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Abstract Primary biliary cholangitis (PBC) is a cholestatic autoimmune liver disease characterized by autoreactive T cell response against intrahepatic small bile ducts. Here, we use Il12b-/-Il2ra-/- mice (DKO mice) as a model of autoimmune cholangitis and demonstrate that Cd8a knockout or treatment with an anti-CD8α antibody prevents/reduces biliary immunopathology. Using single-cell RNA sequencing analysis, we identified CD8+ tissue-resident memory T (Trm) cells in the livers of DKO mice, which highly express activation- and cytotoxicity-associated markers and induce apoptosis of bile duct epithelial cells. Liver CD8+ Trm cells also upregulate the expression of several immune checkpoint molecules, including PD-1. We describe the development of a chimeric antigen receptor to target PD-1-expressing CD8+ Trm cells. Treatment of DKO mice with PD-1-targeting CAR-T cells selectively depleted liver CD8+ Trm cells and alleviated autoimmune cholangitis. Our work highlights the pathogenic role of CD8+ Trm cells and the potential therapeutic usage of PD-1-targeting CAR-T cells.

ABSTRACTRegulatory T (Treg) cells are critical in shaping an immunosuppressive microenvironment to favor tumor progression and resistance to therapies. However, the heterogeneity and function of Treg cells in esophageal squamous cell carcinoma (ESCC) remain underexplored. We identified CD177 as a tumor-infiltrating Treg cell marker in ESCC. Interestingly, expression levels of CD177 and PD-1 were mutually exclusive in tumor Treg cells. CD177+ Treg cells expressed high levels of IL35, in association with CD8+ T cell exhaustion, whereas PD-1+ Treg cells expressed high levels of IL10. Pan-cancer analysis revealed that CD177+ Treg cells display increased clonal expansion compared to PD-1+ and double-negative (DN) Treg cells, and CD177+ and PD-1+ Treg cells develop from the same DN Treg cell origin. Importantly, we found CD177+ Treg cell infiltration to be associated with poor overall survival and poor response to anti-PD-1 immunotherapy plus chemotherapy in ESCC patients. Finally, we found that lymphatic endothelial cells are associated with CD177+ Treg cell accumulation in ESCC tumors, which are also decreased after anti-PD-1 immunotherapy plus chemotherapy. Our work identifies CD177+ Treg cell as a tumor-specific Treg cell subset and highlights their potential value as a prognostic marker of survival and response to immunotherapy and a therapeutic target in ESCC.

Abstract Background Papillary thyroid carcinoma (PTC) is the most common malignant endocrine tumour, and its incidence and prevalence are increasing considerably. Cellular heterogeneity in the tumour microenvironment is important for PTC prognosis. Spatial transcriptomics is a powerful technique for cellular heterogeneity study. Methods In conjunction with a clinical pathologist identification method, spatial transcriptomics was employed to characterise the spatial location and RNA profiles of PTC‐associated cells within the tissue sections. The spatial RNA‐clinical signature genes for each cell type were extracted and applied to outlining the distribution regions of specific cells on the entire section. The cellular heterogeneity of each cell type was further revealed by ContourPlot analysis, monocle analysis, trajectory analysis, ligand–receptor analysis and Gene Ontology enrichment analysis. Results The spatial distribution region of tumour cells, typical and atypical follicular cells (FCs and AFCs) and immune cells were accurately and comprehensively identified in all five PTC tissue sections. AFCs were identified as a transitional state between FCs and tumour cells, exhibiting a higher resemblance to the latter. Three tumour foci were shared among all patients out of the 13 observed. Notably, tumour foci No. 2 displayed elevated expression levels of genes associated with lower relapse‐free survival in PTC patients. We discovered key ligand–receptor interactions, including LAMB3–ITGA2, FN1–ITGA3 and FN1–SDC4, involved in the transition of PTC cells from FCs to AFCs and eventually to tumour cells. High expression of these patterns correlated with reduced relapse‐free survival. In the tumour immune microenvironment, reduced interaction between myeloid‐derived TGFB1 and TGFBR1 in tumour focus No. 2 contributed to tumourigenesis and increased heterogeneity. The spatial RNA‐clinical analysis method developed here revealed prognosis‐associated cellular heterogeneity in the PTC microenvironment. Conclusions The occurrence of tumour foci No. 2 and three enhanced ligand–receptor interactions in the AFC area/tumour foci reduced the relapse‐free survival of PTC patients, potentially leading to improved prognostic strategies and targeted therapies for PTC patients.

