Immune thrombocytopenia (ITP) is a hemorrhagic autoimmune disease characterized by antibody-mediated platelet injury. ITP has complicated immunopathological mechanisms that need further elucidation. It is well known that the costimulatory molecules OX40 ligand (OX40L) and OX40 play essential roles in the immunological mechanisms of autoimmune diseases. Previously, we discovered that the expression of OX40L and OX40 is significantly increased in the peripheral blood mononuclear cells (PBMCs) of ITP patients. In our present study, OX40L-induced follicular helper T (Tfh) cells exhibited an activated phenotype with elevated expression of inducible T-cell costimulator (ICOS), programmed cell death protein-1 (PD-1), and cluster of differentiation 40 ligand (CD40L) in vitro. Moreover, aberrant OX40L‒OX40 expression might promote the Tfh1-to-Tfh2 shift in vivo, inducing the generation of autoantibodies by enhancing the helper function of Tfh cells for B lymphocytes in a mouse model, which might accelerate the progression of ITP. Additionally, signal transduction through the OX40L‒OX40 axis might be related to the activation of tumor necrosis factor receptor-associated factor (TRAF)‒nuclear factor-κB (NF-κB) and Janus kinase (JAK)‒signal transducer and activator of transcription (STAT) signaling pathways. Overall, OX40L‒OX40 signaling is proposed as a potential novel therapeutic target for ITP.
Animals ; OX40 Ligand/physiology* ; Purpura, Thrombocytopenic, Idiopathic/immunology* ; Cell Differentiation ; Mice ; T-Lymphocytes, Helper-Inducer/cytology* ; T Follicular Helper Cells/cytology* ; Signal Transduction ; Receptors, OX40 ; Mice, Inbred C57BL ; Humans ; Female

OBJECTIVES: To investigate the therapeutic effects of cimifugin on Crohn's disease (CD)-like colitis in mice and its possible mechanism. METHODS: Thirty adult male C57BL/6 mice were randomized equally into control group, 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis model group, and cimifugin treatment (daily gavage at 12.5 mg/kg) group. The therapeutic effect of cimifugin was evaluated by observing changes in body weight, disease activity index (DAI) scores, colon length, histopathological inflammation scores, and inflammatory cytokine levels in the colonic mucosa. Intestinal barrier integrity in the mice was assessed using immunofluorescence assay and Western blotting for claudin-1 and ZO-1; T-helper (Th) cell subset ratios in the mesenteric lymph nodes were analyzed with flow cytometry. Network pharmacology, KEGG enrichment analysis and molecular docking were used to predict the targets of cimifugin and analyze the key pathways and cimifugin-MAPK protein interactions, which were validated by Western blotting in the mouse models. RESULTS: In mice with TNBS-induced colitis, cimifugin treatment significantly attenuated body weight loss and colon shortening, lowered DAI and histopathological scores, decreased IFN-γ and IL-17 levels, and increased IL-4 and IL-10 levels in the colonic mucosa. Cimifugin treatment also significantly improved TNBS-induced claudin-1 dislocation and reduction of goblet cells, upregulated claudin-1 and ZO-1 expressions, reduced Th1 and Th17 cell percentages, and increased Th2 and Treg cell percentages in the colonic mucosa of the mice. KEGG analysis suggested a possible connection between the effect of cimifugin and MAPK signaling, and molecular docking showed strong binding affinity between cimifugin and MAPK core proteins. Western blotting demonstrated significantly decreased phosphorylation levels of JNK, ERK, and p38 in the colonic mucosa of cimifugin-treated mouse models. CONCLUSIONS Cimifugin alleviates TNBS-induced CD-like colitis by repairing intestinal barrier damage and restoring Th1/Th2 and Th17/Treg balance via suppressing MAPK pathway activation.
Animals ; Mice, Inbred C57BL ; Male ; Mice ; Crohn Disease/immunology* ; Colitis/immunology* ; MAP Kinase Signaling System/drug effects* ; Trinitrobenzenesulfonic Acid ; T-Lymphocytes, Helper-Inducer/drug effects* ; Intestinal Mucosa ; Disease Models, Animal

