylated in PRCC. Hypomethylated genes in PRCC are associated with the immune function. The gene expression of several novel candidate genes, including interleukin receptor IL17RE and immune checkpoint genes HHLA2, SIRPA and HAVCR2, shows a significant correlation with DNA methylation. We also developed machine learning models using features extracted from single and multi-omics data to distinguish early and late stages of PRCC. A comparative study of different feature selection algorithms, predictive models, data integration techniques and representations of methylation data was performed. Integration of both gene expression and DNA methylation features improved the performance of models in distinguishing tumor stages. In summary, our study identifies PRCC driver genes and proposes predictive models based on both DNA methylation and gene expression. These results on PRCC will aid in targeted experiments and provide a strategy to improve the classification accuracy of tumor stages.

Intrauterine infection caused by viral infection has been implicated to contribute to preterm birth. Hepatitis A virus cellular receptor 2 (HAVCR2) regulates inflammation in non-gestational tissues in response to viral infection.

The aims of this study were to determine the effect of: (i) viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) on HAVCR2 expression; and (ii) HAVCR2 silencing by siRNA (siHAVCR2) in primary amnion and myometrial cells on poly(I:C)-induced inflammation.

In human foetal membranes and myometrium, HAVCR2 mRNA and protein expression was decreased when exposed to poly(I:C). Treatment of primary amnion and myometrial cells with poly(I:C) significantly increased the expression and release of pro-inflammatory cytokines TNF, IL1A, IL1B and IL6; the expression of chemokines CXCL8 and CCL2; the expression and secretion of adhesion molecules ICAM1 and VCAM1; and PTGS2 and PTGFR mRNA expression and the release of prostaglandin PGF2α . This increase was significantly augmented in cells transfected with siHAVCR2. Furthermore, mRNA expression of anti-inflammatory cytokines IL4 and IL10 was significantly decreased.

Collectively, our data suggest that HAVCR2 regulates cytokines, chemokines, prostaglandins and cell adhesion molecules in the presence of viral infection. This suggests a potential for HAVCR2 activators as therapeutics for the management of preterm birth associated with viral infections.

Background: Immune cells account for a large proportion of the tumour microenvironment in anaplastic thyroid carcinomas (ATCs). However, the expression pattern of immune-related genes (IRGs) in ATCs is unclear. Our study aimed to identify an immune-related signature indicating the dedifferentiation of thyroid cells.

Methods: We compared the differences in thyroid differentiation score (TDS), infiltration of immune cells and enriched pathways between ATCs and papillary thyroid carcinomas (PTCs) or normal thyroid tissues in the Gene Expression Omnibus database. Univariate and multivariable Cox analyses were used to screen prognosis-associated IRGs in The Cancer Genome Atlas database. After constructing a risk score, we investigated its predictive value for differentiation and survival by applying receiver operating characteristic and Kaplan-Meier curves. We further explored its associations with important immune checkpoint molecules, infiltrating immune cells and response to immunotherapy.

Results: Compared with PTCs or normal thyroid tissues, ATCs exhibited lower TDS values and higher enrichment of immune cells and activation of the inflammatory response. The quantitative analyses and immunohistochemical staining validated that most ATC cell lines and ATC tissues had higher expression of MMP9 and lower expression of SDC2 than normal thyroid samples and PTC. Higher risk scores indicates dedifferentiation and a worse prognosis. Additionally, the risk score was positively correlated with the immune checkpoint molecules PDL1, CTLA4, IDO1, and HAVCR2 and infiltration of multiple immune cells. Importantly, we found that the samples with higher risk scores tended to have a better response to immunotherapy than those with lower scores.

Conclusion: Our findings indicate that the risk score may not only contribute to the determination of differentiation and prognosis of thyroid carcinomas but also help the prediction of immune cells infiltration and immunotherapy response.

Keywords: Anaplastic thyroid carcinoma; Dedifferentiation; Immune checkpoints; Immune-related genes; Immunotherapy; Infiltrating immune cells; Papillary thyroid carcinoma; Prognosis.

Anti-PD1/PDL1 therapy has proven efficacious against many cancers but only reached modest objective response rates against recurrent ovarian cancer. A deeper understanding of the tumor microenvironment (TME) may reveal other immunosuppressive mechanisms that warrant investigation as immunotherapeutic targets for this challenging disease. Matched primary and recurrent tumors from patients with high-grade serous ovarian carcinoma (HGSC) were analyzed by multicolor immunohistochemistry/immunofluorescence for the presence of T cells, B cells, macrophages, and for the expression of immunosuppressive and HLA molecules. Cancer- and immune-related gene expression was assessed by NanoString analysis. Recurrent tumors showed increased infiltration by immune cells, displayed higher expression of PDL1, IDO, and HLA molecules, and contained more stromal tissue. NanoString analysis demonstrated increased expression of gene signatures related to chemokines and T cell functions in recurrent tumors. The ovarian tumors showed high gene expression of LAG3 and HAVCR2 (TIM3) and enhanced levels of TIGIT and CTLA4 in recurrent tumors compared to primary tumors. The majority of HGSC developed into a more inflamed phenotype during progression from primary to recurrent disease, including indications of adaptive immune resistance. This suggests that recurrent tumors may be particularly sensitive to inhibition of adaptive immune resistance mechanisms.

