Mycobacterial antigen compartmentalization & immunity

结核病仍然是现代世界面临的最重大的公共卫生挑战之一。需要改进疫苗来预防感染，需要改进免疫疗法来对抗现有或复发的疾病。更好的疫苗将由CD4+和CD8+T细胞共同诱导出协同的保护性反应。我们的假设是，CD4+和CD8+T细胞以不同的方式采样不同的细菌隔间。关于哪个隔室最适合有效呈现ClassII和ClassI表位以及产生保护性反应的信息，将有助于创造更好的疫苗。为了做到这一点，我们将使用一种新的方法，同时建立在以前实验系统的优势之上。TCR转基因小鼠将感染重组牛分枝杆菌卡介苗(BCG)，即目前的疫苗株。这些重组卡介苗将在位于细菌不同亚细胞室的相同融合蛋白的背景下表达T细胞表位。将分别使用淋巴脉络膜脑膜炎病毒(LCMV)gp33肽或鸽子细胞色素C肽(PCC)构建一系列平行的rBCG菌株，用于I类或II类呈现。我们将研究每个表位对其各自呈递途径的访问是如何受到其在不同细菌间隔中的位置的影响的。随后，我们将利用过继转移的抗原特异性T细胞，系统地研究不同rBCG诱导的肝肉芽肿中抗原特异性细胞的激活和募集。我们的最终分析将是研究当抗原被呈现在不同的细菌室时，在有或没有先前的多肽特异性免疫的情况下，菌血症是如何影响的。通过这种方式，我们希望确定不同细菌隔间中的不同表位如何影响T细胞的反应和保护。我们选择了PCC(CD4+特异性)和gp33(CD8+特异性)进行这项工作，因为它们都是被广泛研究的模式抗原，并且有多种试剂可用，包括T细胞克隆、杂交瘤、TCR转基因小鼠和MHC四聚体试剂。选择卡介苗感染小鼠模型是因为我们希望提高这种减毒株的疫苗能力，也因为卡介苗感染小鼠已经得到了广泛的应用，而且这种模型的许多特点已经被很好地了解了。这项提议的实验结果应该与改进预防结核病的疫苗设计直接相关，并将提供关于细菌抗原如何进入不同的抗原提呈途径的知识。

Tuberculosis remains one of the most significant public health challenges the modern world faces. Improved vaccines are needed for prevention of infection and improved immunotherapies are needed to combat existing or recurring disease. An better vaccine would induce a concerted protective response by CD4+ and CD8+ T cells together. Our hypothesis is that CD4+ and CD8+ T cells sample different bacterial compartments differently. Information about which compartment is optimal for effective presentation of ClassII and Class I epitopes and generation of protective responses will help create better vaccines. To do this we will use a novel approach while building on the strengths of previous experimental systems. TCR transgenic mice will be infected with recombinant Mycobacterium bovis strain bacille Calmette Guerin (BCG), the current vaccine strain. These rBCG will express T cell epitopes in the context of the same fusion proteins located in different subcellular compartments of the bacteria. A parallel series of rBCG strains will be constructed for both class I or class II presentation using either Lymphochoriomeningitis Virus (LCMV) gp33 peptide or pigeon cytochrome C peptide (PCC) respectively. We will study how access of each epitope to its respective presentation pathway is influenced by its location in different bacterial compartments. Subsequently, we will study the activation and recruitment of antigen specific cells both systemically and in the BCG induced liver granulomas in response to various rBCG using adoptively transferred antigen specific T cells. Our final analysis will be to study how bacteremia is effected when antigen is presented in different bacterial compartments with or without prior peptide specific immunization. In this manner we hope to define how the different epitopes in different bacterial compartments effect T cell responses and protection. We chose PCC (CD4+ specific) and gp33 (CD8+ specific) for this work because they are both widely studied model antigens and a multitude of reagents are available, including T cell clones, hybridomas, TCR transgenic mice, and MHC tetramer reagents. The mouse model of BCG infection was chosen because we wish to improve the vaccine capacity of this attenuated strain and also because infection of mice with BCG has been widely employed and many of the characteristics of this model are well understood. The experimental results from this proposal should have direct relevance to improving vaccine design for protection against tuberculosis, and will also provide knowledge about how bacterial antigen access different antigen presenting pathways.