OBJECTIVES: To investigate the characteristics of immune cell infiltration in tumor samples from Chinese patients with clear cell renal cell carcinoma (ccRCC) and the correlation of immune cell infiltration with tumor stage and response to immunotherapy. METHODS: Tumor samples and clinicopathological data were collected from 154 ccRCC patients treated in Nanfang Hospital, Southern Medical University from October, 2020 to October, 2023. The immune cell types infiltrating the tumor tissues were identified using immunohistochemistry and immunofluorescence staining, and their correlations with the patients' clinicopathological characteristics were analyzed. Patient-derived tumor tissue fragment models (PDTF) models, constructed using tumor tissues from 22 patients, were treated with PD-1 monoclonal antibody, and T cell activation was detected using flow cytometry to assess the patients' responses to immunotherapy. RESULTS: In Chinese ccRCC patients included in this study, CD8⁺ T cells, CD4⁺ T cells, and CD3⁺ T cells were the most abundant in the tumor tissues. Higher infiltration levels of CD3⁺ T cells (P=0.004), PD-1⁺ T cells (P=0.020), CD68⁺ T cells (P=0.049), CD79⁺ T cells (P=0.049), and Tryptase⁺ cells (P=0.049) were all positively correlated with a larger tumor size (≥5 cm). A higher infiltration level of CD4⁺ T cells was associated with a lower tumor stage. Patients with higher International Society of Urological Pathology (ISUP) grades had higher infiltration levels of CD3⁺ T cells (P=0.023), CD8⁺ T cells (P=0.045), PD-1⁺ T cells (P=0.014), CD20⁺ B cells (P=0.020) and CD79⁺ B cells (P=0.049), and lower levels of Tryptase⁺ cells (P=0.001). Patients with abundant infiltrating immune cells tended to have better responses to immunotherapy. CONCLUSIONS The infiltrating immune cells are heterogeneous in Chinese ccRCC patients, and immune cell infiltration characteristics are closely correlated with clinicopathological parameters of the patients.
Humans ; Carcinoma, Renal Cell/pathology* ; Kidney Neoplasms/pathology* ; Immunotherapy ; Male ; Lymphocytes, Tumor-Infiltrating/immunology* ; Female ; Middle Aged ; CD8-Positive T-Lymphocytes/immunology* ; Aged ; T-Lymphocytes/immunology* ; Programmed Cell Death 1 Receptor/immunology* ; Adult ; CD4-Positive T-Lymphocytes/immunology* ; Neoplasm Staging

Infiltration and activation of peripheral immune cells are critical in the progression of multiple sclerosis and its experimental animal model, experimental autoimmune encephalomyelitis (EAE). This study investigates the role of high mobility group box 1 (HMGB1) in oligodendrocyte precursor cells (OPCs) in modulating pathogenic T cells infiltrating the central nervous system through the blood-brain barrier (BBB) by using OPC-specific HMGB1 knockout (KO) mice. We found that HMGB1 released from OPCs promotes BBB disruption, subsequently allowing increased immune cell infiltration. The migration of CD4+ T cells isolated from EAE-induced mice was enhanced when co-cultured with OPCs compared to oligodendrocytes (OLs). OPC-specific HMGB1 KO mice exhibited lower BBB permeability and reduced immune cell infiltration into the CNS, leading to less damage to the myelin sheath and mitigated EAE progression. CD4+ T cell migration was also reduced when co-cultured with HMGB1 knock-out OPCs. Our findings reveal that HMGB1 secretion from OPCs is crucial for regulating immune cell infiltration and provides insights into the immunomodulatory function of OPCs in autoimmune diseases.
Animals ; Encephalomyelitis, Autoimmune, Experimental/metabolism* ; HMGB1 Protein/deficiency* ; Mice, Knockout ; Oligodendrocyte Precursor Cells/immunology* ; Mice, Inbred C57BL ; CD4-Positive T-Lymphocytes/immunology* ; Cell Movement ; Blood-Brain Barrier/immunology* ; Mice ; Myelin Sheath/pathology* ; Disease Models, Animal ; Coculture Techniques ; Oligodendroglia/metabolism* ; Female ; Cells, Cultured

