Immune checkpoint therapy-elicited sialylation of IgG antibodies impairs antitumorigenic type I interferon responses in hepatocellular carcinoma.

The reinvigoration of anti-tumor T cells in response to immune checkpoint blockade (ICB) therapy is well established. Whether and how ICB therapy manipulates antibody-mediated immune response in cancer environments, however, remains elusive. Using tandem mass spectrometric analysis of modification of immunoglobulin G (IgG) from hepatoma tissues, we identified a role of ICB therapy in catalyzing IgG sialylation in the Fc region. Effector T cells triggered sialylation of IgG via an interferon (IFN)-γ-ST6Gal-I-dependent pathway. DC-SIGN+ macrophages represented the main target cells of sialylated IgG. Upon interacting with sialylated IgG, DC-SIGN stimulated Raf-1-elicited elevation of ATF3, which inactivated cGAS-STING pathway and eliminated subsequent type-I-IFN-triggered antitumorigenic immunity. Although enhanced IgG sialylation in tumors predicted improved therapeutic outcomes for patients receiving ICB therapy, impeding IgG sialylation augmented antitumorigenic T cell immunity after ICB therapy. Thus, targeting antibody-based negative feedback action of ICB therapy has potential for improving efficacy of cancer immunotherapies.

Tertiary Lymphoid Structure-Associated B Cells Enhance CXCL13+CD103+CD8+ Tissue-Resident Memory T-Cell Response to Programmed Cell Death Protein 1 Blockade in Cancer Immunotherapy.

BACKGROUND & AIMS: Although the presence of tertiary lymphoid structures (TLS) correlates with positive responses to immunotherapy in many solid malignancies, the mechanism by which TLS enhances antitumor immunity is not well understood. The present study aimed to investigate the underlying cross talk circuits between B cells and tissue-resident memory T (Trm) cells within the TLS and to understand their role in the context of immunotherapy. METHODS: Immunostaining and H&E staining of TLS and chemokine (C-X-C motif) ligand 13 (CXCL13)+ cluster of differentiation (CD)103+CD8+ Trm cells were performed on tumor sections from patients with gastric cancer (GC). The mechanism of communication between B cells and CXCL13+CD103+CD8+ Trm cells was determined in vitro and in vivo. The effect of CXCL13+CD103+CD8+ Trm cells in suppressing tumor growth was evaluated through anti-programmed cell death protein (PD)-1 therapy. RESULTS: The presence of TLS and CXCL13+CD103+CD8+ Trm cells in tumor tissues favored a superior response to anti-PD-1 therapy in patients with GC. Additionally, our research identified that activated B cells enhanced CXCL13 and granzyme B secretion by CD103+CD8+ Trm cells. Mechanistically, B cells facilitated the glycolysis of CD103+CD8+ Trm cells through the lymphotoxin-α/tumor necrosis factor receptor 2 (TNFR2) axis, and the mechanistic target of rapamycin signaling pathway played a critical role in CD103+CD8+ Trm cells glycolysis during this process. Moreover, the presence of TLS and CXCL13+CD103+CD8+ Trm cells correlated with potent responsiveness to anti-PD-1 therapy in a TNFR2-dependent manner. CONCLUSIONS: This study further reveals a crucial role for cellular communication between TLS-associated B cell and CXCL13+CD103+CD8+ Trm cells in antitumor immunity, providing valuable insights into the potential use of the lymphotoxin-α/TNFR2 axis within CXCL13+CD103+CD8+ Trm cells for advancing immunotherapy strategies in GC.

Galectin-7 Induction by EHMT2 Inhibition Enhances Immunity in Microsatellite Stability Colorectal Cancer.

