Expression of tumour necrosis factor (TNF) ligand superfamily co-stimulatory molecules CD30L, CD27L, OX40L, and 4-1BBL in murine hearts with acute myocarditis caused by Coxsackievirus B3.

Antigen-specific T-cells infiltrate the heart and play an important role in the myocardial damage involved in acute myocarditis and dilated cardiomyopathy. To investigate the roles of the co-stimulatory molecules CD30/CD30L, CD27/CD27L, OX40/OX40L, and 4-1BB/4-1BBL, which belong to the tumor necrosis factor (TNF) receptor/ligand superfamily, in the development of acute viral myocarditis, the expression of these molecules was first analysed in the hearts of mice with acute myocarditis induced by Coxsackievirus B3 (CVB3) in vivo. Secondly, the induction of these molecules was evaluated on cultured cardiac myocytes treated with interferon (IFN)-gamma and the interleukin (IL)-6 production by cultured cardiac myocytes was analysed by stimulation with monoclonal antibodies (MAbs) against these molecules in vitro. Thirdly, the effects of in vivo administration of anti-CD30L, anti-CD27L, anti-OX40L, or anti-4-1BBL MAb on the development of acute viral myocarditis were examined. CVB3-induced myocarditis resulted in the induction of CD30L and 4-1BBL on the surface of cardiac myocytes, confirmed by treatment with IFN-gamma in vitro. CD27L and OX40L were constitutively expressed on cardiac myocytes in vivo and in vitro. Anti-CD30L and anti-4-1BBL MAbs stimulated IL-6 production by cardiac myocytes in vitro. Furthermore, in vivo anti-4-1BBL MAb treatment significantly decreased the myocardial inflammation, whereas the other MAbs did not. These findings suggest that TNF ligand superfamily co-stimulatory molecules, especially 4-1BBL, play an important role in the development of acute viral myocarditis and raise the possibility that immunotherapy with anti-4-1BBL MAb may be of benefit in acute viral myocarditis.     

Expression of tumor necrosis factor ligand superfamily costimulatory molecules CD27L, CD30L, OX40L and 4-1BBL in the heart of patients with acute myocarditis and dilated cardiomyopathy.

BACKGROUND: T-cell-mediated myocardial damage is known to be involved in acute myocarditis and dilated cardiomyopathy. Recently, we found that tumor necrosis factor (TNF) ligand superfamily costimulatory molecules, especially 4-1BBL, played an important role in the myocardial damage of murine acute viral myocarditis. METHODS AND RESULTS: To investigate the roles for CD27L, CD30L, OX40L and 4-1BBL, which belong to TNF ligand superfamily, in the development of acute myocarditis and dilated cardiomyopathy, we analyzed the expression of these antigens in the myocardial tissues of patients with acute myocarditis and dilated cardiomyopathy. We also examined expression of the receptors for these molecules, CD27, CD30, OX40 and 4-1BB, which belong to TNF receptor superfamily, on the infiltrating cells. Strong expression of CD27L, CD30L and 4-1BBL and weak to moderate expression of OX40L was found in the cardiac myocytes of patients with acute myocarditis. Moderate expression of CD27L, CD30L and 4-1BBL and weak expression of OX40L was found on the cardiac myocytes of patients with dilated cardiomyopathy. Most of the infiltrating cells expressed CD27, CD30 and 4-1BB and a part of the infiltrating cells expressed OX40. CONCLUSIONS: Our findings suggest that expression of TNF ligand superfamily costimulatory molecules on cardiac myocytes may play a role in the cell-mediated myocardial damage in patients with acute myocarditis and dilated cardiomyopathy as in murine viral myocarditis.     

Effects of in vivo administration of anti-B7-1/B7-2 monoclonal antibodies on the survival of mice with chronic ongoing myocarditis caused by Coxsackievirus B3

