Regulation of B Cell Development by Ras and Raf

Farrar, Michael

Abstract

B cells play an essential role in the establishment of a functional immune response against bacteria and viruses. Therefore, the process of B cell development must produce a population of mature B cells with both sufficient diversity of antigen receptors and sufficient numbers of cells. In order to mount an effective defense against the vast array of potential infectious agents, signals entrained by the Ras/Raf pathway regulate early B cell differentiation at the earliest pre-pro-B stage, in essence determining the number of cells that proceed down this developmental pathway. However, the upstream activators and downstream effectors of this pathway remain undefined. Our hypothesis is that the Ras/Raf pathway is activated by the interleukln-7 or flk2/flt3 receptors, and that it affects downstream responses by altering the expression of transcriptional regulators, specifically members of the Id- and Eta-gene families. Thus the aims of this application are: (1) To determine whether the IL7R or flk2/flt3 entrain early B cell development via Ras activation, (2) To determine whether Ras/Raf-dependent early B cell differentiation involves regulation of Id- or Ets-family proteins, and (3) To identify Ras/Raf-dependent transcription factors involved in regulating pre-pro-B cell differentiation. A number of strategies will be utilized to achieve these goals. First, biochemical approaches will be used to determine whether either lL7 or flt3L can activate the Ras/Raf pathway in pro-B cell lines or in ex vivo pre-pro-B cells. Second, genetic approaches that involve transgenic mice which express constitutively active forms of Raf (Raf-CAAX) will be used to determine whether forced Ref activation can rescue B cell development In IL7R- or flk2/flt3-deflcient mice. Third, transgenic mice which express either dominant negative Ras or constitutively active Rat in developing B cells will be utilized to determine whether this pathway regulates expression of Id-or Ets-family members. These experiments will be extended using inducible approaches for activating the Ref pathway in vivo. Specifically, these include (I) chemical induced dimerization of Raf-GyrB fusion proteins utilizing the symmetrically dimeric small molecule coumermycin, and (ii) doxycycline-regulated transcription of activated forms of Raf (Raf-CAAX). Finally, gene microarray technology will be applied, in conjunction with the mouse mutants described above, to identify novel targets of the Ras/Raf pathway involved in pre-pro-B cell differentiation. These studies should help to illuminate the molecular mechanisms by which the Ras pathway regulates B cell differentiation. The importance of clarifying this process has been highlighted recently by the observation that B cell reconstitution following bone marrow transplantation frequently proceeds poorly, resulting in patient susceptibility to a number of bacterial pathogens. Thus a better understanding of early B cell development should prove useful for optimizing bone marrow transplantation protocols in the future.