BACKGROUND & AIMS: Although immunotherapy shows substantial advancement in colorectal cancer (CRC) with microsatellite instability high, it has limited efficacy for CRC with microsatellite stability (MSS). Identifying combinations that reverse immune suppression and prime MSS tumors for current immunotherapy approaches remains an urgent need. METHODS: An in vitro CRISPR screen was performed using coculture models of primary tumor cells and autologous immune cells from MSS CRC patients to identify epigenetic targets that could enhance immunotherapy efficacy in MSS tumors. RESULTS: We revealed EHMT2, a histone methyltransferase, as a potential target for MSS CRC. EHMT2 inhibition transformed the immunosuppressive microenvironment of MSS tumors into an immunomodulatory one by altering cytokine expression, leading to T-cell-mediated cytotoxicity activation and improved responsiveness to anti-PD1 treatment. We observed galectin-7 up-regulation upon EHMT2 inhibition, which converted a "cold" MSS tumor environment into a T-cell-inflamed one. Mechanistically, CHD4 repressed galectin-7 expression by recruiting EHMT2 to form a cotranscriptional silencing complex. Galectin-7 administration enhanced anti-PD1 efficacy in MSS CRC, serving as a potent adjunct cytokine therapy. CONCLUSIONS: Our findings suggest that targeting the EHMT2/galectin-7 axis could provide a novel combination strategy for immunotherapy in MSS CRC.

NK-cell-elicited gasdermin-D-dependent hepatocyte pyroptosis induces neutrophil extracellular traps that facilitate HBV-related acute-on-chronic liver failure.

BACKGROUND AND AIMS: HBV infection is a major etiology of acute-on-chronic liver failure (ACLF). At present, the pattern and regulation of hepatocyte death during HBV-ACLF progression are still undefined. Evaluating the mode of cell death and its inducers will provide new insights for developing therapeutic strategies targeting cell death. In this study, we aimed to elucidate whether and how immune landscapes trigger hepatocyte death and lead to the progression of HBV-related ACLF. APPROACH AND RESULTS: We identified that pyroptosis represented the main cell death pattern in the liver of patients with HBV-related ACLF. Deficiency of MHC-I in HBV-reactivated hepatocytes activated cytotoxic NK cells, which in turn operated in a perforin/granzyme-dependent manner to trigger GSDMD/caspase-8-dependent pyroptosis of hepatocytes. Neutrophils selectively accumulated in the pyroptotic liver, and HMGB1 derived from the pyroptotic liver constituted an important factor triggering the generation of pathogenic extracellular traps in neutrophils (NETs). Clinically, elevated plasma levels of myeloperoxidase-DNA complexes were a promising prognostic biomarker for HBV-related ACLF. More importantly, targeting GSDMD pyroptosis-HMGB1 release in the liver abrogates NETs that intercept the development of HBV-related ACLF. CONCLUSIONS: Studying the mechanisms that selectively modulate GSDMD-dependent pyroptosis, as well as its immune landscapes, will provide a novel strategy for restoring the liver function of patients with HBV-related ACLF.

miRNome targeting NF-κB signaling orchestrates macrophage-triggered cancer metastasis and recurrence.

Whether and how tumor intrinsic signature determines macrophage-elicited metastasis remain elusive. Here, we show, in detailed studies of data regarding 7,477 patients of 20 types of human cancers, that only 13.8% ± 2.6%/27.9% ± 3.03% of patients with high macrophage infiltration index exhibit early recurrence/vascular invasion. In parallel, although macrophages enhance the motility of various hepatoma cells, their enhancement intensity is significantly heterogeneous. We identify that the expression of malignant Dicer, a ribonuclease that cleaves miRNA precursors into mature miRNAs, determines macrophage-elicited metastasis. Mechanistically, the downregulation of Dicer in cancer cells leads to defects in miRNome targeting NF-κB signaling, which in turn enhances the ability of cancer cells to respond to macrophage-related inflammatory signals and ultimately promotes metastasis. Importantly, transporting miR-26b-5p, the most potential miRNA targeting NF-κB signaling in hepatocellular carcinoma, can effectively reverse macrophage-elicited metastasis of hepatoma in vivo. Our results provide insights into the crosstalk between Dicer-elicited miRNome and cancer immune microenvironments and suggest that strategies to remodel malignant cell miRNome may overcome pro-tumorigenic activities of inflammatory cells.

