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

The remarkable efficacy of chimeric antigen receptor (CAR) T cell therapy in hematological malignancies has provided a solid basis for the therapeutic concept, wherein specific pathogenic cell populations can be eradicated by means of targeted recognition. During the past few years, CAR-based cell therapies have been extensively investigated in preclinical and clinical research across various non-tumor diseases, with particular emphasis in the treatment of autoimmune diseases (ADs), yielding significant advancements. The recent deployment of CD19-directed CAR T cells has induced long-lasting, drug-free remission in patients with systemic lupus erythematosus (SLE) and other systemic ADs, alongside a more profound immune reconstruction of B cell repertoire compared with conventional immunosuppressive agents and B cell-targeting biologics. Despite the initial success achieved by CAR T cell therapy, it is critical to acknowledge the divergences in its application between cancer and ADs. Through examining recent clinical studies and ongoing research, we highlight the transformative potential of this therapeutic approach in the treatment of SLE, while also addressing the challenges and future directions necessary to enhance the long-term efficacy and safety of CAR-based cell therapies in clinical practice.
Humans ; Lupus Erythematosus, Systemic/immunology* ; Receptors, Chimeric Antigen/metabolism* ; Immunotherapy, Adoptive/methods* ; Cell- and Tissue-Based Therapy/methods* ; Animals ; T-Lymphocytes/immunology*

BACKGROUND: Peripheral helper T (T PH ) cells are uniquely positioned within pathologically inflamed non-lymphoid tissues to stimulate B-cell responses and antibody production. However, the phenotype, function, and clinical relevance of T PH cells in hepatocellular carcinoma (HCC) are currently unknown. METHODS: Blood, tumor, and peritumoral liver tissue samples from 39 HCC patients (Sep 2016-Aug 2017) and 101 HCC patients (Sep 2011-Dec 2012) at the Third Affiliated Hospital of Sun Yat-sen University were used. Flow cytometry was used to quantify the expression, phenotype, and function of T PH cells. Log-rank tests were performed to evaluate disease-free survival and overall survival in samples from 39 patients and 101 patients with HCC. T PH cells, CD19 + B cells, and T follicular helper (T FH ) cells were cultured separately in vitro or isolated from C57/B6L mice in vivo for functional assays. RESULTS: T PH cells highly infiltrated tumor tissues, which was correlated with tumor size, early recurrence, and shorter survival time. The tumor-infiltrated T PH cells showed a unique ICOS hi CXCL13 + IL-21 - MAF + BCL-6 - phenotype and triggered naïve B-cell differentiation into regulatory B cells. Triggering programmed cell death protein 1 (PD-1) induced the production of C-X-C motif chemokine ligand 13 (CXCL13) by T PH cells, which then suppressed tumor-specific immunity and promoted disease progression. CONCLUSION Our study reveals a novel regulatory mechanism of T PH cell-regulatory B-cell-mediated immunosuppression and provides an important perspective for determining the balance between the differentiation of protumorigenic T PH cells and that of antitumorigenic T FH cells in the HCC microenvironment.
Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Humans ; T-Lymphocytes, Helper-Inducer/metabolism* ; Animals ; Mice ; Male ; Female ; Mice, Inbred C57BL ; Middle Aged ; B-Lymphocytes, Regulatory/metabolism* ; Flow Cytometry ; Interleukin-21 ; Aged ; Chemokine CXCL13/metabolism*

