ABSTRACT
CD134 (TNFRSF4) is a member of the TNFR superfamily, which is specifically expressed on T cells. Previous studies have shown that blocking of CD134L-CD134 interaction reduces the percentage of activated T cells and prevents effector T cell-mediated graft rejection in heart transplantation. However, the role of microRNA-regulated inhibition of the CD134 signal in cardiac transplantation of T-regulatory (Treg) cells is not clear. In this study, we found microRNA 744 (miR-744) agomir administration enhanced the expression levels of miR-744 in CD4+CD25+ Treg cells from heart transplantation mice. Moreover, miR-744 agomir administration significantly enhanced the expression levels of CD62L and Ki67 in CD4+CD25+ Treg cells from heart transplantation mice and further enhanced immunosuppressive function of Treg cells following coculture with CD4+CD25- T cells for different ratios. In addition, miR-744 agomir treatment significantly prolonged survival time and reduced rejection response of heart allografts in vivo, which are involved in downregulation of TNFRSF4 expression. These results provided a novel molecular mechanism of ameliorating heart allograft rejection in Treg cells, which could be used in the treatment of heart allograft rejection clinically.
Subject(s)
Graft Rejection/metabolism , Heart Transplantation , MicroRNAs/metabolism , Receptors, OX40/biosynthesis , T-Lymphocytes, Regulatory/metabolism , Allografts , Animals , Gene Expression Regulation/immunology , Heart Transplantation/methods , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , T-Lymphocytes, Regulatory/immunologyABSTRACT
The aim of the study was to evaluate the potential role of CD40/CD40 ligand (CD40L) and CD134/CD134 ligand (CD134L) in the development of coronary heart disease (CHD) via the performance of a case-control study.The research objects were 234 cases of CHD patients and 120 cases of well-matched normal controls. Following the separation of peripheral blood mononuclear cells (PBMCs), real-time quantitative PCR (qRT-PCR), Western blot, immunohistochemistry, and flow cytometry were applied for the detection of mRNA levels and expression levels of CD40/CD40L and CD134/CD134L; meanwhile, intercellular adhesion molecule-1 (ICAM-1) and Fas protein mRNA levels were detected using qRT-PCR.There was no statistical difference in the comparison of baseline characteristics between groups, indicating comparability between groups. qRT-PCR and Western blot analysis indicated that CD40/CD40L and CD134/CD134L mRNA and protein expression levels were all increased in the CHD group than those in the control group. Flow cytometry further confirmed the similar tendency. Meanwhile, ICAM-1 and Fas protein mRNA levels were elevated in the CHD group and positively correlated with the above parameters. Furthermore, CD40/CD40L expression rates were negatively correlated with gender and different types of CHD. Meanwhile, CD134/CD134L expressions were also higher in male patients, in patients with family history, previous history of hypertension, diabetes, and cerebrovascular diseases.CD40/CD40L and CD134/CD134L are increased and may have potential correlation with clinical pathological features of patients with CHD. Further in-depth exploration of costimulatory molecules for CHD guidance as well as intrinsic mechanisms are needed combined with in vivo and in vitro experiments.
Subject(s)
CD40 Antigens/biosynthesis , CD40 Ligand/biosynthesis , Coronary Disease/physiopathology , OX40 Ligand/biosynthesis , Receptors, OX40/biosynthesis , Adult , Aged , Case-Control Studies , Female , Flow Cytometry , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Male , Middle Aged , RNA, Messenger , Real-Time Polymerase Chain Reaction , fas Receptor/biosynthesisABSTRACT
BACKGROUND: An increasing amount of evidence shows that the OX40-OX40L interaction serves an important function in atherosclerosis. However, the mechanism of the OX40 signaling pathway remains unclear. This study investigates the effect of OX40-OX40L interaction on the levels of intracellular reactive oxygen species (ROS) and the secretion of Cyclophilin A (CyPA) in C57BL/6J mice atherogenesis. METHODS: The atherosclerotic plaque model was established by placing a rapid perivascular carotid collar on C57BL/6J mice fed with a western-type diet. In vivo, the expressions of CyPA in mouse plaque and lymphocytes were detected by immunohistochemical and Western blot analyses, respectively. In vitro, the expression of CyPA protein in cultured lymphocytes of C57BL/6J mice was assessed by using Western blot analysis. The level of ROS was detected through flow cytometry. RESULTS: CyPA expression was significantly increased in the atherosclerotic lesions and lymphocytes from C57BL/6J mice. The ROS levels in OX40(+)-lymphocytes were increased in vitro and in vivo. After stimulating the OX40-OX40L interaction, the ROS and CyPA levels in lymphocytes were obviously increased in vitro, whereas anti-OX40L mAb significantly down-regulated the anti-OX40 mAb-induced ROS generation and inhibited CyPA secretion in lymphocytes. CONCLUSION: The OX40-OX40L interaction up-regulates intracellular levels of ROS in C57BL/6J mice and increases CyPA secretion in lymphocytes. Increased CyPA secretion may serve an important function in atherosclerotic plaque formation.
