ABSTRACT
Purpose: CD25KO mice are a model of Sjögren disease (SjD) driven by autoreactive T cells. Cathepsin S (CTSS) is a protease crucial for major histocompatibility complex class II presentation that primes T cells. We investigated if a diet containing CTSS inhibitor would improve autoimmune signs in CD25KO mice. Methods: Four-week female CD25KO mice were randomly chosen to receive chow containing a CTSS inhibitor (R05461111, 262.5 mg/kg chow) or standard chow for 4 weeks. Cornea sensitivity was measured. Inflammatory score was assessed in lacrimal gland (LG) histologic sections. Flow cytometry of LG and ocular draining lymph nodes (dLNs) investigated expression of Th1 and Th17 cells. Expression of inflammatory, T- and B-cell, and apoptotic markers in the LG were assessed with quantitative PCR. The life span of mice receiving CTSS inhibitor or standard chow was compared. CD4+ T cells from both groups were isolated from spleens and adoptively transferred into RAG1KO female recipients. Results: Mice receiving CTSS inhibitor had better cornea sensitivity and improved LG inflammatory scores. There was a significant decrease in the frequency of CD4+ immune cells and a significant increase in the frequency of CD8+ immune cells in the dLNs of CTSS inhibitor mice. There was a significant decrease in Th1 and Th17 cells in CTSS inhibitor mice in both LGs and dLNs. Ifng, Ciita, and Casp8 mRNA in CTSS inhibitor mice decreased. Mice that received the CTSS inhibitor lived 30% longer. Adoptive transfer recipients with CTSS inhibitor-treated CD4+ T cells had improved cornea sensitivity and lower inflammation scores. Conclusions: Inhibiting CTSS could be a potential venue for the treatment of SjD in the eye and LG.
Subject(s)
Cathepsins , Disease Models, Animal , Flow Cytometry , Lacrimal Apparatus , Mice, Knockout , Sjogren's Syndrome , Animals , Mice , Sjogren's Syndrome/immunology , Sjogren's Syndrome/drug therapy , Female , Cathepsins/antagonists & inhibitors , Cathepsins/metabolism , Cathepsins/genetics , Lacrimal Apparatus/pathology , Lacrimal Apparatus/metabolism , Mice, Inbred C57BL , Adoptive Transfer , Th17 Cells/immunology , Real-Time Polymerase Chain Reaction , Th1 Cells/immunology , Interleukin-2 Receptor alpha SubunitABSTRACT
BACKGROUND: Human placental mesenchymal stromal cells (hPMSCs) are known to limit graft-versus-host disease (GVHD). CD8+CD122+PD-1+Tregs have been shown to improve the survival of GVHD mice. However, the regulatory roles of hPMSCs in this subgroup remain unclear. Here, the regulatory mechanism of hPMSCs in reducing liver fibrosis in GVHD mice by promoting CD8+CD122+PD-1+Tregs formation and controlling the balance of IL-6 and IL-10 were explored. METHODS: A GVHD mouse model was constructed using C57BL/6J and BALB/c mice and treated with hPMSCs. LX-2 cells were explored to study the effects of IL-6 and IL-10 on the activation of hepatic stellate cells (HSCs). The percentage of CD8+CD122+PD-1+Tregs and IL-10 secretion were determined using FCM. Changes in hepatic tissue were analysed by HE, Masson, multiple immunohistochemical staining and ELISA, and the effects of IL-6 and IL-10 on LX-2 cells were detected using western blotting. RESULTS: hPMSCs enhanced CD8+CD122+PD-1+Treg formation via the CD73/Foxo1 and promoted IL-10, p53, and MMP-8 levels, but inhibited IL-6, HLF, α-SMA, Col1α1, and Fn levels in the liver of GVHD mice through CD73. Positive and negative correlations of IL-6 and IL-10 between HLF were found in liver tissue, respectively. IL-6 upregulated HLF, α-SMA, and Col1α1 expression via JAK2/STAT3 pathway, whereas IL-10 upregulated p53 and inhibited α-SMA and Col1α1 expression in LX-2 cells by activating STAT3. CONCLUSIONS: hPMSCs promoted CD8+CD122+PD-1+Treg formation and IL-10 secretion but inhibited HSCs activation and α-SMA and Col1α1 expression by CD73, thus controlling the balance of IL-6 and IL-10, and alleviating liver injury in GVHD mice.