Keywords: NanoString; TILs; adaptive immune resistance; immune checkpoints; multicolor immunohistochemistry (IHC); ovarian cancer; tumor microenvironment.

Osteosarcoma (OSA) is the most common bone malignancy and displays high heterogeneity of molecular phenotypes. This study aimed to characterize the molecular features of OSA by developing a classification system based on the gene expression profile of the tumor microenvironment. Integrative analysis was performed using specimens and clinical information for OSA patients from the TARGET program. Using a matrix factorization method, we identified two molecular subtypes significantly associated with prognosis, S1 (infiltration type) and S2 (escape type). Both subtypes displayed unique features of functional significance features and cellular infiltration characteristics. We determined that immune and stromal infiltrates were abundant in subtype S1 compare to that in subtype S2. Furthermore, higher expression of immune checkpoint PDCD1LG2 and HAVCR2 was associated with improved prognosis, while a preferable chemotherapeutic response was associated with FAP-positive fibroblasts in subtype S1. Alternatively, subtype S2 is characterized by a lack of effective cytotoxic responses and loss of major histocompatibility complex class I molecule expression. A gene classifier was ultimately generated to enable OSA classification and the results were confirmed using the GSE21257 validation set. Correlations between the percentage of fibroblasts and/or fibrosis and CD8⁺ cells, and their clinical responses to chemotherapy were assessed and verified based on 47 OSA primary tumors. This study established a new OSA classification system for stratifying OSA patient risk, thereby further defining the genetic diversity of OSA and allowing for improved efficiency of personalized therapy.

Keywords: gene expression classifier; molecular subtyping; osteosarcoma; tumor immune microenvironment; tumor-infiltrating lymphocytes.

Background. Anti-PD1/PDL1 immune checkpoint inhibitors (ICIs) showed promising results in breast cancer, and exploration of additional actionable immune checkpoints is ongoing. Inflammatory breast cancer (IBC) is an aggressive form of disease, the immune tumor microenvironment (TME) of which is poorly known. We aimed at providing the first comprehensive immune portrait of IBCs. Methods. From the gene expression profiles of 137 IBC and 252 non-IBC clinical samples, we measured the fractions of 22 immune cell types, expression of signatures associated with tertiary lymphoid structures (TLS) and with the response to ICIs (T cell-inflamed signature: TIS) and of 18 genes coding for major actionable immune checkpoints. The IBC/non-IBC comparison was adjusted upon the clinicopathological variables. Results. The immune profiles of IBCs were heterogeneous. CIBERSORT analysis showed profiles rich in macrophages, CD8+ and CD4 + T-cells, with remarkable similarity with melanoma TME. The comparison with non-IBCs showed significant enrichment in M1 macrophages, γδ T-cells, and memory B-cells. IBCs showed higher expression of TLS and TIS signatures. The TIS signature displayed values in IBCs close to those observed in other cancers sensitive to ICIs. Two-thirds of actionable immune genes (HAVCR2/TIM3, CD27, CD70, CTLA4, ICOS, IDO1, LAG3, PDCD1, TNFRSF9, PVRIG, CD274/PDL1, and TIGIT) were overexpressed in IBCs as compared to normal breast and two-thirds were overexpressed in IBCs versus non-IBCs, with very frequent co-overexpression. For most of them, the overexpression was associated with better pathological response to chemotherapy. Conclusion. Our results suggest the potential higher vulnerability of IBC to ICIs. Clinical trials.

Keywords: Checkpoints; immune checkpoint inhibitors; immune profile; immunotherapy; inflammatory breast cancer.