High-multiplex single-cell imaging analysis reveals tumor immune contexture associated with clinical outcomes after CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has made great progress in treating lymphoma, yet patient outcomes still vary greatly. The lymphoma microenvironment may be an important factor in the efficacy of CAR T therapy. In this study, we designed a highly multiplexed imaging mass cytometry (IMC) panel to simultaneously quantify 31 biomarkers from 13 patients with relapsed/refractory diffuse large B cell lymphoma (DLBCL) who received CAR19/22 T cell therapy. A total of 20 sections were sampled before CAR T cell infusion or after infusion when relapse occurred. A total of 35 cell clusters were identified, annotated, and subsequently redefined into 10 metaclusters. The CD4+ T cell fraction was positively associated with remission duration. Significantly higher Ki67, CD57, and TIM3 levels and lower CD69 levels in T cells, especially the CD8+/CD4+ Tem and Te cell subsets, were seen in patients with poor outcomes. Cellular neighborhood containing more immune cells was associated with longer remission. Fibroblasts and vascular endothelial cells resided much closer to tumor cells in patients with poor response and short remission after CAR T therapy. Our work comprehensively and systematically dissects the relationship between cell composition, state, and spatial arrangement in the DLBCL microenvironment and the outcomes of CAR T cell therapy, which is beneficial to predict CAR T therapy efficacy.

Abnormal global alternative RNA splicing in COVID-19 patients.

Viral infections can alter host transcriptomes by manipulating host splicing machinery. Despite intensive transcriptomic studies on SARS-CoV-2, a systematic analysis of alternative splicing (AS) in severe COVID-19 patients remains largely elusive. Here we integrated proteomic and transcriptomic sequencing data to study AS changes in COVID-19 patients. We discovered that RNA splicing is among the major down-regulated proteomic signatures in COVID-19 patients. The transcriptome analysis showed that SARS-CoV-2 infection induces widespread dysregulation of transcript usage and expression, affecting blood coagulation, neutrophil activation, and cytokine production. Notably, CD74 and LRRFIP1 had increased skipping of an exon in COVID-19 patients that disrupts a functional domain, which correlated with reduced antiviral immunity. Furthermore, the dysregulation of transcripts was strongly correlated with clinical severity of COVID-19, and splice-variants may contribute to unexpected therapeutic activity. In summary, our data highlight that a better understanding of the AS landscape may aid in COVID-19 diagnosis and therapy.

Single-cell profiling reveals kidney CD163+ dendritic cell participation in human lupus nephritis.

OBJECTIVES: The current work aimed to provide a comprehensive single-cell landscape of lupus nephritis (LN) kidneys, including immune and non-immune cells, identify disease-associated cell populations and unravel their participation within the kidney microenvironment. METHODS: Single-cell RNA and T cell receptor sequencing were performed on renal biopsy tissues from 40 patients with LN and 6 healthy donors as controls. Matched peripheral blood samples from seven LN patients were also sequenced. Multiplex immunohistochemical analysis was performed on an independent cohort of 60 patients and validated using flow cytometric characterisation of human kidney tissues and in vitro assays. RESULTS: We uncovered a notable enrichment of CD163+ dendritic cells (DC3s) in LN kidneys, which exhibited a positive correlation with the severity of LN. In contrast to their counterparts in blood, DC3s in LN kidney displayed activated and highly proinflammatory phenotype. DC3s showed strong interactions with CD4+ T cells, contributing to intrarenal T cell clonal expansion, activation of CD4+ effector T cell and polarisation towards Th1/Th17. Injured proximal tubular epithelial cells (iPTECs) may orchestrate DC3 activation, adhesion and recruitment within the LN kidneys. In cultures, blood DC3s treated with iPTECs acquired distinct capabilities to polarise Th1/Th17 cells. Remarkably, the enumeration of kidney DC3s might be a potential biomarker for induction treatment response in LN patients. CONCLUSION: The intricate interplay involving DC3s, T cells and tubular epithelial cells within kidneys may substantially contribute to LN pathogenesis. The enumeration of renal DC3 holds potential as a valuable stratification feature for guiding LN patient treatment decisions in clinical practice.

Targeting inflammatory macrophages rebuilds therapeutic efficacy of DOT1L inhibition in hepatocellular carcinoma.