Subject(s)
Atherosclerosis/metabolism , Cyclophilin A/biosynthesis , OX40 Ligand/biosynthesis , Reactive Oxygen Species/metabolism , Receptors, OX40/biosynthesis , Animals , Atherosclerosis/pathology , Cells, Cultured , Cyclophilin A/metabolism , Lymphocytes/metabolism , Male , Mice , Mice, Inbred C57BL , Protein Binding/physiologyABSTRACT
Elevated levels of the cytokine TL1A is associated with several autoimmune diseases e.g. rheumatoid arthritis and inflammatory bowel disease. However, the exact role of TL1A remains elusive. In this study, we investigated the function of TL1A in a pro-inflammatory setting. We show that TL1A together with IL-12, IL-15 and IL-18 increases expression of the co-stimulatory molecules CD154 (CD40 ligand) and CD134 (OX40) on previously activated CD4+ T cells. This indicates that TL1A functions as a co-stimulatory molecule, decreasing the activation threshold of T-cells. We have previously shown that TL1A co-stimulation strongly induces IL-6 in human healthy leukocytes. Interestingly, the cytokine-activated effector T-cells did not produce IL-6 in response to TL1A, indicating distinct effects of TL1A on different cell populations. We further show that this co-stimulation increases the expression of CD25 (IL-2Rα) and CD11a (α-chain of LFA-1) on CD4 T-cells, likely governing increased IL-2/IL-15 sensitivity and cell-cell contact. Along with this, TL1A co-stimulation caused a specific induction of IL-22 and GM-CSF from the activated T-cells. These results substantially contribute to the explanation of TL1A's role in inflammation. Our results suggest that TL1A should be considered as a target for immunotherapeutic treatment of rheumatoid arthritis and inflammatory bowel disease.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD40 Ligand/biosynthesis , Gene Expression Regulation , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Interleukin-2 Receptor alpha Subunit/biosynthesis , Interleukins/biosynthesis , Lymphocyte Function-Associated Antigen-1/biosynthesis , Receptors, OX40/biosynthesis , Tumor Necrosis Factor Ligand Superfamily Member 15/metabolism , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Arthritis, Rheumatoid/therapy , Humans , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/pathology , Inflammatory Bowel Diseases/therapy , Interleukin-22ABSTRACT
We previously reported the emerging role of OX40-OX40L interaction in inflammation and atherosclerosis. However, the mechanism by which OX40-OX40L interaction contributes to pathogenesis is poorly understood. This study investigated the effects of OX40-OX40L interaction on the nuclear factor of activated T cells c1 (NFATc1) in ApoE(-/-) mice. Atherosclerotic plaque was induced via rapid perivascular carotid collar placement in ApoE(-/-) mice. The expression levels of OX40, OX40L, and NFATc1 in the lymphocytes were measured via real-time polymerase chain reaction and flow cytometry. The presence of NFATc1 in the atherosclerotic plaque was detected via immunohistochemistry, and the level of IL-4 was measured via enzyme-linked immunosorbent assay. The expression level of NFATc1 significantly increased in atherosclerotic lesion and in the leukocytes from the ApoE(-/-) mice. After stimulating OX40-OX40L interaction, the mRNA and protein expression levels of NFATc1 in the lymphocytes significantly increased. Meanwhile, anti-OX40LmAb significantly suppressed the expression of NFATc1 in the leukocytes and substantially elevated the level of IL-4. NFATc1 inhibitor markedly suppressed IL-4 production. This study suggests that OX40-OX40L interaction regulates the expression of NFATc1, which may play a critical role in atherosclerotic plaque formation, and may therefore have implications with pathophysiology of atherosclerosis.