Subject(s)
Forkhead Box Protein O1 , Graft vs Host Disease , Mesenchymal Stem Cells , T-Lymphocytes, Regulatory , Animals , Female , Humans , Mice , Pregnancy , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Forkhead Box Protein O1/metabolism , Graft vs Host Disease/immunology , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/immunology , Interleukin-10/metabolism , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-6/metabolism , Liver/pathology , Liver/immunology , Liver/metabolism , Liver Cirrhosis/immunology , Liver Cirrhosis/therapy , Liver Cirrhosis/metabolism , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/immunology , Mice, Inbred BALB C , Mice, Inbred C57BL , Placenta/cytology , Programmed Cell Death 1 Receptor/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolismABSTRACT
Introduction: Our study investigated the potential of peripheral blood T cell CD25, CD28, and CTLA-4 gene transcription levels as predictive biomarkers for acute graft-versus-host disease (aGVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: Real-time reverse transcription fluorescent quantitative PCR (RT-qPCR) analysis was conducted on day +7, +14, and +21 post-transplantation in patients undergoing allo-HSCT. Results: Elevated levels of CD25 and CTLA-4 mRNA were found to be associated with the occurrence of aGVHD, as well as severe and gastrointestinal aGVHD. Receiver operating characteristic (ROC) curve analysis was utilized to assess the predictive value of each biomarker. Combined analysis of CD25 and CTLA-4 mRNA levels demonstrated promising predictive potential for aGVHD. Conclusion: Our results confirmed that the transcription levels of CD25 and CTLA-4 genes could be used as early predictive biomarkers for aGVHD post-allo-HSCT.
Subject(s)
Biomarkers , CTLA-4 Antigen , Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Interleukin-2 Receptor alpha Subunit , Transcription, Genetic , Graft vs Host Disease/genetics , Graft vs Host Disease/diagnosis , Graft vs Host Disease/immunology , Humans , CTLA-4 Antigen/genetics , Male , Interleukin-2 Receptor alpha Subunit/genetics , Female , Adult , Hematopoietic Stem Cell Transplantation/adverse effects , Middle Aged , Acute Disease , Young Adult , Adolescent , Transplantation, Homologous/adverse effects , PrognosisABSTRACT
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.
Subject(s)
Gene Products, tax , Human T-lymphotropic virus 1 , Protein-Arginine N-Methyltransferases , Humans , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Gene Products, tax/genetics , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/genetics , Cyclin D2/genetics , Cyclin D2/metabolism , Transcriptional Activation , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-2 Receptor alpha Subunit/metabolism , NF-kappa B/metabolism , NF-kappa B/genetics , Histones/metabolism , Histones/genetics , Epigenesis, Genetic , Jurkat CellsABSTRACT
Cytomegalovirus (CMV) infection detrimentally influences graft survival in kidney transplant recipients, with the risk primarily determined by recipient and donor serostatus. However, recipient CD8+ T cells play a crucial role in CMV control. The optimal preventive strategy (prophylaxis vs. pre-emptive treatment), particularly for seropositive (intermediate risk) recipients, remains uncertain. We investigated CD8+ T cell subpopulation dynamics and CMV occurrence (DNAemia ≥ 100 IU/mL) in 65 kidney transplant recipients, collecting peripheral blood mononuclear cells before (T1) and 1 year after transplantation (T2). Comparing the two timepoints, we found an increase in granulocyte, monocyte and CD3+CD8+ T cells numbers, while FoxP3+CD25+, LAG-3+ and PD-1+ frequencies were reduced at T2. CMV DNAemia occurred in 33 recipients (55.8%) during the first year. Intermediate risk patients were disproportionally affected by posttransplant CMV (N = 29/45, 64.4%). Intermediate risk recipients developing CMV after transplantation exhibited lower leukocyte, monocyte, and granulocyte counts and higher FoxP3+CD25+ frequencies in CD3+CD8+ T cells pre-transplantation compared to patients staying CMV negative. Pre-transplant FoxP3+CD25+ in CD3+CD8+ T cells had the best discriminatory potential for CMV infection prediction within the first year after transplantation (AUC: 0.746). The FoxP3+CD25+ CD3+CD8+ T cell subset may aid in selecting intermediate risk kidney transplant recipients for CMV prophylaxis.