BACKGROUND: Regulatory dendritic cell (DCreg) subset exhibits a unique capacity for inducing immune tolerance among the variety subsets of dendritic cells (DCs) within gut-associated lymphoid tissues (GALTs). Fms-like tyrosine kinase 3 ligand (FLT3L) is involved in the differentiation of DCregs and the subsequent expansion of regulatory T-cells (Tregs) mediated by DCregs, though the precise mechanism remains poorly understood. This study aimed to explore the expansion mechanism of Treg induced by DCreg and the role of FLT3L in this process. METHODS: DCregs were distinguished from other DC subsets isolated from GALTs of BALB/c mice through a mixed lymphocyte reaction assay. The functions and mechanisms by which FLT3L promoted Treg expansion via DCregs were investigated in vitro through co-culture experiments involving DCregs and either CD4 + CD25 - T-cells or CD4 + CD25 + T-cells. Additionally, an in vivo experiment was conducted using a dextran sulfate sodium (DSS)-induced colitis model in mice. RESULTS: CD103 + CD11b + DC exhibited DCreg-like functionality and was identified as DCreg for subsequent investigation. Analysis of Foxp3 + Treg percentages within a co-culture system of CD4 + CD25 - T-cells and DCregs, with or without FLT3L, demonstrated the involvement of the FLT3/FLT3L axis in driving the differentiation of precursor T-cells into Foxp3 + Tregs induced by DCregs. Cell migration and co-culture assays revealed that the FLT3/FLT3L axis enhanced DCreg migration toward Tregs via the Rho pathway. Additionally, it was observed that DCregs could promote Treg proliferation through the Notch pathway, as inhibition of Notch signaling by DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) suppressed Treg expansion within the co-culture system of DCregs and CD4 + T-cells or CD4 + CD25 + T-cells. Furthermore, the FLT3/FLT3L axis influenced JAG1 expression in DCregs, indirectly modulating Treg expansion. In vivo experiments further established that FLT3L promoted DCreg expansion and restored Treg balance in DSS-induced colitis models, thereby ameliorating colitis symptoms in mice. CONCLUSION The FLT3/FLT3L axis is integral to the maintenance of DCreg function in Treg expansion.
Animals ; T-Lymphocytes, Regulatory/immunology* ; Dendritic Cells/immunology* ; Mice ; Mice, Inbred BALB C ; Membrane Proteins/metabolism* ; Receptors, Notch/metabolism* ; Lymphoid Tissue/metabolism* ; Signal Transduction/physiology* ; Coculture Techniques ; Flow Cytometry

BACKGROUND: Immunosuppression is closely related to the pathogenesis of sepsis, but the underlying mechanisms have not yet been fully elucidated. In this study, we aimed to examine the role of the Sterile Alpha Motif, Src Homology 3 domain and nuclear localization signal 1 (SAMSN1) in sepsis and elucidate its potential molecular mechanism in sepsis induced immunosuppression. METHODS: RNA sequencing databases were used to validate SAMSN1 expression in sepsis. The impact of SAMSN1 on sepsis was verified using gene knockout mice. Flow cytometry was employed to delineate how SAMSN1 affects immunity in sepsis, focusing on immune cell types and T cell functions. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing in RAW264.7 macrophages enabled interrogation of SAMSN1 's regulatory effects on essential macrophage functions, including cell proliferation and phagocytic capacity. The mechanism of SAMSN1 in the interaction between macrophages and T cells was investigated using the RAW264.7 cell line and primary cell lines. RESULTS: SAMSN1 expression was significantly increased in patients with sepsis and was positively correlated with sepsis mortality. Genetic deletion of Samsn1 in murine sepsis model improved T cell survival, elevated T cell cytolytic activity, and activated T cell signaling transduction. Concurrently, Samsn1 knockout augmented macrophage proliferation capacity and phagocytic efficiency. In macrophage, SAMSN1 binds to Kelch-like epichlorohydrin-associated protein 1 (KEAP1), causing nuclear factor erythroid 2-related factor 2 (NRF2) to dissociate from the KEAP1-NRF2 complex and translocate into the nucleus. This promotes the transcription of the coinhibitory molecules CD48/CD86/carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1), which bind to their corresponding receptors natural killer cell receptor 2B4/CD152/T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) on the surface of T cells, inducing T-cell exhaustion. CONCLUSIONS SAMSN1 deletion augmented adaptive T cell immunity and macrophage phagocytic-proliferative dual function. Furthermore, it mediates the KEAP1-NRF2 axis, which affects the expression of coinhibitory molecules on macrophages, leading to T-cell exhaustion. This novel immunosuppression mechanism potentially provides a candidate molecular target for sepsis immunotherapy.
Animals ; NF-E2-Related Factor 2/metabolism* ; Mice ; Macrophages/immunology* ; Sepsis/metabolism* ; Kelch-Like ECH-Associated Protein 1/genetics* ; T-Lymphocytes/immunology* ; Humans ; Signal Transduction/physiology* ; RAW 264.7 Cells ; Mice, Knockout ; Mice, Inbred C57BL ; Male ; Flow Cytometry ; T-Cell Exhaustion