Neoantigens play a crucial role in cancer immunotherapy. However, the effectiveness and safety of neoantigen-based immunotherapies in patients with colorectal cancer (CRC), particularly in the Chinese population, have not been well studied. This study explored the feasibility and effectiveness of neoantigens in the treatment of CRC. Whole-exome sequencing (WES) and transcriptome sequencing were used to identify somatic mutations, RNA expression, and human leukocyte antigen (HLA) alleles. Neoantigen candidates were predicted, and immunogenicity was assessed. The neoantigens TSHZ3-L523P, RARA-R83H, TP53-R248W, EYA2-V333I, and NRAS-G12D from Patient 4 (PW4); TASP1-P161L, RAP1GAP-S215R, MOSPD1-V63I, and NAV2-D1973N from Patient 10 (PW10); and HAVCR2-F39V, SEC11A-R11L, SMPDL3B-T452M, LRFN3-R118Q, and ULK1-S248L from Patient 11 (HLA-A0201⁺PW11) induced a heightened neoantigen-reactive T cell (NRT) response as compared with the controls in peripheral blood lymphocytes (PBLs) isolated from patients with CRC. In addition, we identified neoantigen-containing peptides SEC11A-R11L and ULK1-S248L from HLA-A0201⁺PW11, which more effectively elicited specific CTL responses than the corresponding native peptides in PBLs isolated from HLA-A0201⁺PW11 as well as in HLA-A2.1/K^b transgenic mice. Importantly, adoptive transfer of NRTs induced by vaccination with two mutant peptides could effectively inhibit tumor growth in tumor-bearing mouse models. These data indicate that neoantigen-containing peptides with high immunogenicity represent promising candidates for peptide-mediated personalized therapy.Abbreviations: CRC: colorectal cancer; DCs: dendritic cells; ELISPOT: enzyme-linked immunosorbent spot; E:T: effector:target; HLA: human leukocyte antigen; MHC: major histocompatibility complex; Mut: mutant type; NGS: next-generation sequencing; NRTs: neoantigen-reactive T cells; PBMCs: peripheral blood mononuclear cells; STR: short tandem repeat; PBLs: peripheral blood lymphocytes; PBS: phosphate-buffered saline; PD-1: programmed cell death protein 1; TILs: tumor-infiltrating lymphocytes; RNA-seq: RNA sequencing; Tg: transgenic; TMGs: tandem minigenes; WES: whole-exome sequencing; WT: wild-type.

Keywords: Colorectal cancer; cancer vaccine; immunotherapy; neoantigens; tumor.

Project High-tech Omics-based Patient Evaluation (HOPE), which used whole-exome sequencing and gene expression profiling, was launched in 2014. A total of ~2,000 patients were enrolled until March 2016, and the survival time was observed up to July 2019. In our previous study, a tumor microenvironment immune type classification based on the expression levels of the programmed death-ligand 1 (PD-L1) and CD8B genes was performed based on four types: A, adaptive immune resistance; B, intrinsic induction; C, immunological ignorance; and D, tolerance. Type A (PD-L1⁺ and CD8B⁺) exhibited upregulated features of T helper 1 antitumor responses. In the present study, survival time analysis at 5 years revealed that patients in type A had a better prognosis than those in other categories [5 year survival rate (%); A (80.5) vs. B (73.9), C (73.4) and D (72.6), P=0.0005]. Based on the expression data of 293 immune response-associated genes, 62 specific genes were upregulated in the type A group. Among these genes, 18 specific genes, such as activated effector T-cell markers (CD8/CD40LG/GZMB), effector memory T-cell markers (PD-1/CD27/ICOS), chemokine markers (CXCL9/CXCL10) and activated dendritic cell markers (CD80/CD274/SLAMF1), were significantly associated with a good prognosis using overall survival time analysis. Finally, multivariate Cox proportional hazard regression analyses of overall survival demonstrated that four genes (GZMB, HAVCR2, CXCL9 and CD40LG) were independent prognostic markers, and GZMB, CXCL9 and CD40LG may contribute to the survival benefit of patients in the immune type A group.

Keywords: TME; immune response-associated gene; immune type classification; prognostic marker; project High-tech Omics-based Patient Evaluation.

Background: Methylation of histone 3 at lysine 9 (H3K9) and DNA methylation are epigenetic marks correlated with genes silencing. The tumor microenvironment significantly influences therapeutic responses and clinical outcomes. The epigenetic-regulation mechanism of the costimulatory factors Tim-3 and galectin-9 in cervical cancer remains unknown.

Methods: The methylation status of HAVCR2 and LGALS9 were detected by MS-PCR in cervical cancer tissues and cell lines. The underlying molecular mechanism of SUV39H1-DNMT3A-Tim-3/galectin-9 regulation was elucidated using cervical cancer cell lines containing siRNA or/and over-expression system. Confirmation of the regulation of DNMT3A by SUV39H1 used ChIP-qPCR.

Results: SUV39H1 up-regulates H3K9me3 expression at the DNMT3A promoter region, which in turn induced expression of DNMT3A in cervical cancer. In addition, the mechanistic studies indicate that DNMT3A mediates the epigenetic modulation of the HAVCR2 and LGALS9 genes by directly binding to their promoter regions in vitro. Moreover, in an in vivo assay, the expression profile of SUV39H1 up-regulates the level of H3K9me3 at the DNMT3A promoter region was found to correlate with Tim-3 and galectin-9 cellular expression level.