Subject(s)
CD8-Positive T-Lymphocytes , Cytomegalovirus Infections , Forkhead Transcription Factors , Interleukin-2 Receptor alpha Subunit , Kidney Transplantation , Humans , Kidney Transplantation/adverse effects , Cytomegalovirus Infections/immunology , Cytomegalovirus Infections/prevention & control , Female , Male , CD8-Positive T-Lymphocytes/immunology , Middle Aged , Forkhead Transcription Factors/metabolism , Adult , Interleukin-2 Receptor alpha Subunit/metabolism , Aged , CD3 Complex/metabolism , Cytomegalovirus/immunology , Risk Factors , Transplant Recipients , Graft Survival/immunologyABSTRACT
CD25, the interleukin-2 receptor α-chain, is expressed on cell surfaces of different immune cells and is commonly used for phenotyping of regulatory T cells (Tregs). CD25 has essential roles in the maintenance of hemostasis and immune tolerance and Treg cell involvement has been shown in human diseases and murine models for allergy, autoimmunity, cancer, chronic inflammation, and many others. In horses, a cross-reactive anti-human CD25 antibody has previously been used for characterizing Tregs. Here, we developed monoclonal antibodies (mAbs) to equine CD25 and compared their staining pattern with the anti-human CD25 antibody by flow cytometry. The comparison of the two reagents was performed by two separate analyses in independent laboratories. Overall, similar staining patterns for equine peripheral blood lymphocytes were obtained with the anti-human CD25 antibody and equine CD25 mAb 15-1 in both laboratories. Both reagents identified comparable CD4+CD25+ and CD4+CD25+FOXP3+ percentages after stimulation of peripheral blood mononuclear cells (PBMC) with pokeweed mitogen. However, when compared to the anti-human CD25 antibody, the equine CD25 mAb 15-1 resulted in a better staining intensity of the equine CD25+ cells and increased the percentages of Tregs and other CD25+ cells ex vivo and after culturing of PBMC without stimulation. In summary, the equine CD25 mAbs provide new, improved reagents for Tregs and CD25+ cell phenotyping in horses.
Subject(s)
Antibodies, Monoclonal , Flow Cytometry , Interleukin-2 Receptor alpha Subunit , T-Lymphocytes, Regulatory , Horses/immunology , Animals , T-Lymphocytes, Regulatory/immunology , Interleukin-2 Receptor alpha Subunit/immunology , Antibodies, Monoclonal/immunology , Flow Cytometry/veterinary , Humans , Leukocytes, Mononuclear/immunologyABSTRACT
Regulatory T cells (Tregs) play a crucial role in mediating immunosuppression in the tumor microenvironment. Furthermore, Tregs contribute to the lack of efficacy and hyperprogressive disease upon Programmed cell death protein 1 (PD-1) blockade immunotherapy. Thus, Tregs are considered a promising therapeutic target, especially when combined with PD-1 blockade. However, systemic depletion of Tregs causes severe autoimmune adverse events, which poses a serious challenge to Treg-directed therapy. Here, we developed a novel treatment to locally and predominantly damage Tregs by near-infrared duocarmycin photorelease (NIR-DPR). In this technology, we prepared anti-CD25 F(ab')2 conjugates, which site-specifically uncage duocarmycin in CD25-expressing cells upon exposure to NIR light. In vitro, CD25-targeted NIR-DPR significantly increased apoptosis of CD25-expressing HT2-A5E cells. When tumors were irradiated with NIR light in vivo, intratumoral CD25+ Treg populations decreased and Ki-67 and Interleukin-10 expression was suppressed, indicating impaired functioning of intratumoral CD25+ Tregs. CD25-targeted NIR-DPR suppressed tumor growth and improved survival in syngeneic murine tumor models. Of note, CD25-targeted NIR-DPR synergistically enhanced the efficacy of PD-1 blockade, especially in tumors with higher CD8+/Treg PD-1 ratios. Furthermore, the combination therapy induced significant anti-cancer immunity including maturation of dendritic cells, extensive intratumoral infiltration of cytotoxic CD8+ T cells, and increased differentiation into CD8+ memory T cells. Altogether, CD25-targeted NIR-DPR locally and predominantly targets Tregs in the tumor microenvironment and synergistically improves the efficacy of PD-1 blockade, suggesting that this combination therapy can be a rational anti-cancer combination immunotherapy.