Extensive epigenetic reprogramming involves in memory CD8+ T-cell differentiation. The elaborate epigenetic rewiring underlying the heterogeneous functional states of CD8+ T cells remains hidden. Here, we profile single-cell chromatin accessibility and map enhancer-promoter interactomes to characterize the differentiation trajectory of memory CD8+ T cells. We reveal that under distinct epigenetic regulations, the early activated CD8+ T cells divergently originated for short-lived effector and memory precursor effector cells. We also uncover a defined epigenetic rewiring leading to the conversion from effector memory to central memory cells during memory formation. Additionally, we illustrate chromatin regulatory mechanisms underlying long-lasting versus transient transcription regulation during memory differentiation. Finally, we confirm the essential roles of Sox4 and Nrf2 in developing memory precursor effector and effector memory cells, respectively, and validate cell state-specific enhancers in regulating Il7r using CRISPR-Cas9. Our data pave the way for understanding the mechanism underlying epigenetic memory formation in CD8+ T-cell differentiation.
CD8-Positive T-Lymphocytes/metabolism* ; Cell Differentiation ; Chromatin/immunology* ; Animals ; Mice ; Immunologic Memory ; Epigenesis, Genetic ; SOXC Transcription Factors/immunology* ; NF-E2-Related Factor 2/immunology* ; Mice, Inbred C57BL ; Gene Regulatory Networks ; Enhancer Elements, Genetic

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy in treating hematological malignancies and is expanding into other indications such as autoimmune diseases, fibrosis, aging and viral infection. However, clinical challenges persist in treating solid tumors, including physical barriers, tumor heterogeneity, poor in vivo persistence, and T-cell exhaustion, all of which hinder therapeutic efficacy. This review focuses on the critical role of tonic signaling in CAR-T therapy. Tonic signaling is a low-level constitutive signaling occurring in both natural and engineered antigen receptors without antigen stimulation. It plays a pivotal role in regulating immune cell homeostasis, exhaustion, persistence, and effector functions. The "Peak Theory" suggests an optimal level of tonic signaling for CAR-T function: while weak tonic signaling may result in poor proliferation and persistence, excessively strong signaling can cause T cell exhaustion. This review also summarizes the recent progress in mechanisms underlying the tonic signaling and strategies to fine-tune the CAR tonic signaling. By understanding and precisely modulating tonic signaling, the efficacy of CAR-T therapies can be further optimized, offering new avenues for treatment across a broader spectrum of diseases. These findings have implications beyond CAR-T cells, potentially impacting other engineered immune cell therapies such as CAR-NK and CAR-M.
Humans ; Immunotherapy, Adoptive/methods* ; Receptors, Chimeric Antigen/immunology* ; Signal Transduction/immunology* ; T-Lymphocytes/immunology* ; Neoplasms/immunology* ; Animals

Postmenopausal osteoporosis (PMOP) is a systemic metabolic bone disease caused by the decrease in estrogen levels after menopause. It leads to bone loss, microstructural damage, and an increased risk of fractures. Studies have found that the gut microbiota and its metabolites can regulate bone metabolism through the gut-bone axis and the gut-brain axis. As research progresses, PMOP has been found to be associated with gut microbiota dysbiosis and Th17/Treg imbalance. The gut microbiota is closely related to the development and differentiation of Treg and Th17 cells. Among them, the metabolites of the gut microbiota such as short-chain fatty acids (SCFAs) can regulate the differentiation of effector T cells by acting on molecular receptors on immune cells, thereby regulating the bone immune process. The multifaceted relationship among the gut microbiota, SCFAs, Th17/Treg cell-mediated bone immunity, and bone metabolism is eliciting attention from researchers. Through a review of existing literature, we have comprehensively summarized the effects of the gut microbiota and SCFAs on PMOP, especially from the perspective of Th17/Treg balance. Regulating this balance may provide new opportunities for PMOP treatment.
Humans ; Gastrointestinal Microbiome/immunology* ; Fatty Acids, Volatile/metabolism* ; Osteoporosis, Postmenopausal/immunology* ; Female ; T-Lymphocytes, Regulatory/metabolism* ; Th17 Cells/metabolism* ; Dysbiosis/immunology* ; Bone and Bones/metabolism*

