The long-term goal of this proposal is to understand the relationship between """"""""naturally-occurring"""""""" and """"""""disease-related"""""""" autoantibodies in man. The former are polyreactive, low affinity, IgM antibodies produced by CD5+ B cells that have non-mutated Ig V genes, presumably because of a lack of T cell-stimulated, antigen-driven maturation. In contrast, disease-related autoantibodies are monoreactive, higher affinity IgM or IgG antibodies whose cellular origin has not been exclusively assigned to either the CD5+ or CD5- subsets. Based on animal experiments, these antibodies are products of cells that are clonally selected and have highly mutated V genes. However, human experiments have failed to demonstrate evidence for V gene somatic mutation. This may relate to the lack of availability of B cell lines making true disease-related autoantibodies. We will try to solve this dilemma by studying the B cells and IgM and IgG rheumatoid factors (RF) from rheumatoid arthritis (RA) and hypergammaglobulinemic purpura (HGGP) patients and comparing these with comparable cells and antibodies from normal neonates and adults. B cells will be taken from umbilical cord blood, and from the major sites of autoantibody synthesis in these diseases: the synovium in RA and the spleen in HGGP. CD5+ and CD5- B cells from these sources will be subcategorized: phenotypically by the expression of mutation and activation antigens; functionally by their T cell dependencies, cytokine responsiveness, and autoantibody synthesis; and genetically by Ig V/H gene family usage and RF cross-reactive idiotype (CRI) expression. Based on these subcategorization data, we will expand those B cell subsets that make disease-related autoantibodies, some of which will be immortalized by EBV transformation and/or somatic cell hybridization. These populations, and those generated during the previous granting period that make natural autoantibodies, will be used for Ig V/H and V/L gene DNA sequencing studies. Sequencing will be facilitated by suing a PCR technique that employs a series of oligonucleotide primers spanning the known upstream leader and downstream C region sequence. Finally, the autoantibodies produced by these cell lines will be analyzed for their antigen-binding specificities, affinities, and CRIs, and these immunologic characteristics compared with the DNA sequences to determine the molecular bases for their expression.
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