Subject(s)
Duocarmycins , Programmed Cell Death 1 Receptor , T-Lymphocytes, Regulatory , Tumor Microenvironment , Animals , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , Mice , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Duocarmycins/pharmacology , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Humans , Cell Line, Tumor , Female , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-2 Receptor alpha Subunit/immunology , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Disease Models, Animal , Mice, Inbred C57BL , Apoptosis/drug effects , Infrared RaysABSTRACT
BACKGROUND: We previously demonstrated that the hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor (statins) play an important role in the regulation of alloimmune responses. However, little is known regarding the effects of statin on allograft protection or donor-specific antibodies (DSA). In this study, we investigated the graft-protective and immunomodulatory effects of rosuvastatin in a model of fully major histocompatibility complex-mismatched murine cardiac allograft transplantation. METHODS: CBA mice underwent transplantation of C57BL/6 (B6) hearts and received 50 and 500 µg/kg/day of rosuvastatin from the day of transplantation until seven days after the completion of transplantation. To confirm the requirement for regulatory T cells (Tregs), we administered an anti-interleukin-2 receptor alpha antibody (PC-61) to rosuvastatin-treated CBA recipients. Additionally, histological and fluorescent staining, cell proliferation analysis, flow cytometry, and DSA measurements were performed. RESULTS: CBA recipients with no treatment rejected B6 cardiac graft acutely (median survival time [MST], 7 days). CBA mice treated with 500 µg/kg/day of rosuvastatin prolonged allograft survival (MSTs, 77 days). Fluorescent staining studies showed that rosuvastatin-treated recipients had strong aggregation of CD4+Foxp3+ cells in the myocardium and around the coronary arteries of cardiac allografts two weeks after grafting. Flow cytometry studies performed two weeks after transplantation showed an increased number of splenic CD4+CD25+Foxp3+ T cells in rosuvastatin-treated recipients. The addition of rosuvastatin to mixed leukocyte cultures suppressed cell proliferation by increasing the number of CD4+CD25+Foxp3+ Tregs. Additionally, Tregs suppressed DSA production in rosuvastatin-treated recipients. CONCLUSION: Rosuvastatin treatment may be a complementary graft-protective strategy for suppressing DSA production in the acute phase, driven by the promotion of splenic and graft-infiltrating CD4+CD25+Foxp3+ Tregs.
Subject(s)
Heart Transplantation , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Mice, Inbred C57BL , Mice, Inbred CBA , Rosuvastatin Calcium , T-Lymphocytes, Regulatory , Animals , Rosuvastatin Calcium/pharmacology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , Mice , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Graft Rejection/prevention & control , Graft Rejection/immunology , Graft Survival/drug effects , Graft Survival/immunology , Interleukin-2 Receptor alpha Subunit/immunology , Male , Forkhead Transcription Factors/metabolism , Disease Models, Animal , Flow CytometryABSTRACT
The importance of neuroinflammation during the ischemic stroke has been extensively studied. The role of CD4+CD25+ regulatory T (Treg) cells during the recovery phase have shown infarct size reduction and functional improvement, possibly through the mitigation of inflammatory immune responses. We aimed to investigate the molecular factors involved in microglia-Treg cell communication that result in Treg trafficking. First, we observed the migration patterns of CD8+ (cytotoxic) T cells and Treg cells and then searched for chemokines released by activated microglia in an oxygen-glucose deprivation (OGD) model. The transwell migration assay showed increased migration into OGD media for both cell types, in agreement with the increase in chemokines involved in immune cell trafficking from the mouse chemokine profiling array. MSCV retrovirus was transduced to overexpress CCR4 in Treg cells. CCR4-overexpressed Treg cells were injected into the mouse transient middle cerebral artery occlusion (tMCAO) model to evaluate the therapeutic potential via the tetrazolium chloride (TTC) assay and behavioral tests. A general improvement in the prognosis of animals after tMCAO was observed. Our results suggest the increased mobility of CCR4-overexpressed Treg cells in response to microglia-derived chemokines in vitro and the therapeutic potential of Treg cells with increased mobility in cellular therapy.
Subject(s)
Cell Movement , Disease Models, Animal , Infarction, Middle Cerebral Artery , Ischemic Stroke , Receptors, CCR4 , T-Lymphocytes, Regulatory , Animals , Receptors, CCR4/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Mice , Ischemic Stroke/immunology , Ischemic Stroke/metabolism , Ischemic Stroke/pathology , Infarction, Middle Cerebral Artery/immunology , Infarction, Middle Cerebral Artery/metabolism , Interleukin-2 Receptor alpha Subunit/metabolism , Microglia/metabolism , Microglia/immunology , Male , Mice, Inbred C57BL , Chemokines/metabolismABSTRACT
Atrial fibrillation (AF) is a prevalent cardiac arrhythmia, with recent research indicating a correlation between immune system characteristics and the development of AF. However, it remains uncertain whether the immunological response is the primary underlying component or a secondary consequence of AF. Initially, we investigated the effect of immune cells on AF by performing forward Mendelian randomization (MR) analyses with immune cells as the exposure variable and their associated genetic variants as instrumental variables. Subsequently, we performed reverse MR analyses with AF as the exposure variable and immune cells as the outcome variable to exclude the interference of reverse causality, to distinguish between primary and secondary effects, and to further elucidate the causal relationship between the immune system and AF. We discovered that membrane proteins on specific immune cells, such as CD25 on memory B cells-which functions as a part of the interleukin-2 receptor-may be risk factors for AF development, with odds ratios of 1.0233 (95% confidence interval: 1.0012-1.0458, Pâ =â .0383). In addition, certain immune cell counts, such as the CD4 regulatory T cell Absolute Count, play a protective factor in the development of AF (odds ratio: 0.9513, 95% confidence interval: 0.9165-0.9874; Pâ =â .0086). More detailed results are elaborated in the main text. Our MR study has yielded evidence that substantiates a genetically inferred causal association between the immune system and AF. Identifying the risk factors associated with AF is vital to facilitate the development of innovative pharmaceutical treatments.