BACKGROUND: Pre-transplant exposure to immune checkpoint inhibitors (ICIs) significantly increases the risk of allograft rejection after liver transplantation (LT); however, whether ICI-related rejection leads to increased graft loss remains controversial. Therefore, this study aimed to investigate the association between ICI-related allograft rejection and perioperative graft loss. METHODS: This was a retrospective analysis of adult liver transplant recipients with early biopsy-proven T-cell-mediated rejection (TCMR) at Liver Transplantation Center of Sun Yat-sen Memorial Hospital from June 2019 to September 2024. The pathological features, clinical characteristics, and perioperative graft survival were analyzed. RESULTS: Twenty-eight patients who underwent early TCMR between June 2019 and September 2024 were included. Based on pre-LT ICI exposure, recipients were categorized into ICI-related TCMR (irTCMR, n = 12) and conventional TCMR (cTCMR, n = 16) groups. Recipients with irTCMR had a higher median Banff rejection activity index (RAI) (6 vs . 5, P = 0.012) and more aggressive tissue damage and inflammation. Recipients with irTCMR showed higher proportion of treatment resistance, achieving a complete resolution rate of only 8/12 compared to 16/16 for cTCMR. Graft loss occurred in 5/12 of irTCMR recipients within 90 days after LT, with no graft loss in cTCMRs recipients. Cox analysis demonstrated that irTCMR with an ICI washout period of <30 days was an independent risk factor for perioperative graft loss (hazard ratio [HR], 6.540; 95% confidence interval [CI], 1.067-40.067, P = 0.042). CONCLUSION IrTCMR is associated with severe pathological features, increased resistance to treatment, and higher graft loss in adult liver transplant recipients.
Humans ; Liver Transplantation/adverse effects* ; Male ; Female ; Middle Aged ; Retrospective Studies ; Graft Rejection/immunology* ; Immune Checkpoint Inhibitors/therapeutic use* ; Adult ; T-Lymphocytes/drug effects* ; Graft Survival/immunology* ; Aged

Chimeric antigen receptor T (CAR-T) cells have reshaped the treatment landscape of hematological malignancies, offering a potentially curative option for patients. Despite these major milestones in the field of immuno-oncology, growing experience with CAR-T cells has also highlighted several limitations of this strategy. The production process of CAR-T cells is complex, time-consuming, and costly, thus leading to poor drug accessibility. The potential carcinogenic risk of viral transfection systems remains a matter of controversy. Treatment-related side effects, such as cytokine release syndrome, can be life-threatening. And the biggest challenge is the inadequate efficacy related to poor infiltration and retention of CAR-T cells in tumor tissues and impaired T cell activation caused by the immunosuppressive tumor microenvironment (TME). Innovative strategies are urgently needed to address these problems, and nanomedicine offers good solutions to these challenges. In this review, we provide a comprehensive summary of recent advancements in the application of nanomaterials to enhance CAR-T cell therapy. We examine the role of innovative nanoparticle-based delivery systems in the production of CAR-T cells, with a particular focus on polymeric delivery systems and lipid nanoparticles (LNPs). Furthermore, we explore various strategies for delivering immune stimulators, which significantly enhance the efficacy of CAR-T cells by modulating T cell viability and functionality or by reprogramming the immunosuppressive TME. In addition, we discuss several novel therapeutic approaches aimed at mitigating the adverse effects associated with CAR-T therapies. Finally, we offer an integrated perspective on the future challenges and opportunities facing CAR-T therapies.
Humans ; Nanomedicine/methods* ; Receptors, Chimeric Antigen/metabolism* ; Immunotherapy, Adoptive/methods* ; T-Lymphocytes/immunology* ; Nanoparticles/chemistry* ; Animals