Subject(s)
Apolipoproteins E/genetics , Membrane Glycoproteins/immunology , NFATC Transcription Factors/genetics , Plaque, Atherosclerotic/pathology , Receptors, OX40/immunology , Tumor Necrosis Factors/immunology , Animals , Antibodies, Monoclonal/immunology , Atherosclerosis/genetics , Atherosclerosis/immunology , Atherosclerosis/pathology , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Interleukin-4/biosynthesis , Lymphocyte Activation/immunology , Male , Membrane Glycoproteins/biosynthesis , Mice , Mice, Knockout , NFATC Transcription Factors/antagonists & inhibitors , NFATC Transcription Factors/biosynthesis , OX40 Ligand , Plaque, Atherosclerotic/genetics , Plaque, Atherosclerotic/immunology , RNA, Messenger/biosynthesis , Receptors, OX40/biosynthesis , Tumor Necrosis Factors/biosynthesisABSTRACT
CD4(+) helper T cells orchestrate protective immunity against pathogens, yet can also induce undesired pathologies including allergies, transplant rejection and autoimmunity. Non-depleting CD4-specific antibodies such as clone YTS177.9 were found to promote long-lasting T cell tolerance in animal models. Thus, CD4 blockade could represent a promising therapeutic approach for human autoimmune diseases. However, the mechanisms underlying anti-CD4-induced tolerance are incompletely resolved. Particularly, multiple immune cells express CD4 including Foxp3(+) regulatory T cells (Tregs) and dendritic cells (DCs), both controlling the activation of CD4(+)Foxp3(-) helper T cells. Utilizing mixed leukocyte reactions (MLRs) reflecting physiological interactions between T cells and DCs, we report that anti-CD4 treatment inhibits CD4(+)Foxp3(-) T cell proliferation in an IL-2-independent fashion. Notably, YTS177.9 binding induces a rapid internalization of CD4 on both CD4(+)Foxp3(-) T cells and Foxp3(+) Tregs. However, no expansion or activation of immunosuppressive CD4(+)Foxp3(+) Tregs was observed following anti-CD4 treatment. Additionally, cytokine production, maturation and T cell priming capacity of DCs are not affected by anti-CD4 exposure. In line with these data, the selective ablation of Foxp3(+) Tregs from MLRs by the use of diphtheria toxin (DT)-treated bacterial artificial chromosome (BAC)-transgenic DEREG mice completely fails to abrogate the suppressive activity of multiple anti-CD4 antibodies. Instead, tolerization is associated with the defective expression of various co-stimulatory receptors including OX40 and CD30, suggesting altered signaling through the TCR complex. Consistent with our findings in mice, anti-CD4 treatment renders human CD4(+) T cells tolerant in the absence of Tregs. Thus, our results establish that anti-CD4 antibodies can directly tolerize pathogenic CD4(+)Foxp3(-) helper T cells. This has important implications for the treatment of human inflammatory diseases.
Subject(s)
Antilymphocyte Serum/immunology , CD4 Antigens/immunology , Forkhead Transcription Factors/immunology , Immune Tolerance/immunology , Lymphocyte Depletion , T-Lymphocytes, Regulatory/microbiology , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , Cells, Cultured , Dendritic Cells/immunology , Humans , Interleukin-2/immunology , Ki-1 Antigen/biosynthesis , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, OX40/biosynthesisABSTRACT
Bronchiolitis obliterans syndrome (BOS) is associated with lack of immunosuppression of T cell proinflammatory cytokines and increased T cell granzyme B. Repeated antigen-driven proliferation down-regulates T cell CD28. We hypothesized that down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules (CD134, CD137, CD152 and CD154) on T cells may be associated with BOS. Co-stimulatory molecules, granzyme B, perforin and intracellular cytokines were measured by flow cytometry on T cells from stable lung transplant patients (n = 38), patients with BOS (n = 20) and healthy controls (n = 10). There was a significant increase in the percentage of CD4/28(null) and CD8/28(null) T cells producing granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in BOS compared with stable patients. Down-regulation of CD28 was associated with steroid resistance and up-regulation of CD134, CD137, CD152 and CD154 on CD4(+) T cells and CD137 and CD152 on CD8(+) T cells. There was a significant correlation between increased CD28(null) /CD137 T cells producing IFN-γ, TNF-α with BOS grade (r = 0·861, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8) and time post-transplant (r = 0·698, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8). BOS is associated with down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules on steroid-resistant peripheral blood proinflammatory CD4(+) and CD8(+) T cells. Therapeutic targeting of alternate co-stimulatory molecules on peripheral blood CD28(null) T cells and monitoring response using these assays may help in the management of patients with BOS.
