The role of CD40 and CD154/CD40L in dendritic cells

In this review, we focus on the function of CD40–CD40L (CD154) interactions in the regulation of dendritic cell (DC)–T cell and DC–B cell crosstalk. In addition, we examine differences and similarities between the CD40 signaling pathway in DCs and other innate immune cell receptors, and how these pathways integrate DC functions. As research into DC vaccines and immunotherapies progresses, further understanding of CD40 and DC function will advance the applicability of DCs in immunotherapy for human diseases.     

Cellular and molecular mechanisms of atherogenesis. CD40-CD40L immunoregulatory signal

Contact-dependent interactions of CD40 receptor and its ligand CD40L are regarded as a stimulator of atheroma-associated cells. A new T lymphocyte-dependent pathway of activation of immune inflammation in the vascular wall is discussed, which is presumably maintained by self-regulation of antiinflammatory cytokine production. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 10, pp. 364–379, October, 1999     

CD40/CD40L dyad in the inflammatory and immune responses in the central nervous system

CD40 and its cognate ligand （CD40L） are a pair of regulators of pro-inflammatory and immune responses. In the central nervous system （CNS）, CD40 is expressed on a variety of cells, including vascular endothelial cells, smooth muscle cells, astrocytes and microglia （the brain macrophages, being the most sensitive cell type to respond to CD40 ligand）. Interaction between CD40 on microglia and CD40L presented by infiltrating T lymphocytes and other resident CNS cells triggers a series of intracellular signaling events that promote the production of a wide array of cytokines, chemokines and neurotoxins. Thus, both molecules serve as amplifiers of pro-inflammatory and immune responses in the CNS and constitute important molecular targets for therapeutic intervention of diseases.Cellular ＆ Molecular Immunology. 2006;3（3）：163-169.     

CD40-CD40L在强直性脊柱炎患者外周血淋巴细胞上的表达

目的探讨共刺激分子CD40CD40L在强直性脊柱炎(AS)患者的外周血淋巴细胞亚群上的异常表达与免疫功能紊乱的关系。方法用流式细胞仪采用直接免疫荧光法测定30例AS患者和20例健康对照人外周血淋巴细胞表面标志CD3、CD4、CD8、CD19的表达情况,CD40L在CD4 T和CD8 T细胞上的表达及CD40在CD19 B细胞上的表达。用速率散射比浊法测定血清中免疫球蛋白IgG,IgA和IgM的水平。结果①AS患者CD3 、CD3 CD4 、CD19 细胞较正常对照组显著增高(P<0.05),CD3 CD8 细胞较正常对照组显著降低(P<0.05);②AS患者CD4 T细胞和CD8 T细胞上的CD40L、CD19 B细胞上CD40的表达都较对照组显著增高(P<0.05);③AS患者血清中2种免疫球蛋白IgG、IgA的水平均较对照组显著增高(P<0.05)。结论CD40CD40L途径在AS免疫功能紊乱中起了重要作用。     

Differential responses of human B-lymphocyte subpopulations to graded levels of CD40-CD154 interaction

Naïve and memory B‐lymphocyte populations are activated by CD154 interaction through cell‐surface CD40. This interaction plays an important role in the regulation of the humoral immune response, and increasing evidence indicates that fine variation in CD40 binding influences B lymphocytes, macrophages and dendritic cells in murine models. Here we have investigated whether and how variations in the intensity of the CD40–CD154 interaction could contribute to differential regulation of human B‐lymphocyte populations. Proliferation and differentiation of B lymphocytes were monitored in response to graded levels of CD40 stimulation in the presence of interleukin (IL)‐2, IL‐4 and IL‐10. Our results show that the level of CD154 binding to CD40 on B lymphocytes can directly influence the evolution of CD19⁺ CD27^– and CD19⁺ CD27⁺ cell populations. Furthermore, proliferation, global expansion of CD19⁺ cells and emergence of CD38⁺⁺ CD138⁺ cells, as well as immunoglobulin G (IgG) and IgM secretion, were affected by the level of exposure of B lymphocytes to CD154. These results suggest that the CD40–CD154 interaction is more like a rheostat than an on/off switch, and its variation of intensity may play a role in the regulation of B‐lymphocyte activation following the primary and/or secondary humoral immune response.     

