Objectives To investigate interleukin 36γ (IL-36γ) expression, and analyze the influence of IL-36γ to CD8⁺ T cell activity in chronic obstructive pulmonary diseases (COPD) patients with secondary fungal pneumonia. Methods Peripheral blood was collected from 47 COPD patients, 39 COPD patients with secondary fungal pneumonia, and 20 controls. Bronchial alveolar lavage fluid (BALF) was isolated from 27 COPD patients with secondary fungal pneumonia. CD8⁺ T cells were purified. The levels of four IL-36 isoforms in plasma and BALF were measured by enzyme linked immunosorbent assay (ELISA). CD8⁺ T cells were stimulated with recombinant human IL-36γ. The levels of interferon γ(IFN-γ), tumor necrosis factor α(TNF-α), perforin and granzyme B in the cultured supernatants were measured by ELISA. Recombinant human IL-36γ-stimulated CD8⁺ T cells were co-cultured with NCI-H1882 cells in either direct cell-to-cell contact or Transwell^TM manner. The levels of IFN-γ, TNF-α, and lactate dehydrogenase in the cultured supernatants were assessed. The percentage of target cell death was calculated. Results Plasma IL-36α, IL-36β, and IL-36γ levels were significantly elevated in both COPD group and COPD with secondary fungal pneumonia group compared with those in control group. However, only plasma IL-36γ level was higher in COPD with secondary fungal pneumonia group than that in COPD group [(200.11±99.95)pg/mL vs (53.03±87.18)pg/mL, P=0.023]. There was no remarkable difference in plasma IL-36 receptor antagonist level among three groups. IL-36γ level in BALF from infectious site was higher than that from non-infectious site in COPD with secondary fungal pneumonia group [(305.82±59.60)pg/mL vs (251.93±76.01)pg/mL, P=0.011]. IL-36γ stimulation enhanced IFN-γ, TNF-α, perforin and granzyme B secreted by CD8⁺ T cells. When IL-36γ-stimulated CD8⁺ T cells were directly mixed with NCI-H1882 cells for co-culture, the percentage of cell death was increased [(16.06±3.67)% vs (11.47±2.36)%, P=0.002]. When using Transwell^TM plate for non-contact co-culture, IL-36γ-stimulated CD8⁺ T cell-mediated death of NCI-H1882 cells showed no significant difference compared to that without stimulation [(4.77±0.78)% vs (4.99±0.92)%, P=0.554]. Conclusion IL-36γ level in plasma and infectious site is elevated in COPD patients with secondary fungal pneumonia, which enhances the cytotoxicity of CD8⁺ T cells in peripheral blood and infectious microenviroment.
Humans ; Pulmonary Disease, Chronic Obstructive/complications* ; CD8-Positive T-Lymphocytes/metabolism* ; Male ; Female ; Aged ; Middle Aged ; Interferon-gamma/metabolism* ; Interleukin-1/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Lung Diseases, Fungal/complications* ; Bronchoalveolar Lavage Fluid/chemistry* ; Perforin/metabolism* ; Pneumonia/immunology* ; Granzymes/metabolism*

Objective To investigate the differences of type 2 innate lymphocytes (ILC2) and interlukin 9 (IL-9) between chronic lymphocytic leukemia (CLL) patients and healthy controls, and to understand the effects of ILC2 on the function of regulatory T cells (Tregs), CD8⁺ T cells and CLL cells through IL-9. Methods Flow cytometry was used to detect the levels of ILC2 and Tregs in the peripheral blood of 45 newly diagnosed CLL patients and 24 healthy controls, and the expressions of granzyme B and perforin in CD8⁺ T cells in the peripheral blood of 28 patients and 15 healthy controls; ELISA was used to detect the level of IL-9 in the serum. ILC2 of patients and healthy controls was sorted by immunomagnetic beads and cultured separately, and the level of IL-9 in the culture supernatant was measured by ELISA. ILC2 sorted from CLL patients and healthy control-derived peripheral blood mononuclear cells(PBMCs) were co-cultured with the B cell leukemia MEC-1 cells, one group was supplemented with IL-9 antibody and the other group was not. After 72 hours of culture, the ratio of Tregs, programmed death 1 (PD-1), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), cytotoxic T lymphocyte antigen 4 (CTLA-4) on Tregs, granzyme B and perforin in CD8⁺ T cells were measured by flow cytometry, IL-9 level of the culture supernatant was measured by ELISA, the apoptosis of MEC-1 cells was measured by Annexin V-PI. Results Compared with the healthy control group, the levels of ILC2, Tregs and IL-9 in the CLL group increased significantly. The levels of granzyme B and perforin in CD8⁺ T cells were positively correlated in the peripheral blood of CLL patients. Compared with the healthy control group, IL-9 levels in the supernatant of sorted ILC2 from CLL patients increased. In the anti-IL9 antibody group, the level of PD-1 and TIGIT on Tregs decreased, and the level of granzyme B in CD8⁺ T cells increased significantly. The level of IL-9 in the anti-IL9 antibody group decreased statistically. And MEC-1 cells showed increased early apoptotic rate in the anti-IL9 antibody group statistically. Conclusion In CLL, ILC2 affects CD8⁺ T cells and Tregs through IL-9, which weakens the anti-tumor effect of CD8⁺ T cells, enhances the immunosuppressive effect of Tregs, and plays a role in the occurrence and development of CLL disease.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/immunology* ; CD8-Positive T-Lymphocytes/immunology* ; T-Lymphocytes, Regulatory/immunology* ; Middle Aged ; Male ; Female ; Interleukin-9/blood* ; Aged ; Granzymes/metabolism* ; Perforin/metabolism* ; Immunity, Innate ; Adult ; Lymphocytes/immunology*

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

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*

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: 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