BACKGROUND: Cluster of differentiation 8 positive (CD8 + ) T cells play a crucial role in the response against tumors, including hepatocellular carcinoma (HCC), where their dysfunction is commonly observed. While the association between elevated peroxisome proliferator-activated receptor alpha (PPARα) expression in HCC cells and exosomes and unfavorable prognosis in HCC patients is well-established, the underlying biological mechanisms by which PPARα induces CD8 + T cell exhaustion mediated by HCC exosomes remain poorly understood. METHODS: Bioinformatics analyses and dual-luciferase reporter assays were used to investigate the regulation of microRNA-27b-3p ( miR-27b-3p ) and thymocyte selection-associated high mobility group box ( Tox ) by Pparα . In vitro and in vivo experiments were conducted to validate the effects of HCC-derived exosomes, miR-27b-3p overexpression, and Pparα on T cell function. Exosome characterization was confirmed using transmission electron microscopy, Western blotting, and particle size analysis. Exosome tracing was performed using small animal in vivo imaging and confocal microscopy. The expression levels of miR-27b-3p , Pparα , and T cell exhaustion-related molecules ( Tox , Havcr2 , and Pdcd1 ) were detected using quantitative reverse transcription polymerase chain reaction analysis, Western blotting analysis, immunofluorescence staining, and flow cytometry analysis. RESULTS: Pparα expression was significantly increased in HCC and negatively correlated with prognosis. It showed a positive correlation with Tox and a negative correlation with miR-27b-3p . The overexpressed Pparα from HCC cells was delivered to CD8 + T cells via exosomes, which absorbed miR-27b-3p both in vitro and in vivo , acting as "miRNA sponges". Further experiments demonstrated that Pparα can inhibit the negative regulation of Tox mediated by miR-27b-3p through binding to its 3'untranslated regions. CONCLUSIONS HCC-derived exosomes deliver Pparα to T cells and promote CD8 + T cell exhaustion and malignant progression of HCC via the miR-27b-3p /TOX regulatory axis. The mechanisms underlying T-cell exhaustion in HCC can be utilized for the advancement of anticancer therapies.
MicroRNAs/metabolism* ; PPAR alpha/genetics* ; Carcinoma, Hepatocellular/genetics* ; Humans ; Liver Neoplasms/genetics* ; CD8-Positive T-Lymphocytes/immunology* ; Exosomes/metabolism* ; Animals ; Cell Line, Tumor ; Mice ; High Mobility Group Proteins/genetics* ; Male ; T-Cell Exhaustion

This article reviews the role of different types of T lymphocyte subpopulations in pathological cardiac fibrosis remodeling. T helper 17 (Th17) cells are implicated in promoting the development of pathological cardiac fibrosis remodeling, while regulatory T (Treg) cells exert an immunosuppressive functions as negative regulators, attributing to their interleukin-10 (IL-10) secretion and functional phenotype. Th1 and Th2 cells are involved in different stages of the inflammatory response in pathological cardiac fibrosis remodeling, and their influence varies according to the pathological mechanisms of different cardiac diseases. In addition, CD8⁺ T cells regulate the activation and polarization of macrophages, promote the secretion of granzyme B, induce cardiomyocyte apoptosis, and aggravate cardiac fibrosis post-myocardial infarction. Considering the limitation of cytokine modulation in clinical therapy of heart failure, targeting T-cell co-stimulatory molecules emerges as a promising strategy for treating pathologic cardiac remodeling. Future research will explore chimeric antigen receptor modified T cells (CAR-T cells) technology and targeted regulation of Treg cells quantity and phenotype, for both of which have the potential to become effective methods for treating heart disease.
Humans ; Fibrosis ; T-Lymphocytes, Regulatory/immunology* ; Ventricular Remodeling/immunology* ; Myocardium/immunology* ; Animals ; Th17 Cells/immunology* ; Interleukin-10/metabolism* ; Th1 Cells/immunology* ; Th2 Cells/immunology*