In situ demonstration of CD40- and CD154-positive cells in psoriatic lesions and keratinocyte production of chemokines by CD40 ligation in vitro

In psoriatic lesions, T cells and keratinocytes are in an activated state. Ligation of CD40 expressed on activated keratinocytes with CD154 expressed on activated T cells is thought to be involved in the pathogenesis of psoriasis. However, the presence of CD40(+) and CD154(+) cells in psoriatic skin has not been thoroughly studied. The present study has therefore examined their presence by immunohistochemistry in the lesional and non-lesional skin of ten patients. The influence of CD154-CD40 ligation on the release of chemokines (IL-8, RANTES, and MCP-1) and complement components (C3 and factor B) from keratinocytes was also investigated in vitro. Studies using single and double staining showed that clusters of CD40(+) keratinocytes were present in both lesional and non-lesional skin; CD40(+)CD1a(+) Langerhans cells in lesional, non-lesional, and normal skin; and numerous CD40(+)CD83(+) cells in lesional skin. CD1a(+) and CD83(+) cells always expressed CD40 strongly. Numerous T cells were seen in lesional skin. A small number of T cells expressed CD154. CD154(+) T cells were seen in the lesional epidermis of seven of ten patients-in six, in juxtaposition to CD40(+) cells including keratinocytes. In non-lesional epidermis, CD154(+) T cells were seen in two patients-in one, in juxtaposition to CD40(+) keratinocytes. In vitro studies showed that IFN-gamma-treated keratinocytes released small amounts of IL-8, RANTES, and MCP-1; ligation of these cells with CD154-transfected J558 cells or soluble CD154 greatly enhanced the release. This ligation did not enhance the release of C3 and factor B. These results warrant further studies on the role of CD40 ligation in the pathogenesis of psoriasis.     

CD40/CD40L交联在CD4+T细胞诱导肿瘤细胞凋亡中的机制研究

目的探讨CD40/CD40L交联在CIK细胞中CD4 T细胞(CD4 CIK)诱导肿瘤细胞凋亡中的作用机制.方法体外扩增CIK细胞并纯化CD4 T细胞亚群,AnnexinV染色法观察CD4 CIK诱导肿瘤细胞凋亡的作用;半定量PCR、流式细胞法及ELISA法比较CD4 CIK激活前后CD40L的表达变化;将转染质粒pIRES2-EGFP-sCD40L的CHO细胞(CHO-sCD40L)与乳腺癌细胞T47D共孵育,监测24小时后其表面分子Fas的表达变化及对Fas介导凋亡的敏感性.结果CD4 CIK细胞可诱导肿瘤细胞凋亡,凋亡率随孵育时间和效靶比的升高而增加,且肿瘤细胞表面分子Fas水平升高,可从1.98%±0.23%升高到31.62%±7.07%;CD4 CIK细胞被激活后,CD40L表达水平均较激活前明显增加;成功转染的CHO-sCD40L细胞与T47D共培养后,T47D表面分子Fas可被诱导升高,加入CH-11 24小时后可观察到明显T47D细胞的凋亡.结论CD4 CIK可能通过CD40/CD40L交联提升肿瘤细胞表面功能性Fas表达来诱导其凋亡.     

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。     

CD40/CD40L expression in leukocytes from chronic granulomatous disease patients

Chronic granulomatous disease (CGD) is an inherited disorder caused by defects in the NADPH oxidase complex, which generates superoxide, the precursor of hydrogen peroxide (H(2)O(2)) and other reactive oxygen derivatives with microbicidal activity. Because CGD patients are at risk of chronic inflammatory manifestations, including inflammatory bowel disease and autoimmune diseases, and it is not clear whether these pathologies are exclusively secondary to altered superoxide production, or whether distinct immunologic defects are involved, we explored cell proliferation, lymphocyte cell counts, immunoglobulin levels, presence of autoimmune antibodies and expression of costimulatory molecules in leukocytes from CGD patients. We found that CGD patients have a diminished phytohemagglutinin-induced proliferation of blood mononuclear cells. Following stimulation with PMA plus ionomycin, a reduced percentage of CD40L expression in T lymphocytes and a diminished expression of CD40 molecules in neutrophils were observed on leukocytes from these patients. Our results suggest an altered interplay between elements of innate and adaptive immunity in CGD patients, which may be reflected in an increased susceptibility to opportunistic infections.     