Xiaohuang Qudan decoction (XHQDD) is a classical traditional Chinese medicine (TCM) formula widely used in the treatment of cholestatic liver injury. Despite its widespread use, the protective mechanism of XHQDD against cholestatic liver injury remains incompletely understood. The aim of this study was to investigate whether XHQDD mediates its beneficial effects by inhibiting the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway and regulating TH17/Treg balance. To this end, the researchers used Sprague-Dawley (SD) rats and established a cholestatic liver injury model by oral administration of alpha-naphthylisothiocyanate (ANIT). The experimental group was divided into six groups: Control (CON), ANIT, ursodeoxycholic acid (UDCA), XHQDD-low dose (XHQDD-L) group, XHQDD-medium dose (XHQDD-M) group, and XHQDD-high dose (XHQDD-H) groups. Then, after 7 d of treatment, various tests were performed to verify the results. Firstly, XHQDD and its drug-containing serum were analyzed by ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS), and 14 blood-entry components were identified. Then, bile flow was monitored and found to be significantly reduced in the model group, which was significantly reversed in the UDCA and XHQDD groups. To further assess ANIT-induced liver injury, hematoxylin and eosin (H&E) and Sirius red staining, alongside transmission electron microscopy (TEM), were employed to observe liver tissues, revealing hepatocellular injury, cholestasis, and hepatic fibrotic changes. Serum inflammatory factors and liver injury indicators were assessed using enzyme-linked immunosorbent assay (ELISA), indicating an inflammatory state in ANIT-induced liver injury rats. The expression levels of JAK2/STAT3-related genes and proteins in liver and intestinal tissues were measured via quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, immunofluorescence (IF) staining, and Western blottting (WB) assays. These studies revealed that the inflammatory state of liver-injured rats was inextricably linked to the inflammatory cascade associated with the JAK2/STAT3 pathway and that XHQDD may exert anti-inflammatory efficacy by inhibiting the JAK2/STAT3 pathway. Flow cytometry was used to determine the percentage of T helper 17 (Th17)/regulatory T (Treg) cells in serum and hepatocytes, and it was further found that XHQDD was able to regulate Th17/Treg immune homeostasis in liver-injured rats. The findings suggest that XHQDD markedly alleviates inflammation in ANIT rats, potentially treating cholestasis and liver injury through JAK2/STAT3 inhibition and Th17/Treg balance regulation.
Animals ; STAT3 Transcription Factor/immunology* ; Janus Kinase 2/immunology* ; Drugs, Chinese Herbal/pharmacology* ; Rats, Sprague-Dawley ; 1-Naphthylisothiocyanate/adverse effects* ; Male ; Rats ; Th17 Cells/immunology* ; Cholestasis/immunology* ; Signal Transduction/drug effects* ; T-Lymphocytes, Regulatory/immunology* ; Chemical and Drug Induced Liver Injury/immunology* ; Liver/drug effects*

