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: 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

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*

Asthma is a chronic inflammatory disease involving multiple inflammatory cells and cytokines. Its pathogenesis is complex, involving various cells and cytokines. Traditional Chinese medicine(TCM) theory suggests that the pathogenesis of asthma is closely related to the dysfunction of internal organs such as the lungs, spleen, and kidneys. In contrast, modern immunological studies have revealed the central role of T helper 1(Th1)/T helper 2(Th2) and T helper 17(Th17)/regulatory T(Treg) cellular immune imbalance in the pathogenesis of asthma. Th1/Th2 imbalance is manifested as hyperfunction of Th2 cells, which promotes the synthesis of immunoglobulin E(IgE) and the activation of eosinophil granulocytes, leading to airway hyperresponsiveness and inflammation.Meanwhile, Th17/Treg imbalance exacerbates the inflammatory response in the airways, further contributing to asthma pathology.Currently, therapeutic strategies for asthma are actively exploring potential targets for regulating the balance of Th1/Th2 and Th17/Treg immune responses. These targets include cytokines, transcription factors, key proteins, and non-coding RNAs. Precisely regulating the expression and function of these targets can effectively modulate the activation and differentiation of immune cells. In recent years,traditional Chinese medicine active ingredients have shown unique potential and prospects in the field of asthma treatment. Based on this, the present study systematically summarizes the efficacy and specific mechanisms of TCM active ingredients in treating asthma by regulating Th1/Th2 and Th17/Treg immune balance through literature review and analysis. These active ingredients, including flavonoids, terpenoids, polysaccharides, alkaloids, and phenolic acids, exert their effects through various mechanisms, such as inhibiting the activation of inflammatory cells, reducing the release of cytokines, and promoting the normal differentiation of immune cells. This study aims to provide a solid foundation for the widespread application and in-depth development of TCM in asthma treatment and to offer new ideas for clinical research and drug development of asthma.
Asthma/genetics* ; Humans ; Drugs, Chinese Herbal/chemistry* ; Th2 Cells/drug effects* ; Th17 Cells/drug effects* ; T-Lymphocytes, Regulatory/drug effects* ; Th1 Cells/drug effects* ; Animals ; Cytokines/immunology* ; Medicine, Chinese Traditional

This study investigated the therapeutic effects of Yiguanjian on dry eye in rats and its mechanisms involving the T helper cell 17(Th17)/regulatory T cell(Treg) balance. The rat model of dry eye was established by administrating 0.2% benzalkonium chloride solution in eye drops. After successful modeling, the rats were treated with Yiguanjian for 4 consecutive weeks. The Schirmer test was carried out to assess the lacrimal gland function, corneal fluorescence staining to detect corneal injury, hematoxylin-eosin staining to observe corneal histopathology, enzyme-linked immunosorbent assay to measure serum levels of interleukin(IL)-6, IL-8, IL-17A, IL-21, and tumor necrosis factor-α(TNF-α), RT-qPCR to analyze mRNA levels of retinoic acid receptor-related orphan receptor gamma t(RORγt) and forkhead box protein p3(Foxp3) in the corneal tissue, immunofluorescence double staining to evaluate RORγt and Foxp3 expression in the lacrimal gland tissue, and Western blot to quantify the protein levels of signal transducer and activator of transcription 3(STAT3), phosphorylated STAT3(p-STAT3), Janus kinase 2(Jak2), phosphorylated Jak2(p-Jak2), RORγt, and Foxp3 in the corneal tissue. The results demonstrated that Yiguanjian increased tear secretion(P<0.01), alleviated corneal damage and pathological changes, and lowered the serum levels of IL-6, IL-8, IL-17A, IL-21, and TNF-α(P<0.05) in model rats. Additionally, Yiguanjian decreased the ratio of RORγt to Foxp3 in the corneal and lacrimal gland tissue(P<0.01), downregulated the protein levels of STAT3, Jak2, and RORγt(P<0.05), upregulated the protein level of Foxp3(P<0.05), and inhibited phosphorylation of STAT3 and Jak2(P<0.01). These findings indicate that Yiguanjian ameliorates ocular surface dysfunction in dry eye rats by restoring Th17/Treg balance in the corneal and lacrimal gland tissue and suppressing systemic inflammatory cytokine release, thus mitigating ocular surface inflammation.
Animals ; Rats ; T-Lymphocytes, Regulatory/immunology* ; Drugs, Chinese Herbal/administration & dosage* ; Th17 Cells/immunology* ; Male ; Rats, Sprague-Dawley ; Dry Eye Syndromes/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 3/immunology* ; Lacrimal Apparatus/immunology* ; Humans ; STAT3 Transcription Factor/immunology*