脱氢表雄酮对人脐静脉内皮细胞CD40/CD40L表达的影响

目的:探讨脱氢表雄酮(DHEA)对干扰素-γ(I NF-γ)刺激下的人脐静脉内皮细胞(HUVECs)CD40/CD40L表达的影响。方法:原代培养人脐静脉内皮细胞,给予I NF-γ刺激和不同浓度DHEA干预。采用流式细胞术检测CD40/CD40L在细胞表面的表达,通过反转录-聚合酶链反应(RT-PCR)检测CD40/CD40L mRNA的表达。结果:I NF-γ刺激HUVECs表达CD40/CD40L,DHEA下调I NF-γ诱导的HUVECs表面CD40/CD40L的表达,同时对I NF-γ刺激下的CD40/CD40L mRNA的表达有抑制作用,并且呈剂量依赖性。结论:DHEA能减轻I NF-γ刺激下的人脐静脉内皮细胞CD40/CD40L的表达。     

Analysis of CD154 and CD40 expression in native coronary atherosclerosis and transplant associated coronary artery disease

T cells have roles in the pathogenesis of native coronary atherosclerosis (CA) and transplant-associated coronary artery disease (TCAD). The mechanisms by which T cells interact with other cells in these lesions are not fully known. CD154 is an activation-induced CD4⁺ T cell surface molecule that interacts with CD40⁺ target cells, including macrophages and endothelial cells, and induces the production of pro-inflammatory molecules, including CD54 (ICAM-1) and CD106 (VCAM-1). To investigate whether CD154-CD40 interactions might be involved in the pathogenesis of CA or TCAD we performed immunohistochemical studies of CD154 and CD40 expression on frozen sections of coronary arteries obtained from cardiac allograft recipients with CA (n=10) or TCAD (n=9). Utilizing four different anti-CD154 mAb we found that CD154 expression was restricted to infiltrating lymphocytes in CA and TCAD. CD40 expression was markedly up-regulated on intimal endothelial cells, foam cells, macrophages and smooth muscle cells in both diseases. Dual immunolabeling demonstrated many CD40⁺ cells co-expressed CD54 and CD106. The extent of CD40, CD54 and CD106 expression showed statistical significant correlation with the severity of disease and the amount of intimal lymphocytes. Together these studies demonstrate the presence of activated CD154⁺ and CD40⁺ cells in both CA and TCAD lesions and suggest that CD154-mediated interactions with CD40⁺ macrophages, foam cells, smooth muscle cells and/or endothelial cells may contribute to the pathogenesis of these diseases. Received: 17 December 1999 / Accepted: 20 January 2000     

Molecular mechanism and function of CD40/CD40L engagement in the immune system

Summary:  During the generation of a successful adaptive immune response, multiple molecular signals are required. A primary signal is the binding of cognate antigen to an antigen receptor expressed by T and B lymphocytes. Multiple secondary signals involve the engagement of costimulatory molecules expressed by T and B lymphocytes with their respective ligands. Because of its essential role in immunity, one of the best characterized of the costimulatory molecules is the receptor CD40. This receptor, a member of the tumor necrosis factor receptor family, is expressed by B cells, professional antigen-presenting cells, as well as non-immune cells and tumors. CD40 binds its ligand CD40L, which is transiently expressed on T cells and other non-immune cells under inflammatory conditions. A wide spectrum of molecular and cellular processes is regulated by CD40 engagement including the initiation and progression of cellular and humoral adaptive immunity. In this review, we describe the downstream signaling pathways initiated by CD40 and overview how CD40 engagement or antagonism modulates humoral and cellular immunity. Lastly, we discuss the role of CD40 as a target in harnessing anti-tumor immunity. This review underscores the essential role CD40 plays in adaptive immunity.     

