Development of Conformer-Specific Anti-I-A Class II mAbs

Summary Major histocompatibility (MHC) class II molecules mediate important interactions between CD4 T cells and antigen presenting cells such as dendritic cells, B cells, macrophages, thymic epithelium and certain tumors. Conventional wisdom posits that class II molecules exhibit a single conformation with a foundation of two Ig-like domains supporting a membrane distal peptide binding groove. However, work from this and other laboratories supports a more refined model of MHC class II structure where class II molecules adopt two distinct conformational states based on alternative pairing of transmembrane domains GxxxG dimerization motifs (i.e., M1- and M2-paired class II molecules). Moreover, these distinct conformers bind different sets of peptides and have different immunological functions. Advances in biomedical science can be limited by the lack of important investigative tools. Our previous work identified two mAbs, 11-5.2 and Tü36, that selectively bind M1-paired I-Ak and HLA-DR molecules, respectively. While this was an important advance, murine I-Ak molecules are not expressed by many commonly used mouse strains, limiting the ability to study I-A class II conformers using sophisticated engineered mouse strains and in vivo approaches. The goal of the proposal is to address this barrier by identifying and characterizing additional conformer-specific anti-I-A mAbs. We will identify conformer-specific anti-I-A mAbs by an established two-step approach. First, we will use sequential immunoprecipitation to identify a set of anti-I-A mAbs that recognize a subset of class II molecules. We will then use cells expressing I-A transmembrane domain GxxxG mutants impaired for conformer formation to screen this set of mAbs and identify conformer-specific anti-I-A mAbs. We will then use our established approaches to further characterize the role of mAb- recognized I-A conformers in TCR-class II interactions, class II lipid raft partitioning and MHC class II signaling. This laboratory is uniquely placed to perform these studies for two reasons. First, we previously identified and extensively characterized both of the know M1/M2 conformer-specific mAbs. Second, included preliminary data demonstrates that we have an additional conformer-specific anti-I-Ad mAb in hand. Characterization of mAbs that differentiate M1- from M2-paired I-A molecules beyond I-Ak will allow the field to test the paradigm of M1- vs. M2-paired MHC class II in a wider range of model systems such as the Balb/c or C57Bl/6 mouse strains and all of their genetically engineered derivatives.