In acute myocarditis and dilated cardiomyopathy, it has previously been reported that antigen-specific T-cells infiltrate the heart and play an important role in the myocardial damage involved. For antigen-specific T-cell activation to occur, it is necessary for the T-cell to receive co-stimulatory signals provided by co-stimulatory molecules expressed on the antigen-presenting cell (APC), as well as the main signal provided by binding of the T-cell receptor (TCR) to the antigen. To investigate the roles for the co-stimulatory molecules B7-1 and B7-2 in the development of chronic ongoing viral myocarditis, firstly the expression of B7-1/B7-2 was analysed in the hearts of A/J mice with myocarditis induced by Coxsackievirus B3 (CVB3). Secondly the induction of B7-1/B7-2 on cultured cardiac myocytes treated with interferon (IFN)-γ was evaluated. Thirdly the effects of the in vivo administration of anti-B7-1/B7-2 monoclonal antibodies (MAbs) on the survival of mice with viral myocarditis were examined. CVB3-induced myocarditis resulted in enhanced expression of B7-1/B7-2 on cardiac myocytes. The expression of B7-1/B7-2 on cardiac myocytes could be induced by IFN-γ in vitro. In vivo anti-B7-1 MAb treatment significantly prolonged the survival of mice with myocarditis, whereas anti-B7-2 MAb treatment abrogated the protective effect of anti-B7-1. These findings indicate that distinct roles for B7-1 and B7-2 antigens are involved in the development of viral myocarditis and raise the possibility of immunotherapy with anti-B7-1 MAb to prevent T-cell-mediated cardiac myocyte injury and to improve the prognosis of viral myocarditis. Copyright © 1999 John Wiley & Sons, Ltd.     

Requirements for the functional expression of OX40 ligand on human activated CD4+ and CD8+ T cells

Interaction between OX40 expressed on activated T cells and its ligand (OX40L) on antigen presenting cells (APC) provides a co-stimulatory signal for T cells to promote acquired immunity. In the present study, we have examined various culture conditions for optimum OX40L expression on T cells stimulated with immobilized anti-CD3/CD28 monoclonal antibodies (mAbs). Although the day 3 primed T cells expressed minimal OX40L, after repeated stimulations both the CD4+ and CD8+ T cells became OX40L positive as determined by flow cytometry. Interleukin (IL)-12 interfered with the OX40L expression. Among activated T cells, a higher frequency of CD8+ T cells expressed OX40L than CD4+ T cells. By blocking OX40L-OX40 interaction by an anti-OX40 mAb, the number of OX40L+ T cells significantly increased. Screening of various cytokines showed that transforming growth factor (TGF)-beta1 was capable of induction of OX40L on the activated T cells within 3 days. The OX40L expressed on T cells was functional, as they bound soluble OX40 and stimulated human immunodeficiency virus-1 (HIV-1) production from cell lines chronically infected with HIV-1 and expressing OX40. Altogether the present study findings indicate that functional OX40L is inducible on human activated CD4+ and CD8+ T cells, and that the expression is enhanced by TGF-beta1.     

Nasal cardiac myosin peptide treatment and OX40 blockade protect mice from acute and chronic virally-induced myocarditis

Myocarditis poses a severe health problem, can lead to dilated cardiomyopathy (DCM) and death, and is thought to be triggered by infections. Enteroviruses such as Coxsackie virus B 3 (CVB3) have been implicated as a culprit, since they can cause acute and chronic heart disease in susceptible mice. CVB was detected in human cardiac myocytes in some cases, whereas acute CVB infection was thought to have caused death. Here we studied, whether nasal administration of cardiac myosin (CM) major histocompatibility class (MHC) II peptides CM_947–960 and CM_735–747 and OX40 blockade would be able to ameliorate immunopathology and heart disease in BALB/C mice infected with CVB3. We found that nasal CM-peptide prophylactic treatment significantly reduced myocarditis and mortality by enhancing Treg and IL-10 induction and that blockade of OX40 signaling could reduce heart inflammation when administered late during pathogenesis. Altogether, these results chart the way for novel prevention and intervention strategies for viral myocarditis.     

大鼠心脏移植排斥反应过程中外周血CD28、CD40通路相关分子的变化

目的探讨CD28、CD40共刺激通路与排斥反应的关系,同时也为排斥反应的诊断寻找一种新的检测指标。方法采用大鼠异位心脏移植模型,用免疫组化方法动态检测外周血单核细胞（PBMC）CD28、CTLA4、CD40及CD40L分子的表达。结果在0～4级排斥反应中,外周血细胞CD28分子阳性表达率在各组间的差异无统计学意义。外周血细胞表达CTLA4分子的阳性率随排斥反应增强而升高（P〈0.01）。CD40及CD40L在PBMC中的表达强度也随排斥反应的分级逐渐增强（P〈0.01）。结论外周血CTLA4、CD40及CD40L分子的表达与排斥反应有密切关系,动态检测这些分子有助于对排斥反应状态的评价。     

