論文題目重組卡介苗誘導T細胞免疫應答的分子、細胞機理研究

1、論文題目：重組卡介苗誘導T細胞免疫應答的分子、細胞機理研究 作者簡介：焦新安，男，1964年09月出生，1997年02月師從于揚州大學劉秀梵教授，于1999年12月獲博士學位。  摘 要牛結核分枝桿菌BCG是世界上廣泛使用的疫苗，亦被認為是表達與傳遞外源抗原的理想活載體之一，然而迄今對BCG或重組BCG(rBCG)與宿主免疫系統(tǒng)相互作用機理尚未闡明，而從免疫調節(jié)生物學角度探討B(tài)CG或rBCG誘導T細胞應答的規(guī)律亦未涉足。為此，本研究以表達大腸桿菌麥芽糖結合蛋白(MalE)的rBCG (rBCG·MalE)為模型，以期闡明rBCG與抗原提呈細胞相互作用的規(guī)

2、律、rBCG表達產(chǎn)物抗原表位多樣性、rBCG和BCG誘導的T細胞應答規(guī)律與調控因素。1重組卡介苗表達的MalE蛋白T細胞表位的鑒定在體外抗原提呈試驗中，證實rBCG·MalE成功表達MalE蛋白的四種T細胞抗原表位，即p68-82、p100-114、p151-165和p277-291。T細胞增殖試驗和ELISPOT試驗結果均表明這種表達是功能性的，rBCG·MalE可刺激小鼠產(chǎn)生MalE蛋白及其多肽特異的T細胞增殖和IFN-應答。表位作圖顯示，BCG表達的MalE未形成新的抗原表位或使隱蔽表位暴露出來，并進一步證實MalE p68-82多肽表位是MalE H-2d限制性主要

3、T細胞表位，而p100-114、p151-165和p277-291多肽表位為次要表位。本研究結果表明，作為表達和運送外源抗原的BCG載體，它功能性地表達了外源抗原MalE蛋白，并能向宿主免疫系統(tǒng)有效地傳遞，這進一步證明BCG是一種優(yōu)良的疫苗載體。2重組卡介苗感染早期樹突細胞的作用具有抗原提呈細胞(APC)功能的吞噬性細胞，通過刺激T細胞應答可促進細菌清除，從而在抵抗細菌感染中起重要作用。巨噬細胞(MØ)是胞內(nèi)寄生菌的優(yōu)選宿主細胞，并貯存大量的抗原物質，而樹突細胞(DC)卻是非常強勢的APC。然而在體內(nèi)針對細菌的T細胞應答中，這兩種有吞噬活性的細胞之作用還未闡明。為此，本研究以rBCG

4、·MalE為材料，對MØ和DC誘導抵抗細菌的T細胞應答中的相對作用進行了探討。在小鼠i.v.接種rBCG·MalE 12小時后，脾臟MØ和DC的感染率相當，但在來自體內(nèi)的體外試驗(Ex vivo)中，用針對MalE蛋白的特異T細胞雜交瘤僅測出DCS表面存在免疫原性的MalE多肽/MHC復合物，而MØ卻沒有。同樣，在rBCG·MalE感染后，僅在DCs觀察到CD40、 B7.1(CD80)和B7.2(CD86)分子表達水平升高，并分泌IL-12 p40，因而首次證實DC在激發(fā)針對分枝桿菌T細胞應答中的關鍵作用。進一步試驗還顯示，脾臟DC

5、 CD8和CD8亞群在體內(nèi)是相同勢能的APC，但IL-12的產(chǎn)生主要來自CD8亞群。這表明在rBCG感染早期，DC在體內(nèi)條件下不僅在針對分枝桿菌獲得性免疫中發(fā)揮主導作用，而且還通過分泌IL-12在自然免疫中起重要作用。研究中還首次發(fā)現(xiàn)，rBCG在其感染早期能在DC中存活，但數(shù)量沒有增長。鑒于DC提呈rBCG抗原能力的迅速喪失，表明除MØ外DC亦是rBCG或BCG 感染早期的貯存宿主細胞。這些結果為BCG感染與免疫機理提供了新認識，對結核病控制有重要價值，同時，為以BCG作載體研制新型疫苗提供了重要理論依據(jù)。3表達MalE重組卡介苗誘導T細胞應答的動力學應用免疫磁性分離技術去除免疫小鼠

6、脾臟中CD4和CD8T 細胞后，在ELISPOT試驗中證實，rBCG·MalE誘導的T細胞應答是CD4 T 細胞依賴的。對MalE、 PPD特異T細胞應答的動態(tài)分析結果表明，·MalE、 BCG·wt誘導的特異CD4T細胞應答存在Th1/Th2平衡轉換現(xiàn)象，即起始階段為Th1應答，一段時間后出現(xiàn)Th2應答，并逐步形成Th1/Th2混合應答。在此基礎上，進一步分析了rBCG·MalE誘導針對MalE不同T細胞表位的應答規(guī)律，結果發(fā)現(xiàn)，隨著感染與免疫過程的發(fā)展，針對MalE p68-82和p277-291的特異T細胞應答從起初的Th1應答向Th1/Th2混合

7、類型變遷，非常有趣的是，針對p100-114的特異T細胞應答呈現(xiàn)典型的Th1/Th2平衡轉換，而針對p151-165的T細胞應答僅是Th2類型，而且在免疫后較長時間才出現(xiàn)。這些結果不僅進一步驗證rBCG·MalE誘導的T細胞應答規(guī)律，而且還揭示MalE蛋白不同T細胞表位的功能多樣性。此外，研究中證實rBCG不同表達構建產(chǎn)生的MalE及其表達量亦直接影響它們誘導免疫應答的質和量。這些結果亦為疫苗的分子設計提供了新思路。 關鍵詞 重組BCG， MalE， T細胞表位， 樹突細胞， 巨噬細胞， IL-12， Th1/Th2應答 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