CD40单克隆抗体的制备、筛选及功能鉴定

CD40是肿瘤坏死因子受体超家族成员之一,属于Ⅰ型跨膜糖蛋白,其自然配体是CD40L。CD40-CD40L信号通路在许多重要的生理和病理过程中都起到关键的调节作用。基于其在激活免疫应答中的重要作用,在肿瘤免疫治疗中,具有激活CD40-CD40L信号的CD40激动剂抗体可用于提高抗肿瘤免疫应答;在移植排斥和炎症反应中,CD40-CD40L信号也起重要作用,阻断CD40信号通路是目前治疗风湿性关节炎、肠炎、系统性红斑狼疮、多发性硬化和糖尿病的一个重要靶点。制备CD40阻断性抗体或激动剂抗体不仅可用于检测CD40分子表达,还可以开发用于炎症性疾病或肿瘤治疗的免疫疗法。本研究表达CD40胞外段蛋白,通过免疫制备14株CD40单克隆抗体,筛选发现全部可作为western blotting检测CD40蛋白表达的抗体,9株CD40抗体可作为一抗用于流式细胞术检测人CD40表达,1株CD40抗体能阻断人CD40L和CD40相互作用。有意义的是,8株CD40抗体能激活人CD40分子信号通路,另外有10株能激活小鼠脾脏细胞CD40下游基因的表达,7株能上调小鼠脾脏B细胞表面共刺激分子CD86的表达。本研究制备一系列CD40抗体,可用于CD40检测、CD40信号的阻断或激活。     

淋巴细胞表面CD40分子的表达和作用

CD40和CD40L(CD40 ligand,CD154)都属于肿瘤超家族成员。CD40-CD40L(CD40 ligand,CD154)的相互作用一直是与APC和T细胞的活化相关联,并且在肿瘤免疫、自身免疫性疾病、炎症反应等中起重要的作用。目前,有研究发现,CD40不仅表达在APC、内皮细胞以及某些肿瘤细胞上,也会在T细胞中有表达。CD40+CD8T细胞的研究主要集中在细胞信号通路和过继免疫治疗方面,CD40+CD4T细胞主要集中在自身免疫性疾病的研究。本文将对CD40在T细胞表面的表达及其作用做一综述。     

肿瘤抗原肽研究新进展

肿瘤抗原肽的寻找已成为当前肿瘤研究领域的一大热点。它包括癌基因蛋白抗原肽、致癌病毒基因抗原肽、肿瘤特异性抗原肽和组织特异性分化抗原肽等。这些抗原肽在与MHC类分子结合后 ,提呈到细胞表面被相应T细胞受体识别 ,从而激活细胞毒性T淋巴细胞的活性 ,使机体产生特异性抗肿瘤免疫应答。因而它们在肿瘤免疫接种和免疫治疗等方面有着广阔的应用前景。     

CD40-CD40L共刺激途径在体液免疫和细胞免疫中的作用

CD40与CD40配体(CD40L)是体内炎症和免疫反应系统的一对共同刺激分子,参与机体的体液免疫和细胞免疫反应.CD40-CD40L共刺激途径能促进T细胞活化并放大T淋巴细胞依赖的免疫应答,是产生记忆性CD8+T细胞的基础,能够促进CD8+T细胞的扩增和多能性,在CD4+T细胞分化的过程中起重要作用,介导更强的抗肿瘤效应和杀伤靶细胞的能力.CD40-CD40L共刺激途径参与T淋巴细胞依赖的B淋巴细胞应答过程、生发中心的形成、长期记忆性R细胞的产生、抗体的产生及抗体类别转换,并可诱导和扩增CD4+ CD25+ Treg细胞.CD40-CD40L共刺激途径在多种疾病的发生、发展中起作用,有广阔的临床应用前景.     

CIITA调控MHC-Ⅱ类分子表达分子机制的研究进展

MHC-Ⅱ类分子抗原处理、提呈通路在调控CD4 T细胞激活及特异性应答的过程中发挥重要的作用,T细胞的功能在很大程度上取决于MHC-Ⅱ类分子的表达水平.     