Abstract Autoimmune liver diseases (ALDs) are chronic inflammatory hepatobiliary diseases in which the autoimmune responses directed against liver tissue result in inflammation and tissue damage. There are several types of ALDs, including autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, immunoglobulin G4‐related sclerosing cholangitis, and overlap syndromes. Metabolism and epigenetics are both critical components of the development and progression of ALDs. Immunometabolism addresses how metabolic processes influence immune cell development, fate and behavior, and how immune responses impact metabolism. In the context of immunity, epigenetics involves regulating gene expression without altering the DNA sequence. Epigenetic modifications can ultimately result in changes in the immunophenotype. The interplay between the immune system, metabolism, and epigenetic factors is highly complex. A better understanding of this interplay and the regulatory mechanisms involved is crucial to uncover the disease pathogenesis of ALDs and exploring novel therapeutic options. This article provides a comprehensive review of the dysregulation of immunometabolism and epigenetics, as well as the multilevel regulatory mechanisms, underlying the autoimmunity in ALDs.

Abstract Background The Notch signaling mutation is associated with enhanced anti-tumor immune response in colorectal cancer (CRC). In this study, we aim to investigate the underlying mechanism and the predictive potential of Notch signaling mutation for responding to immunotherapy in CRC. Methods We analyzed the immune response associated genes in CRC with Notch signaling mutation concomitant with or without microsatellite instability (MSI) using TCGA dataset and investigated the mutation profiles of the Notch signaling pathway using cBioPortal. The Notch signaling scores and immune cell infiltration scores in different groups were calculated. We applied the Kaplan–Meier method for survival analysis in CRC patients who underwent immunotherapy, and the log-rank test to determine the statistically significant differences in survival. Notch1-knock-down cell line was constructed to detect the pathway and gene variations. Results We found that Notch signaling pathway mutation was associated with activated immune response, especially in those with MSI. Such association is useful for predicting a prolonged overall survival of CRC patients who underwent immune checkpoint inhibitor treatment. The mutation resulted in the functional loss of Notch signaling and may modulate the tumor immune microenvironment by increasing the expression of chemokines that are important for recruiting immune cells. Conclusions The Notch signaling mutation can modulate the chemotaxis of immune cells by upregulating the chemokine levels of the tumor immune microenvironment, and CRC patients with Notch signaling pathway mutation have better overall survival after immune checkpoint inhibitor treatment.

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune inflammatory disease mainly defined by T cell–dominated destruction of exocrine glands. Currently, CD8+ T cells are thought to be involved in the pathogenesis of pSS. However, the single-cell immune profiling of pSS and molecular signatures of pathogenic CD8+ T cells have not been well elucidated. Our multiomics investigation showed that both T cells and B cells, especially CD8+ T cells, were undergoing significant clonal expansion in pSS patients. TCR clonality analysis revealed that peripheral blood granzyme K+ (GZMK+) CXCR6+CD8+ T cells had higher a proportion of clones shared with CD69+CD103–CD8+ tissue-resident memory T (Trm) cells in labial glands in pSS. CD69+CD103–CD8+ Trm cells featured by high expression of GZMK were more active and cytotoxic in pSS compared with their CD103+ counterparts. Peripheral blood GZMK+CXCR6+CD8+ T cells with higher CD122 expression were increased and harbored a gene signature similar to Trm cells in pSS. Consistently, IL-15 was significantly elevated in pSS plasma and showed the capacity to promote differentiation of CD8+ T cells into GZMK+CXCR6+CD8+ T cells in a STAT5-dependent manner. In summary, we depicted the immune profile of pSS and further conducted comprehensive bioinformatics analysis and in vitro experimental investigations to characterize the pathogenic role and differentiation trajectory of CD8+ Trm cells in pSS.

