MHCII Cross-presentation as a Driver of CD4+ T Cell Responses to Poxviruses

 描述（由申请人提供）：通过识别外源肽（表位）与主要组织相容性复合体 II 类分子 (MHCII) 的组合，CD4 T 淋巴细胞 (TCD4) 在对大多数病毒（包括痘病毒）的适应性反应中发挥着关键作用，痘病毒继续对全世界人类构成相当大的威胁。根据使用标称蛋白抗原制定的惯例，MHCII 结合肽源自内化物质，该内化物质在内吞网络中分解代谢并加载到晚期内涵体中的新生 MHCII 上，该复合物随后转移到质膜。艾森洛尔实验室之前和正在进行的工作表明，这种经典方案对流感 (flu) 特异性 TCD4 的激活贡献最小。整个病毒粒子的催化效率低下，相反，在受感染的抗原呈递细胞（APC）内合成的病毒蛋白是肽的主要来源，通过内源性加工途径网络产生并直接呈递给流感特异性TCD4。我们最近将工作扩展到痘病毒，以探索这些发现的普遍性。对牛痘 (VACV) 和 ectromelia (ECTV) 的调查表明情况要复杂得多。与流感一样，外源提供的痘病毒粒子也难以加工，因此需要病毒蛋白的生物合成。然而，与流感相比，TCD4 激活似乎主要取决于 MHCII 交叉呈递，其中生物合成的抗原从受感染的细胞转移到未受感染的 APC。这要归功于深刻的 抑制受感染细胞的直接呈递，特别是在 ECTV 的情况下，ECTV 是一种天然的 小鼠病原体。虽然直接破坏 MHC I 类限制性抗原加工和呈递是众所周知的，但很少报道 MHCII 的情况，而且痘病毒抑制的程度和特异性是前所未有的。 Roper 实验室的工作已确定 VACV 和 ECTV 的 A35 基因产物是抑制作用的主要介质，而 Hersperger 实验室的初步数据表明 B22 基因产物是一个有效的共谋者。 该 Co-PI RO1 应用将抗原加工和呈递方面的专业知识与痘病毒生物学和发病机制相结合，将扩展这些初步发现如下：1) 在体外（目标 1）和体内（目标 2）严格测试假设，即 MHCII 交叉呈递是 TCD4 对 VACV 和 ECTV 特异性 TCD4 反应的主要驱动因素，2) 确定 A35 和 B22 对直接呈递中的块的贡献并识别其他可能提供补充活性的痘病毒产品（目标 3）。 3) 确定 A35 和 B22 抑制活性的机制（目标 4）。 这些研究汇集了三个具有重大影响的领域——TCD4 对病毒抗原的识别、痘病毒毒力以及病毒对 MHCII 抗原加工和呈递的颠覆。这些目标的实现将大大增强对痘病毒发病机制和合理疫苗设计的理解，并可能带来新的实验和临床免疫调节策略。

 DESCRIPTION (provided by applicant): By recognizing foreign peptides (epitopes) in combination with Major Histocompatibility Complex class II molecules (MHCII), CD4+ T lymphocytes (TCD4+) play a critical role in the adaptive responses to most viruses, including the poxviruses, which continue to pose a considerable threat to humans worldwide. According to a convention developed with nominal protein antigens, MHCII-bound peptides are derived from internalized material that is catabolized in the endocytic network and loaded onto nascent MHCII in the late endosome, the complex subsequently transiting to the plasma membrane. Previous and ongoing work in the Eisenlohr lab has demonstrated that this classical scheme contributes minimally to the activation of influenza (flu)-specific TCD4+. Catalysis of whole virions is inefficient and, instead, viral proteins synthesized within the infected antigen- presenting cell (APC) are the primary source of peptides, generated via a network of endogenous processing pathways and directly presented to flu-specific TCD4+. We have recently extended the work to poxviruses in order to explore the generality of these findings. Investigation of vaccinia (VACV) and ectromelia (ECTV) reveals the landscape to be far more complex. As with flu, exogenously provided poxvirions are also resistant to processing, necessitating biosynthesis of viral proteins. However, in contrast to flu, TCD4+ activation appears to depend primarily upon MHCII cross-presentation, in which the biosynthesized antigen is transferred from an infected cell to an uninfected APC. This is attributable to profound inhibition of direct presentation by the infected cell, particularly in the case of ECTV, a natural mouse pathogen. While direct disruption of MHC class I-restricted antigen processing and presentation is well known, it has rarely been reported for MHCII, and the degree and specificity of inhibition by poxviruses is unprecedented. Established work by the Roper lab has identified the A35 gene product of both VACV and ECTV as a major mediator of the inhibition, and preliminary data from the Hersperger lab implicates the B22 gene product as a potent co-conspirator. This Co-PI RO1 application, which combines expertise in antigen processing and presentation with that in poxvirus biology and pathogenesis, will extend these preliminary findings as follows: 1) Rigorously test the hypothesis both in vitro (Aim 1) and in vivo (Aim 2) that MHCII cross-presentation is the principal driver of TCD4+ responses to VACV- and ECTV-specific TCD4+ responses, 2) Determine the contributions of A35 and B22 to the block in direct presentation and identify other poxviral products that might provide complementary activity (Aim 3). 3) Identify the mechanisms underlying A35 and B22 inhibitory activities (Aim 4). These studies bring together three areas of high impact - TCD4+ recognition of viral antigens, poxvirus virulence, and viral subversion of MHCII antigen processing and presentation. Achievement of the aims will substantially enhance understanding of poxvirus pathogenesis and rational vaccine design, and could lead to novel experimental and clinical immunomodulatory strategies.