Objective Zinc finger protein 335 (Zfp335) plays a crucial role in the early development of thymic T cells and the differentiation of peripheral T cell subpopulations. The objective of this study is to investigate the role and underlying mechanisms of Zfp335 in the regulation of regulatory T cell (Treg) within tumor immunity. Methods The Zfp335 gene was specifically knocked out in Treg using tamoxifen (Zfp335^fl/fl FOXP3^creERT2), and the MC38 tumor model was established. On the 7th day after tumor inoculation, tumor size was observed and measured. Tumor size was monitored and recorded daily starting from day 7 post-inoculation. On day 12, tumors were harvested, and the proportions of CD4⁺ T cells, CD8⁺ T cells, and Treg were analyzed by flow cytometry. Additionally, the mitochondrial function of effector regulatory T cell (eTreg) was assessed. Results From day 10 post-tumor inoculation, tumor volume in the Zfp335^CKO group was significantly reduced compared to that of the wild-type (WT) group. Furthermore, the infiltration of CD4⁺ and CD8⁺ T cells, along with their respective effector cells, was significantly higher in the Zfp335^CKO group than in the WT group. The proportions of CD4⁺ and CD8⁺ T cells producing interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were also significantly increased in the Zfp335^CKO group compared to that of the WT group. In addition, the percentage of CD8⁺ T cells secreting granzyme B (GzmB) was significantly higher in the Zfp335^CKO group than that in the WT group. In contrast, the proportion of Treg and inducible T cell co-stimulator (ICOS)⁺ Treg in the Zfp335^CKO group was significantly lower than that in the WT group. Finally, the expression level of Mitotracker Deep Red in eTreg from the Zfp335^CKO group was significantly reduced compared to that in the WT group. Conclusion During tumorigenesis, the specific deletion of Zfp335 impairs Treg activation, which is related to decreased mitochondrial function in eTreg. In Zfp335^CKO mice. Tumors exhibit increased infiltration of effector T cells, accompanied by elevated levels of cytotoxic cytokines, ultimately enhancing resistance to tumor progression.
Animals ; T-Lymphocytes, Regulatory/metabolism* ; Mice ; CD8-Positive T-Lymphocytes/immunology* ; Neoplasms/genetics* ; Cell Line, Tumor ; Mice, Inbred C57BL ; Mice, Knockout ; DNA-Binding Proteins/genetics* ; Female