Blockade of CD40-CD40 ligand pathway induces tolerance in murine contact hypersensitivity

Interactions between CD40 on antigen-presenting cells and its ligand (CD40L) on T cells has been implicated in T cell-mediated immune responses. Previously, we have shown that contact hypersensitivity (CHS), a cell-mediated cutaneous immune response in reaction to haptens, could be subclassified based on whether the hapten primed for Th1 or Th2 cytokines in cells isolated from draining lymph nodes. We also found that tolerance to a Th2-priming hapten could be induced only by simultane blockade of the CD40-CD40L and B7-CD28 at the time of sensitization. Here we demonstrate that blockade of CD40-CD40L signaling alone induces long-lasting unresponsiveness to the Th1 hapten 2,4-dinitrofluorobenzene (DNFB), and inhibits antigen-specific T cell proliferation in vitro. We find that CD40-CD40L signaling is required in the sensitization but not elicitation phase of DNFB-induced CHS, as treatment of mice with anti-CD40L monoclonal antibody (mAb) does not affect the response to hapten challenge in previously sensitized and untreated animals. Examination of cytokine production shows that anti-CD40L mAb decreases interferon-γ production by draining lymph node cells from DNFB-sensitized mice, and reciprocally increases interleukin (IL)-4 production. Consistent with this Th1 to Th2 immune deviation, anti-CD40L mAb prevents the induction of IL-12 mRNA in regional lymph nodes, an event which is normally seen within 12 h following hapten sensitization. In contrast, suppression of CHS by CTLA4Ig decreased the production of all cytokines by draining lymph node cells. Together, these data show that blockade of the CD40-CD40L pathway by itself is sufficient to induce tolerance to DNFB-induced CHS, and that this is associated with blockade of IL-12 induction and Th1 to Th2 immune deviation.     

CD40 and autoimmunity: The dark side of a great activator

CD40 is a tumor necrosis factor receptor superfamily member expressed by immune and non-immune cells. CD40:CD154 interactions mediate T-dependent B cell responses and efficient T cell priming. Thus, CD40 is a likely candidate to play roles in autoimmune diseases in which activated T and B cells cause pathology. Diseases in which CD40 plays a pathogenic role include autoimmune thyroiditis, type 1 diabetes, inflammatory bowel disease, psoriasis, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. This review discusses the role of CD40:CD154 interaction in human and mouse autoimmunity, human polymorphisms associated with disease incidence, and disrupting CD40:CD154 interactions as an autoimmune therapy.     

RSV毛细支气管炎患儿血液CD4~+CD25~+Foxp3~+Treg与CD40/CD40L关系的研究

探讨CD40/CD40L信号通路对不同病原体毛细支气管炎(以下简称:毛支炎)患儿血液CD4~+CD25~+Foxp3~+ Treg的增殖分化及其分泌抑制性细胞因子TGF-β1、IL-10的影响。取呼吸道合胞病毒(RSV)及非RSV感染毛支炎患儿血液并检测CD4~+CD25~+Foxp3~+Treg的百分率;并将血液中分离的单个核细胞(PBMC)接种于24孔板内,加入CD40L McAb进行阻断,作用72h后采用流式细胞仪检测培养板内CD4~+CD25~+Foxp3~+Treg的百分率,激光共聚焦检测CD4~+CD25~+Foxp3~+Treg细胞表面Foxp3的平均密度,酶联免疫吸附法检测培养上清中TGF-β1、IL-10的含量。RSV毛支炎患儿血液中CD4~+CD25~+Foxp3~+Treg百分率显著低于非RSV毛支炎患儿及正常对照组(P<0.05);RSV毛支炎患儿体外培养PBMC中CD4~+CD25~+Foxp3~+Treg百分率均显著低于非RSV毛支炎患儿、正常对照组和抗CD40L McAb组(P<0.05)。毛支炎患儿PBMC经过体外培养72h后,抗CD40L McAb组培养的细胞中CD4~+CD25~+Foxp3~+Treg表面Foxp3的平均密度显著高于RSV毛支炎患儿组(P<0.05)。PBMC培养上清中IL-10和TGF-β1的水平抗CD40L McAb组明显高于非RSV毛支炎组,非RSV毛支炎组明显高于RSV毛支炎组,差异有统计学意义(P<0.05)。RSV毛支炎患儿体内存在严重的CD4~+CD25~+Foxp3~+Treg数量不足;体外用抗CD40L McAb阻断CD40/CD40L通路可促进RSV毛支炎PBMC中CD4~+CD25~+Foxp3~+Treg的增殖。     

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.     