Conclusion: These results indicate that SUV39H1-DNMT3A is a crucial Tim-3 and galectin-9 regulatory axis in cervical cancer.

Keywords: Cervical cancer; DNMT3A; Galectin-9; H3K9me3; SUV39H1; Tim-3.

Long noncoding RNAs (lncRNAs) have multiple functions with regard to the cancer immunity response and the tumor microenvironment. The prognosis of head and neck squamous cell carcinoma (HNSCC) is still poor currently, and it may be effective to predict the clinical outcome and immunotherapeutic response of HNSCC by immunogenic analysis. Therefore, by using univariate COX analysis and Lasso Cox regression, we identified a signature consisting of 21 immune-related lncRNA pairs (IRLPs) that predicted clinical outcome and Immunotherapeutic response in HNSCC. Specifically, it was associated with immune cell infiltration (i.e., T cells CD4 memory resting, CD8 T cells, macrophages M0, M2, and NK cells), and more importantly this signature was strongly related with immune checkpoint inhibitors (ICIs) [such as PDCD1 (r = -0.35, P < 0.001), CTLA4 (r = -0.26, P < 0.001), LAG3 (r = -0.22, P < 0.001) and HAVCR2 (r = -0.2, P < 0.001)] and immunotherapy-related biomarkers (MMR and HLA). The present study highlighted the value of the 21 IRLPs signature as a predictor of prognosis and immunotherapeutic response in HNSCC.

Keywords: head and neck squamous cell carcinoma (HNSCC); immune checkpoint inhibitors (ICIs); immune-related lncRNA pairs (IRLPs); immunotherapies; prognosis.

Natural Killer (NK) cells play an essential role in antiviral and anti-tumoral immune responses. In peripheral blood, NK cells are commonly classified into two major subsets: CD56^brightCD16^neg and CD56^dimCD16^pos despite the characterization of a CD56^negCD16^pos subset 25 years ago. Since then, several studies have described the prevalence of an CD56^negCD16^pos NK cell subset in viral non-controllers as the basis for their NK cell dysfunction. However, the mechanistic basis for their cytotoxic impairment is unclear. Recently, using a strict flow cytometry gating strategy to exclude monocytes, we reported an accumulation of CD56^negCD16^pos NK cells in Plasmodium falciparum malaria-exposed children and pediatric cancer patients diagnosed with endemic Burkitt lymphoma (eBL). Here, we use live-sorted cells, histological staining, bulk RNA-sequencing and flow cytometry to confirm that this CD56^negCD16^pos NK cell subset has the same morphological features as the other NK cell subsets and a similar transcriptional profile compared to CD56^dimCD16^pos NK cells with only 120 genes differentially expressed (fold change of 1.5, p < 0.01 and FDR<0.05) out of 9235 transcripts. CD56^negCD16^pos NK cells have a distinct profile with significantly higher expression of MPEG1 (perforin 2), FCGR3B (CD16b), FCGR2A, and FCGR2B (CD32A and B) as well as CD6, CD84, HLA-DR, LILRB1/2, and PDCD1 (PD-1), whereas Interleukin 18 (IL18) receptor genes (IL18RAP and IL18R1), cytotoxic genes such as KLRF1 (NKp80) and NCR1 (NKp46), and inhibitory HAVCR2 (TIM-3) are significantly down-regulated compared to CD56^dimCD16^pos NK cells. Together, these data confirm that CD56^negCD16^pos cells are legitimate NK cells, yet their transcriptional and protein expression profiles suggest their cytotoxic potential is mediated by pathways reliant on antibodies such as antibody-dependent cell cytotoxicity (ADCC), antibody-dependent respiratory burst (ADRB), and enhanced by complement receptor 3 (CR3) and FAS/FASL interaction. Our findings support the premise that chronic diseases induce NK cell modifications that circumvent proinflammatory mediators involved in direct cytotoxicity. Therefore, individuals with such altered NK cell profiles may respond differently to NK-mediated immunotherapies, infections or vaccines depending on which cytotoxic mechanisms are being engaged.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the head and neck with a poor prognosis. Oral cancer development is a multistep process involving carcinogenesis. Though significant advances in cancer immunotherapy over the years, there is lack of evidence for T cell exhaustion during oral carcinogenesis. Clinical specimens from healthy donors and patients diagnosed with oral leukoplakia (OLK) or OSCC were collected for immunohistochemical staining with PD-L1, CD86, CD8, PD-1 and CTLA-4 antibodies. Meanwhile, chemically induced mouse models of oral carcinogenesis were constructed with 4-NQO induction. Exhaustion status of T cells was measured by flow cytometry for spleens and by multiplex immunohistochemistry (mIHC) for formalin-fixed paraffin-embedded lesions in multiple stages of oral carcinogenesis. The efficacy of PD-1 blockade with or without cisplatin treatment was evaluated on the mice in precancerous and OSCC stages. We observed higher expression of PD-1 in the human OLK and OSCC tissues compared with the normal, while low expression CTLA-4 in all oral mucosa tissues. Animal experiments showed that the exhausted CD4 + T cells existed much earlier than exhausted CD8 + T cells, and an increased ratio of stem-like exhausted T cells and partially exhausted T cells were detected in the experimental groups. Besides, the expression of immune checkpoint markers (PDCD1, CTLA4, HAVCR2) were strongly positively correlated with cytokines (IFNG, IL-2). In summary, T cell exhaustion plays a vital role in oral carcinogenesis, and PD-1 blockade can prevent the progression of oral carcinogenesis.