Epigenetic reprogramming is a promising therapeutic strategy for aggressive cancers, but its limitations in vivo remain unclear. Here, we showed, in detailed studies of data regarding 410 patients with human hepatocellular carcinoma (HCC), that increased histone methyltransferase DOT1L triggered epithelial-mesenchymal transition-mediated metastasis and served as a therapeutic target for human HCC. Unexpectedly, although targeting DOT1L in vitro abrogated the invasive potential of hepatoma cells, abrogation of DOT1L signals hardly affected the metastasis of hepatoma in vivo. Macrophages, which constitute the major cellular component of the stroma, abrogated the anti-metastatic effect of DOT1L targeting. Mechanistically, NF-κB signal elicited by macrophage inflammatory response operated via a non-epigenetic machinery to eliminate the therapeutic efficacy of DOT1L targeting. Importantly, therapeutic strategy combining DOT1L-targeted therapy with macrophage depletion or NF-κB inhibition in vivo effectively and successfully elicited cancer regression. Moreover, we found that the densities of macrophages in HCC determined malignant cell DOT1L-associated clinical outcome of the patients. Our results provide insight into the crosstalk between epigenetic reprogramming and cancer microenvironments and suggest that strategies to influence the functional activities of inflammatory cells may benefit epigenetic reprogramming therapy.

Distinct single-cell immune ecosystems distinguish true and de novo HBV-related hepatocellular carcinoma recurrences.

OBJECTIVE: Revealing the single-cell immune ecosystems in true versus de novo hepatocellular carcinoma (HCC) recurrences could help the optimal development of immunotherapies. DESIGN: We performed 5'and VDJ single-cell RNA-sequencing on 34 samples from 20 recurrent HCC patients. Bulk RNA-sequencing, flow cytometry, multiplexed immunofluorescence, and in vitro functional analyses were performed on samples from two validation cohorts. RESULTS: Analyses of mutational profiles and evolutionary trajectories in paired primary and recurrent HCC samples using whole-exome sequencing identified de novo versus true recurrences, some of which occurred before clinical diagnosis. The tumour immune microenvironment (TIME) of truly recurrent HCCs was characterised by an increased abundance in KLRB1+CD8+ T cells with memory phenotype and low cytotoxicity. In contrast, we found an enrichment in cytotoxic and exhausted CD8+ T cells in the TIME of de novo recurrent HCCs. Transcriptomic and interaction analyses showed elevated GDF15 expression on HCC cells in proximity to dendritic cells, which may have dampened antigen presentation and inhibited antitumour immunity in truly recurrent lesions. In contrast, myeloid cells' cross talk with T cells-mediated T cell exhaustion and immunosuppression in the TIME of de novo recurrent HCCs. Consistent with these findings, a phase 2 trial of neoadjuvant anti-PD-1 immunotherapy showed more responses in de novo recurrent HCC patients. CONCLUSION: True and de novo HCC recurrences occur early, have distinct TIME and may require different immunotherapy strategies. Our study provides a source for genomic diagnosis and immune profiling for guiding immunotherapy based on the type of HCC recurrence and the specific TIME.

Tubular cell transcriptional intermediary factor 1γ deficiency exacerbates kidney injury-induced tubular cell polyploidy and fibrosis.

Tubulointerstitial fibrosis is considered the final convergent pathway of progressive chronic kidney diseases (CKD) regardless of etiology. However, mechanisms underlying kidney injury-induced fibrosis largely remain unknown. Recent studies have indicated that transcriptional intermediary factor 1γ (TIF1γ) inhibits the progression of fibrosis in other organs. Here, we found that TIF1γ was highly expressed in the cytoplasm and nucleus of the kidney proximal tubule. Interestingly, we found tubular TIF1γ expression was decreased in patients with CKD, including those with diabetes, hypertension, and IgA nephropathy, and in mouse models with experimental kidney fibrosis (unilateral ureteral obstruction [UUO], folic acid nephropathy [FAN], and aristolochic acid-induced nephrotoxicity). Tubule-specific knock out of TIF1γ in mice exacerbated UUO- and FAN-induced tubular cell polyploidy and subsequent fibrosis, whereas overexpression of kidney TIF1γ protected mice against kidney fibrosis. Mechanistically, in tubular epithelial cells, TIF1γ exerted an antifibrotic role via transforming growth factor-ß (TGF-ß)-dependent and -independent signaling. TIF1γ hindered TGF-ß signaling directly by inhibiting the formation and activity of the transcription factor Smad complex in tubular cells, and we discovered that TIF1γ suppressed epidermal growth factor receptor (EGFR) signaling upstream of TGF-ß signaling in tubular cells by ubiquitylating EGFR at its lysine 851/905 sites thereby promoting EGFR internalization and lysosomal degradation. Pharmacological inhibition of EGFR signaling attenuated exacerbated polyploidization and the fibrotic phenotype in mice with tubule deletion of TIF1γ. Thus, tubular TIF1γ plays an important role in kidney fibrosis by suppressing profibrotic EGFR and TGF-ß signaling. Hence, our findings suggest that maintaining homeostasis of tubular TIF1γ may be a new therapeutic option for treating tubulointerstitial fibrosis and subsequent CKD.