Expression of OX40 (CD134) on CD4+ T-cells from patients with myasthenia gravis

Myasthenia gravis (MG) is commonly regarded as the prototype of an antibody-mediated, organ-specific autoimmune disease. Antibodies against the acetylcholine receptor (AChR) on the muscle endplate trigger its typical clinical manifestations of weakness and fatiguability. T-B cell interactions are thought to play a crucial role in the pathogenesis of MG. OX40 (CD134), a costimulatory molecule that is expressed on activated CD4+ T-cells, might contribute to the development or pathogenesis of immune-mediated diseases such as rheumatoid arthritis and graft-versus-host disease. In the present study, we investigated the expression of OX40 on CD4+ T-cells from patients with MG and healthy individuals. Results from 36 MG patients and 28 healthy controls revealed that more freshly isolated CD4+ T-cells from MG patients expressed OX40 than cells from healthy individuals. High levels of antibodies against the AChR, thymic hyperplasia and onset at an early age were associated with elevated expression of OX40. Upon activation by various concentrations of anti-CD3 antibodies, CD4+ T-cells from MG patients showed a tendency toward higher levels of OX40 expression than cells from healthy individuals. Given the role of OX40 in the immune system, we conclude that OX40 might contribute to the development of MG.     

Critical contribution of CD80 and CD86 to induction of anterior chamber-associated immune deviation

Intraocular inoculation of antigens induces anterior chamber-associated immune deviation (ACAID), which is mediated by development of regulatory T cells in response to antigen-presenting cells (APC) pre-conditioned by intraocular transforming growth factor-beta (TGF-beta). In this study, we examined the involvement of T-cell co-stimulatory molecules in this process. To mimic the intraocular APC, thioglycollate-elicited peritoneal exudate cells (PEC) were pre-treated with TGF-beta in vitro. Expression of CD80, CD86, OX40 ligand (OX40L) and CD70 was analyzed by flow cytometry. Contribution of these molecules to co-stimulatory activity of TGF-beta-treated PEC on antigen-stimulated T-cell proliferation and cytokine production was determined by inhibition with blocking antibodies in vitro. Contribution of CD80 and CD86 to induction of ACAID was determined by the administration of blocking antibodies at intraocular antigen inoculation in vivo. TGF-beta-treated PEC expressed CD80 and CD86 but not OX40L or CD70. Antigen-stimulated T cells proliferated and produced IL-10, but not IFN-gamma, in response to co-stimulation by TGF-beta-treated PEC, which was abrogated by blocking antibodies against CD80 and CD86. Induction of regulatory cells mediating ACAID was abolished by in vivo blockade of CD80 and CD86. The present results indicated that CD80 and CD86 play a critical role in induction of ACAID, possibly by co-stimulating expansion and IL-10 production of regulatory T cells in response to TGF-beta-conditioned APC.     

The significance of OX40 and OX40L to T-cell biology and immune disease

Summary:  OX40 (CD134) and its binding partner, OX40L (CD252), are members of the tumor necrosis factor receptor/tumor necrosis factor superfamily and are expressed on activated CD4⁺ and CD8⁺ T cells as well as on a number of other lymphoid and non-lymphoid cells. Costimulatory signals from OX40 to a conventional T cell promote division and survival, augmenting the clonal expansion of effector and memory populations as they are being generated to antigen. OX40 additionally suppresses the differentiation and activity of T-regulatory cells, further amplifying this process. OX40 and OX40L also regulate cytokine production from T cells, antigen-presenting cells, natural killer cells, and natural killer T cells, and modulate cytokine receptor signaling. In line with these important modulatory functions, OX40–OX40L interactions have been found to play a central role in the development of multiple inflammatory and autoimmune diseases, making them attractive candidates for intervention in the clinic. Conversely, stimulating OX40 has shown it to be a candidate for therapeutic immunization strategies for cancer and infectious disease. This review provides a broad overview of the biology of OX40 including the intracellular signals from OX40 that impact many aspects of immune function and have promoted OX40 as one of the most prominent costimulatory molecules known to control T cells.     

