Targeting OXCT1-mediated ketone metabolism reprograms macrophages to promote antitumor immunity via CD8+ T cells in hepatocellular carcinoma.

BACKGROUND & AIMS: The liver is the main organ of ketogenesis, while ketones are mainly metabolized in peripheral tissues via the critical enzyme OXCT1. We previously found that ketolysis is reactivated in hepatocellular carcinoma (HCC) cells through OXCT1 expression to promote tumor progression; however, whether OXCT1 regulates antitumor immunity remains unclear. METHODS: To investigate the expression pattern of OXCT1 in hepatocellular carcinoma in vivo, we conducted multiplex immunohistochemistry (mIHC) experiments on human HCC specimens. To explore the role of OXCT1 in mouse hepatocellular carcinoma tumor-associated macrophages (TAMs), we generated LysMcreOXCT1f/f (OXCT1 conditional knockout in macrophages) mice. RESULTS: Here, we found that inhibiting OXCT1 expression in tumor-associated macrophages reduced CD8+ T-cell exhaustion through the succinate-H3K4me3-Arg1 axis. Initially, we found that OXCT1 was highly expressed in liver macrophages under steady state and that OXCT expression was further increased in TAMs. OXCT1 deficiency in macrophages suppressed tumor growth by reprogramming TAMs toward an antitumor phenotype, reducing CD8+ T-cell exhaustion and increasing CD8+ T-cell cytotoxicity. Mechanistically, high OXCT1 expression induced the accumulation of succinate, a byproduct of ketolysis, in TAMs, which promoted Arg1 transcription by increasing the H3K4 trimethylation (H3K4me3) level in the Arg1 promoter. In addition, Pimozide, an inhibitor of OXCT1, suppressed Arg1 expression as well as TAM polarization toward the protumor phenotype, leading to decreasing CD8+ T-cell exhaustion and deceleration of tumor growth. Finally, high expression of OXCT1 in macrophages was positively associated with poor survival in HCC patients. CONCLUSIONS: In conclusion, our results demonstrate that OXCT1 epigenetically suppresses antitumor immunity, suggesting that suppressing OXCT1 activity in TAMs is an effective approach for treating liver cancer. IMPACT AND IMPLICATIONS: The intricate metabolism of liver macrophages plays a critical role in shaping HCC progression and immune modulation. Targeting macrophage metabolism to counteract immune suppression presents a promising avenue for HCC. Here, we found that ketogenesis gene OXCT1 was highly expressed in tumor-associated macrophages and promoted tumor growth by reprogramming TAMs toward a protumor phenotype. And the strategic pharmacological intervention or genetic downregulation of OXCT1 in TAMs enhances the antitumor immunity and decelerated tumor growth. Our results suggest that suppressing OXCT1 activity in TAMs is an effective approach for treating liver cancer.

Deficiency of CBL and CBLB ubiquitin ligases leads to hyper T follicular helper cell responses and lupus by reducing BCL6 degradation.

Recent evidence reveals hyper T follicular helper (Tfh) cell responses in systemic lupus erythematosus (SLE); however, molecular mechanisms responsible for hyper Tfh cell responses and whether they cause SLE are unclear. We found that SLE patients downregulated both ubiquitin ligases, casitas B-lineage lymphoma (CBL) and CBLB (CBLs), in CD4+ T cells. T cell-specific CBLs-deficient mice developed hyper Tfh cell responses and SLE, whereas blockade of Tfh cell development in the mutant mice was sufficient to prevent SLE. ICOS was upregulated in SLE Tfh cells, whose signaling increased BCL6 by attenuating BCL6 degradation via chaperone-mediated autophagy (CMA). Conversely, CBLs restrained BCL6 expression by ubiquitinating ICOS. Blockade of BCL6 degradation was sufficient to enhance Tfh cell responses. Thus, the compromised expression of CBLs is a prevalent risk trait shared by SLE patients and causative to hyper Tfh cell responses and SLE. The ICOS-CBLs axis may be a target to treat SLE.