Keywords: Immune checkpoint blockade; Immunotherapy; Oral squamous cell carcinoma; T cell exhaustion; ：Oral carcinogenesis.

Lung adenocarcinoma (LUAD) needs to be stratified for its heterogeneity. Oncogenic driver alterations such as EGFR mutation, ALK translocation, ROS1 translocation, and BRAF mutation predict response to treatment for LUAD. Since oncogenic driver alterations may modulate immune response in tumor microenvironment that may influence prognosis in LUAD, the effects of EGFR, ALK, ROS1, and BRAF alterations on tumor microenvironment remain unclear. Immune-related prognostic model associated with oncogenic driver alterations is needed. In this study, we performed the Cox-proportional Hazards Analysis based on the L1-penalized (LASSO) Analysis to establish an immune-related prognostic model (IPM) in stage I-II LUAD patients, which was based on 3 immune-related genes (PDE4B, RIPK2, and IFITM1) significantly enriched in patients without EGFR, ALK, ROS1, and BRAF alterations in The Cancer Genome Atlas (TCGA) database. Then, patients were categorized into high-risk and low-risk groups individually according to the IPM defined risk score. The predicting ability of the IPM was validated in GSE31210 and GSE26939 downloaded from the Gene Expression Omnibus (GEO) database. High-risk was significantly associated with lower overall survival (OS) rates in 3 independent stage I-II LUAD cohorts (all P < 0.05). Moreover, the IPM defined risk independently predicted OS for patients in TCGA stage I-II LUAD cohort (P = 0.011). High-risk group had significantly higher proportions of macrophages M1 and activated mast cells but lower proportions of memory B cells, resting CD4 memory T cells and resting mast cells than low-risk group (all P < 0.05). In addition, the high-risk group had a significantly lower expression of CTLA-4, PDCD1, HAVCR2, and TIGIT than the low-risk group (all P < 0.05). In summary, we established a novel IPM that could provide new biomarkers for risk stratification of stage I-II LUAD patients.

Keywords: Gene Expression Omnibus; The Cancer Genome Atlas; immune-related prognostic model; lung adenocarcinoma; oncogenic driver alterations; tumor microenvironment.

Germline HAVCR2 mutations, recently identified in a large subset of patients with subcutaneous panniculitis-like T-cell lymphoma (SPTCL), are associated with an increased risk of hemophagocytic lymphohistiocytosis (HLH). Discovery of this heritable HLH/SPTCL diathesis has expanded our understanding of a rare and molecularly heterogeneous lymphoma. Furthermore, patients with SPTCL have excellent survival unless they develop HLH. Therefore, through compiling data on SPTCL-related conditions that predispose patients to HLH, we are better able to predict which patients with SPTCL have the greatest risk of mortality. We present the first case of SPTCL with concomitant HLH and autoimmune lymphoproliferative syndrome (ALPS) in a patient who was subsequently diagnosed with familial HLH (F-HLH) attributable to a germline STXBP2 splice-site mutation. She had wildtype HAVCR2. Reports including ours demonstrate how SPTCL can evolve in the setting of an exaggerated host inflammatory response attributable to a variety of unusual underlying etiologies. This article is protected by copyright. All rights reserved.

Keywords: cutaneous lymphoma; hemophagocytic syndrome; lymphoproliferative disease associated with primary immune disorder; subcutaneous panniculitis-like T-cell lymphoma.

Objective: To screen out the potential key genes of endotoxin tolerance (ET), and to provide theoretical and experimental evidence for treatment and prognosis of sepsis.

