NEGATIVE SELECTION AT THE PRO-B TO PRE-B CELL TRANSITION

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DESCRIPTION (provided by applicant): Preliminary studies have identified an unusual group of human mu heavy chains that can be expressed on the cell surface as part of a pre-BCR in the absence of surrogate or conventional light chains. These aberrant mu heavy chains, which were first identified in a patient with a defect in pre-BCR signaling, appear to be able to activate cells and induce rearrangement of light chain genes but they do not promote expansion or long-term survival of B-cell precursors. The light chain independent chains are of the expected molecular weight and have intact VH and CH1 domains. Transcripts for these muheavy chains can be found in both the VH3 and VH4 families. They constitute approximately 10 percent of the rearranged VH3 muheavy chain genes in normal human pro-B-cells; however, they are not found in normal pre-B-cells or mature B-cells. The proposed experiments will biochemically characterize these aberrant heavy chains and examine the responses elicited by them. The specific aims are 1) determine the particular sequences within the V(D)J rearrangement that allow cell surface expression or secretion in the absence of light chains. The binding of the aberrant muheavy chains to the ER chaperone BiP will also be examined; 2) use two culture systems, retroviral infection of RAG2 deficient pro-B-cells and a tetracycline inducible reconstitution of the BCR in Jurkat T-cells, to evaluate the effects of expression of the aberrant muheavy chain on cell surface expression of activation markers, alterations in phosphorylation of signal transduction molecules and shifts in the amount of transcripts that characterize the pro-B-cell or pre-B-cell stage of differentiation; and 3) produce transgenic mice expressing the normal or aberrant mu heavy chain and characterize B-cell development in these mice. The transgenic mice will be crossed with mice that are null or transgenic for signal transduction proteins or molecules involved in apoptosis. A better understanding of the biochemical nature of these unusual mu heavy chains and the mechanisms by which they are removed should elucidate requirements for the normal pro-B-cell to pre-B-cell transition. The proposed studies will also provide valuable information about the development of the normal and abnormal antibody repertoire.