Switching antigens by the exchange catalyst HLA-DM
Understanding of the intracellular processes that tailor the MHC class II bound peptide repertoire for CD4 T cell surveillance is pivotal for understanding cellular adaptive immunity and serves as a basis for manipulation of T cell responses in the context of infectious diseases, autoimmunity and cancer. While the principal players of antigen processing within antigen-presenting cells (APC) have been identified, the interplay of molecular events that lead to surface display of a subset of proteolytically processed protein antigens, the MHCII immunopeptidome, has become the subject of intense research. Efficient peptide loading and exchange in the late endosome is one of the major events that shifts the antigenic repertoire towards high-affinity ligands, leading to potentially immunodominant epitopes. At the acidic pH of this compartment, protease activity is tuned and the exchange catalyst HLA-DM is activated. HLA-DM then promotes the exchange of the MHCII bound placeholder class II-associated invariant chain peptide (CLIP) against higher affine antigenic peptides and also the flipping of CLIP in the MHCII binding groove. As a result, the immunopeptidomes presented by cells that experience high levels of DM activity are very different from those presented in its absence. However, how a multitude of biophysical parameters, including peptide abundance and affinity, endosomal pH and redox potential, as well as localization and molecular regulators of HLA-DM shape the resulting immunopeptidome remains incompletely understood. The molecular switches that control or that are the consequence of MHCII peptide exchange are the topic of this research grant. These involve (i) the pH and redox control of HLA-DM catalyzed peptide exchange (ii) the orientational flipping of MHCII-bound CLIP and other antigenic peptides, and (iii) the regulation of HLA-DM via the non-canonical MHCII molecule HLA-DO. We will combine detailed mechanistic and kinetic investigations in an in vitro reconstitution system with model antigens with the probing of full peptide repertoires of intact cells by mass spectrometry. Mathematical modelling of peptide exchange will play a pivotal role for integrating the biophysical and biochemical data, generating mechanistic hypotheses and designing experiments. Our results will help understand how antigen-presenting cells tune endosomal peptide exchange to control their MHCII immunopeptidomes.