Methods: (1) Experiment 1 (gene chip and bioinformatics analysis): ET related data set GSE47783 was downloaded from the Gene Expression Omnibus (GEO). The data set was obtained from lipopolysaccharide (LPS) stimulated mouse macrophages to establish sepsis model (LPS group) and ET model (ET group). IDEP 0.92 software was used to screen differential expressed gene (DEG) between the two groups, analyze gene ontology (GO), and locate the main functions and signaling pathways of differential genes. The protein-protein interaction (PPI) network of DEG was constructed by the Search Tool for the Retrieval of Interacting Genes Database (STRING) to screen core genes hepatitis A virus cell membrane protein receptor 2 (HAVCR2) for following up validation study. (2) Experiment 2 (reproduction of mouse macrophage RAW264.7 model): RAW264.7 cells were cultured in vitro, the ET model (ET group, cells were cultured with 10 μg/L LPS for 24 hours and then with 100 μg/L LPS for 4 hours) and sepsis model (LPS group, cells were cultured with 100 μg/L LPS for 4 hours) were reproduced by LPS stimulation. Phosphate buffer saline (PBS) group was given equal volume of solvent PBS for 4 hours. The mRNA and protein expressions of HAVCR2 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. (3) Experiment 3 (RAW264.7 cells transfected with HAVCR2 lentiviral vector): to further clarify whether HAVCR2 was involved in the formation of ET, after knockdown of HAVCR2 in RAW264.7 cells by lentiviral short hairpin RNA (shRNA) technology, the ET model (HAVCR2^--ET group) was constructed again, and the control group (ET group) without knockdown of HAVCR2 was set up. RT-qPCR method was used to detect the mRNA expressions of macrophage polarization key proteins [arginase 1 (ARG1), CD206, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide synthase 2 (NOS2)] in cells.

Results: (1) Experiment 1: a total of 1 013 DEG were identified, compared with LPS group, 521 genes were up-regulated and 492 genes were down-regulated in ET group. The function of these DEG was to increase biosynthesis and reduce inflammatory reaction. Signal pathways were mainly enriched in Janus kinase/signal transducers and activators of transcription (JAK/STAT), NOD like receptor, Toll-like receptor (TLR), TNF, hypoxia inducible factor-1 (HIF-1). The first up-regulated HAVCR2 in the ET group was selected as the target of the study. (2) Experiment 2: the results of in vitro experiment showed that the mRNA expression of HAVCR2 after high-dose LPS stimulation was down-regulated as compared with PBS group, and the mRNA expression of HAVCR2 in ET group was significantly higher than that in LPS group (2^-ΔΔCT: 1.10±0.10 vs. 0.60±0.10, P < 0.05). The results of Western blotting were consistent with RT-qPCR results. (3) Experiment 3: the mRNA expressions of ARG1 and CD206 in HAVCR2^--ET group were significantly lower than those in ET group [ARG1 mRNA (2^-ΔΔCT): 0.50±0.10 vs. 1.00±0.10, CD206 (2^-ΔΔCT): 0.73±0.10 vs. 1.00±0.10], and the mRNA expressions of TNF-α and IL-1β were significantly higher than those in ET group [TNF-α mRNA (2^-ΔΔCT): 2.20±0.10 vs. 1.00±0.10, IL-1β mRNA (2^-ΔΔCT): 9.00±0.10 vs. 1.00±0.10], with significant differences (all P < 0.05). There was no significant difference in the expression of NOS2 mRNA between the two groups.

Conclusions: HAVCR2 is involved in the regulation of inflammatory factors downstream of sepsis and the formation of ET, which is expected to become a new therapeutic target of sepsis.

Background: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma (RCC), which accounts for majority of RCC-related deaths. It is clearly essential to further identify more novel prognostic signatures and therapeutic targets.

Material and methods: We identified differentially expressed genes (DEGs) between ccRCC and adjacent normal tissues in GEO database using a Robust Rank Aggregation (RRA) method. An mRNA signature (mRNASig) based on DEGs was developed using Cox and LASSO analysis in the TCGA database and validated in the ICGC database. Afterward, the influence of mRNASig mRNAs on the immune microenvironment in ccRCC was explored using comprehensive bioinformatics analysis.

Results: A total of 957 robust DEGs were identified using the RRA method. mRNASig comprised CEP55, IFI44, NCF4, and TCIRG1 and was developed and validated to identify high-risk patients who had poorer prognosis than low-risk patients. A nomogram was also constructed based on mRNASig, AJCC stage, and tumor grade. The mRNASig were closely related to a variety of tumor-infiltrating lymphocytes, especially including CD8+ T cells, activated CD4+ memory T cells, regulatory T cells, activated NK cells, and resting NK cells. The mRNASig were also correlated positively with the expression of CTLA4, LAG3, PDCD1, TIGIT, and HAVCR2.

Conclusion: We developed and validated mRNASig to assist clinicians in making personalized treatment decisions. Furthermore, CEP55, IFI44, NCF4, and TCIRG1 may be novel potential targets for future treatment of ccRCC.

Keywords: GEO; ICGC; Robust Rank Aggregation method; TCGA; clear cell renal cell carcinoma; nomogram.