Bone marrow CD8+ Trm cells induced by IL-15 and CD16+ monocytes contribute to HSPC destruction in human severe aplastic anemia.

Idiopathic severe aplastic anemia (SAA) is a disease of bone marrow failure caused by T-cell-induced destruction of hematopoietic stem and progenitor cells (HSPCs), however the mechanism remains unclear. We performed single-cell RNA sequencing of PBMCs and BMMCs from SAA patients and healthy donors and identified a CD8+ T cell subset with a tissue residency phenotype (Trm) in bone marrow that exhibit high IFN-γ and FasL expression and have a higher ability to induce apoptosis in HSPCs in vitro through FasL expression. CD8+ Trm cells were induced by IL-15 presented by IL-15Rα on monocytes, especially CD16+ monocytes, which were increased in SAA patients. CD16+ monocytes contributed to IL-15-induced CD38+CXCR6+ pre-Trm differentiation into CD8+ Trm cells, which can be inhibited by the CD38 inhibitor 78c. Our results demonstrate that IL-15-induced CD8+ Trm cells are pathogenic cells that mediate HSPC destruction in SAA patients and are therapeutic targets for future treatments.

Targeting pathogenic CD8+ tissue-resident T cells with chimeric antigen receptor therapy in murine autoimmune cholangitis.

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.

Spatial transcriptomics reveals prognosis-associated cellular heterogeneity in the papillary thyroid carcinoma microenvironment.

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.

Lymphatic endothelial cell-mediated accumulation of CD177+Treg cells suppresses antitumor immunity in human esophageal squamous cell carcinoma.

Regulatory 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.

Immunophenotyping with high-dimensional flow cytometry identifies Treg cell subsets associated with recurrence in papillary thyroid carcinoma.

The activation of Treg cell subsets is critical for the prognosis of tumor patients; however, their heterogeneity and disease association in papillary thyroid carcinoma (PTC) need further investigation. We performed high-dimensional flow cytometry for immunophenotyping on thyroid tissues and matched peripheral blood samples from patients with multinodular goiters or PTC. We analyzed CD4+ T cell and Treg cell phenotypes and compared the recurrence-free survival of PTC patients with different Treg cell subset characteristics using TCGA. Furthermore, PTC recurrent and non-recurrent group were compared by multiplex immunohistochemistry. High-dimensional flow cytometry and bioinformatics analysis revealed an enrichment of Tregs in tumors compared with multinodular goiters and peripheral blood specimens. Moreover, effector Tregs (e-Tregs) as well as FOXP3+ non-Tregs were enriched in tumor samples, and the expression of CD39, PD-1, and CD103 increased on tumor Tregs. TCGA data analysis showed that individuals with CD39hi PD-1loCD103loe-Treghi and CD39loPD-1loCD103hie-Treghi expression patterns had a high recurrence rate. According to the multiplex immunohistochemistry and analysis, compared with non-recurrent group, the proportion of high recurrence rate effector Treg clusters (CD39+PD-1-CD103- plus CD39-PD-1-CD103+) was increased in recurrent patients. Overall, our results highlight the potential of e-Treg subsets as future immunotherapy targets for PTC recurrence.

Dynamic single-cell mapping unveils Epstein‒Barr virus-imprinted T-cell exhaustion and on-treatment response.

Epstein‒Barr virus (EBV)-associated gastric cancer (GC) manifests an intriguing immunotherapy response. However, the cellular basis for EBV-imprinted tumour immunity and on-treatment response remains undefined. This study aimed to finely characterize the dynamic tumour immune contexture of human EBV (+) GC treated with immunochemotherapy by longitudinal scRNA-seq and paired scTCR/BCR-seq. EBV (+) GC exhibits an inflamed-immune phenotype with increased T-cell and B-cell infiltration. Immunochemotherapy triggers clonal revival and reinvigoration of effector T cells which step to determine treatment response. Typically, an antigen-specific ISG-15+CD8+ T-cell population is highly enriched in EBV (+) GC patients, which represents a transitory exhaustion state. Importantly, baseline intratumoural ISG-15+CD8+ T cells predict immunotherapy responsiveness among GC patients. Re-emerged clonotypes of pre-existing ISG-15+CD8+ T cells could be found after treatment, which gives rise to a CXCL13-expressing effector population in responsive EBV (+) tumours. However, LAG-3 retention may render the ISG-15+CD8+ T cells into a terminal exhaustion state in non-responsive EBV (+) tumours. In accordance, anti-LAG-3 therapy could effectively reduce tumour burden in refractory EBV (+) GC patients. Our results delineate a distinct implication of EBV-imprinted on-treatment T-cell immunity in GC, which could be leveraged to optimize the rational design of precision immunotherapy.

