Elucidation of the role of 2 novel cross presentation genes

 描述(申请人提供)：树突状细胞(DC)和巨噬细胞能够通过一种称为交叉呈现的过程从外部抗原获得并展示多肽。这一过程是允许免疫系统识别并动员CD8 T细胞对癌症、许多病毒感染和细胞内病原体做出反应的关键机制。因此，这一途径对于免疫监测是重要的，也是使疫苗能够诱导CD8 T细胞免疫的一个有吸引力的靶点。有鉴于此，重要的是阐明TE机制，使发展中国家能够履行这一关键职能，这也是这笔赠款的总体目标。为此，我们建议表征和阐明两个关键的和先前未被怀疑的新的交叉呈现基因的功能，这两个基因是我们在无偏见的全基因组正向遗传筛查中发现的。我们的第一个目标是阐明Rab39A在交叉呈现中的作用。非常有趣的是，虽然交叉演示需要Rab39A，但传统的MHC I或MHC II演示不需要。RAB蛋白是一种GTP酶，它控制囊泡在细胞内的形成、含量、运输和最终融合，从而决定内细胞室的组成。我们支持的假设是，Rab39A通过运输XPT途径的基本成分，在DC中帮助形成一个专门的XPT隔室。此外，鉴于RABS及其招募的效应器在确定囊泡组成方面的作用，它们可以作为标记来识别和分离独特的内吞隔室(例如，早期内小体、晚期内小体、溶酶体、黑素体等)。在这种情况下，非常有趣的是，Rab39A只存在于DC中的吞噬小体的一部分中。鉴于此，我们还假设Rab39A将标记XPT隔间。这些假说的重要性在于，Rab39A可以解开MHC I和/或内质网其他成分如何到达吞噬小体的谜团；澄清XPT在哪里进行及其与其他途径的关系，如MHC II；和/或允许对Rab39A隔室的特性来确定途径中的新成分。这一目的将检验这些假说，并更广泛地探索和阐明Rab39A在XPT中的作用。我们的第二个目标是阐明Loc547349(Loc)在交叉呈现中的作用。我们的初步数据显示，这种分子是交叉递呈所必需的，而不是经典的MHC I或MHC II抗原递呈所必需的。非常有趣的是，Loc是一种功能未知的MHC编码的MHC I类分子。这一发现非常耐人寻味，因为MHC I和MHC II抗原呈递的许多关键基因都在MHC基因座上编码。此外，还有一个类似的分子，HLA-DM，在MHC II途径中作为肽编辑，到目前为止在MHC I途径中还没有明确的对应分子，但理论上需要这样的活性来交叉呈现。我们支持的假设是，Loc在交叉呈现中发挥作用，帮助MHC I分子运输到胞内室，稳定它们，和/或将多肽加载到它们。这一目的的目的是检验这一假说以及其他潜在功能，并阐明Loc在交叉呈现中的作用。

 DESCRIPTION (provided by applicant): Dendritic cells (DCs) and macrophages are capable of acquiring and then displaying peptides from external antigens through a process called cross presentation. This process is the key mechanism that allows the immune system to recognize and then mobilize a CD8 T cell response to cancers, many viral infections and intracellular pathogens. Consequently, this pathway is important for immune surveillance and is an attractive target to enable vaccines to elicit CD8 T cell immunity. Given this, it is important to elucidate te mechanisms that allow DCs to carry out this critical function and this is the overall goal of this grant. To this end, we propose to characterize and elucidate the function of two key and previously unsuspected novel cross presentation genes that we discovered in an unbiased, genome-wide forward genetic screen. Our first Aim will elucidate the role of Rab39A in cross presentation. Very interestingly, while Rab39A is required for cross presentation but not conventional MHC I or MHC II presentation. Rab proteins are GTPases that control the formation, content, trafficking and ultimate fusion of vesicles in cells and thereby determine the composition of endocytic compartments. Our favored hypothesis is that Rab39A is functioning to help form a specialized XPT compartment in DCs by trafficking essential components of the XPT pathway. Furthermore, given their role in determining the composition of vesicles, Rabs and their recruited effectors serve as markers to identify and isolate unique endocytic compartments (e.g. early endosomes, late endosomes, lysosomes, melanosomes, etc.). In this context, and very interestingly, Rab39A is only present in a subset of phagosomes in DCs. Given this, we also hypothesize that Rab39A will mark the XPT compartment. The importance of these hypotheses is that Rab39A could solve the mystery as to how MHC I and/or other components from the ER get to phagosomes; clarify where XPT is carried out and its relationship to other pathways such as MHC II; and/or allow the characterization of the Rab39A+ compartment to identify novel components in the pathway. This Aim will test these hypotheses and more generally explore and elucidate the role of Rab39A in XPT. Our second Aim will elucidate the role of Loc547349 (Loc) in cross presentation. Our preliminary data show that this molecule is required for cross presentation but not classical MHC I or MHC II antigen presentation. Very interestingly, Loc is an MHC-encoded MHC I-like molecule of unknown function. This discovery is very intriguing because many of the key genes for MHC I and MHC II antigen presentation are encoded in the MHC locus. Moreover, there is a similar molecule, HLA-DM, that operates as a peptide editor in the MHC II pathway and for which up until now has not had a clear counterpart in the MHC I pathway, yet there is a theoretical need for such an activity for cross presentation. Our favored hypotheses are that Loc functions in cross presentation to help traffic MHC I molecules to endocytic compartments, stabilize them, and/or load them with peptides. The goal of this aim is to test this hypothesis as well as other potential functions and elucidate the role of Loc in cross presentation.