Hepatitis A virus cellular receptor2 (Havcr2) also named T-cell immunoglobulin and mucin domain containing-3 (Tim-3) was initially described as a T helper 1-specific cell surface protein, a member of Tim family implicated in the regulating process of adaptive and innate immune responses. Here, medaka (Oryzias latipes) Havcr2 (OlHavcr2) was isolated and characterized. Unlike other Havcr2 proteins, OlHavcr2 possesses two Ig-like domains but lacks cytoplasmic and transmembrane domains. RT-PCR results revealed that OlHavcr2 mRNA was expressed strongly in the liver, moderately in the intestine, heart and ovary, and weakly in the muscle, gill, brain, eye, spleen, and testis. OlHavcr2 expression begun from gastrula stage and was maintained until hatching. The signal of OlHavcr2 was mainly identified in the blood system in the yolk sac by in situ hybridization. These results indicated that OlHavcr2 is expressed ubiquitously in adult tissues, and is a zygotic gene expressed from gastrula onwards in embryogenesis. OlHavcr2 may play a significant role in the blood system of medaka. In the immune organs, OlHavcr2 expression was affected by the immune stimulants, lipopolysaccharide and poly I:C, suggesting that OlHavcr2 was involved in innate immunity and adaptive immunity in medaka.

The tumor immune microenvironment of lung cancer is associated with prognosis and immunotherapy efficacy. Long noncoding RNAs are identified as prognostic biomarkers associated with immune functions. We constructed a signature comprising differentially expressed immune-related lncRNAs to predict the prognosis of patients with lung adenocarcinoma. We established the immune-related lncRNA signature by pairing immune-related lncRNAs regardless of expression level and lung adenocarcinoma patients were divided into high- and low-risk groups. The prognosis of patients in the two groups was significantly different; The immune-related lncRNA signature could serve as an independent lung adenocarcinoma prognostic indicator. The signature correlated negatively with B cell, CD4+ T cell, M2 macrophage, neutrophil, and monocyte immune infiltration. Patients with low risk scores had a higher abundance of immune cells and stromal cells around the tumor. Gene set enrichment analysis showed that samples from low-risk group were more active in the IgA production in intestinal immune network and the T and B cell receptor signaling pathway. High-risk groups had significant involvement of the cell cycle, DNA replication, adherens junction, actin cytoskeleton regulation, pathways in cancer, and TGF-β signaling pathways. High risk scores correlated significantly negatively with high CTLA-4 and HAVCR2 expression and higher median inhibitory concentration of common anti-tumor chemotherapeutics (e.g., cisplatin, paclitaxel, gemcitabine) and targeted therapy (e.g., erlotinib and gefitinib). We identified a reliable immune-related lncRNA lung adenocarcinoma prognosis model, and the immune-related lncRNA signature showed promising clinical prediction value.

Keywords: infiltration of immune cell; long noncoding RNA; lung adenocarcinoma; prognostic signature; tumor immune microenvironment.

Purpose: CXCR5-positive (CXCR5⁺) tumor cell infiltration has different prognostic values in different types of cancer. The objective was to evaluate the effect of CXCR5⁺ cell infiltration in head and neck squamous cell carcinoma (HNSCC).

Patients and methods: The study included two patient cohorts: The Cancer Genome Atlas cohort (TCGA, n = 472) and the Renji Hospital cohort (RJHC, n = 201). The TCGA and RJHC cohorts were analyzed for CXCR5-related mRNAs and CXCR5+ cell infiltration, respectively. We then evaluated the correlation between CXCR5 mRNA and CXCR5⁺ cell infiltration in terms of overall survival and the immune contexture.

Results: The 5-year overall survival rate was significantly correlated with high CXCR5 mRNA expression and CXCR5⁺ cell infiltration in the TCGA and RJHC cohorts, respectively (p < 0.01), even after adjusting for confounders. Moreover, high CXCR5 mRNA expression was associated with more CD4⁺ T cells, CD8⁺ T cells, plasma cells, and less dendritic cells. A high CXCR5 mRNA expression was also correlated with increased expression of cytotoxic IFNG, TNFSF11 (RANKL), GZMA, GZMB, GZMK, GZMM, and PRF1 and increased expression of the immunosuppressive gene PDCD1 (PD-1), CD274 (PD-L1), CTLA4, LAG3, HAVCR2 (TIM-3), BTLA, and TIGIT.

Conclusion: HNSCC patients with a high intratumoral CXCR5 expression had a better prognosis than those with low intratumoral CXCR5 expression. Moreover, CXCR5⁺ cell infiltration could be used as an independent prognostic biomarker or as a potential therapeutic target. The presence of CXCR5⁺ cells affects the infiltration of immunocytes in head and neck cancer, differently from what was reported in other cancer types. Further randomized controlled trials or studies with more patients are needed to validate our results.