Abstract Background Finding correlation patterns is an important goal of analyzing biological data. Currently available methods for correlation analysis mainly use non-direct associations, such as the Pearson correlation coefficient, and focus on the interpretation of networks at the level of modules. For biological objects such as genes, their collective function depends on pairwise gene-to-gene interactions. However, a large amount of redundant results from module level methods often necessitate further detailed analysis of gene interactions. New approaches of measuring direct associations among variables, such as the part mutual information (PMI), may help us better interpret the correlation pattern of biological data at the level of variable pairs. Results We use PMI to calculate gene co-expression networks of cancer mRNA transcriptome data. Our results show that the PMI-based networks with fewer edges could represent the correlation pattern and are robust across biological conditions. The PMI-based networks recall significantly more important parts of omics defined gene-pair relationships than the Pearson Correlation Coefficient (PCC)-based networks. Based on the scores derived from PMI-recalled copy number variation or DNA methylation gene-pairs, the patients with cancer can be divided into groups with significant differences on disease specific survival. Conclusions PMI, measuring direct associations between variables, extracts more important biological relationships at the level of gene pairs than conventional indirect association measures do. It can be used to refine module level results from other correlation methods. Particularly, PMI is beneficial to analysis of biological data of the complicated systems, for example, cancer transcriptome data.

Abstract Activation of adipose tissue macrophages (ATMs) contributes to chronic inflammation and insulin resistance in obesity. However, the transcriptional regulatory machinery involved in ATM activation during the development of obesity is not fully understood. Here, we profiled the chromatin accessibility of blood monocytes and ATMs from obese and lean mice using assay for transposase-accessible chromatin sequencing (ATAC-seq). We found that monocytes and ATMs from obese and lean mice exhibited distinct chromatin accessibility status. There are distinct regulatory elements that are specifically associated with monocyte or ATM activation in obesity. We also discovered several transcription factors that may regulate monocyte and ATM activation in obese mice, specifically a predicted transcription factor named ETS translocation variant 5 (ETV5). The expression of ETV5 was significantly decreased in ATMs from obese mice and its downregulation was mediated by palmitate stimulation. The decrease in ETV5 expression resulted in macrophage activation. Our results also indicate that ETV5 suppresses endoplasmic reticulum (ER) stress and Il6 expression in macrophages. Our work delineates the changes in chromatin accessibility in monocytes and ATMs during obesity, and identifies ETV5 as a critical transcription factor suppressing ATM activation, suggesting its potential use as a therapeutic target in obesity-related chronic inflammation.

BackgroundThyroid tissue has a special immune microenvironment that is not well characterized. Whether immune cells have a prognostic value in the recurrence of papillary thyroid cancer (PTC) needs further investigation.MethodsMultinodular non-toxic goiter (MNG) was taken as normal tissue for the difficulty in obtaining completely normal thyroid tissue (normal thyroid function, no thyroiditis, and no nodules). We compared the composition of mononuclear cells (MNCs) in peripheral blood and thyroid tissues from MNG and PTC patients by high-dimensional flow cytometry profiling and verified the results by multiplex immunohistochemistry. The recurrence rates of PTC patients with different CD8+T cell subset signatures were compared using TCGA database.ResultsWe observed that the immune cell composition of MNG was different from that in peripheral blood. Thyroid tissue contains higher percentages of T cells and NK cells. Moreover, the percentages of memory T cells and Treg cells were higher in thyroid than in peripheral blood and increased in PTC tumors. We further focused on the antitumoral CD8+T cells and found that the expression patterns of PD-1, CD39, and CD103 on CD8+T cells were different between MNG and PTC. Importantly, we found higher percentages of PD-1+CD39+CD103+CD8+T and PD-1+CD39+CD103-CD8+T cells in PTC tumor tissues from recurrent patients than non-recurrent patients. By analyzing PTC data from TCGA database, we found that the expression patterns of these molecules were associated with different pathologic types and genders among PTC patients. Moreover, patients with PD-1hiCD39loCD103hiCD8hi, PD-1hiCD39hiCD103loCD8hi, and PD-1loCD39hiCD103hiCD8hi expression patterns have a higher 10-year recurrence-free survival.ConclusionThe immune microenvironment in MNG tissue is distinct from that in peripheral blood and paratumor tissue. More memory CD8+T cells were detected in PTC, and expression patterns of PD-1, CD39, and CD103 on CD8+T cells were significantly different in physiology and gender and associated with the recurrence rate of PTC. These observations indicate that CD8+T cell signatures may be useful prognostic markers for PTC recurrence.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis, characterized by glomerular deposition of IgA immune complexes, mainly produced by B cells under the regulation of CD4+T cells. However, the alterations of specific CD4+T cell subsets and the mechanism of B cells activation in IgAN remain unclear. Therefore, we aimed to investigate the landscape characteristics and role of CD4+T cells in the progression of IgAN. We identified that the proportion of Th2, Th17 and Tfh (follicular helper T) cells in patients with IgAN was significantly higher than that of healthy controls (P < 0.05). Single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) showed that Th cells and B cells in patients with IgAN were more activated. Correspondingly, multiplex immunohistochemistry staining of renal biopsy showed increased infiltration of CD4+T and B cells in the kidneys of patients with IgAN. The degree of infiltration was positively correlated with the degree of renal damage. Interestingly, the proportion of Tfh cells in peripheral blood was positively correlated with the severity of proteinuria. Moreover, the proximity position of Tfh cells and B cells suggested that cell-cell interactions between Tfh and B cells were happening in situ. Intercellular communication analysis also showed enhanced interaction between Tfh cells and B cells in IgAN. Our findings suggested that Tfh cells of patients possibly contributed to the progression of IgAN by activating B cells via cell-cell interactions and TNFSF14-TNFRSF14 may be an underlying signaling pathway.