Single-cell and spatial transcriptome analysis reveals the cellular heterogeneity of liver metastatic colorectal cancer.

In this study, we comprehensively charted the cellular landscape of colorectal cancer (CRC) and well-matched liver metastatic CRC using single-cell and spatial transcriptome RNA sequencing. We generated 41,892 CD45- nonimmune cells and 196,473 CD45+ immune cells from 27 samples of six CRC patients, and found that CD8_CXCL13 and CD4_CXCL13 subsets increased significantly in liver metastatic samples that exhibited high proliferation ability and tumor-activating characterization, contributing to better prognosis of patients. Distinct fibroblast profiles were observed in primary and liver metastatic tumors. F3+ fibroblasts enriched in primary tumors contributed to worse overall survival by expressing protumor factors. However, MCAM+ fibroblasts enriched in liver metastatic tumors might promote generation of CD8_CXCL13 cells through Notch signaling. In summary, we extensively analyzed the transcriptional differences of cell atlas between primary and liver metastatic tumors of CRC by single-cell and spatial transcriptome RNA sequencing, providing different dimensions of the development of liver metastasis in CRC.

Single-cell profiling reveals pathogenic role and differentiation trajectory of granzyme K+CD8+ T cells in primary Sjögren's syndrome.

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.

Single-Cell Analysis of Patients with Axial Spondyloarthritis After Anti-TNFα Treatment: Experimental Data and Review of the Literature.

Axial spondyloarthritis (Ax-SpA) is a chronic inflammatory disease that predominantly affects the axial joints and is most common in young men. However, the precise immune cell subset involved in Ax-SpA remains unclear. Our study characterized the periphery immune landscape of Ax-SpA patients before and after anti-TNFα treatment using single-cell transcriptomics and proteomics sequencing and elucidated the effects of anti-TNFα treatment at the single-cell level. First, we found that peripheral granulocytes and monocytes significantly increased in Ax-SpA patients. Second, we identified a more functional subtype of regulatory T cells, which was present in synovial fluid and increased in patients after treatment. Third, we identified a cluster of inflammatory monocyte subset with stronger inflammatory and chemotactic characteristics. A potential interaction between classical monocytes and granulocytes via the CXCL8/2-CXCR1/2 signaling pathway was observed, which decreased after treatment. Together, these results defined the complex expression profiles and advanced our understanding of the immune atlas in Ax-SpA patients before and after anti-TNFα treatment.

Spontaneous development of an autoimmune hepatitis - primary biliary cholangitis overlap syndrome in dnTGFßRII Aire-/- mice.

Autoimmune regulator (Aire) and TGF-ß signaling play important roles in central tolerance and peripheral tolerance, respectively, by eliminating or suppressing the activity of autoreactive T cells. We previously demonstrated that dnTGFßRII mice develop a defect in peripheral tolerance and a primary biliary cholangitis (PBC)-like disease. We hypothesized that by introducing the Aire gene to this model, we would observe a more severe PBC phenotype. Interestingly, however, we demonstrated that, while dnTGFßRII Aire-/- mice do manifest key histological and serological features of autoimmune cholangitis, they also develop mild to moderate interface hepatitis and show high levels of alanine transaminase (ALT) and antinuclear antibodies (ANA), characteristics of autoimmune hepatitis (AIH). To further understand this unique phenotype, we performed RNA sequencing (RNA-seq) and flow cytometry to explore the functional pathways and immune cell pathways in the liver of dnTGFßRII Aire-/- mice. Our data revealed enrichments of programmed cell death pathways and predominant CD8+ T cell infiltrates. Depleting CD8+ T cells using an anti-CD8α antibody significantly alleviated hepatic inflammation and prolonged the life span of these mice. Finally, RNA-seq data indicated the clonal expansion of hepatic CD8+ T cells. In conclusion, these mice developed an autoreactive CD8+ T-cell-mediated autoimmune cholangitis with concurrent hepatitis that exhibited key histological and serological features of the AIH-PBC overlap syndrome, representing a novel model for the study of tolerance and autoimmune liver disease. © 2023 The Pathological Society of Great Britain and Ireland.