Keywords: CXCR5-producing cells; head and neck squamous cell carcinoma; immune contexture; prognosis.

Background: Although immunotherapy for colon cancer has made promising progress, only a few patients currently benefit from it. A recent study revealed that infiltrating immune cells are highly relevant to tumor prognosis and influence the expression of immune-related genes. However, the characterization of immune cell infiltration (ICI) has not yet been comprehensively analyzed and quantified in colon adenocarcinoma (COAD).

Methods: The multiomic data of COAD samples were downloaded from TCGA. ESTIMATE algorithm, ssGSEA method and CIBERSORT analysis were conducted to estimate the subpopulations of infiltrating immune cells. COAD subtypes based on ICI pattern were identified by consensus clustering then principal-component analysis was performed to obtain ICI scores to quantify the ICI patterns in individual tumors. Kaplan-Meier analysis was employed to validate prognostic value. Gene set enrichment analysis (GSEA) was applied for functional annotation. Finally, the mutation data was analyzed by employing "maftools" package.

Results: Three bioinformatics algorithms were used to evaluate the ICI patterns from 538 patients with COAD. Two ICI subtypes were determined using consensus clustering, and the ICI score was constructed by performing principal component analysis. Our findings showed that a higher ICI score often indicated a more advanced tumor and worse prognosis. The high-ICI score subgroup had a higher stromal score and more M0 macrophages but fewer plasma cells and decreased CD8 T cell infiltration. In addition, patients with high ICI scores had significantly higher expression levels of HAVCR2 and PCDC1LG2. Real-time polymerase chain reaction (PCR) was conducted to determine the prognostic significances of ICI-related genes.

Conclusions: In conclusion, ICI score may be considered as an original and useful indicator for independent prognostic prediction and individual immune-related therapy.

Keywords: Colon adenocarcinoma; Immune cell infiltration; Immunotherapy; Prognosis; Tumor microenvironment.

Background: Previous studies have shown that RNA Polymerase III Subunit G (POLR3G) has oncogenic effects in cultured cells and mice. However, the role of POLR3G in transitional cell carcinoma (TCC) has not been reported. This study explores the potential of POLR3G as a novel molecular marker for TCC.

Methods: The RNA sequencing data and clinical information of patients with TCC were downloaded from The Cancer Genome Atlas official website. Transcriptome analysis was performed as implemented in the edgeR package to explore whether POLR3G was up-regulated in TCC tissues compared to normal bladder tissues. The expression of POLR3G in bladder cancer cell line T24 and human uroepithelial cell line SV-HUC-1 were detected via quantitative real time polymerase chain reaction (qRT-PCR). Correlations between POLR3G expression and clinicopathological characteristics were analyzed using Mann-Whitney U test or Kruskal-Wallis H test. Clinicopathological characteristics associated with overall survival were explored using the Kaplan-Meier method and Cox regression analyses. Gene set enrichment analysis (GSEA) was performed to explore the associated gene sets enriched in different POLR3G expression phenotypes and the online tool Tumor IMmune Estimation Resource (TIMER) was used to explore the correlation between POLR3G expression and tumor immune infiltration in TCC.

Results: Transcriptome analysis showed that POLR3G was significantly up-regulated in TCC tissues compared to normal bladder tissues. Furthermore, qRT-PCR revealed high expression of POLR3G in T24 cells compared to SV-HUC-1 cells. Overall, POLR3G expression was associated with race, tumor status, tumor subtype, T classification, and pathological stage. Kaplan-Meier survival analysis revealed that higher POLR3G expression was associated with lower overall survival. The univariate Cox regression model revealed that age at diagnosis, pathological stage, and POLR3G expression were associated with prognosis of TCC patients. Further multivariate analyses identified these three clinicopathological characteristics as independent prognostic factors for overall survival. GSEA analysis showed that several gene sets associated with tumor development and metastasis, including TGF-β signaling, PI3K-AKT-mTOR signaling, and IL6-JAK-STAT3 signaling, were significantly enriched in POLR3G high expression phenotype. Immune infiltration analysis revealed that the expression of POLR3G was significantly correlated with infiltrating levels of immune cells, including CD8+ T cells, neutrophils, and dendritic cells; and the expression of POLR3G was also significantly correlated with the expression of immune checkpoint molecules, such as PD1, PD-L1, PD-L2, CTLA4, LAG3, HAVCR2, and TIGIT.

Conclusions: POLR3G was up-regulated in TCC and high POLR3G expression correlated with poor prognosis. POLR3G can potentially be used as a prognostic marker for TCC and might be of great value in predicting the response to immunotherapy.

Keywords: Biomarker; POLR3G; Prognosis; TCGA; Transitional cell carcinoma; Tumor immue infiltration.