Ag85B调节小鼠髓样树突状细胞的成熟和TSLP介导下TSLPR和OX40L的表达

目的:研究抗原85B(Ag85B)体外诱导小鼠未成熟的髓样树突状细胞(m DCs)的成熟以及对胸腺基质淋巴细胞生成素(TSLP)介导下m DCs表达TSLP受体(TSLPR)和OX40L的影响,探究Ag85B抑制哮喘气道炎症的可能机制。方法:应用重组小鼠GM-CSF和IL-4体外诱生C57BL/6小鼠未成熟的m DCs,并运用免疫磁珠分离的方法纯化,采用光镜和扫描电镜、流式细胞术进行形态学观察和细胞表型鉴定;分别用0、50、100、200μg/L不同浓度的Ag85B或TSLP作用于纯化并鉴定后的m DCs,培养24 h,流式细胞术检测细胞表面分子CD80、CD86、TSLPR和OX40L的表达,选取最佳的Ag85B或TSLP处理浓度。随后将m DCs随机分为空白对照组、Ag85B处理组、TSLP处理组和Ag85B+TSLP处理组,培养24 h后检测m DCs的促炎表面分子TSLPR和OX40L的表达。结果:体外诱导培养7 d,倒置相差显微镜下可见细胞表面呈现不规则树突样突起,扫描电镜下见细胞类圆形,表面有少量皱褶和较少分叉的树突状突起,符合未成熟m DCs的形态学特点;纯化后的m DCs表达表面分子CD11c的细胞较表达共刺激分子CD80和CD86的细胞多,符合未成熟m DCs的表型特征。与空白对照组比较,50~200μg/L的Ag85B处理组m DCs表达CD80和CD86的细胞比率显著增高(P0.05),表达TSLPR和OX40L的细胞比率无显著差异。与空白对照组相比较,50、100和200μg/L浓度的TSLP处理组的m DCs表达CD80和CD86的细胞比率均显著增加(P0.05);与空白对照组和50μg/L TSLP处理组相比较,100μg/L和200μg/L TSLP处理组的m DCs表达TSLPR和OX40L的细胞比率均显著升高(P0.05)。选取200μg/L作为Ag85B和TSLP的优化作用浓度,结果发现Ag85B处理组和Ag85B+TSLP处理组的m DCs表达TSLPR和OX40L的细胞比率较TSLP处理组均显著降低(P0.05),与空白对照组比较差异不显著。结论:Ag85B可通过上调m DCs表达共刺激分子CD80和CD86促进其成熟,同时下调TSLP介导的m DCs表达促炎表面分子TSLPR和OX40L,推测Ag85B可能通过TSLP介导的m DCs途径抑制气道炎症。     

Graves病患者外周血OX40和OX40L分子表达的研究

探讨OX40和OX40L分子在Graves病患者外周血T淋巴细胞和单核细胞上的表达及其在Graves病发病机制中的可能作用。采用流式细胞仪检测技术对50例初发Graves病患者和30例健康志愿者外周血T细胞上OX40和单核细胞上OX40L的表达水平进行分析,并对28例治疗前后病人淋巴细胞上OX40/OX40L的表达进行了比较。结果:与正常对照组比,初发Graves病患者外周血CD4+T细胞上OX40表达水平明显升高,而CD8+T细胞上OX40表达水平无显著变化;同时,还检测到单核细胞上OX40L表达水平也明显增加。治疗后,FT3(游离三碘甲腺原氨酸)值恢复至健康对照组表达范围的Graves病患者其CD4+T细胞上OX40和单核细胞上OX40L的表达均显著降低至正常水平;而FT3值仍偏高的患者单核细胞上OX40L的表达降低,但未降至健康对照组表达范围,CD4+T细胞上OX40的表达则无显著变化。Graves病患者外周血T细胞和单核细胞上分别存在OX40和OX40L的异常表达,且与治疗效果密切相关,提示OX40/OX40L信号在T细胞与单核细胞相互作用过程中可能有助于自身反应性T细胞的持续活化,从而参与Graves病的免疫发生发展。     

CD40L, but not CD40, is required for allergen-induced bronchial hyperresponsiveness in mice