Immune profiling identifies CD8+ T-cell subset signatures as prognostic markers for recurrence in papillary thyroid cancer.

Background: Thyroid 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. Methods: Multinodular 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. Results: We 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. Conclusion: The 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.

HBV immune tolerance of HBs-transgenic mice observed through parabiosis with WT mice.

It was extensively recognized that central tolerance to HBV exists in HBs-transgenic (Tg) mice, however, the immune response to HBV vaccine may be inspired in adult HBs-Tg mice after boosting with potent adjuvants, leaving a mystery to explore its immune tolerance. Here, WT-HBs-Tg parabiotic mice model was generated by conjoining WT (donor) and HBs-Tg (host) mouse via parabiotic surgery, in order to see how immunocompetent WT mice naturally respond to HBV, and how tolerant HBs-Tg mice influence the anti-HBV immunity from WT mice. It was found that WT CD8+ T cells markedly accumulated into the liver of HBs-Tg parabionts, and importantly, almost all HBsAg-specific CD8+ T cells derived from WT but not HBs-Tg mice, making a clear separation of a normal immune response from WT donor and a tolerant response by recipient host. Further, in the absence of host but not donor spleen, HBsAg-specific CD8+ T cells disappeared, indicating that host spleen was the indispensable site for donor HBsAg-specific CD8+ T cell priming though its mechanisms need further study. We found that donor CD4+ T helper cells were necessary for donor HBsAg-specific CD8+ T cell response by CD4-deficiency in WT or in HBs-Tg mice, indicating that an immune response was elicited between CD4+ T helper cells and CD8+ cytotoxic T cells of donor in the host but not donor spleen. It was noted that compared to donor CD4+ T cells, host CD4+ T cells were characterized with more tolerant features by harboring more CD25+Foxp3+ Tregs with higher expression of PD-1 and TIGIT in the spleen of HBs-Tg parabionts, which exhibited suppressive function on CD8+ T cells directly. Moreover, the Th1/Treg ratio was enhanced after parabiosis, suggesting that donor T helper cells may overcome the negative regulation of host Tregs in host spleen. In conclusion, both incompetent anti-HBV CD8+ T cells and insufficient help from CD4+ T cells are the major mechanisms underlying immune tolerance in HBs-Tg mice which helps explain HBV persistence.

XBP1-mediated activation of the STING signalling pathway in macrophages contributes to liver fibrosis progression.