Subject(s)
Bronchiolitis Obliterans/immunology , Costimulatory and Inhibitory T-Cell Receptors/biosynthesis , Postoperative Complications/immunology , T-Lymphocyte Subsets/metabolism , Adult , Bronchiolitis Obliterans/etiology , CD28 Antigens/analysis , CD40 Ligand/biosynthesis , CD40 Ligand/genetics , CTLA-4 Antigen/biosynthesis , CTLA-4 Antigen/genetics , Case-Control Studies , Costimulatory and Inhibitory T-Cell Receptors/genetics , Cyclosporine/therapeutic use , Female , Granzymes/analysis , Humans , Immunosuppressive Agents/therapeutic use , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Lung Transplantation , Lymphocyte Activation , Male , Middle Aged , Perforin/analysis , Postoperative Complications/etiology , Receptors, OX40/biosynthesis , Receptors, OX40/genetics , T-Lymphocyte Subsets/immunology , Tacrolimus/therapeutic use , Tumor Necrosis Factor Receptor Superfamily, Member 9/biosynthesis , Tumor Necrosis Factor Receptor Superfamily, Member 9/genetics , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Up-RegulationABSTRACT
OBJECTIVE: To examine whether both OX40 and its ligand OX40L are expressed in idiopathic inflammatory myopathies and to investigate the types of inflammatory cells expressing OX40L. STUDY DESIGN: Immunohistochemistry was performed in limb muscle specimens from dermatomyositis, polymyositis and inclusion body myositis patients to analyze the expression of OX40 and its ligand OX40L. Double immunofluorescence labeling was performed to clarify the phenotype of inflammatory cells expressing OX40L. RESULTS: OX40 and OX40L expressing cells were observed in all subsets of inflammatory myopathies following a similar pattern of distribution mainly in the perimysium. In polymyositis and inclusion body myositis inflammatory cells expressing the receptors invaded non-necrotic muscle fibers. OX40L expression was also found in endothelial blood cells in all dermatomyositis and some polymyositis specimens. In all subsets of inflammatory myopathies OX40L was expressed by T cells (CD4+ and CD8+), macrophages (CD68+), B cells (CD20+) and myeloid dendritic cells (BDCA1+). Plasmacytoid dendritic cells (BDCA2+) expressing OX40L were found only in dermatomyositis and polymyositis. CONCLUSION: The simultaneous expression of both OX40 and its ligand OX40L in idiopathic inflammatory myopathies suggests that they might participate in disease pathogenesis. Expression of OX40L by different types of cells within the inflamed muscle implies that OX40-OX40L interaction may contribute in disease mechanisms through different pathways.
Subject(s)
Myositis/metabolism , OX40 Ligand/biosynthesis , Receptors, OX40/biosynthesis , Adolescent , Adult , Aged , Child , Child, Preschool , Female , Humans , Immunohistochemistry , Male , Middle Aged , Myositis/immunology , Myositis/pathology , OX40 Ligand/immunology , Receptors, OX40/immunology , Young AdultABSTRACT
Hepatitis C virus (HCV)-specific CD4(+) effector T cell responses are likely to play a key role in the immunopathogenesis of HCV infection by promoting viral clearance and maintaining control of viraemia. As the precursor frequency of HCV-specific CD4(+) T cells in peripheral blood is low, favoured assay systems such as intracellular cytokine (ICC) or tetramer staining have limited utility for ex vivo analyses. Accordingly, the traditional lymphocyte proliferation assay (LPA) remains the gold standard, despite detecting responses in only a minority of infected subjects. Recently, we reported development and validation of a novel whole blood CD4(+) effector T cell assay based on ex vivo antigen stimulation followed by co-expression of CD25 and CD134 on CD4(+) T cells. Here we report adaptation of this assay to assessment of HCV-specific responses in cryopreserved peripheral blood mononuclear cells using standardised antigens, including peptide pools, viral supernatants and recombinant viral proteins. The assay allowed detection of HCV-specific CD4 responses in donors with both resolved and chronic infection. Responses were highly correlated with those revealed by LPA. Application of this assay will further define the role of CD4(+) T cells in the immunopathogenesis of HCV infection.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Hepacivirus/immunology , Hepatitis C Antigens/immunology , Hepatitis C/immunology , Interleukin-2 Receptor alpha Subunit/immunology , Receptors, OX40/immunology , Adult , CD4-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Cells, Cultured , Cryopreservation , Cytokines/immunology , Cytokines/metabolism , DNA-Directed RNA Polymerases/immunology , DNA-Directed RNA Polymerases/metabolism , Female , Hepacivirus/metabolism , Hepatitis C/metabolism , Hepatitis C/virology , Humans , Interleukin-2 Receptor alpha Subunit/biosynthesis , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/immunology , Male , Receptors, OX40/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Viral Nonstructural Proteins/immunology , Viral Nonstructural Proteins/metabolismABSTRACT
CARMA1 is a lymphocyte-specific scaffold protein necessary for T cell activation. Deletion of CARMA1 prevents the development of allergic airway inflammation in a mouse model of asthma due to a defect in naive T cell activation. However, it is unknown if CARMA1 is important for effector and memory T cell responses after the initial establishment of inflammation, findings that would be more relevant to asthma therapies targeted to CARMA1. In the current study, we sought to elucidate the role of CARMA1 in T cells that have been previously activated. Using mice in which floxed CARMA1 exons can be selectively deleted in T cells by OX40-driven Cre recombinase (OX40(+/Cre)CARMA1(F/F)), we report that CD4(+) T cells from these mice have impaired T cell reactivation responses and NF-κB signaling in vitro. Furthermore, in an in vivo recall model of allergic airway inflammation that is dependent on memory T cell function, OX40(+/Cre)CARMA1(F/F) mice have attenuated eosinophilic airway inflammation, T cell activation, and Th2 cytokine production. Using MHC class II tetramers, we demonstrate that the development and maintenance of Ag-specific memory T cells is not affected in OX40(+/Cre)CARMA1(F/F) mice. In addition, adoptive transfer of Th2-polarized OX40(+/Cre)CARMA1(F/F) Ag-specific CD4(+) T cells into wild-type mice induces markedly less airway inflammation in response to Ag challenge than transfer of wild-type Th2 cells. These data demonstrate a novel role for CARMA1 in effector and memory T cell responses and suggest that therapeutic strategies targeting CARMA1 could help treat chronic inflammatory disorders such as asthma.
