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

 描述(申请人提供)：通过识别外来肽(表位)和主要组织相容性复合体II类分子(MHCII)，CD4T淋巴细胞(TCD4)在对包括痘病毒在内的大多数病毒的适应性反应中发挥关键作用，这些病毒继续对世界各地的人类构成相当大的威胁。根据与标称蛋白抗原发展的惯例，MHCII结合的多肽来自内化的物质，这些物质在内吞体网中分解并加载到晚期内吞体内的新生MHCII上，随后复合体过渡到质膜。艾森洛尔实验室以前和正在进行的工作已经证明，这一经典方案对流感(流感)特异性TCD4的激活作用最小。整个病毒粒子的催化是低效的，相反，在受感染的抗原提呈细胞(APC)内合成的病毒蛋白是多肽的主要来源，通过内源性加工途径网络产生并直接呈递到流感特异性TCD4。我们最近将这项工作扩展到痘病毒，以探索这些发现的一般性。对牛痘(VACV)和皮疹(ECTV)的调查显示，情况要复杂得多。与流感一样，外源性提供的痘病毒也对加工产生抵抗力，需要病毒蛋白质的生物合成。然而，与流感不同的是，TCD4的激活似乎主要依赖于MHCII交叉呈现，在这种交叉呈现中，生物合成的抗原从感染的细胞转移到未感染的APC。这要归功于深刻的 抑制受感染细胞的直接呈现，特别是在ECTV的情况下，这是一种天然的 老鼠病原体。虽然直接干扰MHC-I类限制性抗原的处理和提呈是众所周知的，但对于MHCII来说，这种情况很少有报道，而且痘病毒抑制的程度和特异性是前所未有的。罗珀实验室建立的工作已经确定VACV和ECTV的A35基因产物是抑制作用的主要介体，而赫斯佩格实验室的初步数据表明B22基因产物是一个有效的共谋者。这项Co-PI RO1应用结合了抗原处理和提呈方面的专业知识与痘病毒生物学和发病机制方面的专业知识，将对这些初步发现进行如下扩展：1)在体外(目标1)和体内(目标2)严格检验MHCII交叉提呈是VACV和ECTV特异性TCD4应答的主要驱动因素的假说；2)确定A35和B22在直接提呈中对阻断作用的贡献，并寻找其他可能提供互补活性的痘病毒产品(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高影响领域-病毒抗原的TCD4识别，痘病毒毒力，以及病毒对MHCII抗原的处理和呈递。这些目标的实现将大大提高对痘病毒发病机制的了解和合理的疫苗设计，并可能导致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.