CD40L/CD40轴的生物学功能及其与疾病的关系研究进展

CD40L／CD40为体内免疫反应系统中一对重要的共刺激分子,参与机体的体液和细胞免疫反应。CD40L主要表达在CD4^＋T细胞、CD8^＋T细胞和B细胞,CD40主要表达于B细胞、活化的单核／巨噬细胞、树突状细胞（DC）、上皮细胞等。CD40L／CD40轴对B细胞的活化、增殖、细胞因子的分泌及免疫球蛋白的类别转换起到了非常重要的作用。CD40L／CIMO相互作用也对细胞毒性T细胞前体的活化,维持调节性T细胞的动态平衡及诱导辅助性T细胞的分化等方面起重要作用。CIMOL／CIMO轴还参与多种免疫相关疾病的致病过程,如自身免疫性疾病、肝炎等,且在抗肿瘤免疫中起到重要作用。多种动物模型的建立为探究疾病病因、研究CIMOL／CIMO轴在疾病发生过程中的作用提供了重要的平台。     

The Janus faces of CD40 in cancer

CD40 is a TNF receptor family member that is widely recognized for its prominent role in immune regulation and homeostasis. Expression of CD40 is not restricted to normal lymphoid cells but is also evident in the majority of haemopoietic and epithelial malignancies where it has been implicated in oncogenic events. Accumulating evidence, however, suggests that the CD40 pathway can be exploited for cancer therapy by virtue of its ability to stimulate the host anti-tumor immune response, normalize the tumor microenvironment and directly suppress the growth of CD40-positive tumors. Here, we provide an overview of the multifaceted functions of the CD40 pathway in cancer and its emerging role in the treatment of malignancy.     

CD40L is transferred to antigen‐presenting B cells during delivery of T‐cell help

The delivery of T‐cell help to B cells is antigen‐specific, MHC‐restricted, and CD40L (CD154) dependent. It has been thought that when a T cell recognizes an antigen‐presenting B cell, CD40L expressed on the T‐cell surface engages with CD40 on the surface of B cells as long as the cells remain conjugated. By adding fluorescently labeled anti‐CD40L antibody during overnight incubation of antigen‐presenting B cells with antigen‐specific T cells, we discovered that CD40L does not remain on the surface of the T cell, but it is transferred to and endocytosed by B cells receiving T‐cell help. In the presence of anti‐CD40L antibody, transferred CD40L is nearly absent on bystander B cells that are not presenting antigen, and the bystander cells do not become activated. Because transfer of CD40L to B cells correlates with B‐cell activation, we speculate that persistence of helper T‐cell‐derived CD40L on or in B cells could permit sustained CD40 signaling enabling survival and proliferation of antigen‐presenting B cells following brief interactions with helper T cells in vivo in germinal centers.     

CD40-CD154 interactions between macrophages and natural killer cells during sepsis are critical for macrophage activation and are not interferon gamma dependent

Natural killer (NK) cell interactions with macrophages have been shown to be important during bacterial sepsis in activating macrophages to improve bacterial clearance. The mechanism for this increased activation, however, is unclear. This study determines the relative roles of interferon (IFN)-gamma and CD40/CD154 direct cell interactions on macrophage and NK cell activation in an experimental model of sepsis. Splenic NK cells and peritoneal macrophages were isolated and cultured alone or in coculture, with and without LPS. CD69 expression on NK cells, phagocytosis ability of macrophages, and cell cytokine production was assessed at 24 and 48 h. Coculture of NK cells and macrophages significantly increased activation levels of both cell types, and through experiments culturing NK cells with supernatants from stimulated macrophages and macrophages with supernatants from stimulated NK cells, this activation was determined to be cell-contact-dependent. Similar experiments were conducted using NK cells from IFN-gamma deficient (-/-) mice, as well as anti-IFN-gamma neutralizing antibody. These experiments determined that IFN-gamma is not required for NK or macrophage activation, although it did augment activation levels. Experiments were again repeated using peritoneal macrophages from CD40-/- mice or splenic NK cells from CD154-/- mice. CD40/CD154 interactions were important in the ingestion of bacteria by macrophages, but did not affect NK cell activation at 24 h. There was, however, a protective effect of CD40/CD154 interactions on NK cell activation-induced cell death that occurred at 48 h. CD40/CD154 interactions between macrophages and NK cells are therefore important in macrophage phagocytosis, and are not dependent on IFN-gamma.