Conformational variants of I-A^k MHC class II molecules carry distinct immunopeptidomes

Major histocompatibility (MHC) class II molecules can exist in two distinct conformational states based on alternative pairing of transmembrane domain GxxxG dimerization motifs (i.e., M1- and M2-paired MHC class II). M1- and M2-paired MHC class II molecules drive different levels of T cell activation and B cell signaling; consequently, differential peptide loading would impact the level of immune response elicited by various antigens/epitopes. In previous studies of a single model antigen, we show that while peptide from BCR-bound antigen is selectively loaded onto M1-paired I-A^k class II, peptide from fluid phase processing of the same antigen is loaded onto both M1- and M2-paired I-A^k. To expand this analysis, we determined the immunnopeptidomes of M1-paired vs. total I-A^k class II molecules isolated from murine B cells. By comparing the two immunopeptidomes as well as the source proteins (antigens), a picture emerges highlighting the unique access each class II conformer has to antigens from different subcellular compartments. Sequence analysis of the two immunopeptidomes suggests a high degree of similarity between the peptide binding grooves of the two class II conformers. Analysis of class II-associated invariant chain (Ii)-derived peptides reveals the robust presence of a nested set of non-CLIP peptides that associate primarily with M1-paired class II, likely outside of the canonical peptide binding groove. In total, these results further highlight the differential peptide loading of M1- vs. M2-paired MHC class II molecules and support the idea that differential peptide loading could impact overall immune responsiveness.