Background & Aims: XBP1 modulates the macrophage proinflammatory response, but its function in macrophage stimulator of interferon genes (STING) activation and liver fibrosis is unknown. X-box binding protein 1 (XBP1) has been shown to promote macrophage nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) activation in steatohepatitis. Herein, we aimed to explore the underlying mechanism of XBP1 in the regulation of STING signalling and the subsequent NLRP3 activation during liver fibrosis. Methods: XBP1 expression was measured in the human fibrotic liver tissue samples. Liver fibrosis was induced in myeloid-specific Xbp1-, STING-, and Nlrp3-deficient mice by carbon tetrachloride injection, bile duct ligation, or a methionine/choline-deficient diet. Results: Although increased XBP1 expression was observed in the fibrotic liver macrophages of mice and clinical patients, myeloid-specific Xbp1 deficiency or pharmacological inhibition of XBP1 protected the liver against fibrosis. Furthermore, it inhibited macrophage NLPR3 activation in a STING/IRF3-dependent manner. Oxidative mitochondrial injury facilitated cytosolic leakage of macrophage self-mtDNA and cGAS/STING/NLRP3 signalling activation to promote liver fibrosis. Mechanistically, RNA sequencing analysis indicated a decreased mtDNA expression and an increased BCL2/adenovirus E1B interacting protein 3 (BNIP3)-mediated mitophagy activation in Xbp1-deficient macrophages. Chromatin immunoprecipitation (ChIP) assays further suggested that spliced XBP1 bound directly to the Bnip3 promoter and inhibited the transcription of Bnip3 in macrophages. Xbp1 deficiency decreased the mtDNA cytosolic release and STING/NLRP3 activation by promoting BNIP3-mediated mitophagy activation in macrophages, which was abrogated by Bnip3 knockdown. Moreover, macrophage XBP1/STING signalling contributed to the activation of hepatic stellate cells. Conclusions: Our findings demonstrate that XBP1 controls macrophage cGAS/STING/NLRP3 activation by regulating macrophage self-mtDNA cytosolic leakage via BNIP3-mediated mitophagy modulation, thus providing a novel target against liver fibrosis. Lay summary: Liver fibrosis is a typical progressive process of chronic liver disease, driven by inflammatory and immune responses, and is characterised by an excess of extracellular matrix in the liver. Currently, there is no effective therapeutic strategy for the treatment of liver fibrosis, resulting in high mortality worldwide. In this study, we found that myeloid-specific Xbp1 deficiency protected the liver against fibrosis in mice, while XBP1 inhibition ameliorated liver fibrosis in mice. This study concluded that targeting XBP1 signalling in macrophages may provide a novel strategy for protecting the liver against fibrosis.

Notch signaling mutations increase intra-tumor chemokine expression and predict response to immunotherapy in colorectal cancer.

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.

Increased proportion of follicular helper T cells is associated with B cell activation and disease severity in IgA nephropathy.

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.

The gut microbiome contributes to splenomegaly and tissue inflammation in a murine model of primary biliary cholangitis.

Background: Splenomegaly is not just a consequence of numerous chronic and acute conditions but may also contribute to their severity, due to the interaction of the spleen with the gut microbiome. This study aimed to explore the effect of the gut microbiome on splenomegaly. Methods: We used p40-/-IL-2Rα-/- mice as a murine model of primary biliary cholangitis (PBC) as per our previous study. Splenomegaly was evaluated by spleen weight. Severity of liver inflammation was evaluated by hepatic mononuclear cell (MNCs) number and pathological score. Changes of immune cells in the spleen and liver were detected by flow cytometry. The effects of the gut microbiome on splenomegaly and liver inflammation were observed by combined antibiotic treatment in p40-/-IL-2Rα-/- mice. Results: A proportion of p40-/-IL-2Rα-/- mice developed splenomegaly. The results revealed that liver mononuclear cells infiltration, histological scores of hepatic inflammation, and bile duct damage were positively correlated with the degree of splenomegaly. Hepatic CD4+ and CD8+ T cells numbers were significantly higher in mice with splenomegaly, and this was particularly observed in activated effector memory CD4+ T and CD8+ T cells. A proportion of some other immune cells including granulocytes, B, natural killer (NK), and CD8+ T effector memory cells were also altered in the enlarged spleen. More importantly, administration of quadruple antibiotics to deplete gut microbiota relieved the splenomegaly of p40-/-IL-2Rα-/- mice, significantly alleviated liver inflammation, and caused a significant reduction of liver and spleen T cell accumulation and activation; however, single antibiotics did not induce these changes. Conclusions: Splenomegaly was associated with more severe liver inflammation in our PBC murine model, and this effect was reversed by quadruple antibiotic treatment.

Bioorthogonal Equipping CAR-T Cells with Hyaluronidase and Checkpoint Blocking Antibody for Enhanced Solid Tumor Immunotherapy.