Subject(s)
Atrial Fibrillation , Mendelian Randomization Analysis , Atrial Fibrillation/genetics , Atrial Fibrillation/immunology , Atrial Fibrillation/epidemiology , Humans , Interleukin-2 Receptor alpha Subunit/genetics , Risk Factors , B-Lymphocytes/immunologyABSTRACT
Aberrant neuronal excitability in the anterior cingulate cortex (ACC) is implicated in cognitive and affective pain processing. Such excitability may be amplified by activated circulating immune cells, including T lymphocytes, that interact with the central nervous system. Here, we conducted a study of individuals with chronic pain using magnetic resonance spectroscopy (MRS) to investigate the clinical evidence for the interaction between peripheral immune activation and prefrontal excitatory-inhibitory imbalance. In thirty individuals with chronic musculoskeletal pain, we assessed markers of peripheral immune activation, including soluble interleukin-2 receptor alpha chain (sCD25) levels, as well as brain metabolites, including Glx (glutamate + glutamine) to GABA+ (γ-aminobutyric acid + macromolecules/homocarnosine) ratio in the ACC. We found that the circulating level of sCD25 was associated with prefrontal Glx/GABA+. Greater prefrontal Glx/GABA+ was associated with higher pain catastrophizing, evaluative pain ratings, and anxiodepressive symptoms. Further, the interaction effect of sCD25 and prefrontal Glx/GABA+ on pain catastrophizing was significant, indicating the joint association of these two markers with pain catastrophizing. Our results provide the first evidence suggesting that peripheral T cellular activation, as reflected by elevated circulating sCD25 levels, may be linked to prefrontal excitatory-inhibitory imbalance in individuals with chronic pain. The interaction between these two systems may play a role as a potential mechanism underlying pain catastrophizing. Further prospective and treatment studies are needed to elucidate the specific role of the immune and brain interaction in pain catastrophizing.
Subject(s)
Chronic Pain , Interleukin-2 Receptor alpha Subunit , Prefrontal Cortex , Humans , Male , Female , Chronic Pain/metabolism , Prefrontal Cortex/metabolism , Adult , Middle Aged , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-2 Receptor alpha Subunit/blood , Gyrus Cinguli/metabolism , Glutamic Acid/metabolism , Glutamic Acid/blood , Proton Magnetic Resonance Spectroscopy , Glutamine/metabolism , Glutamine/blood , gamma-Aminobutyric Acid/metabolism , Catastrophization/metabolismABSTRACT
While Interleukin 2 (IL2) has the capability to activate both NK and T cells robustly, its limited in vivo half-life, considerable toxicity, and tendency to boost Treg cells pose significant challenges, restricting its widespread application in cancer therapy. In this investigation, we engineered a novel IL2 variant (IL2-4M-PEG) with reduced CD25 binding activity and an extended half-life by substituting amino acids associated with CD25 binding and implementing site-directed PEGylation. IL2-4M-PEG notably amplifies effector cells over Treg cells. Furthermore, our findings reveal that IL2-4M-PEG, characterized by an extended half-life, exhibits anti-tumor effects in a mouse model. Consequently, this innovative IL2 holds the potential for enhancing combined cancer therapies in the future.
Subject(s)
Immunotherapy , Interleukin-2 Receptor alpha Subunit , Interleukin-2 , Polyethylene Glycols , Animals , Interleukin-2/metabolism , Polyethylene Glycols/chemistry , Immunotherapy/methods , Humans , Mice , Interleukin-2 Receptor alpha Subunit/metabolism , Cell Line, Tumor , Neoplasms/therapy , Neoplasms/immunology , T-Lymphocytes, Regulatory/immunology , Protein Binding , Mice, Inbred C57BL , Female , Mice, Inbred BALB C , Killer Cells, Natural/immunologyABSTRACT
The involvement of γδT cells, Th17 cells, and CD4+CD25+ regulatory T cells (Tregs) is crucial in the progression of pulmonary fibrosis (PF), particularly in maintaining immune tolerance and homeostasis. However, the dynamics of these cells in relation to PF progression, especially under pharmacological interventions, remains poorly understood. This study aims to unravel the interplay between the dynamic changes of these cells and the effect of pharmacological agents in a mouse model of PF induced by intratracheal instillation of bleomycin. We analyzed changes in lung histology, lung index, hydroxyproline levels, and the proportions of γδT cells, Th17 cells, and Tregs on the 3rd, 14th, and 28th days following treatment with Neferine, Isoliensinine, Pirfenidone, and Prednisolone. Our results demonstrate that these drugs can partially or dynamically reverse weight loss, decrease lung index and hydroxyproline levels, and ameliorate lung histopathological damage. Additionally, they significantly modulated the abnormal changes in γδT, Th17, and Treg cell proportions. Notably, on day 3, the proportion of γδT cells increased in the Neferine and Prednisolone groups but decreased in the Isoliensinine and Pirfenidone groups, while the proportion of Th17 cells decreased across all treated groups. On day 14, the Neferine group showed an increase in all three cell types, whereas the Pirfenidone group exhibited a decrease. In the Isoliensinine group, γδT and Th17 cells increased, and in the Prednisolone group, only Tregs increased. By day 28, an increase in Th17 cell proportion was observed in all treatment groups, with a decrease in γδT cells noted in the Neferine group. These shifts in cell proportions are consistent with the pathogenesis changes induced by these anti-PF drugs, suggesting a correlation between cellular dynamics and pharmacological interventions in PF progression. Our findings imply potential strategies for assessing the efficacy and timing of anti-PF treatments based on these cellular changes.