BACKGROUND: T cell dysfunction, which includes exhaustion, anergy, and senescence, is a distinct T cell differentiation state that occurs after antigen exposure. Although T cell dysfunction has been a cornerstone of cancer immunotherapy, its potential in transplant research, while not yet as extensively explored, is attracting growing interest. Interferon regulatory factor 4 (IRF4) has been shown to play a pivotal role in inducing T cell dysfunction. METHODS: A novel ultra-low-dose combination of Trametinib and Rapamycin, targeting IRF4 inhibition, was employed to investigate T cell proliferation, apoptosis, cytokine secretion, expression of T-cell dysfunction-associated molecules, effects of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways, and allograft survival in both in vitro and BALB/c to C57BL/6 mouse cardiac transplantation models. RESULTS: In vitro , blockade of IRF4 in T cells effectively inhibited T cell proliferation, increased apoptosis, and significantly upregulated the expression of programmed cell death protein 1 (PD-1), Helios, CD160, and cytotoxic T lymphocyte-associated antigen (CTLA-4), markers of T cell dysfunction. Furthermore, it suppressed the secretion of pro-inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17. Combining ultra-low-dose Trametinib (0.1 mg·kg -1 ·day -1 ) and Rapamycin (0.1 mg·kg -1 ·day -1 ) demonstrably extended graft survival, with 4 out of 5 mice exceeding 100 days post-transplantation. Moreover, analysis of grafts at day 7 confirmed sustained IFN regulatory factor 4 (IRF4) inhibition, enhanced PD-1 expression, and suppressed IFN-γ secretion, reinforcing the in vivo efficacy of this IRF4-targeting approach. The combination of Trametinib and Rapamycin synergistically inhibited the MAPK and mTOR signaling network, leading to a more pronounced suppression of IRF4 expression. CONCLUSIONS Targeting IRF4, a key regulator of T cell dysfunction, presents a promising avenue for inducing transplant immune tolerance. In this study, we demonstrate that a novel ultra-low-dose combination of Trametinib and Rapamycin synergistically suppresses the MAPK and mTOR signaling network, leading to profound IRF4 inhibition, promoting allograft acceptance, and offering a potential new therapeutic strategy for improved transplant outcomes. However, further research is necessary to elucidate the underlying pharmacological mechanisms and facilitate translation to clinical practice.
Animals ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Interferon Regulatory Factors/metabolism* ; Heart Transplantation/methods* ; T-Lymphocytes/immunology* ; Sirolimus/therapeutic use* ; Pyridones/therapeutic use* ; Graft Survival/drug effects* ; Pyrimidinones/therapeutic use* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Male ; Signal Transduction/drug effects*

BACKGROUND: G protein-coupled receptor kinase 2 (GRK2) could participate in the regulation of diverse cells via interacting with non-G-protein-coupled receptors. In the present work, we explored how paroxetine, a GRK2 inhibitor, modulates the differentiation and activation of immune cells in rheumatoid arthritis (RA). METHODS: The blood samples of healthy individuals and RA patients were collected between July 2021 and March 2022 from the First Affiliated Hospital of Anhui Medical University. C57BL/6 mice were used to induce the collagen-induced arthritis (CIA) model. Flow cytometry analysis was used to characterize the differentiation and function of dendritic cells (DCs)/T cells. Co-immunoprecipitation was used to explore the specific molecular mechanism. RESULTS: In patients with RA, high expression of GRK2 in peripheral blood lymphocytes, accompanied by the increases of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In animal model, a decrease in regulatory T cells (T regs ), an increase in the cluster of differentiation 8 positive (CD8 + ) T cells, and maturation of DCs were observed. Paroxetine, when used in vitro and in CIA mice, restrained the maturation of DCs and the differentiation of CD8 + T cells, and induced the proportion of T regs . Paroxetine inhibited the secretion of pro-inflammatory cytokines, the expression of C-C motif chemokine receptor 7 in DCs and T cells. Simultaneously, paroxetine upregulated the expression of programmed death ligand 1, and anti-inflammatory cytokines. Additionally, paroxetine inhibited the PI3K-AKT-mTOR metabolic pathway in both DCs and T cells. This was associated with a reduction in mitochondrial membrane potential and changes in the utilization of glucose and lipids, particularly in DCs. Paroxetine reversed PI3K-AKT pathway activation induced by 740 Y-P (a PI3K agonist) through inhibiting the interaction between GRK2 and PI3K in DCs and T cells. CONCLUSION Paroxetine exerts an immunosuppressive effect by targeting GRK2, which subsequently inhibits the metabolism-related PI3K-AKT-mTOR pathway of DCs and T cells in RA.
G-Protein-Coupled Receptor Kinase 2/metabolism* ; Arthritis, Rheumatoid/immunology* ; Animals ; Dendritic Cells/metabolism* ; Paroxetine/therapeutic use* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mice ; Humans ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Male ; Phosphatidylinositol 3-Kinases/metabolism* ; Lymphocyte Activation/drug effects* ; Female ; T-Lymphocytes/metabolism* ; Middle Aged