B lymphocytes develop from hematopoietic stem cells in a highly regulated series of steps, characterized by ordered rearrangements of immunoglobulin (Ig) genes. These rearrangements are required for subsequent progression to the next developmental stage. In particular, B cell precursors arrest at an S7+ c-kit+ CD2+ stage if they are unable to make a functional membrane mu heavy chain protein. Although these pre-B cells have not yet rearranged their Ig light chain genes, they do express two proteins, called gamma5 and Vpre=B1, which serve as surrogate light chains to combine with mu chain and forms an Ig-type structure. Mice deficient for gamma5 expression also exhibit a block in B cell development, although it is not as severe. Thus, it has been proposed that this pre-B cell form of membrane Ig sends a signal to induce developmental progression from the S7+ c-kit+ CD2+ stage to the subsequent S7+ c-Kit+ stage. The proposed experiments will test this hypothesis. We have recently developed a system for culturing pre-B cells in vitro and efficiently introducing proteins into them with retroviral vectors.
In Aim 1, we shall determine whether introducing mu heavy chain into cultured Rag1-deficient pre=B cells will induce developmental maturation, as expected. Next, the structural requirements for mu chain to have developmental function will be determined. Mutant forms of muchain with characterized associations with the Ig-alpha/Ig-beta accessory proteins and with characterized signaling properties will be tested for developmental function. If these studies support eh hypothesis that the signaling function of mu chain is important for developmental progression, then in Aim 2 we shall tests the abilities of various chimeric proteins that have signaling regions of lg-alpha or Ig- beta grafted onto them. In particular, we hope to be able to introduce into the pre-B cells chimeric molecules with developmental function that is activated by exogenous crosslinking. Such molecules will allow us to study the signaling events activated in the pre-B cell.
In Aims 3 and 4, alterations in signaling components will be analyzed for their effects on B cell development. Dr. Lowell' Project 3 in this SCOR application will generate Lyn-deficient mice. Given the likely role of Lyn in membrane Ig signaling, the properties of pre-B cells from these mice will be quite interesting and will be analyzed in this project. These cells will be cultured and, if they exhibit may defect in development, we shall complement that by introducing wild type and altered forms of Lyn. In addition, we shall introduce a variety of dominant negative mutant forms of signaling components (including Lyn, Syk, and Ras) into cultured pre-B to examine their effect on mu heavey chain-induced developmental maturation.
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