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.

Narrative MHC class II molecules exist in two distinct conformational states, which have distinct immunobiological properties and each class II conformer presents peptides derived from distinct pools of intracellular antigen. The goal of this research proposal is to define the coincident molecular pathways that lead to peptide loading onto these two distinct forms of MHC class II molecules. The results of the studies will provide important insight that will allow us to therapeutically target these pathways to augment beneficial immune responses and/or attenuate harmful responses.