Autoantibody to a cryptic determinant on erythrocytes is predominantly encoded by the germline gene VH11 together with a particular Vk9 light chain in BALB/c mice. Expression of a VH11 heavy chain transgene (Tg) results in an expanded population of autoreactive CD5+ B cells bearing endogenous Vk9 rearrangements. The overall goal of this proposal is to understand the mechanisms that act to enrich this specificity in CD5+ B cells and determine why such cells are not subject to deletion or anergy as seen in other systems. Interestingly, VH11 heavy chain in pre-B cells fails to induce expected changes in gene expression (downregulation of Rag-1/2) and migrates differently from conventional heavy chains in SDS PAGE. Because the interaction of immunoglobulin components is critical for progression of B lineage development, the investigator will ask in this study whether the expansion of VH11 Tg B cells results from a novel B cell developmental mechanism. In addition, since this specificity normally arises in B cells generated during fetal/neonatal B lymphopoiesis, we will investigate whether such interactions differ between fetal and adult development. Specifically, in Aim 1, for B cells bearing VH11 alone or as an autoreactive combination with the Vk9 light chain, we will define the precise stage in B cell development at which positive/negative selection occurs by analyzing fetal and adult sorted fractions for: VH and Jk biases, extent of endogenous Ig expression, susceptibility to apoptosis, and the importance of surrogate light chain.
In Aim 2, using mice with VH11 alone, the investigator will test the role of Ig signaling complex in creating this bias by: measuring regulation of Rag-1/2 expression, analyzing the composition of the receptor, and determining the subcellular localization of the receptor constituents. Finally, in Aim 3, in order to test the role of N-addition in biasing against this type of natural autoreactive VH gene, the investigator will determine whether VH11-encoded CD5+ B cells are generated in bone marrow from mice lacking TdT and also generate another set of Ig Tg mice bearing an N-region containing VH gene cloned from an anti-thymocyte hybridoma. The results of these studies will help to establish the types of selection that shape the expressed B cell repertoire, fetally and in the adult, with implications for modulating both normal and autoimmune responses.
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