Adoptive cellular therapy utilizing chimeric antigen receptor redirected T (CAR-T) cells has shown impressive therapeutic effects on hematological malignancies. In contrast, the efficacy of CAR-T therapies in treating solid tumors is still poor, which is largely due to inefficient penetration into solid tumors and the immunosuppressive tumor microenvironment. Herein, we engineered hyaluronidase (HAase) and the checkpoint blocking antibody α-PDL1 on the CAR-T cell surface via highly efficient and biocompatible bioorthogonal click chemistry to improve their therapeutic effects on solid tumors. The modified HAase degrades hyaluronic acid and destroys the tumor extracellular matrix, allowing CAR-T cells to penetrate deeply into solid tumors, as evidenced by in vitro infiltration experiments and in vivo biodistribution studies. In addition, in vitro cytotoxicity studies showed stronger antitumor activity of α-PDL1-decorated cells than traditional CAR-T cells. Importantly, HAase- and α-PDL1-engineered CAR-T cells showed better therapeutic efficacy on two solid tumor models and did not cause significant systemic side effects. In this work, we provide a simple, efficient, and biologically safe chemical strategy to engineer traditional CAR-T cells for enhanced therapeutic efficacy on solid tumors, which can be extended to other adoptive cellular immunotherapies and holds great potential for clinical application.

NUDT1 promotes the accumulation and longevity of CD103+ TRM cells in primary biliary cholangitis.

BACKGROUND & AIMS: Pyruvate dehydrogenase (PDC)-E2 specific CD8+ T cells play a leading role in biliary destruction in PBC. However, there are limited data on the characterization of these autoantigen-specific CD8+ T cells, particularly in the liver. Herein, we aimed to identify pathogenic intrahepatic CD8+ T-cell subpopulations and investigate their immunobiology in PBC. METHODS: Phenotypic and functional analysis of intrahepatic T-cell subsets were performed by flow cytometry. CD103+ TRM cell frequency was evaluated by histological staining. The transcriptome and metabolome were analyzed by RNA-seq and liquid chromatography-mass spectrometry, respectively. Cytotoxicity of TRM cells against cholangiocytes was assayed in a 3D organoid co-culture system. Moreover, the longevity (long-term survival) of TRM cells in vivo was studied by 2-octynoic acid-BSA (2OA-BSA) immunization, Nudt1 conditional knock-out and adoptive co-transfer in a murine model. RESULTS: Intrahepatic CD103+ TRM (CD69+CD103+CD8+) cells were significantly expanded, hyperactivated, and potentially specifically reactive to PDC-E2 in patients with PBC. CD103+ TRM cell frequencies correlated with clinical and histological indices of PBC and predicted poor ursodeoxycholic acid response. NUDT1 blockade suppressed the cytotoxic effector functions of CD103+ TRM cells upon PDC-E2 re-stimulation. NUDT1 overexpression in CD8+ T cells promoted tissue-residence programming in vitro; inhibition or knockdown of NUDT1 had the opposite effect. Pharmacological blockade or genetic deletion of NUDT1 eliminated CD103+ TRM cells and alleviated cholangitis in mice immunized with 2OA-BSA. Significantly, NUDT1-dependent DNA damage resistance potentiates CD8+ T-cell tissue-residency via the PARP1-TGFßR axis in vitro. Consistently, PARP1 inhibition restored NUDT1-deficient CD103+ TRM cell durable survival and TGFß-Smad signaling. CONCLUSIONS: CD103+ TRM cells are the dominant population of PDC-E2-specific CD8+ T lymphocytes in the livers of patients with PBC. The role of NUDT1 in promoting pathogenic CD103+ TRM cell accumulation and longevity represents a novel therapeutic target in PBC. LAY SUMMARY: Primary biliary cholangitis (PBC) is a rare inflammatory condition of the bile ducts. It can be treated with ursodeoxycholic acid, but a large percentage of patients respond poorly to this treatment. Liver-infiltrating memory CD8+ T cells recognizing the PDC-E2 immunodominant epitope are critical in the pathogenesis of PBC. We identifed the key pathogenic CD8+ T cell subset, and worked out the mechanisms of its hyperactivation and longevity, which could be exploited therapeutically.