Asthma is characterized by immunoglobulin (Ig) E production, infiltration of the respiratory mucosa by eosinophils (EOSs) and mononuclear cells, and bronchial hyperresponsiveness (BHR). Interaction of CD40 on B cells and antigen presenting cells, with its ligand (CD40L) expressed transiently on activated T cells, is known to augment both T cell-driven inflammation and humoral immune responses, especially IgE production. Considering both the prominent role of inflammation in asthma and the association of the disease with IgE, we hypothesized that CD40-CD40L interactions would be important in pathogenesis. To test this hypothesis, we subjected wild-type (WT) mice and animals lacking either CD40 or CD40L to repeated inhalation of Aspergillus fumigatus (Af ) antigen. Af-treated WT mice displayed elevated IgE levels, bronchoalveolar lavage and pulmonary tissue eosinophilic inflammation, and BHR. IgE production was markedly suppressed in both the CD40 -/- and CD40L -/- strains. However, pulmonary inflammation did not appear to be inhibited by either of these mutations. Paradoxically, development of BHR was prevented by the lack of CD40L but not by the absence of CD40. We conclude that CD40/CD40L interactions, although critical in the induction of IgE responses to inhaled allergen, are not required for the induction of EOS-predominant inflammation. CD40L, but not CD40, is necessary for the development of allergen-induced BHR.     

OX40/OX40L interaction induces the expression of CXCR5 and contributes to chronic colitis induced by dextran sulfate sodium in mice

Interactions between APC and T lymphocytes have been implicated as a major factor contributing to inflammatory bowel disease. To test whether OX40/OX40L interaction plays a role in chronic intestinal inflammation, we induced chronic colitis using dextran sulfate sodium and treated the mice with a murine fusion protein (OX40-IgG). Treatment resulted in a dose-dependent and significant reduction of intestinal inflammation (46%) as measured by a histologic score. IL-10 and IL-5 production from mesenteric lymph node cells increased 20-fold and 18-fold, respectively. In colonic tissue, IL-10 mRNA levels increased and the expression of T-bet was decreased to 30%. IL-10 neutralization partly inhibited the beneficial effects of OX40-IgG treatment. Surprisingly, despite the reduction of inflammation we found the number and size of colonic lymphoid follicles increased, with an accumulation of CD4(+) cells in the mantle area. In contrast, the number of CD4(+) cells infiltrating the mucosa was significantly reduced, as was their CXCR5 expression (24-fold). We conclude that OX40/OX40L interaction contributes to the perpetuation of chronic colitis partly by suppressing IL-10 production. Furthermore, our data suggest that the OX40/OX40L-induced CXCR5 expression on CD4(+) cells may be important for the inflammatory process by allowing migration to the germinal center for further differentiation of CD4(+) cells before they infiltrate the chronically inflamed mucosa.     

Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice

Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3⁺ regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3⁺CD103⁺CD38^? stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3⁺CD103^?CD38⁺ labile-phenotype Tregs in the thymus and increased autoreactive CD4⁺ T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4⁺ T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using ex vivo cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.     

Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue

Functional interactions between B and T lymphocytes are known to depend on the expression of co-stimulatory molecules B7.1/CD80, B7.2/CD86 and their counter-receptors CD28 and CTLA4, as well as CD40 and its ligand CD40L. To study the role of these molecules in situ, an immunohistochemical analysis was carried out on normal human lymphoid tissue. In the germinal centers (GC), B7.1 and B7.2 were differentially expressed. In the dark zone, centroblasts were predominantly B7.1⁺, while centrocytes in the light zone were B7-2⁺, resulting in reversed gradients of both markers in GC. Follicle mantle cells were negative for B7.1 and B7.2. Macrophages and interdigitating dendritic cells (IDC) in T cell zones both expressed B7.1 and B7.2. Moreover, clusters of B7.2⁺ T cells were demonstrated in interfollicular areas. Intrafollicular CD4⁺ T cells in GC, predominantly in the apical light zone, expressed CD28 and CTLA4, as did the majority of interfollicular T cells. CTLA4 showed a striking excentric cytoplasmic staining, which was also seen on T cells activated in vitro. CD40 was expressed on all B cells and more strongly on macrophages and IDC. Moreover, small clusters of T cells in a rim outside the GC showed CD40 expression. CD40L was expressed both on intrafollicular CD4⁺ T cells as well as on T cells in T cell zones. The differential distribution of co-stimulatory molecules in different compartments of normal human lymphoid tissue in situ indicates that these interactions play a distinctive role in different stages of B cell differentiation and in the immune response.