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.

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.

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.

Chromatin accessibility analysis identifies the transcription factor ETV5 as a suppressor of adipose tissue macrophage activation in obesity.

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.

Cage-like La4B24 and Core-Shell La4B290/+/- : perfect spherically aromatic tetrahedral metallo-borospherenes.

Cage-like and core-shell metallo-borospherenes exhibit interesting structures and bonding. Based on extensive global searches and first-principles theory calculations, we predict herein the perfect tetrahedral cage-like Td La4B24 (1) and core-shell Td La4B29 (2), Td La4B29+ (3), and Td La4B29- (4) which all possess the same geometrical symmetry as their carbon fullerene counterpart Td C28, with four equivalent interconnected B6 triangles on the cage surface and four nona-coordinate La centers in four conjoined η9-B9 rings. In these tetra-La-doped boron complexes, La4[B@B4@B24]0/+/- (2/3/4) in the structural motif of 1 + 4 + 28 contain a B-centered tetrahedral Td B@B4 core in a La-decorated tetrahedral La4B24 shell, with the negatively charged tetra-coordinate B- at the center being the boron analog of tetrahedral C in Td CH4 (B- ~ C). Detailed orbital and bonding analyses indicate that these Td lanthanide boride complexes are spherically aromatic in nature with a universal La--B9 (d-p) σ and (d-p) δ coordination bonding pattern. The IR, Raman, and UV-Vis or photoelectron spectra of these novel metallo-borospherenes are computationally simulated to facilitate their spectral characterizations. Graphical abstract.

Multi-Omics Profiling Identifies Pathways Associated With CD8+ T-Cell Activation in Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is an autoimmune disease characterized by immune-mediated destruction of hematopoietic stem and progenitor cells. Autoreactive CD8+ T cells have been reported as the effector cells; however, the mechanisms regulating their cell activation in SAA remain largely unknown. Here, we performed proteomics and metabolomics analyses of plasma and bone marrow supernatant, together with transcriptional analysis of CD8+ T cells from SAA patients and healthy donors, to find key pathways that are involved in pathogenic CD8+ T-cell activation. We identified 21 differential proteins and 50 differential metabolites in SAA patients that were mainly involved in energy metabolism, complement and coagulation cascades, and HIF-1α signaling pathways. Interestingly, we found that these pathways are also enriched in T cells from SAA patients by analyzing available single-cell RNA sequencing data. Moreover, CD8+ T cells from SAA patients contain a highly activated CD38+ subset, which was increased in the bone marrow of SAA patients and a murine model of SAA. This subset presented enriched genes associated with the glycolysis or gluconeogenesis pathway, HIF-1α signaling pathway, and complement associated pathways, all of which were of importance in T-cell activation. In conclusion, our study reveals new pathways that may regulate CD8+ T-cell activation in SAA patients and provides potential therapeutic targets for SAA treatment.

Immunobiology of T Cells in Sjögren's Syndrome.

Sjögren's syndrome (SjS) is a systemic autoimmune disease marked by xerostomia (dry mouth), keratoconjunctivitis sicca (eye dryness), and other systematic disorders. Its pathogenesis involves an inflammatory process that is characterized by lymphocytic infiltration into exocrine glands and other tissues. Although the development of ectopic lymphoid tissue and overproduction of autoantibodies by hyperactive B cells suggest that they may promote SjS development, treatment directed towards them fails to induce significant laboratory or clinical improvement. T cells are overwhelming infiltrators in most phases of the disease, and the involvement of multiple T cell subsets of suggests the extraordinary complexity of SjS pathogenesis. The factors, including various cellular subtypes and molecules, regulate the activation and suppression of T cells. T cell activation induces inflammatory cell infiltration, B cell activation, tissue damage, and metabolic changes in SjS. Knowledge of the pathways that link these T cell subtypes and regulation of their activities are not completely understood. This review comprehensively summarizes the research progress and our understanding of T cells in SjS, including CD4+ T cells, CD8+ TRM cells, and innate T cells, to provide insights into for clinical treatment.

Multi-omics analysis identifies FoxO1 as a regulator of macrophage function through metabolic reprogramming.

