In this application we seek continuing support of our studies on the developmental origins an the mechanism of repertoire selection of CD5+ B cells. Although these cells comprise only a minor subset in adult immune system, they appear to play significant roles in the neonatal immune system and in the pathogenesis of certain autoimmune disease and chronic leukemias. previously we showed that expression of CD5 during B lymphopoiesis and enrichment for certain types of autoreactivities are unique characteristics of B cells generated early in ontogeny """"""""fetal"""""""" B cells, in contrast to typical """"""""adult B cells. Here we focus on determining further how fetal B lymphopoiesis differs from that of the adult and on how autoreactive B cells might be enriched in five specific aims. We will address issues relating to distinctive developmental pathways by:
Aim 1) Determining the multilineage capacity (T/B/myeloid) of phenotypically defined subsets from fetal liver and bone marrow using single cell assays for B/myeloid and T/B potential;
and Aim 2). Identifying genes and surface proteins differentially expressed by B precursors present in fetal liver and adult bone marrow using differential display of RNA and flow cytometric selection of phage display antibody clones. We will investigate mechanisms that could result in distinctive fetal reactivity by:
Aim 3) Determining whether susceptibility to apoptosis during fetal B lymphopoiesis differs from that in adult bone marrow by using a sensitive assay for apoptosis an with particular focus on the interleukin-1 beta converting enzyme, generating transgenic mice that constitutively express high levels of this gene during fetal B lymphopoiesis;
and Aim 4) Testing the effect of VH gene usage on Pre-B proliferation and progression, determining whether this effect is less critical for fetal B lymphopoiesis by functional Pro/Pre-B proliferation assays and by measuring alterations in gene expression in Ig- transfected Abelson lines derived from Rag+1-mice. Finally, in Aim 5 we will ask whether the presence of N-addition in VH genes of a portion of the CD5+ B cell subset in adult mice is evidence that these cells are selected for this feature from infrequent fetal-generated B cells or else generated in the adult. We will accomplish this by determining the extent of N- addition in VH sequences from individual CD5+ B cells generated from B progenitor populations isolated at distinct ontogenic times. Cumulatively, these studies will provide a clearer picture of fetal and adult B lymphopoiesis and distinguish whether the modes of selection affecting developing B cells are similar or different during these two ontogenic timi s.
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