Subject(s)
Asthma/immunology , CARD Signaling Adaptor Proteins/physiology , Th2 Cells/immunology , Acute Disease , Adoptive Transfer , Animals , Asthma/genetics , Asthma/pathology , CARD Signaling Adaptor Proteins/deficiency , CARD Signaling Adaptor Proteins/genetics , Cell Polarity/genetics , Cell Polarity/immunology , Cells, Cultured , Chronic Disease , Disease Models, Animal , Immunologic Memory/genetics , Immunologic Memory/immunology , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/physiology , Integrases/genetics , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/physiology , Receptors, OX40/biosynthesis , Receptors, OX40/genetics , Respiratory Hypersensitivity/genetics , Respiratory Hypersensitivity/immunology , Respiratory Hypersensitivity/prevention & control , Th2 Cells/cytology , Th2 Cells/transplantationABSTRACT
Memory T cells are known to play a key role in prevention of allograft tolerance in alloantigen-primed mice. Here, we used an adoptively transferred memory T cell model and an alloantigen-primed model to evaluate the abilities of different combinations of monoclonal antibodies (mAb) to block key signaling pathways involved in activation of effector and memory T cells. In the adoptively transferred model, the use of anti-CD134L mAb effectively prevented activation of CD4(+) memory T cells and significantly prolonged islet survival, similar to the action of anti-CD122 mAb to CD8(+) memory T cells. In the alloantigen-primed model, use of anti-CD134L and anti-CD122 mAbs in addition to co-stimulatory blockade with anti-CD154 and anti-LFA-1 prolonged secondary allograft survival and significantly reduced the proportion of memory T cells; meanwhile, this combination therapy increased the proportion of regulatory T cells (Tregs) in the spleen, inhibited lymphocyte infiltration in the graft, and suppressed alloresponse of recipient splenic T cells. However, we also detected high levels of alloantibodies in the serum which caused high levels of damage to the allogeneic spleen cells. Our results suggest that combination of four mAbs can significantly suppress the function of memory T cells and prolong allograft survival in alloantigen primed animals.
Subject(s)
Immunologic Memory , Isoantigens/chemistry , Lymphocyte Activation , Animals , Diabetes Complications/immunology , Female , Interleukin-2 Receptor beta Subunit/biosynthesis , Islets of Langerhans/cytology , Isoantibodies/chemistry , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Receptors, OX40/biosynthesis , Time Factors , Transplantation Tolerance/immunology , Transplantation, Homologous/methodsABSTRACT
Ag-specific human CD4(+) memory T lymphocytes have mostly been studied using assays of proliferation in vitro. Intracellular cytokine and ELISPOT assays quantify effector cell populations but barely detect responses to certain recall Ags that elicit strong proliferative responses, e.g., tetanus toxoid, that comprise non-Th1 CD4(+) cells. We have found that culturing whole blood with Ag for 40-48 h induces specific CD4(+) T cells to simultaneously express CD25 and CD134. This new technique readily detects responses to well-described CD4(+) T cell recall Ags, including preparations of mycobacteria, CMV, HSV-1, influenza, tetanus toxoid, Candida albicans, and streptokinase, as well as HIV-1 peptides, with high specificity. The assay detects much higher levels of Ag-specific cells than intracellular cytokine assays, plus the cells retain viability and can be sorted for in vitro expansion. Furthermore, current in vitro assays for human CD4(+) memory T lymphocytes are too labor-intensive and difficult to standardize for routine diagnostic laboratories, whereas the whole-blood CD25(+)CD134(+) assay combines simplicity of setup with a straightforward cell surface flow cytometry readout. In addition to revealing the true extent of Ag-specific human CD4(+) memory T lymphocytes, its greatest use will be as a simple in vitro monitor of CD4(+) T cell responses to Ags such as tuberculosis infection or vaccines.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/immunology , Interleukin-2 Receptor alpha Subunit/blood , Lymphocyte Activation/immunology , Receptors, OX40/blood , Adult , Amino Acid Sequence , Animals , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , Chronic Disease , Epitopes, T-Lymphocyte/blood , Fluoresceins , HIV Infections/immunology , HIV Infections/pathology , Humans , Interleukin-2 Receptor alpha Subunit/biosynthesis , Longitudinal Studies , Macaca nemestrina , Molecular Sequence Data , Receptors, OX40/biosynthesis , Succinimides , Thymidine , TritiumABSTRACT