Objective To investigate the therapeutic effects of interleukin 23p19(IL-23p19) monoclonal antibody in the MRL/lpr lupus-like mouse model. Methods A total of 36 female MRL/lpr mice aged 8 weeks were randomly divided into 6 groups: PBS group (blank control), IgG group (isotype IgG), dexamethasone (DEX) group (positive control), and three IL-23p19 monoclonal antibody treatment groups with different dose gradients: low dose (LD, 1 mg/kg), medium dose (MD, 3 mg/kg), and high dose (HD, 10 mg/kg). Drug intervention began at 12 weeks of age via tail vein injection. Urine protein levels were measured using urine protein test strips; serum anti-dsDNA antibody levels were detected by ELISA; serum creatinine and blood urea nitrogen levels were measured using an automatic biochemical analyzer; renal histopathological changes were analyzed by H&E and PAS staining; immunofluorescence was used to assess IgG and C3 immune complex deposition in kidney tissues; flow cytometry was employed to examine the expression of T helper 1(Th1), Th2, Th17, T follicular helper (Tfh), and regulatory T cells(Treg) cell subsets in the spleen; and RT-qPCR was used to detect the expression of related transcription factors in the spleen. Results IL-23p19 monoclonal antibody reduced urine protein levels, alleviated splenomegaly, improved renal function, and decreased anti-dsDNA antibody levels in MRL/lpr mice. It also mitigated glomerulonephritis and reduced renal immune complex deposition. Furthermore, IL-23p19 monoclonal antibody significantly suppressed the proportion of Th1 and Th17 cells while upregulating Treg cell proportion in the spleen. Additionally, it downregulated T-bet and retinoic acid receptor-related orphan receptor γt (RORγt) mRNA levels and upregulated forkhead box P3(FOXP3) mRNA levels in the spleen. Conclusions IL-23p19 monoclonal antibody demonstrates significant therapeutic effects in MRL/lpr mice, likely through modulation of the Th17/Treg cell balance.
Animals ; Female ; Mice, Inbred MRL lpr ; T-Lymphocytes, Regulatory/drug effects* ; Th17 Cells/drug effects* ; Antibodies, Monoclonal/therapeutic use* ; Interleukin-23 Subunit p19/immunology* ; Mice ; Lupus Nephritis/drug therapy* ; Kidney/drug effects* ; Antibodies, Antinuclear/blood*

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*

Rheumatoid arthritis (RA) is an autoimmune disease characterized by complex pathogenesis, with its development closely linked to immune dysregulation. Regulatory T cells (Tregs), as specialized immunomodulatory cells, play a pivotal role in negatively regulating immune responses and maintaining autoimmune tolerance. In recent years, extensive research has focused on the relationship between Tregs and RA pathogenesis, with the functional role of CD8⁺ Tregs as a critical area of investigation. This review summarizes the alterations in CD8⁺ Tregs during RA progression and their potential mechanisms of action. By elucidating the diversity of CD8⁺ Treg subsets and their intricate roles in modulating immune responses, this analysis provides novel insights and therapeutic strategies for RA diagnosis and treatment.
Arthritis, Rheumatoid/pathology* ; Humans ; T-Lymphocytes, Regulatory/immunology* ; CD8-Positive T-Lymphocytes/immunology* ; Animals

OBJECTIVE: To investigate the expression of co-inhibitory molecules TIGIT/CD155 and PD-1 on CD4⁺T cells and Treg cells in peripheral blood of patients with chronic lymphocytic leukemia (CLL) and analyze their clinical significance. METHODS: The expression of PD-1 and TIGIT on CD4⁺T cells and Treg cells was detected by flow cytometry in 40 CLL patients and 20 healthy controls. Additionally, the expression of CD155 on peripheral blood B cells and DC cells of the enrolled subjects was detected. RESULTS: The proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells and CD155⁺DC cells in peripheral blood of CLL patients were significantly higher than those of healthy controls ( P < 0.05). The proportions of PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells in CLL patients were significantly higher than those of PD-1⁺TIGIT^-CD4⁺T cells and PD-1⁺TIGIT^-Treg cells, respectively ( P < 0.05). Both PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells were positively correlated with the level of CD155⁺DC cells (r =0.742, r =0.766). With the progression of Binet stage, the proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells, and CD155⁺DC cells gradually increased ( P < 0.05), and the aforementioned three types cells were all increased in patients with CD38≥30%, IGVH unmutated, or poor prognosis due to chromosomal abnormalities ( P < 0.05). CONCLUSION Co-inhibitory molecules PD-1 and TIGIT may be involved in immunodepletion in patients with advanced CLL, which has clinical prognostic value. Dual inhibitor molecular targeted therapy provides a new direction for the individualized treatment of CLL.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/immunology* ; Receptors, Immunologic/metabolism* ; Programmed Cell Death 1 Receptor/metabolism* ; T-Lymphocytes, Regulatory/metabolism* ; Receptors, Virus/metabolism* ; CD4-Positive T-Lymphocytes/metabolism* ; Male ; Female ; Middle Aged ; Flow Cytometry ; Clinical Relevance