O. elatus Nakai is a traditional medicine that has been confirmed to exert effective antioxidant and anti-inflammatory functions, and is used for the treatment of different disorders. However, its potential beneficial effects on drug induced hepatotoxicity and relevant molecular mechanisms remain unclear. This study investigated the protective effect and further elucidated the mechanisms of action of O. elatus on liver protection. O. elatus chlorogenic acids-enriched fraction (OEB), which included chlorogenic acid and isochlorogenic acid A, were identified by HPLC-MS/MS. OEB was administrated orally daily for seven consecutive days, followed by a single intraperitoneal injection of an overdose of APAP after the final OEB administration. The effects of OEB on immune cells in mice liver were analyzed using flow cytometry. APAP metabolite content in serum was detected using HPLC-MS/MS in order to investigate whether OEB affects CYP450 activities. The intestinal content samples were processed for 16 s microbiota sequencing. Results demonstrated that OEB decreased alanine aminotransferase, aspartate aminotransferase contents, affected the metabolism of APAP, and decreased the concentrates of APAP, APAP-CYS and APAP-NAC by inhibiting CYP2E1 and CYP3A11 activity. Furthermore, OEB pretreatment regulated lipid metabolism by affecting the peroxisome proliferator-activated receptors (PPAR) signaling pathway in mice and also increased the abundance of Akkermansia and Parabacteroides. This study indicated that OEB is a potential drug candidate for treating hepatotoxicity because of its ability to affect drug metabolism and regulate lipid metabolism.

Primary biliary cholangitis (PBC), an organ-specific autoimmune disease, is characterized by injury to small bile ducts, inflammatory cell infiltrates within the liver, progressive cholestasis, and in some cases, cirrhosis with unclear pathogenesis. We aimed to clarify the importance role of hepatic immunce cells in the pathogenesis of human and experimental PBC.The dominant-negative TGFβ receptor type II transgenic (dnTGFβRII) mice, a well-studied and established murine model of PBC were used to identify changes of immune cells, especially the pathogenic CD8+ T cells. The high-throughput single-cell RNA sequencing technology were applied and found functional heterogeneity among the hepatic CD8+ T cells subsets in dnTGFβRII mice. CD8+ T cells were confirmed the key cells leading to the pathogenesis of PBC in dnTGFβRII mice, and identified the terminally differentiated CD8αα T cells and CD8αβ T cell subsets in the liver of dnTGFβRII mice. While terminally differentiated CD8αα T cells have higher cytokine production ability and cytotoxicity, the terminally differentiated CD8αβ T cells retain their proliferative profile. Our work suggests that there are developmental and differentiated trajectories of pathogenic CD8+ T cell subsets in the pathogenesis of PBC. A further clarification of their roles would be helpful to our understanding of the pathogenesis of PBC and may potentially lead to identifying novel therapeutic modalities.