Subject(s)
Bleomycin , Pulmonary Fibrosis , T-Lymphocytes, Regulatory , Th17 Cells , Animals , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/drug therapy , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , Th17 Cells/drug effects , Th17 Cells/immunology , Mice , Pyridones/pharmacology , Male , Prednisolone/pharmacology , Disease Progression , Mice, Inbred C57BL , Disease Models, Animal , Lung/pathology , Lung/immunology , Lung/drug effects , Interleukin-2 Receptor alpha Subunit/metabolism , Isoquinolines/pharmacology , Benzylisoquinolines/pharmacologyABSTRACT
Regulatory T cells (Tregs) are central in controlling immune responses, and dysregulation of their function can lead to autoimmune disorders or cancer. Despite extensive studies on Tregs, the basis of epigenetic regulation of human Treg development and function is incompletely understood. Long intergenic noncoding RNAs (lincRNA)s are important for shaping and maintaining the epigenetic landscape in different cell types. In this study, we identified a gene on the chromosome 6p25.3 locus, encoding a lincRNA, that was up-regulated during early differentiation of human Tregs. The lincRNA regulated the expression of interleukin-2 receptor alpha (IL2RA), and we named it the lincRNA regulator of IL2RA (LIRIL2R). Through transcriptomics, epigenomics, and proteomics analysis of LIRIL2R-deficient Tregs, coupled with global profiling of LIRIL2R binding sites using chromatin isolation by RNA purification, followed by sequencing, we identified IL2RA as a target of LIRIL2R. This nuclear lincRNA binds upstream of the IL2RA locus and regulates its epigenetic landscape and transcription. CRISPR-mediated deletion of the LIRIL2R-bound region at the IL2RA locus resulted in reduced IL2RA expression. Notably, LIRIL2R deficiency led to reduced expression of Treg-signature genes (e.g., FOXP3, CTLA4, and PDCD1), upregulation of genes associated with effector T cells (e.g., SATB1 and GATA3), and loss of Treg-mediated suppression.
Subject(s)
Forkhead Transcription Factors , Interleukin-2 Receptor alpha Subunit , RNA, Long Noncoding , T-Lymphocytes, Regulatory , Humans , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-2 Receptor alpha Subunit/metabolism , Epigenesis, Genetic , Gene Expression Regulation , Cell Differentiation/geneticsABSTRACT
Introduction: IL-2Rα knock out (KO) mice have been instrumental to discovering the immunoregulatory properties of IL-2Rα. While initially thought of only as a stimulatory cytokine, IL-2 and IL-2Rα KO mice revealed that this cytokine-receptor system controls immune responses through restimulation-induced cell death and by promoting the survival of T regulatory cells. Although described mostly in the context of lymphocytes, recent studies by our laboratory showed that IL-2R is expressed in smooth muscle cells. Given this finding, we sought to use IL-2Rα KO to determine the function of this receptor in vascular smooth muscle cells. Surprisingly, we found that IL-2Rα KO vascular smooth muscle cells had detectable IL-2Rα. Methods: We used multiple gene and protein-based methods to determine why IL-2Rα KO vascular smooth muscle cells exhibited IL-2Rα protein. These methods included: genomic sequencing, assessing cells and tissues for evidence of maternal microchimerism, and determining the half-life of IL-2Rα protein. Results: Our studies demonstrated the following: (1) in addition to the cell surface, IL-2Rα is localized to the nucleus; (2) the genetic deletion of IL-2Rα is intact in IL-2Rα KO mice; (3) both IL-2Rα KO and WT tissues show evidence of maternal microchimerism, the likely source of IL-2Rα (4) IL-2Rα is transmitted between cells; (5) IL-2Rα has a long half-life; and (6) nuclear IL-2Rα contributes to the regulation of cell proliferation and size. Conclusion: Our findings suggest that the phenotype of complete IL-2Rα loss is more severe than demonstrated by IL-2Rα KO mice, and that IL-2Rα plays a here-to-fore unrecognized role in regulating cell proliferation in non-lymphoid cells.