Macrophages are plastic cells that can switch among different states according to bioenergetic or biosynthetic requirements. Our previous work demonstrated that the transcription factor Forkhead Box Protein 1 (FoxO1) plays a pivotal role in regulating the function of macrophages, but the underlying mechanisms are still unclear. Here we identify FoxO1 as a regulator of macrophage function through metabolic reprogramming. Transcriptomic and proteomic analyses showed that the deficiency of FoxO1 results in an alternatively activated (M2) phenotype of macrophages, with lower expression of inflammatory response- and migration-associated genes. Using the high content screening and analysis technology, we found that deletion of FoxO1 in macrophages slows their migration rate and impairs their function to limit tumor cell growth in vitro. Next, we demonstrated that glycolysis is inhibited in FoxO1-deficient macrophages, which leads to the observed functional changes and the reduced tumor suppression capability. This prospective study shows that FoxO1 serves as a bridge between metabolism and macrophage function.

Tissue-Resident Memory CD8+ T Cells Acting as Mediators of Salivary Gland Damage in a Murine Model of Sjögren's Syndrome.

OBJECTIVE: Although a role for CD4+ T cells in the pathogenesis of Sjögren's syndrome (SS) has been documented, the pathogenic significance of CD8+ T cells is unclear. The aim of this study was to investigate the role of CD8+ T cells in the development of SS. METHODS: Flow cytometry and immunofluorescence analyses were utilized to detect T cell infiltration within the labial salivary glands of patients with primary SS. In parallel, p40-/- CD25-/- mice were used as a murine model of SS. In addition, mice with genetic knockout of CD4, CD8a, or interferon-γ (IFNγ) were crossed with p40-/- CD25-/- mice to study the pathogenic significance of specific lineage subpopulations, including functional salivary gland tests as well as histopathologic and serologic data. A CD8+ T cell-specific depletion antibody was used in this murine SS model to evaluate its potential as a therapeutic strategy. RESULTS: CD8+ T cells with a tissue-resident memory phenotype outnumbered CD4+ T cells in the labial salivary glands of patients with SS, and were primarily colocalized with salivary duct epithelial cells and acinar cells. Furthermore, infiltrating CD8+ T cells with a CD69+CD103+/- tissue-resident phenotype and with a significant elevation of IFNγ production were dominant in the submandibular glands of mice in this murine SS model. CD8a knockout abrogated the development of SS in these mice. Knockout of IFNγ decreased CD8+ T cell infiltration and gland destruction. More importantly, depletion of CD8+ T cells fully protected mice against the pathologic manifestations of SS, even after the onset of disease. CONCLUSION: These data reveal the pathogenic significance of CD8+ T cells in the development and progression of SS in the salivary glands. Treatment directed against CD8+ T cells may be a rational therapy for the management of SS in human subjects.

Gut microbiota translocation promotes autoimmune cholangitis.

Gut microbiota and bacterial translocation have been implicated as significant contributors to mucosal immune responses and tolerance; alteration of microbial molecules, termed pathogen-associated molecular patterns (PAMP) and bacterial translocation are associated with immune pathology. However, the mechanisms by which dysregulated gut microbiota promotes autoimmunity is unclear. We have taken advantage of a well-characterized murine model of primary biliary cholangitis, dnTGFßRII mice, and an additional unique construct, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice coined dnTGFßRIITLR2-/- mice to investigate the influences of gut microbiota on autoimmune cholangitis. Firstly, we report that dnTGFßRII mice manifest altered composition of gut microbiota and that alteration of this gut microbiota by administration of antibiotics significantly alleviates T-cell-mediated infiltration and bile duct damage. Second, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice demonstrate significant exacerbation of autoimmune cholangitis when their epithelial barrier integrity was disrupted. Further, TLR2-deficiency mediates downregulated expression of tight junction-associated protein ZO-1 leading to increased gut permeability and bacterial translocation from gut to liver; use of antibiotics reduces microbiota translocation to liver and also decreases biliary pathology. In conclusion, our data demonstrates the important role of gut microbiota and bacterial translocation in the pathogenesis of murine autoimmune cholangitis.

