The goal of this proposal is to define mechanisms that regulate V(D)J recombination in the earliest B lymphocyte precursors. The continual production of B cells in the bone marrow (BM) is essential for the proper function of the immune system, and BM stromal cell factors are critical to support this production. The initiation of V(D)J recombination of immunoglobulin (Ig) genes is a limiting step in B cell development, and efficient recombination is required to generate the immense diversity of Igs needed to combat an ever expanding number of pathogens. V(D)J recombination is strictly regulated: in particular, expression of V(D)J recombinase components RAG1 and RAG2 is tightly controlled. We hypothesize that extrinsic signals from stromal cells such as the cytokine IL-7 regulate the recombinase in the earliest B progenitors through as yet undefined mechansisms.
Specific Aim 1 is to determine the mechanism by the IL7R:JAK3:pSTAT5 pathway regulates the V(D)J recombinase. We detected V(D)J recombinase activity in uncommitted common lymphoid progenitors (CLPs) but not in upstream progenitors. Therefore, we hypothesize that signals to the - CLP mediate critical control mechanisms for recombinase activity. We have demonstrated that the essential kinase in the IL-7 pathway, JAK3, is required for recombinase activity in CLPs. pSTATS is the transcription factor activated by JAK3, and therefore we will investigate the mechanisms by which STATS may regulate recombinase activity, including direct regulation of rag gene expression.
Specific Aim 2 is to define the specific role of the transcription factor E47 in the regulation of recombinase activity and investigate regulation of E47. We demonstrated that E47 KO mice lack recombinase activity in CLPs, and will now determine if this reflects direct regulation of expression of both ragl and rag2. Flt3 is the tyrosine kinase receptor for flt3- ligand;because both flt3- and E47-deficient mice have reduced numbers of CLPs, we will examine the role of flt3 in the regulation of E47 expression. >:.; Relevance to human health: IL-7, FL and their receptors are well-conserved between mice and humans.,., . Thus, findings using murine models should aid investigations aimed at generating new human lymphocytes for therapeutic purposes including transplantation, recovery from chemotherapy, gene therapy, curing , inherited and acquired immunodeficiencies, and promoting new B cell development in aging individuals.
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