Transcriptional and proteomic insights into the host response in fatal COVID-19 cases.

Coronavirus disease 2019 (COVID-19), the global pandemic caused by SARS-CoV-2, has resulted thus far in greater than 933,000 deaths worldwide; yet disease pathogenesis remains unclear. Clinical and immunological features of patients with COVID-19 have highlighted a potential role for changes in immune activity in regulating disease severity. However, little is known about the responses in human lung tissue, the primary site of infection. Here we show that pathways related to neutrophil activation and pulmonary fibrosis are among the major up-regulated transcriptional signatures in lung tissue obtained from patients who died of COVID-19 in Wuhan, China. Strikingly, the viral burden was low in all samples, which suggests that the patient deaths may be related to the host response rather than an active fulminant infection. Examination of the colonic transcriptome of these patients suggested that SARS-CoV-2 impacted host responses even at a site with no obvious pathogenesis. Further proteomics analysis validated our transcriptome findings and identified several key proteins, such as the SARS-CoV-2 entry-associated protease cathepsins B and L and the inflammatory response modulator S100A8/A9, that are highly expressed in fatal cases, revealing potential drug targets for COVID-19.

A novel diagnostic approach for the classification of small B-cell lymphoid neoplasms based on the NanoString platform.

Small B-cell lymphoid neoplasms (SBCLNs) are a heterogeneous group of diseases characterized by malignant clonal proliferation of mature B-cells. However, the classification of SBCLNs remains a challenge, especially in cases where histopathological analysis is unavailable or those with atypical laboratory findings or equivocal pathologic data. In this study, gene expression profiling of 1039 samples from 27 gene expression omnibus (GEO) datasets was first investigated to select highly and differentially expressed genes among SBCLNs. Samples from 57 SBCLN cases and 102 nonmalignant control samples were used to train a classifier using the NanoString platform. The classifier was built by employing a cascade binary classification method based on the random forest algorithm with 35 refined gene signatures. Cases were successively classified as chronic lymphocytic leukemia/small lymphocytic lymphoma, conventional mantle cell lymphoma, follicular lymphoma, leukemic non-nodal mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia, and other undetermined. The classifier algorithm was then validated using an independent cohort of 197 patients with SBCLNs. Under the distribution of our validation cohort, the overall sensitivity and specificity of proposed algorithm model were >95%, respectively, for all the cases with tumor cell content greater than 0.72. Combined with additional genetic aberrations including IGH-BCL2 translocation, MYD88 L265P mutation, and BRAF V600E mutation, the optimal sensitivity and specificity were respectively found at 0.88 and 0.98. In conclusion, the established algorithm demonstrated to be an effective and valuable ancillary diagnostic approach for the sub-classification and pathologic investigation of SBCLN in daily practice.

Visualizing dynamic changes in PD-L1 expression in non-small cell lung carcinoma with radiolabeled recombinant human PD-1.