CD8+ T cells and IFN-γ induce autoimmune myelofibrosis in mice.

Myelofibrosis usually occurs either as a part of a myelodysplastic syndrome or in conjunction with neoplasia. It is not commonly thought of an autoimmune disease. We reported that p40-/-IL-2Rα-/- (interleukin-12p40 and interleukin-2 receptor alpha double knockout) mice, a mouse model of human primary biliary cholangitis, exhibited features consistent with autoimmune myelofibrosis, including anemia associated with bone marrow fibrosis, and extramedullary hematopoiesis (EMH) including LSK (Lineage-c-Kit+Sca-1+) cells in spleen, liver and peripheral blood. There were also increased LSK cells in bone marrow but they demonstrated impaired hematopoiesis. Importantly effector memory T cells that infiltrated the bone marrow of p40-/-IL-2Rα-/- mice manifested a higher ability to produce IFN-γ. CD8+ T cells, already known to play a dominate role in portal inflammation, were also key for bone marrow dysregulation and EMH. IFN-γ was the key cytokine that induced bone marrow fibrosis, bone marrow failure and EMH. Finally anti-CD8α antibody therapy fully protected p40-/-IL-2Rα-/- mice from autoimmune myelofibrosis. In conclusion, our results demonstrate that CD8+ T cells and IFN-γ are associated with autoimmune myelofibrosis, a finding that may allow targeting of CD8+ T cells and IFN-γ as a therapeutic targets.

The molecular basis of immune regulation in autoimmunity.

Autoimmune diseases can be triggered and modulated by various molecular and cellular characteristics. The mechanisms of autoimmunity and the pathogenesis of autoimmune diseases have been investigated for several decades. It is well accepted that autoimmunity is caused by dysregulated/dysfunctional immune susceptible genes and environmental factors. There are multiple physiological mechanisms that regulate and control self-reactivity, but which can also lead to tolerance breakdown when in defect. The majority of autoreactive T or B cells are eliminated during the development of central tolerance by negative selection. Regulatory cells such as Tregs (regulatory T) and MSCs (mesenchymal stem cells), and molecules such as CTLA-4 (cytotoxic T-lymphocyte associated antigen 4) and IL (interleukin) 10 (IL-10), help to eliminate autoreactive cells that escaped to the periphery in order to prevent development of autoimmunity. Knowledge of the molecular basis of immune regulation is needed to further our understanding of the underlying mechanisms of loss of tolerance in autoimmune diseases and pave the way for the development of more effective, specific, and safer therapeutic interventions.

IFN-γ-STAT1-iNOS Induces Myeloid Progenitors to Acquire Immunosuppressive Activity.

Autoimmune diseases often induce dysregulated hematopoiesis with altered number and function of hematopoietic stem and progenitor cells (HSPCs). However, there are limited studies on the direct regulation of HSPCs on T cells, which are often detrimental to autoimmunity. Here, we found that in a murine model of Concanavalin A-induced autoimmune hepatitis, LSK (Lineage-Sca-1+c-Kit+)-like cells accumulated in liver, spleen, and bone marrow (BM), which were myeloid progenitors (Lineage-Sca-1-c-Kit+) that upregulated Sca-1 expression upon T cell-derived IFN-γ stimulation. Strikingly, BM LSK-like cells from mice induced by Con A to develop autoimmune hepatitis or alternatively myeloid progenitors from wild-type mice possessed strong in vitro suppressive ability. Their suppressive function depended on T cell-derived IFN-γ in a paracrine fashion, which induced STAT1 phosphorylation, inducible nitric oxide synthase expression, and nitric oxide production. Blocking IFN-γ/IFN-γ receptor interaction, knockout of STAT1, or iNOS inhibition abrogated their suppressive function. In addition, the suppressive function was independent of differentiation; mitomycin C-treated myeloid progenitors maintained T cell suppressive ability in vitro. Our data demonstrate a mechanism of inflammation induced suppressive function of myeloid progenitors, which may participate directly in suppressing T cell-mediated immunopathology.