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

摘要： 目前关于外源抗原加工的主流观点假定存在一个蛋白酶加工池。 内体内的外源抗原，具有结构上均一的MHC II类(MHCII)分子 随机捕获生成的多肽。然而，这种观点至少在两个层面上过于简单化了。第一, 包括我们自己在内的多个实验室的研究表明，MHCII分子存在于两个不同的 基于跨膜结构域GxxxG二聚基序的交替配对的构象状态(即， M1和M2配对的MHCII)，这两个MHCII构象具有不同的生物学和免疫学 属性。第二，我们已经报道了在B淋巴细胞中，配对的MHCII分子M1和M2是 选择性地负载来自不同外源抗原池的多肽。M1 MHCII加载了 来自B细胞受体(BCR)结合同源抗原的多肽，而来自非同源抗原的多肽 抗原被装载到M1和M2 MHCII上。新的初步数据还显示，~50%的多肽 结合到每个构象上的分子是那组分子所特有的。作为M1和M2多肽-MHCII的结合 驱动不同水平的B和T细胞活化，不同的MHCII构象多肽负荷会影响 免疫反应的重点、强度和质量。 这一框架导致假设，在一个抗原提呈细胞内，有两个重合的 但不同的外源抗原处理途径导致选择性多肽加载到M1上而不是 M2配对的MHCII分子。这项提议的目标是定义外源抗原的两条途径。 为两个MHCII异构体提供饲料的加工。这一目标将通过完成两个具体目标来实现。 第一个目的是定义/分析从中国分离的M1和M2配对MHCII的多肽。 静息B细胞和抗原致敏B细胞。该信息将指向M1的亚细胞处理位置 通过定义每个MHCII构象的源抗原库，比较M2MHCII结合肽。结果是 还将揭示抗原靶向的影响以及BCR信号诱导的抗原变化 M1和M2 MHCII多肽的加工途径。第二个目标的目标是；1)采取一个 分子遗传学方法确定MHCII肽负载复合体(PLC)的结构和动力学 它选择性地将来自BCR结合同源抗原的多肽带入M1 MHCII和2)利用一项发现- 识别和表征新的PLC组件的样式方法。 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如何 (例如，疫苗)提供给免疫系统会影响M1和M2 MHCII多肽负荷水平 因此，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限制性抗原提呈可能导致选择性地 控制/阻止自身免疫，同时保持对病原体的免疫反应相对不受损害。

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.