PURPOSE: Tumor heterogeneity limits the predictive value of PD-L1 expression and influences the outcomes of the immunohistochemical assay for therapy-induced changes in PD-L1 levels. This study aimed to determine the predictive value of PD-L1 for non-small cell lung carcinoma (NSCLC), thereby developing imaging agents to non-invasively image and examine the effect of the therapeutic response to PD-L1 blockade therapy. METHODS: A cohort of 102 patients with lung cancer was analyzed, and the prognostic significance of PD-L1 expression level was investigated. Recombinant human PD-1 ECD protein (rhPD1) was expressed, purified, and labeled with 64Cu for the evaluation of PD-L1 status in tumors. Mice subcutaneously bearing PD-L1 high-expressing tumor HCC827 and PD-L1 low-expressing tumor A549 were used to determine tracer-target specificity and examine the effect of therapeutic response to PD-L1 blockade therapy. RESULTS: PD-L1 was proved to be a good prognosis marker for NSCLC, and its expression was correlated with the histology of NSCLC. PET imaging revealed high tumor accumulation of 64Cu-NOTA-rhPD1 in HCC827 tumors (9.0 ± 0.5%ID/g), whereas it was 3.2 ± 0.4%ID/g in A549 tumors at 3 h post-injection. The lower tumor uptake (3.1 ± 0.3%ID/g) of 64Cu-labeled denatured rhPD1 in HCC827 tumors at 3 h post-injection (p < 0.001) demonstrated the target specificity of 64Cu-NOTA-rhPD1. Furthermore, PET showed that 64Cu-NOTA-rhPD1 sensitively monitored treatment-related changes in PD-L1 expression, and seemed to be superior to [18F]FDG. CONCLUSION: We identified PD-L1 as a good prognosis marker for surgically resected NSCLC and developed the PET tracer 64Cu-NOTA-rhPD1 with high target specificity for PD-L1.

Multiparametric MRI-Based Radiomic Signature for Preoperative Evaluation of Overall Survival in Intrahepatic Cholangiocarcinoma After Partial Hepatectomy.

BACKGROUND: The clinical outcomes of patients with intrahepatic cholangiocarcinoma (ICC) after partial hepatectomy remain suboptimal. Identifying patients with poor outcomes before surgery is urgently required. PURPOSE: To develop a multiparametric magnetic resonance imaging (MRI)-based radiomic signature to evaluate overall survival (OS) preoperatively and to investigate its incremental value for disease stratification. STUDY TYPE: Retrospective. SUBJECTS: One hundred and sixty-three patients with pathologically defined ICC, divided into training (N = 115) and validation sets (N = 48). SEQUENCE: Three-dimensional T1-weighted gradient-echo sequence with and without contrast agent, T2-weighted fast spin-echo sequence, and diffusion-weighted imaging with single-shot echo-planar sequence at 1.5 T or 3.0 T. ASSESSMENT: OS was defined as the time from the date of surgery to death or last contact. The radiomic signature was built based on the least absolute shrinkage and selection operator regression model. A clinicopathologic-radiographic (CPR) model and a combined model integrating radiomic signature with CPR factors were developed with multivariable Cox regression models. STATISTICAL TESTS: Harrell's concordance index (C-index) was used to compare the discrimination of different models. Net reclassification index (NRI) and integrated discrimination improvement (IDI) were used to quantify the improvement of prognostic accuracy after adding radiomic signature. RESULTS: The high-risk patients of death defined by the radiomic signature showed significantly lower OS compared with low-risk patients in validation set (3-year OS 17.1% vs. 56.4%, P < 0.001). Integrating radiomic signature into tumor, node, and metastasis (TNM) staging system significantly improved the prognostic accuracy compared with TNM stage alone (validation set C-index 0.745 vs. 0.649, P = 0.039, NRI improvement 39.9%-43.8%, IDI improvement 16.1%-19.4%). The radiomic signature showed no significant difference of C-index with postoperative CPR model (validation set, 0.698 vs. 0.674, P = 0.752). Incorporating the radiomic signature into CPR model significantly improved prognostic accuracy (NRI improvement 32.5%-34.3%, IDI improvement 8.1%-12.9%). DATA CONCLUSION: Multiparametric MRI-based radiomic signature is a potential biomarker for preoperative prognostic evaluation of ICC patients. LEVEL OF EVIDENCE: 4 TECHNICAL EFFICACY: Stage 4.

Immune landscape and therapeutic strategies: new insights into PD-L1 in tumors.

PD-1/PD-L1 axis represents an important target for renormalizing and resetting anti-tumor immunity in cancer patients. Currently, anti-PD-1/PD-L1 therapy has been applied in a broad spectrum of tumors and has yielded durable remission in patients. However, how to further broaden the application, guide personalized therapeutic strategies, and improve clinical responses remains a vital task. At present, PD-L1 expression is an important parameter of clinical indications for immune checkpoint blockade in many types of cancers, a strategy based on the supposition that positive PD-L1 expression reflects local T cell response. Recent studies have revealed that PD-L1 expression is regulated by multiple layers of complicated factors, during which the host immune microenvironment exerts a pivotal role and determines the clinical efficacy of the therapy. In this review, we will summarize recent findings on PD-1/PD-L1 in cancer, focusing on how local immune landscape participates in the regulation of PD-L1 expression and modification. Importantly, we will also discuss these topics in the context of clinical treatment and analyze how these fundamental principles might inspire our efforts to develop more precise and effective immune therapeutics for cancer.

