The long range goal of this project is to understand the signals regulating antigen-specific, class II mediated differentiation of B lymphocytes. The model system is a clone of murine B lymphoma cells, CHl2.LX, which can be induced to secrete IgM in response to a known antigen plus Ek-restricted T cells or Ek-specific monoclonal antibodies (Mabs). The proposed experiments will address the detailed biological and structural requirements for the function of the class II major histocompatibility complex (MHC) molecule as a signal receptor in antigen-specific B cell differentiation. The biological requirements will be determined by: 1) Determining the optimal temporal sequence of antigen and class II-specific signals. 2) Determining the role of class II crosslinking in the delivery of B cell differentiative signals, using Mab fragments. 3) Testing the role of receptor-mediated endocytosis in the delivery of class II specific signals, using Mabs coupled to solid supports. To examine the relationship between the primary structure of the class II molecule and its B cell signaling function, CH12.LX cells will be transfected with altered A-beta genes. Stable A-beta transfectants of CH12.LX have already been made, which respond to differentiative signals delivered via the transfected molecules. A-beta mutants will be constructed using site-directed mutagenesis, site-directed truncation, and random oligonucleotide directed mutagenesis. Transfectants of CH12.LX will be tested for a differentiative response to signals delivered via altered class II molecules. The features of the primary sequence of class II which are most important to its function as a B cell receptor for differentiative signals can thus be defined. It has been shown using CH12.LX and other B lymphoma cells that differentiation to Ig secretion is incompatible with continued cell proliferation. Thus, understanding the signals which regulate this process is of vital interest to understanding how the growth of B cell lymphomas can be controlled. In addition, understanding how B cells are induced to secrete specific antibody is important to therapeutic manipulation of the antibody response in infectious and autoimmune disease.