CD40分子在树突状细胞中的信号转导通路

树突状细胞 (DC )作为体内功能最强的抗原提呈细胞 (APC ) ,是启动机体免疫应答的中心环节[1] 。未成熟DC定居在外周组织 ,具有极强的捕获抗原能力 ,通过多种方式捕获入侵的病原体、损伤或恶变的组织后迁移至淋巴结 ,将加工过的抗原以MHC 抗原肽的形式提呈给T细胞启动机体免疫应答。在此过程中 ,DC逐渐发育成熟 ,伴随着膜表面黏附分子表达的变化以及MHCII类分子和共刺激分子如CD4 0、OX4 0L、CD80、CD86表达的上调。然而 ,DC抗原提呈功能的完全成熟需要T细胞提供的共刺激信号 ,其中CD4 0相关的信号通路在此过程中发挥了重要作用     

PD-1及PD-L1与肿瘤适应性免疫抵抗的研究进展

程序性死亡蛋白-1(programmed death protein 1,PD-1)与其配体程序性死亡配体1(programmed cell death-ligand 1,PD-L1)属于CD28/B7家族,是近年研究比较透彻的免疫检查点分子。PD-1/PD-L1通过调节外周组织中免疫反应的持续性和效价避免组织损伤并维持对于自身抗原的耐受。肿瘤细胞主动性抑制T细胞的机制称为适应性免疫抵抗(adaptive immune resistance),即肿瘤抗原特异性T细胞企图攻击肿瘤,但肿瘤细胞发生反应性改变(诱导表达PD-L1)从而避免免疫攻击。适应性免疫抵抗过程中的关键分子即为PD-1/PD-L1。PD-1/PD-L1抗体治疗已在临床试验中显示出良好的疗效,使阻断适应性免疫抵抗有望成为重要的肿瘤免疫治疗手段。分析肿瘤活检样本的基线免疫信息能指导制定个体化的免疫治疗方案。本文就PD-1/PD-L1的生物学结构、适应性免疫抵抗机制及相关临床决策等进行综述。     

129 CD40-CD40L共刺激分子与自身免疫性疾病

大量研究证实CD40分子与其配体CD40L结合所产生的共刺激信号在B细胞的增殖、分化、抗体的分泌和类型转换以及T细胞活化、效应性细胞因子分泌中起重要作用.近年来发现,CD40、CD40L异常表达与一些疾病的免疫病理密切相关.本文仅就CD40-CD40L的生物学特性、功能和在自身免疫性疾病中的致病作用及其可能的治疗策略作一综述.     

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.     

自然杀伤T细胞在抗感染免疫中的作用研究进展

自然杀伤T细胞（NKT）是一类同时表达T细胞受体（TCR）dB和NK细胞表面标志的T细胞亚群,能识别由抗原提呈细胞（APC）表面CD1d分子提呈的脂类抗原。NKT细胞经抗原刺激后能迅速分泌大量的细胞因子（如IFN-1和IL-4）,调节固有免疫和适应性免疫,还能直接杀伤靶细胞,具有效应细胞的功能,在机体抗感染、肿瘤和自身免疫病方面发挥重要作用。     

In this issue: harnessing immunity against difficult targets, through the CD40-CD40L pathway

This issue highlights translational aspects related to the CD40-CD40L pathway, a veritable master-switch at the interface between the innate and adaptive immunity. From direct targeting of CD40 onto tumor cells to licensing professional antigen presenting cells (APC), and from in vivo activation of immune effectors to enabling adoptive cell immune interventions, this pathway already shows translational promise in oncology and infectious diseases. In light of the pivotal nature and versatility of the target, the authors discuss a diverse range of options to design and advance novel, safe and effective immune interventions or combinatorial therapies involving CD40 agonistic engagement.     

