論文題目重組卡介苗誘導(dǎo)T細(xì)胞免疫應(yīng)答的分子、細(xì)胞機(jī)理研究

1、論文題目：重組卡介苗誘導(dǎo)T細(xì)胞免疫應(yīng)答的分子、細(xì)胞機(jī)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(yáng)州大學(xué)劉秀梵教授，于1999年12月獲博士學(xué)位。  摘 要牛結(jié)核分枝桿菌BCG是世界上廣泛使用的疫苗，亦被認(rèn)為是表達(dá)與傳遞外源抗原的理想活載體之一，然而迄今對(duì)BCG或重組BCG(rBCG)與宿主免疫系統(tǒng)相互作用機(jī)理尚未闡明，而從免疫調(diào)節(jié)生物學(xué)角度探討B(tài)CG或rBCG誘導(dǎo)T細(xì)胞應(yīng)答的規(guī)律亦未涉足。為此，本研究以表達(dá)大腸桿菌麥芽糖結(jié)合蛋白(MalE)的rBCG (rBCG·MalE)為模型，以期闡明rBCG與抗原提呈細(xì)胞相互作用的規(guī)

2、律、rBCG表達(dá)產(chǎn)物抗原表位多樣性、rBCG和BCG誘導(dǎo)的T細(xì)胞應(yīng)答規(guī)律與調(diào)控因素。1重組卡介苗表達(dá)的MalE蛋白T細(xì)胞表位的鑒定在體外抗原提呈試驗(yàn)中，證實(shí)rBCG·MalE成功表達(dá)MalE蛋白的四種T細(xì)胞抗原表位，即p68-82、p100-114、p151-165和p277-291。T細(xì)胞增殖試驗(yàn)和ELISPOT試驗(yàn)結(jié)果均表明這種表達(dá)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(xì)胞增殖和IFN-應(yīng)答。表位作圖顯示，BCG表達(dá)的MalE未形成新的抗原表位或使隱蔽表位暴露出來，并進(jìn)一步證實(shí)MalE p68-82多肽表位是MalE H-2d限制性主要

3、T細(xì)胞表位，而p100-114、p151-165和p277-291多肽表位為次要表位。本研究結(jié)果表明，作為表達(dá)和運(yùn)送外源抗原的BCG載體，它功能性地表達(dá)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(jìn)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(xì)胞的作用具有抗原提呈細(xì)胞(APC)功能的吞噬性細(xì)胞，通過刺激T細(xì)胞應(yīng)答可促進(jìn)細(xì)菌清除，從而在抵抗細(xì)菌感染中起重要作用。巨噬細(xì)胞(MØ)是胞內(nèi)寄生菌的優(yōu)選宿主細(xì)胞，并貯存大量的抗原物質(zhì)，而樹突細(xì)胞(DC)卻是非常強(qiáng)勢的APC。然而在體內(nèi)針對(duì)細(xì)菌的T細(xì)胞應(yīng)答中，這兩種有吞噬活性的細(xì)胞之作用還未闡明。為此，本研究以rBCG

4、·MalE為材料，對(duì)MØ和DC誘導(dǎo)抵抗細(xì)菌的T細(xì)胞應(yīng)答中的相對(duì)作用進(jìn)行了探討。在小鼠i.v.接種rBCG·MalE 12小時(shí)后，脾臟MØ和DC的感染率相當(dāng)，但在來自體內(nèi)的體外試驗(yàn)(Ex vivo)中，用針對(duì)MalE蛋白的特異T細(xì)胞雜交瘤僅測出DCS表面存在免疫原性的MalE多肽/MHC復(fù)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(dá)水平升高，并分泌IL-12 p40，因而首次證實(shí)DC在激發(fā)針對(duì)分枝桿菌T細(xì)胞應(yīng)答中的關(guān)鍵作用。進(jìn)一步試驗(yàn)還顯示，脾臟DC

5、 CD8和CD8亞群在體內(nèi)是相同勢能的APC，但I(xiàn)L-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(duì)分枝桿菌獲得性免疫中發(fā)揮主導(dǎo)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(xì)胞。這些結(jié)果為BCG感染與免疫機(jī)理提供了新認(rèn)識(shí)，對(duì)結(jié)核病控制有重要價(jià)值，同時(shí)，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(dá)MalE重組卡介苗誘導(dǎo)T細(xì)胞應(yīng)答的動(dòng)力學(xué)應(yīng)用免疫磁性分離技術(shù)去除免疫小鼠

6、脾臟中CD4和CD8T 細(xì)胞后，在ELISPOT試驗(yàn)中證實(shí)，rBCG·MalE誘導(dǎo)的T細(xì)胞應(yīng)答是CD4 T 細(xì)胞依賴的。對(duì)MalE、 PPD特異T細(xì)胞應(yīng)答的動(dòng)態(tài)分析結(jié)果表明，·MalE、 BCG·wt誘導(dǎo)的特異CD4T細(xì)胞應(yīng)答存在Th1/Th2平衡轉(zhuǎn)換現(xiàn)象，即起始階段為Th1應(yīng)答，一段時(shí)間后出現(xiàn)Th2應(yīng)答，并逐步形成Th1/Th2混合應(yīng)答。在此基礎(chǔ)上，進(jìn)一步分析了rBCG·MalE誘導(dǎo)針對(duì)MalE不同T細(xì)胞表位的應(yīng)答規(guī)律，結(jié)果發(fā)現(xiàn)，隨著感染與免疫過程的發(fā)展，針對(duì)MalE p68-82和p277-291的特異T細(xì)胞應(yīng)答從起初的Th1應(yīng)答向Th1/Th2混合

7、類型變遷，非常有趣的是，針對(duì)p100-114的特異T細(xì)胞應(yīng)答呈現(xiàn)典型的Th1/Th2平衡轉(zhuǎn)換，而針對(duì)p151-165的T細(xì)胞應(yīng)答僅是Th2類型，而且在免疫后較長時(shí)間才出現(xiàn)。這些結(jié)果不僅進(jìn)一步驗(yàn)證rBCG·MalE誘導(dǎo)的T細(xì)胞應(yīng)答規(guī)律，而且還揭示MalE蛋白不同T細(xì)胞表位的功能多樣性。此外，研究中證實(shí)rBCG不同表達(dá)構(gòu)建產(chǎn)生的MalE及其表達(dá)量亦直接影響它們誘導(dǎo)免疫應(yīng)答的質(zhì)和量。這些結(jié)果亦為疫苗的分子設(shè)計(jì)提供了新思路。 關(guān)鍵詞 重組BCG， MalE， T細(xì)胞表位， 樹突細(xì)胞， 巨噬細(xì)胞， IL-12， Th1/Th2應(yīng)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