The goal of this project is to determine whether two newly described murine Major Histocompatibility Complex class II genes, A beta 2 and E beta 2, encode for cell surface proteins and to determine the roles of such molecules. First, a cDNA library will be constructed from size selected LK35.2 RNA and cDNAs for the A beta 2 and E beta 2 genes will be cloned and sequenced. Based on this, peptides corresponding to the intracytoplasmic portions of these beta chains will be synthesized and used to immunize rabbits in order to raise anti-peptide antisera capable of detecting the A beta 2 ad E beta 2 molecules. To guide the use of this antisera, first the cellular range of transcription of A beta 2 and E beta 2 in both resting and stimulated cells and their expression during ontogeny will be determine by Northern blot analysis and by in situ hybridization. This will help determine which cells should be analyzed for expression of A beta 2 and E beta 2 molecules, will provide clues to the function of A beta 2 and E beta 2, and may extend our understanding of the control of class II gene transcription. Second, we will determine if A beta 2 and E beta 2 are capable of cell surface expression with one of the known alpha chains and if anti-alpha monocolonal antibodies can detect such molecules by analyzing L cells cotransfected with recombinant A beta 2 and E beta 2 genes and various alpha genes. Subsequently, the antisera raised will be used in immunoprecipitation experiments to biochemically analyze the products of the A beta 2 and E beta 2 genes, determine if these polypeptides achieve cell surface expression, and determine the identity of their partner alpha chain(s) (if any). In addition, reagents capable of directly reacting with cell surface A beta 2 and E beta 2 molecules will be raised by immunizing rats or hamsters with transfectants expressing these molecules on their surface or normal cells expressing high levels of A beta 2 and E beta 2. Immune spleen cells will be fused to the mouse myeloma SP2/0 and monoclonal antibodies will be screened by ELISA or flow cytometry for those reactive with the immunizing cells but not with control cells which express conventional class II molecules but not A beta 2 and E beta 2. Finally, to begin investigating the potential function of these molecules, L cell transfectants, normal cells, or tumor cells expressing the A beta 2 and E beta 2 chains (paired with the appropriate alpha chain) will be used to determine if T cells expressing alpha beta or gamma delta receptor molecules can be directly activated by A beta 2 and/or E beta 2-expressing cells or if A beta 2 and E beta 2 have an accessory function in the activation of T cells. The monoclonal antibodies raised to A beta and E beta 2 will be used to try to inhibit such responses. While it is difficult to predict the results of these experiments, the previous demonstration of several unique properties of the A beta 2 and E beta 2 genes and the conservation of homologous genes in man suggests that whatever their role, it is likely to be significant.