In this issue: Harnessing Immunity against Difficult Targets,through the CD40-CD40L Pathway

This issue highlights translational aspects related to the CD40-CD40L pathway, a veritable master-switch at the interface between the innate and adaptive immunity. From direct targeting of CD40 onto tumor cells to licensing professional antigen presenting cells (APC), and from in vivo activation of immune effectors to enabling adoptive cell immune interventions, this pathway already shows translational promise in oncology and infectious diseases. In light of the pivotal nature and versatility of the target, the authors discuss a diverse range of options to design and advance novel, safe and effective immune interventions or combinatorial therapies involving CD40 agonistic engagement.     

CAR T cells for brain tumors: Lessons learned and road ahead

Malignant brain tumors, including glioblastoma, represent some of the most difficult to treat of solid tumors. Nevertheless, recent progress in immunotherapy, across a broad range of tumor types, provides hope that immunological approaches will have the potential to improve outcomes for patients with brain tumors. Chimeric antigen receptors (CAR) T cells, a promising immunotherapeutic modality, utilizes the tumor targeting specificity of any antibody or receptor ligand to redirect the cytolytic potency of T cells. The remarkable clinical response rates of CD19‐targeted CAR T cells and early clinical experiences in glioblastoma demonstrating safety and evidence for disease modifying activity support the potential of further advancements ultimately providing clinical benefit for patients. The brain, however, is an immune specialized organ presenting unique and specific challenges to immune‐based therapies. Remaining barriers to be overcome for achieving effective CAR T cell therapy in the central nervous system (CNS) include tumor antigenic heterogeneity, an immune‐suppressive microenvironment, unique properties of the CNS that limit T cell entry, and risks of immune‐based toxicities in this highly sensitive organ. This review will summarize preclinical and clinical data for CAR T cell immunotherapy in glioblastoma and other malignant brain tumors, including present obstacles to advancement.     

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.     

Inhibition of Fas-mediated Apoptosis by Antigen: Implications for Lymphomagenesis

Apoptotic deletion of expanded B cell populations is essential in avoidance of autoimmune disease and immune regulation of some B cell malignancies. The role of CD4+ T cells in B cell apoptosis is evident from the high incidence of B cell tumors and autoimmunity in patients with T cell diseases such as the acquired immune deficiency syndrome (AIDS). We have previously demonstrated that in Epstein-Barr Virus (EBV) negative Burkitt's lymphoma (BL), a tumor derived from proliferating centroblasts of the germinal center, the malignant lymphocytes can be induced to express Fas (CD95) by ligation of CD40 at the B cell surface. Upon CD40 engagement, BL cells are sensitized to T-cell derived death signals provided by Fas ligand (FasL, CD95L). HBL-3 is a cell line derived from an AIDS-related BL in which the tumor IgM binds the human erythrocyte "i" antigen. To determine whether Fas-mediated apoptosis of BL cells is reduced in the context of antigen to which the tumor IgM binds, we stimulated HBL-3 cells with CD40 ligand (CD40L, CD154) in the presence and absence of human erythrocytes expressing the "i" antigen, and measured Fas-mediated apoptosis upon exposure to an agonistic anti-Fas antibody. We observed that HBL-3 cells were sensitized to Fas-mediated death by exposure to CD40L. When i+ RBCs were present, Fas-mediated apoptosis in HBL-3 cells was reduced by greater than 30%. In contrast, there was no reduction in Fas-mediated apoptosis in the presence of i &#109 (I+) RBCs. These findings demonstrate that Fas-mediated deletion of BL cells is inhibited upon surface IgM engagement by antigen for which the malignant clone has affinity.     