Subject(s)
Cell Nucleus , Interleukin-2 Receptor alpha Subunit , Mice, Knockout , Animals , Female , Mice , Cell Nucleus/metabolism , Chimerism , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-2 Receptor alpha Subunit/genetics , Lymphocytes/immunology , Lymphocytes/metabolism , Mice, Inbred C57BL , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/immunology , Myocytes, Smooth Muscle/metabolismABSTRACT
CD20+ T cells constitute a small subset of T cells. These are found among CD4+, CD8+, CD4+CD8+, CD4-CD8- T, and TCRγδ+ T cells, and have been poorly characterized. The aim of this study was to characterize peripheral blood (PB) CD20+ T cells and compare them to their PB CD20- T cell counterparts. PB from 17 healthy individuals was collected. The distribution of CD20+ T cells among maturation-associated T cells compartments (naïve, central memory, transitional memory, effector memory, and effector T cells), their polarization, activation status, and expression of immune-regulatory proteins were evaluated by flow cytometry. Their function was also assessed, by measuring IFN-γ, TNF-α, and IL-17 production. Compared with CD20- T cells, CD20+ T cells represent a higher proportion of transitional memory cells. Furthermore, CD20+ T cells display a proinflammatory phenotype, characterized by the expansion of Th1, Th1/17, and Tc1 cell subsets , associated to a high expression of activation (CD25) and exhaustion (PD-1) markers. In addition, the simultaneous production of the proinflammatory cytokines IFN-γ, TNF-α, and IL-17 was also detected in CD4+CD20+ T cells. Our results show that CD20+ T cells are phenotypically and functionally different from CD20- T cells, suggesting that these cells are a distinct subset of T cells.
Subject(s)
Antigens, CD20 , Flow Cytometry , Humans , Antigens, CD20/immunology , Male , Female , Adult , Interferon-gamma , Tumor Necrosis Factor-alpha , Interleukin-17/blood , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Lymphocyte Activation/immunology , Middle Aged , Immunologic Memory/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Programmed Cell Death 1 Receptor/metabolism , Programmed Cell Death 1 Receptor/immunology , Cytokines/metabolism , Memory T Cells/immunology , Interleukin-2 Receptor alpha Subunit/immunologyABSTRACT
Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: ⢠A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. ⢠C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. ⢠The frequency of Treg cells was positively correlated with the viral load during orf virus infection.
Subject(s)
Antibodies, Monoclonal , Flow Cytometry , Goats , T-Lymphocytes, Regulatory , Viral Load , Animals , T-Lymphocytes, Regulatory/immunology , Antibodies, Monoclonal/immunology , Ecthyma, Contagious/diagnosis , Ecthyma, Contagious/immunology , Interleukin-2 Receptor alpha Subunit/immunology , Orf virus/immunology , Fluorescent Antibody Technique/methods , CD4 Antigens/immunology , Goat Diseases/immunology , Goat Diseases/virology , Goat Diseases/diagnosisABSTRACT
Mycosis fungoides (MF) has been widely reported to mimick a considerable number of different dermatoses, including scarring alopecia, bullous dermatoses or cysts, and comedones. In atypical presentations, histopathology is essential for the diagnosis. We present two cases of MF with clinical urticarial lesions and a striking blood involvement that responded to mogamulizumab treatment. Histopathologically, both cases had classic MF features and shared a peculiar immunophenotype, with positivity for CD25 and FOXP3. Differential diagnoses included urticarial lymphomatoid drug reactions and other lymphomas, like T-cell prolymphocytic leukemia, atypical Sézary syndrome, or adult T-cell lymphocytic leukemia. A low suspicion threshold is necessary for the diagnosis of atypical presentations of MF.