Influence of gut and intratumoral microbiota on the immune microenvironment and anti-cancer therapy.

Microbiota has been implicated in the regulation of tumor progression and therapeutic efficacy. However, the effect of microbiota on disease progression is context dependent, differing according to tumor types, therapeutic regimens, and composition of the microbiota, calling for a deeper understanding of host-microbiome interactions. Previous studies have demonstrated that gut microbiota influences disease progression by regulating local and systemic immunity. Notably, with the advent of next-generation sequencing technology, intratumoral microbiota has also been found and constitutes an important component of the tumor microenvironment. In this review, we summarize recent knowledge about the identification of intra-tumor microbiota and discuss the role of gut and intratumoral microbiota in solid tumors in the angle of immune microenvironment interaction. Furthermore, we discuss how these findings may benefit current anti-cancer approaches. Key problems to be solved in ongoing and future research are highlighted.

Bmi1 drives the formation and development of intrahepatic cholangiocarcinoma independent of Ink4A/Arf repression.

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most prevalent types of primary liver cancer. Compared with HCC, for which several drugs have been approved, ICC is associated with shorter survival, and no drug has been approved for this type. Previously, we reported that Bmi1 drives HCC and is required for HCC development and growth. However, whether Bmi1 plays a critical role in ICC is not clear, although it reportedly is highly expressed in ICC. Therefore, we investigated its role in ICC. Here, we report that Bmi1 promotes ICC initiation and progression independent of the Ink4A/Arf pathway, a canonical downstream pathway of Bmi1. We found that Bmi1 is overexpressed in human ICC. Co-expression of Bmi1 and NRas induced ICC formation in mice. Knockdown or inactivation of Bmi1 inhibited ICC growth in vitro. Liver-specific knockout or inactivation of Bmi1 remarkably suppressed ICC tumor formation and development in vivo. Mechanistically, no correlation between Bmi1 and Ink4A/Arf levels was found in mouse and human ICC tissues. Together, our data indicate that Bmi1 functions as an oncogene independent of repression of the Ink4A/Arf locus in ICC and that it can serve as a target for ICC treatment.

Plasma Cell Polarization to the Immunoglobulin G Phenotype in Hepatocellular Carcinomas Involves Epigenetic Alterations and Promotes Hepatoma Progression in Mice.