CD4+ T cell dysfunction in subjects with chronic HIV infection is in part due to an imbalance of costimulatory and coinhibitory receptors. We report that virus-specific CD4+ T cells expressing 4-1BB (CD137) or OX40 (CD134) produced more IL-2 than cells lacking these costimulatory receptors (P<0.05) and that 4-1BB was expressed at a lower level on HIV- than CMV-specific IFN-gamma and IL-2 producing CD4+ T cells (P<0.0001 and P<0.01, respectively). Suppression of viral replication with antiretroviral therapy was associated with increased 4-1BB expression on HIV- and CMV-specific IL-2 producing CD4+ T cells (P<0.05 and P<0.01, respectively) and the percentage of IL-2 producing HIV-specific CD4+ T cells that expressed 4-1BB was inversely correlated with HIV plasma viral load (r=-0.75, P=0.007). These findings indicate that the loss of 4-1BB on HIV-specific CD4+ T cells is associated with viral replication and that it may contribute to reduced IL-2 production observed during chronic infection.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , HIV Infections/metabolism , Interleukin-2/biosynthesis , Receptors, OX40/biosynthesis , Tumor Necrosis Factor Receptor Superfamily, Member 9/biosynthesis , Virus Replication , 4-1BB Ligand/biosynthesis , Analysis of Variance , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dendritic Cells/virology , Flow Cytometry , Fluorescent Antibody Technique , HIV Infections/blood , HIV Infections/immunology , Humans , Interferon-gamma/biosynthesis , Monocytes/immunology , Monocytes/metabolism , Monocytes/virology , Myeloid Cells/immunology , Myeloid Cells/metabolism , Myeloid Cells/virology , OX40 Ligand/biosynthesisABSTRACT
Disrupting the CD40-CD40L costimulation pathway promotes allograft acceptance in many settings. Herein, we demonstrate that stimulating OX40 overrides cardiac allograft acceptance induced by disrupting CD40-CD40L interactions. This effect of OX40 stimulation was dependent on CD4(+) T cells, which in turn provided help for CD8(+) T cells and B cells. Allograft rejection was associated with donor-reactive Th1 and Th2 responses and an unconventional granulocytic infiltrate and thrombosis of the arteries. Interestingly, OX40 stimulation induced a donor-reactive IgG class switch in the absence of CD40-CD40L interactions, and the timing of OX40 stimulation relative to transplantation affected the isotype of donor-reactive Ab produced. Inductive OX40 stimulation induced acute graft rejection, which correlated with both IgG1 and IgG2a deposition within the graft. Once graft acceptance was established following CD40-CD40L blockade, delayed OX40 stimulation did not induce acute allograft rejection despite priming of graft-reactive Th1 and Th2. Rather, chronic rejection was induced, which was characterized by IgG1 but not IgG2a deposition within the graft. These studies reveal both redundancy and key differences in function among costimulatory molecules that manifest in distinct pathologies of allograft rejection. These findings may help guide development of therapeutics aimed at promoting graft acceptance in transplant recipients.
Subject(s)
CD40 Antigens/antagonists & inhibitors , CD40 Ligand/antagonists & inhibitors , Graft Rejection/immunology , Graft Survival/immunology , Heart Transplantation/immunology , Receptors, OX40/biosynthesis , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacology , CD40 Antigens/deficiency , CD40 Antigens/genetics , CD40 Ligand/deficiency , CD40 Ligand/genetics , Cell Movement/genetics , Cell Movement/immunology , Cells, Cultured , Coronary Thrombosis/immunology , Coronary Thrombosis/pathology , Female , Graft Rejection/metabolism , Graft Rejection/pathology , Graft Survival/genetics , Granulocytes/immunology , Granulocytes/pathology , Heart Transplantation/pathology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Receptors, OX40/genetics , Receptors, OX40/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism , Transplantation, Heterotopic , Up-Regulation/genetics , Up-Regulation/immunologySubject(s)
Bone Marrow Transplantation/methods , Graft vs Host Disease/therapy , Granulomatous Disease, Chronic/therapy , Liver Transplantation/methods , Liver/pathology , Living Donors , Child , Graft Survival , Heterozygote , Humans , Liver/immunology , Male , Receptors, OX40/biosynthesis , Time Factors , Treatment OutcomeABSTRACT
Feline immunodeficiency virus (FIV) causes progressive immunodeficiency in domestic cats, with clinical course dependent on virus strain. For example, clade A FIV-PPR is predominantly neurotropic and causes a mild disease in the periphery, whereas clade C FIV-C36 causes fulminant disease with CD4(+) T-cell depletion and neutropenia but no significant pathology in the central nervous system. In order to map pathogenic determinants, chimeric viruses were prepared between FIV-C36 and FIV-PPR, with reciprocal exchanges involving (i) the 3' halves of the viruses, including the Vif, OrfA, and Env genes; (ii) the 5' end extending from the 5' long terminal repeat (LTR) to the beginning of the capsid (CA)-coding region; and (iii) the 3' LTR and Rev2-coding regions. Ex vivo replication rates and in vivo replication and pathologies were then assessed and compared to those of the parental viruses. The results show that FIV-C36 replicates ex vivo and in vivo to levels approximately 20-fold greater than those of FIV-PPR. None of the chimeric FIVs recapitulated the replication rate of FIV-C36, although most replicated to levels similar to those of FIV-PPR. The rates of chloramphenicol acetyltransferase gene transcription driven by the FIV-C36 and FIV-PPR LTRs were identical. Furthermore, the ratios of surface glycoprotein (SU) to capsid protein (CA) in the