Subject(s)
Immunophenotyping , Mycosis Fungoides , Skin Neoplasms , Humans , Mycosis Fungoides/pathology , Mycosis Fungoides/diagnosis , Skin Neoplasms/pathology , Male , Immunophenotyping/methods , Diagnosis, Differential , Middle Aged , Female , Urticaria/pathology , Interleukin-2 Receptor alpha Subunit/metabolism , Aged , Forkhead Transcription FactorsABSTRACT
The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the clinical treatment for tumor. However, the low response rate of ICIs remains the major obstacle for curing patients and effective approaches for patients with primary or secondary resistance to ICIs remain lacking. In this study, immune stimulating agent unmethylated CG-enriched (CpG) oligodeoxynucleotide (ODN) was locally injected into the tumor to trigger a robust immune response to eradicate cancer cells, while anti-CD25 antibody was applied to remove immunosuppressive regulatory T cells, which further enhanced the host immune activity to attack tumor systematically. The combination of CpG and anti-CD25 antibody obtained notable regression in mouse melanoma model. Furthermore, rechallenge of tumor cells in the xenograft model has resulted in smaller tumor volume, which demonstrated that the combinational treatment enhanced the activity of memory T cells. Remarkably, this combinational therapy presented significant efficacy on multiple types of tumors as well and was able to prevent relapse of tumor partially. Taken together, our combinational immunotherapy provides a new avenue to enhance the clinical outcomes of patients who are insensitive or resistant to ICIs treatments.
Subject(s)
Oligodeoxyribonucleotides , T-Lymphocytes, Regulatory , Animals , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , Oligodeoxyribonucleotides/therapeutic use , Oligodeoxyribonucleotides/pharmacology , Mice , Mice, Inbred C57BL , Female , Humans , Cell Line, Tumor , Cancer Vaccines/immunology , Cancer Vaccines/therapeutic use , Interleukin-2 Receptor alpha Subunit/immunology , Melanoma, Experimental/immunology , Melanoma, Experimental/drug therapy , Melanoma, Experimental/therapy , Immunotherapy/methods , Neoplasms/immunology , Neoplasms/drug therapy , Neoplasms/therapy , Vaccination , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic useABSTRACT
OBJECTIVE: To carry out a preliminary analysis on the Treg lymphocyte counts present in the peripheral blood of allergic asthmatic children from the city of Cartagena, Colombia, compared to healthy controls. METHODS: We compared cytometry counts of ten asthmatic patients (age 7-16 years) and seven healthy controls (6-12 years), recruited in the city of Cartagena. Peripheral blood samples were stained using Cytek's 14-color cFluor Immunoprofiling kit (Cytek® cFluor® Immunoprofiling Kit 14 Color RUO kit), and analyzed on a Northern Lights™ spectral cytometer (Cytek® Biosciences, Fremont, CA, USA), to read 50.000 events per sample. The data obtained were analyzed in SpectroFlo® and FlowJo. The study was approved by the ethics committee of the University of Cartagena (SGR, Grant BPIN2020000100405). RESULTS: The frequency of CD3+, CD4+, CD25+, CD127- Tregs was 11% of all CD4+ T cells, with a range of minimum 8,1% and maximum 17,7%. There was no significant difference in the proportion of Tregs between allergic asthmatic patients and healthy controls (P = 0,2). CONCLUSIONS: With this preliminary sample size, no significant differences were found in the Treg lymphocyte population between allergic asthmatic patients and healthy controls. The 14-color multiplexed panel is a useful tool not only to count CD3+ and CD4+ populations, but also to obtain the percentage of regulatory T cells using cell surface markers.
OBJETIVO: Realizar un análisis preliminar sobre los conteos de linfocitos Tregs presentes en sangre periférica de niños asmáticos alérgicos de la ciudad de Cartagena, comparado con controles sanos. MÉTODOS: Se compararon los conteos de citometría de diez pacientes asmáticos (entre 7 y16 años) y siete controles sanos (entre 6 y12 años), reclutados en la ciudad de Cartagena. La muestra de sangre periférica fue teñida empleando el kit de inmunofenotipo multiplexado de 14 colores de Cytek (Cytek® cFluor® Immunoprofiling Kit 14 Color), y analizada en un citómetro espectral Northern Lights™ (Cytek® Biosciences, Fremont, CA, USA), a lectura de 50.000 eventos por muestra. Los datos obtenidos fueron analizados en SpectroFlo® y FlowJo. El estudio fue aprobado por el Comité de Ética de la Universidad de Cartagena. RESULTADOS: El panel de tinción funcionó apropiadamente y dentro de los parámetros apropiados. Se obtuvo un promedio de células Tregs CD3+, CD4+, CD25+ y CD127- del 11% de todos los CD4+ en las muestras estudiadas, con un rango de mínimo de 8,1% y un máximo de 17,7%. No hubo diferencias significativas en la proporción de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos (P = 0.2). CONCLUSIONES: Con este tamaño de muestra preliminar, no se encontraron diferencias significativas en la población de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos. El panel multiplexado de 14 colores es una herramienta útil no solo para derivar las poblaciones CD3+ y CD4+, sino también para obtener el porcentaje de células T reguladoras empleando marcadores de superficie celular.