论文题目重组卡介苗诱导T细胞免疫应答的分子、细胞机理研究

1、论文题目：重组卡介苗诱导T细胞免疫应答的分子、细胞机理研究 作者简介：焦新安，男，1964年09月出生，1997年02月师从于扬州大学刘秀梵教授，于1999年12月获博士学位。  摘 要牛结核分枝杆菌BCG是世界上广泛使用的疫苗，亦被认为是表达与传递外源抗原的理想活载体之一，然而迄今对BCG或重组BCG(rBCG)与宿主免疫系统相互作用机理尚未阐明，而从免疫调节生物学角度探讨BCG或rBCG诱导T细胞应答的规律亦未涉足。为此，本研究以表达大肠杆菌麦芽糖结合蛋白(MalE)的rBCG (rBCG·MalE)为模型，以期阐明rBCG与抗原提呈细胞相互作用的规

2、律、rBCG表达产物抗原表位多样性、rBCG和BCG诱导的T细胞应答规律与调控因素。1重组卡介苗表达的MalE蛋白T细胞表位的鉴定在体外抗原提呈试验中，证实rBCG·MalE成功表达MalE蛋白的四种T细胞抗原表位，即p68-82、p100-114、p151-165和p277-291。T细胞增殖试验和ELISPOT试验结果均表明这种表达是功能性的，rBCG·MalE可刺激小鼠产生MalE蛋白及其多肽特异的T细胞增殖和IFN-应答。表位作图显示，BCG表达的MalE未形成新的抗原表位或使隐蔽表位暴露出来，并进一步证实MalE p68-82多肽表位是MalE H-2d限制性主要

3、T细胞表位，而p100-114、p151-165和p277-291多肽表位为次要表位。本研究结果表明，作为表达和运送外源抗原的BCG载体，它功能性地表达了外源抗原MalE蛋白，并能向宿主免疫系统有效地传递，这进一步证明BCG是一种优良的疫苗载体。2重组卡介苗感染早期树突细胞的作用具有抗原提呈细胞(APC)功能的吞噬性细胞，通过刺激T细胞应答可促进细菌清除，从而在抵抗细菌感染中起重要作用。巨噬细胞(MØ)是胞内寄生菌的优选宿主细胞，并贮存大量的抗原物质，而树突细胞(DC)却是非常强势的APC。然而在体内针对细菌的T细胞应答中，这两种有吞噬活性的细胞之作用还未阐明。为此，本研究以rBCG

4、·MalE为材料，对MØ和DC诱导抵抗细菌的T细胞应答中的相对作用进行了探讨。在小鼠i.v.接种rBCG·MalE 12小时后，脾脏MØ和DC的感染率相当，但在来自体内的体外试验(Ex vivo)中，用针对MalE蛋白的特异T细胞杂交瘤仅测出DCS表面存在免疫原性的MalE多肽/MHC复合物，而MØ却没有。同样，在rBCG·MalE感染后，仅在DCs观察到CD40、 B7.1(CD80)和B7.2(CD86)分子表达水平升高，并分泌IL-12 p40，因而首次证实DC在激发针对分枝杆菌T细胞应答中的关键作用。进一步试验还显示，脾脏DC

5、 CD8和CD8亚群在体内是相同势能的APC，但IL-12的产生主要来自CD8亚群。这表明在rBCG感染早期，DC在体内条件下不仅在针对分枝杆菌获得性免疫中发挥主导作用，而且还通过分泌IL-12在自然免疫中起重要作用。研究中还首次发现，rBCG在其感染早期能在DC中存活，但数量没有增长。鉴于DC提呈rBCG抗原能力的迅速丧失，表明除MØ外DC亦是rBCG或BCG 感染早期的贮存宿主细胞。这些结果为BCG感染与免疫机理提供了新认识，对结核病控制有重要价值，同时，为以BCG作载体研制新型疫苗提供了重要理论依据。3表达MalE重组卡介苗诱导T细胞应答的动力学应用免疫磁性分离技术去除免疫小鼠

6、脾脏中CD4和CD8T 细胞后，在ELISPOT试验中证实，rBCG·MalE诱导的T细胞应答是CD4 T 细胞依赖的。对MalE、 PPD特异T细胞应答的动态分析结果表明，·MalE、 BCG·wt诱导的特异CD4T细胞应答存在Th1/Th2平衡转换现象，即起始阶段为Th1应答，一段时间后出现Th2应答，并逐步形成Th1/Th2混合应答。在此基础上，进一步分析了rBCG·MalE诱导针对MalE不同T细胞表位的应答规律，结果发现，随着感染与免疫过程的发展，针对MalE p68-82和p277-291的特异T细胞应答从起初的Th1应答向Th1/Th2混合

7、类型变迁，非常有趣的是，针对p100-114的特异T细胞应答呈现典型的Th1/Th2平衡转换，而针对p151-165的T细胞应答仅是Th2类型，而且在免疫后较长时间才出现。这些结果不仅进一步验证rBCG·MalE诱导的T细胞应答规律，而且还揭示MalE蛋白不同T细胞表位的功能多样性。此外，研究中证实rBCG不同表达构建产生的MalE及其表达量亦直接影响它们诱导免疫应答的质和量。这些结果亦为疫苗的分子设计提供了新思路。 关键词 重组BCG， MalE， T细胞表位， 树突细胞， 巨噬细胞， IL-12， Th1/Th2应答 Molecular and Cellula