BACKGROUND & AIMS: Little is known about the composition and generation of plasma cell subsets in patients with hepatocellular carcinoma (HCC) and how these associate with outcomes. We investigated whether, or how, plasma cells differentiate and function in patients with HCC and mice with liver tumors. METHODS: We analyzed subset composition and distribution of plasma cells in HCC samples from 342 patients who underwent curative resection at the Cancer Center of Sun Yat-sen University in China; samples of non-tumor liver tissue were used as controls. We associated plasma cell profiles with patient outcomes. Tissue-derived leukocytes were analyzed by flow cytometry and real-time polymerase chain reaction. The ability of macrophages to regulate plasma cell differentiation was determined in ex vivo cultures of cells from human HCC tissues. C57BL/6 and BALB/c mice were given injections of Hepa1-6 cells, which formed hepatomas, or H22 cells, which formed ascitic hepatomas. Gene expression patterns were analyzed in human HCC, mouse hepatoma, and non-tumor tissues by real-time polymerase chain reaction. Mice with hepatomas were given injections of GSK126 (an inhibitor of histone H3 lysine 27 methyltransferase [EZH2]) and 5-AZA-dC (an inhibitor of DNA methyltransferases); tumor tissues were analyzed by immunofluorescence and immunohistochemistry for the presence of immune cells and cytokines. RESULTS: B cells isolated from HCCs had somatic hypermutations and class-switch recombinations to the IgG phenotype that were not observed in non-tumor tissues. Increased level of plasma cells correlated with poor outcomes of patients. Activated CD4+ T cells from HCCs stimulated C-X-C motif chemokine 10 (CXCL10) production by macrophages. CXCL10 bound CXC chemokine receptor 3 on B cells and signaled via extracellular signal-regulated kinase to cause them to become IgG-producing plasma cells. IgG activated Fc receptors on macrophages and induced them to produce interleukin 6, interleukin 10, and C-C motif chemokine ligand 20 (CCL20). In mice with hepatomas, depletion of B cells prevented generation of these macrophage, increased the anti-tumor T cell response, and reduced growth of hepatomas. However, these effects were lost after injection of CXC chemokine receptor 3-positive plasma cells. Human HCC and mouse hepatoma tissues had increased expression of DNA methyltransferase 1 and EZH2 compared with non-tumor tissues. Injection of mice with GSK126 and 5-AZA-dC induced expression of CXCL10 by tumor cells and caused plasma cell polarization, suppression of the anti-tumor T cell response, and hepatoma growth. CONCLUSIONS: Human HCC tissues contain B cells with class-switch recombinations to the IgG phenotype. Activated CD4+ T cells from HCCs stimulate CXCL10 production by macrophages; CXCL10 binds CXC chemokine receptor 3 on B cells and causes them to become IgG-producing plasma cells. IgG activates Fc receptor in macrophages to produce cytokines that reduce the anti-tumor immune response. In mice with hepatomas, depletion of B cells prevented generation of these macrophages, increased the anti-tumor T cell response, and reduced growth of hepatomas. This pathway involves increased expression of DNA methyltransferase 1 and EZH2 by HCC and hepatoma cells.

Glycolytic activation of peritumoral monocytes fosters immune privilege via the PFKFB3-PD-L1 axis in human hepatocellular carcinoma.

BACKGROUND & AIMS: Programmed cell death 1 ligand 1 (PD-L1) expression on antigen-presenting cells is essential for T cell impairment, and PD-L1-expressing macrophages may mechanistically shape and therapeutically predict the clinical efficacy of PD-L1 or programmed cell death 1 blockade. We aimed to elucidate the mechanisms underlying PD-L1 upregulation in human tumor microenvironments, which remain poorly understood despite the clinical success of immune checkpoint inhibitors. METHODS: Monocytes/macrophages were purified from peripheral blood, non-tumor, or paired tumor tissues of patients with hepatocellular carcinoma (HCC), and their possible glycolytic switch was evaluated. The underlying regulatory mechanisms and clinical significance of metabolic switching were studied with both ex vivo analyses and in vitro experiments. RESULTS: We found that monocytes significantly enhanced the levels of glycolysis at the peritumoral region of human HCC. The activation of glycolysis induced PD-L1 expression on these cells and subsequently attenuated cytotoxic T lymphocyte responses in tumor tissues. Mechanistically, tumor-derived soluble factors, including hyaluronan fragments, induced the upregulation of a key glycolytic enzyme, PFKFB3, in tumor-associated monocytes. This enzyme not only modulated the cellular metabolic switch but also mediated the increased expression of PD-L1 by activating the nuclear factor kappa B signaling pathway in these cells. Consistently, the levels of PFKFB3+CD68+ cell infiltration in peritumoral tissues were negatively correlated with overall survival and could serve as an independent prognostic factor for survival in patients with HCC. CONCLUSIONS: Our results reveal a mechanism by which the cellular metabolic switch regulates the pro-tumor functions of monocytes in a specific human tumor microenvironment. PFKFB3 in both cancer cells and tumor-associated monocytes is a potential therapeutic target in human HCC. LAY SUMMARY: Programmed cell death 1 ligand 1 (PD-L1) expressed on antigen-presenting cells, rather than tumor cells, has been reported to play an essential role in checkpoint blockade therapy. A fundamental understanding of mechanisms that regulate the expression of PD-L1 on tumor-infiltrating monocytes/macrophages will undoubtedly lead to the possibility of developing novel PD-L1 blockade strategies with high specificity and efficiency. The current study unveils a novel mechanism by which metabolic switching links immune activation responses to immune tolerance in the tumor milieu, identifying potential targets for future immune-based anti-cancer therapies.