released particles were essentially the same in the wild-type and chimeric FIVs. Tests were performed in vivo on the wild-type FIVs and chimeras carrying the 3' half of FIV-C36 or the 3' LTR and Rev2 regions of FIV-C36 on the PPR background. Both chimeras were infectious in vivo, although replication levels were lower than for the parental viruses. The chimera carrying the 3' half of FIV-C36 demonstrated an intermediate disease course with a delayed peak viral load but ultimately resulted in significant reductions in neutrophil and CD4(+) T cells, suggesting potential adaptation in vivo. Taken together, the findings suggest that the rapid-growth phenotype and pathogenicity of FIV-C36 are the result of evolutionary fine tuning throughout the viral genome, rather than being properties of any one constituent.
Subject(s)
Feline Acquired Immunodeficiency Syndrome/virology , Immunodeficiency Virus, Feline/chemistry , Animals , Capsid/metabolism , Cats , Chloramphenicol O-Acetyltransferase/metabolism , Genome, Viral , Immunodeficiency Virus, Feline/metabolism , Infections , Kidney/metabolism , Kinetics , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/virology , Phenotype , Protein Binding , Receptors, OX40/biosynthesis , Terminal Repeat SequencesABSTRACT
Feline immunodeficiency virus (FIV) causes a natural infection of domestic cats that resembles HIV-1 in pathogenesis and disease progression. Feline AIDS is characterized by depression of the CD4+ T cell population and fatal opportunistic infections. Maternal-fetal transmission of FIV readily occurs under experimental conditions, resulting in infected viable kittens and resorbed or arrested fetal tissues. Although both FIV and HIV use the chemokine receptor CXCR4 as a co-receptor, FIV does not utilize CD4 as the primary receptor. Rather, CD134 (OX40), a T cell activation antigen and co-stimulatory molecule, is the primary receptor for FIV. We hypothesized that placental expression of CD134 and CXCR4 may render the placenta vulnerable to FIV infection, possibly facilitating efficient vertical transmission of FIV, and impact pregnancy outcome. The purpose of this project was to quantify the relative expression of CD134 and CXCR4 mRNA from the term placentas of three groups of cats: uninfected queens producing viable offspring, experimentally-infected queens producing only viable offspring, and experimentally-infected queens producing viable offspring among mostly non-viable fetuses. Total RNA was extracted from term placental tissues from all groups of cats. Real-time one-step reverse transcriptase-PCR was used to measure gene expression. The FIV receptors CD134 and CXCR4 were expressed in all late term feline placental tissues. Placentas from FIV-infected queens producing litters of only viable offspring expressed more CD134 and CXCR4 mRNA than those from uninfected queens, suggesting that infection may cause upregulation of the receptors. On the other hand, placentas from FIV-infected cats with non-successful pregnancies expressed similar levels of CD134 mRNA and slightly less CXCR4 mRNA than those from uninfected queens. Thus, it appears that cells expressing these receptors may play a role in pregnancy maintenance.
Subject(s)
Feline Acquired Immunodeficiency Syndrome/metabolism , Immunodeficiency Virus, Feline/immunology , Placenta/immunology , Pregnancy Complications, Infectious/veterinary , Receptors, CXCR4/biosynthesis , Receptors, OX40/biosynthesis , Animals , Animals, Newborn , Antibodies, Viral/blood , Cats , Enzyme-Linked Immunosorbent Assay/veterinary , Feline Acquired Immunodeficiency Syndrome/genetics , Feline Acquired Immunodeficiency Syndrome/transmission , Feline Acquired Immunodeficiency Syndrome/virology , Female , Immunodeficiency Virus, Feline/genetics , Infectious Disease Transmission, Vertical , Litter Size , Male , Placenta/virology , Pregnancy , Pregnancy Complications, Infectious/immunology , Pregnancy Complications, Infectious/virology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, CXCR4/genetics , Receptors, OX40/genetics , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Specific Pathogen-Free OrganismsABSTRACT
Major problems in the analysis of CD4+ effector cell and regulatory T cell (Treg) populations in an activated immune system are caused by the facts that both cell types can express CD25 and that the discriminatory marker forkhead box p3 can only be analyzed in nonviable (permeabilized) cells. Here, we show that CD134 (OX40) can be used as a discriminatory marker combined with CD25 to isolate and characterize viable CD4+ effector cells and Tregs. Before and during adjuvant arthritis in rats, coexpression of CD134 and CD25 identified activated Tregs consistently, as these T cells proliferated poorly to disease-associated antigens and were suppressive in vitro and in vivo. Depending on the time of isolation and location, CD4+ T cell populations expressing CD134 or CD25 contained effector/memory T cells. Analysis of the function, phenotype, and amount of the CD4+ T cell subsets in different lymph node stations revealed spatiotemporal differences in effector cell and Treg compartments during experimental arthritis.