8、r Mechanisms of T Cell ImmuneResponses Induced by Recombinant Mycobacterium bovis BCGAbstractMycobacterium bovis BCG is the most widely used vaccine all over the world, and represents one of the most promising live vectors to deliver foreign antigens to the immune system. However, until now, little

9、is known about the mechanisms of interaction between BCG or recombinant BCG(rBCG) and host immune system, and the detailed characteristics of cell-mediated immunity induced by rBCG or BCG. To address these questions, the rBCG expressing MalE protein of Escherichia coli (rBCG·MalE) was used as m

10、odel strain to study the mechanisms by which MalE protein is presented by major histocompatibility complex class (MHC )molecules to T cells, to define the functional diversity of T cell epitopes of expressed MalE from rBCG, and to demonstrate the characteristics and regulation mechanisms of Th respo

11、nses initiated by rBCG·MalE.1. Identification of T cell epitopes of MalE protein expressed by recombinant Mycobacterium bovis BCGThe epitope repertoire of expressed MalE from rBCG was analyzed in vitro by antigen presentation assay using dendritic cells (DCs) as antigen presenting cell (APC) pu

12、lsed with rBCG·MalE、 BCG·wt or purified MalE protein. Four T cell epitopes of MalE protein presented on the surface of those DCs pulsed with rBCG·MalE and purified MalE protein were recognized by CD4 T hybridomas specific for MalE peptides p68-82, p100-114, p151-165 and p277-291,respe

13、ctively, whereas DCs pulsed with BCG·wt failed to stimulate these T hybridomas. The results also showed that rBCG·MalE induced in vivo T cell proliferation and IFN- responses against MalE protein and its peptides or PPD antigen while BCG·wt only triggered responses specific for PPD. r

14、BCG·MalE functionally expressed the four T cell epitopes of MalE protein and epitope mapping from mice immunized with rBCG·MalE showed that no novel epitopes or cryptic epitopes were revealed in rBCG, and the p68-82 was immunodominant epitope and the others were subdominant epitopes. It wa

15、s further shown that BCG is one of the most promising live vectors to express and deliver foreign antigens to the immune system.2. The role of dendritic cells during the early stages of a recombinant Mycobacterium bovis BCG infection Phagocytic cells with APC functions play an important role in resi

16、stance to bacterial infection in turn that they can stimulate T cell responses enhancing bacterial clearance. Macrophages(MØ) are privileged host cells for intracellular bacteria and thus represent a large reservoir of antigenic material, but DCs are much more potent APC. Therefore, in T cell i

17、mmunity to bacteria the role of these two phagocytic cells is not clearly established. Here, we investigated in vivo the relative contribution of MØ and DC subsets to the antibacterial T cell response to Mycobacterium bovis BCG. Twelve hours after i.v. administration of rBCG·MalE, the rate

18、 of infection in the spleen was comparable for MØ and DC. However,the presence of immunogenic MalE peptides/MHC complexes was detected ex vivo on DC, but not on MØ, using T cell hybridomas specific for the MalE protein. Likewise,upregulation of CD40, B7.1 and B7.2 molecules and production

19、of IL-12 p40 following infection was only observed for DC, confirming the exclusive role of DC in stimulating T cell responses. CD8 and CD8 spleen DC were equally potent antigen presenting cells in vivo, but the production of IL-12 was mainly associated with the CD8 subset. Altogether, these data in

20、dicated that in vivo DC playeda primary role not only in acquired immunity to mycobacteria in the early times of infection, but also in innate immunity through IL-12 secretion. Strikingly, BCG bacillus survive but remain stable in number in the DC during the early stages of infection. As antigen pre

21、sentation by DC is rapidly lost, this suggests that DC may represent a hidden reservoir for mycobacteria. These results were very useful for the prevention of tuberculosis and related infections, and for the development of new vaccines based on BCG vector.3. Kinetics of T cell immune responses induc

22、ed by recombinant Mycobacterium bovis BCG expressing MalE proteinIn order to determine the characteristics of T cell responses induced by rBCG·MalE, the splenocytes from immunized mice were treated to deplete CD4 or CD8+ T cells by an immunomagnetic selection, then these cells were used to meas

23、ure the IFN- or IL-2 responses to MalE protein or its peptides in ELISPOT test.No IFN- or IL-2 responses against MalE and its peptides were detected after depletion of CD4 T cells, in contrast, high level of IFN- or IL-2 responses against MalE and its peptides were still detected after removing CD8

24、T cells. These results clearly indicated that T cell responses induced by rBCG·MalE were CD4 T cell-dependent. The results of analyzing T cell responses to MalE protein or PPD after rBCG·MalE immunization showed that initial Th1 responses to BCG or rBCG were modulated during the course of

25、infection to become a mixed Th1/Th2 responses. To verify this phenomenon of Th1/Th2 shift, we further analyzed the kinetic responses to MalE peptides after rBCG·MalE immunization. T cell responses against p68-82 and p277-291 were shift from Th1 type to Th2 type, then become a mixed Th1/Th2 resp

26、onse. Intriguingly, T cell responses to p100-114 were typical shift of Th1/Th2 responses, while only Th2 responses to p151-165 were detected at later time points.This revealed that MalE protein had functional diversity of T cell epitopes. All the results in this study suggested that the characteristics of CD4 T cell responses induced by rBCG·MalE had