Coincident Antigen Processing Pathways Feed M1 and M2 MHC Class II Conformers

总结： 目前外源性抗原加工的主流观点假定有一个蛋白酶加工池 内体内的外源性抗原，具有结构均一的MHC II类（MHCII）分子 随机捕获所得肽。然而，这种观点至少在两个层面上过于简单化。第一、 包括我们自己在内的多个实验室的工作表明，MHCII分子存在于两种不同的 基于跨膜结构域GxxxG二聚化基序的交替配对的构象状态（即， M1和M2配对MHCII），这两种MHCII构象异构体具有不同的生物学和免疫学特性， 特性.第二，我们已经报道了在B淋巴细胞中M1和M2配对的MHCII分子， 选择性地装载来自不同外源抗原库的肽。M1 MHCII装载有 源自B细胞受体（BCR）结合的同源抗原的肽，而源自非同源抗原的肽 将抗原装载到M1和M2 MHCII上。新的初步数据还显示， 与每个构象异构体结合的分子都是独一无二的。由于M1和M2肽-MHCII的接合 驱动不同水平的B和T细胞活化，不同的MHCII构象肽加载将影响 免疫反应的焦点、强度和质量。 这一框架导致了这样的假设，即在抗原呈递细胞内，存在两个重合的 但是导致选择性肽装载到M1上的外源抗原加工的不同途径， M2配对MHCII分子。本提案的目标是明确外源性抗原的两种途径 加工，以供给两种MHCII构象异构体。这一目标将通过完成两个具体目标来实现。 第一个目的的目的是定义/分析分离自MHCII的M1和M2配对的肽组。 静息和抗原脉冲B细胞。该信息将指向M1的亚细胞加工位点 vs. M2 MHCII-结合肽通过定义每个MHCII构象异构体的源抗原库。结果 还将揭示抗原靶向以及BCR信号诱导的抗原变化的影响 M1和M2 MHCII肽体上的加工途径。第二个目标的目标是：1）采取 分子遗传学方法来确定MHCII肽加载复合物（PLC）的结构和动力学 其选择性地将来自BCR结合的同源抗原的肽装载到M1 MHCII上，以及2）使用发现- 风格的方法来识别和表征新的PLC组件。 这些研究的潜在影响是多方面的。例如，结果将揭示抗原的形式 (e.g.,疫苗）将影响M1和M2 MHCII肽负载的水平 从而影响随后的免疫激活水平。在治疗上，选择性干扰M1的能力 或M2 MHCII限制性抗原呈递可能导致新的治疗方法， 控制/阻断自身免疫，同时使对病原体的免疫应答相对不受损害。

Summary: The current mainstream view of exogenous antigen processing postulates a pool of proteases processing exogenous antigen within the endosome, with structurally homogeneous MHC class II (MHCII) molecules randomly capturing resultant peptides. However, this view is an oversimplification on at least two levels. First, work from multiple laboratories including our own has shown that MHCII molecules exist in two distinct conformational states based on alternative pairing of transmembrane domain GxxxG dimerization motifs (i.e., M1 and M2 paired MHCII) and that these two MHCII conformers have distinct biological and immunological properties. Second, we have reported that in B lymphocytes M1 and M2 paired MHCII molecules are selectively loaded with peptide derived from different pools of exogenous antigen. M1 MHCII is loaded with peptide derived from B cell receptor (BCR)-bound cognate antigen, whereas peptide derived from non-cognate antigen is loaded onto both M1 and M2 MHCII. New preliminary data also reveals that ~50% of the peptides bound to each conformer are unique to that set of molecules. As engagement of M1 and M2 peptide-MHCII drive different levels of B and T cell activation, differential MHCII conformer peptide loading will impact the focus, strength and quality of an immune response. This framework leads to the hypothesis that within an antigen presenting cells, there are two coincident but distinct pathways of exogenous antigen processing that leads to selective peptide loading onto M1 versus M2 paired MHCII molecules. The goal of this proposal is to define the two pathways of exogenous antigen processing that feed the two MHCII conformers. This goal will be attained by completing two Specific Aims. The objective of the first aim is to define/analyze the peptidomes of M1 and M2 paired MHCII isolated from both resting and antigen-pulsed B cells. This information will point to the subcellular sites of processing for M1 vs. M2 MHCII-bound peptides by defining the pool of source antigens for each MHCII conformer. The results will also reveal the impact of antigen targeting as well as BCR signaling-induced changes in the antigen processing pathway on the M1 and M2 MHCII peptidomes. The objectives of the second aim are to; 1) take a molecular genetics approach to define the structure and dynamics of the MHCII peptide loading complex (PLC) that selectively charges peptides from BCR-bound cognate antigen onto M1 MHCII and 2) to use a discovery- style approach to identify and characterize new PLC components. The potential impact of these studies is many-fold. For example, results will reveal how the form of antigen (e.g., vaccine) provided to the immune system would impact the levels of M1 and M2 MHCII peptide loading and thus the level of subsequent immune activation. Therapeutically, the ability to selectively interfere with M1 or M2 MHCII-restricted antigen presentation may lead to new therapeutic approaches to selectively control/block autoimmunity, while leaving the immune response to pathogens comparatively unimpaired.