Inhibition of Fas-mediated apoptosis by antigen: implications for lymphomagenesis

Apoptotic deletion of expanded B cell populations is essential in avoidance of autoimmune disease and immune regulation of some B cell malignancies. The role of CD4+ T cells in B cell apoptosis is evident from the high incidence of B cell tumors and autoimmunity in patients with T cell diseases such as the acquired immune deficiency syndrome (AIDS). We have previously demonstrated that in Epstein-Barr Virus (EBV) negative Burkitt's lymphoma (BL), a tumor derived from proliferating centroblasts of the germinal center, the malignant lymphocytes can be induced to express Fas (CD95) by ligation of CD40 at the B cell surface. Upon CD40 engagement, BL cells are sensitized to T-cell derived death signals provided by Fas ligand (FasL, CD95L). HBL-3 is a cell line derived from an AIDS-related BL in which the tumor IgM binds the human erythrocyte "i" antigen. To determine whether Fas-mediated apoptosis of BL cells is reduced in the context of antigen to which the tumor IgM binds, we stimulated HBL-3 cells with CD40 ligand (CD40L, CD154) in the presence and absence of human erythrocytes expressing the "i" antigen, and measured Fas-mediated apoptosis upon exposure to an agonistic anti-Fas antibody. We observed that HBL-3 cells were sensitized to Fas-mediated death by exposure to CD40L. When i+ RBCs were present, Fas-mediated apoptosis in HBL-3 cells was reduced by greater than 30%. In contrast, there was no reduction in Fas-mediated apoptosis in the presence of i- (I+) RBCs. These findings demonstrate that Fas-mediated deletion of BL cells is inhibited upon surface IgM engagement by antigen for which the malignant clone has affinity.     

Modulation of the CD40-CD40 ligand interaction using human anti-CD40 single-chain antibody fragments obtained from the n-CoDeR phage display library

CD40 plays a central regulatory role in the immune system and antibodies able to modulate CD40 signalling may consequently have a potential in immunotherapy, in particular for treatment of lymphomas and autoimmune disease like multiple sclerosis. As a first step to achieve this goal, we describe the selection and characterization of a novel set of fully human anti-CD40 antibody fragments (scFv) from a phage display library (n-CoDeR). In order to determine their biological potential, these antibody fragments have been analysed for their ability to promote B-cell activation, rescue from apoptosis and to block the CD40-CD40 ligand (CD40L) interaction. The selected cohort of human scFv could be subcategorized, each expressing a distinct functional signature. Thus scFv were generated that induced B-cell proliferation, rescued B cells from apoptosis and blocked the CD40-CD40L interaction to different extents. In particular, one of the scFv clones (F33) had the ability to abrogate completely this interaction. The epitope recognition patterns as well as individual rate constants were also determined and the affinity was shown to vary from low to high nanomolar range. In conclusion, this panel of human anti-CD40 scFv fragments displays a number of distinct properties, which may constitute a valuable source when evaluating candidates for in vivo trials.     

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.     

Adeno-associated virus mediated gene transfer into lung cancer cells promoting CD40 ligand-based immunotherapy

Expression of the CD40 ligand (CD40L) on tumors can activate host immune systems and produce antitumor effects against the tumors. To deliver the CD40L gene efficiently, we evaluated the efficiency of transduction of different serotypes of adeno-associated virus (AAV) vectors in lung cancer A549 cells and compared the transduction efficiency of a conventional AAV vector with that of self-complementary AAV (scAAV) vectors as well. We determined that serotype AAV2/5 transduced A549 cells much more efficiently than serotypes AAV2/1, AAV2/2, AAV2/6, AAV2/7, AAV2/8, AAV2/9 and AAV2/10. And the transduction efficiency of scAAV2/5 was significantly higher than conventional AAV2/5. Furthermore, pre-treatment with carboplatin, which is a chemotherapeutic agent used in lung cancer chemotherapy, substantially increased AAV-mediated transgene expression. The scAAV2/5 vectors encoding human CD40L were used to tranduce CD40L into A549 cells, which were then co-cultivated with immature human dendritic cells (DCs). Interleukin 12 (IL-12) that was released was measured in the culture supernatant. Specificity of the immunostimulatory effect of CD40L was confirmed by blocking with a monoclonal antibody binding to human CD40L. The in vivo antitumor activity was evaluated in BALB/c nude mice bearing A549 lung cancer. scAAV2/5-CD40L showed significant inhibitory effects on the growth of transplanted tumor cells as compared with control group. These studies suggest that recombinant AAV2/5-CD40L may provide an effective form of therapy for lung cancer.