Subject(s)
Arthritis, Experimental/immunology , Interleukin-2 Receptor alpha Subunit/biosynthesis , Receptors, OX40/biosynthesis , T-Lymphocytes, Regulatory/immunology , Animals , Disease Models, Animal , Disease Progression , Forkhead Transcription Factors/immunology , Interleukin-2 Receptor alpha Subunit/immunology , Male , Rats , Rats, Inbred Lew , Receptors, OX40/immunologyABSTRACT
Perforin is a membrane-disrupting protein that allows the entry of granzymes into a target cell inducing degradation of target substances in the cytoplasm and nucleus thus leading to programmed cell death or apoptosis. CD134 was originally described as an activation antigen found on activated T cells. In this work Flowcytometry was used to evaluate the expression of perforin and CD134, as a costimulatory molecule on T cells, in patients with Systemic Lupus Erythematosus (SLE) to elucidate their role in the pathogenesis of SLE and disease severity. The study was conducted on 15 patients with SLE, 6 patients out of the 15 patients were suffering from lupus nephritis, 10 healthy subjects were included as controls. The results revealed that absolute number of circulating CD3+ lymphocytes in the patients was significantly lower than the controls (P = 0.013). The percentage of CD8+ CD3+ T cells was significantly increased in the SLE group when compared to that of CD4+ CD3+ T cells in same group (P = 0.001) Perforin expression on both CD4+ and CD8+ cells was significantly increased in patients compared to controls. (P = 0.002 & P = 0.001, respectively). In addition, a significant increase was observed in the percent of pf+CD8+CD3+ in the patient group compared to that of pf+CD4+CD3+ in the same group (P = 0.001). There was a significant increase in the expression of CD134 on CD4+ and CD8+ cells (P = 0.001 & P = 0.001 respectively). Also, in the same group of patients a significant increase was detected in the frequency of CD134+CD4+CD3+ T cells compared to that of CD134+CD8+CD3+ T cells (P = 0.032). A significant positive correlation was detected in the patient group between CD134 and perforin expression on both CD4+ and CD8+ T cells (p = 0.045, r = 0.523). Moreover, CD134+CD4+CD3+ was also correlated positively with urinary proteinuria (P = 0.023, r = 0.524). Our data suggest the role of Perforin + cytotoxic T lymphocytes and CD134+ cells in the pathogenesis of autoimmunity of SLE. Thus, inhibition of perforin could be beneficial for SLE patients. Targeting pf and CD134 could be a new therapeutic approach in patients with SLE.
Subject(s)
Flow Cytometry/methods , Lupus Erythematosus, Systemic/metabolism , Perforin/biosynthesis , Receptors, OX40/biosynthesis , Adolescent , Adult , CD3 Complex/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Female , Humans , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology , Male , Proteinuria/immunology , Proteinuria/metabolism , Proteinuria/pathology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Young AdultABSTRACT
Naive and central memory CD8 T cells use CCR7 to recirculate through T cell zones of secondary lymphoid organs where they can encounter antigen. Here we describe a subset of human CD8 T cells expressing CXCR5 which enables homing in response to CXCL13 produced within B cell follicles. CXCR5+ CD8 T cells were found in tonsil B cell follicles, and isolated cells migrated towards CXCL13 in vitro. They expressed CD27, CD28, CD45RO, CD69, and were CD7low, and produced IFN-gamma and granzyme A but lacked perforin, a functional profile suggesting that these cells are early effector memory cells in the context of contemporary T cell differentiation models. Receptors important in the interaction with B cells, including CD70, OX40 and ICOS, were induced upon activation, and CXCR5+ CD8 T cells could to some extent support survival and IgG production in tonsil B cells. Furthermore, CXCR5+ CD8 T cells expressed CCR5 but no CCR7, suggesting a migration pattern distinct from that of follicular CD4 T cells. The finding that a subset of early effector memory CD8 T cells use CXCR5 to locate to B cell follicles indicates that MHC class I-restricted CD8 T cells are part of the follicular T cell population.