PROJECT 1) ? The CDR3, which conveys the antigen specificity of the B cell, is the most variable region of Ig due to hypermutation and extensive molecular processing in the junctions during recombination. The development by our laboratory of a web based software analysis program (JoinSolver) has provided a tool to perform high throughput quantitative and qualitative analysis of the molecular mechanisms that contribute to CDR3 specificity and diversity. This year JoinSolver software improvements include the analysis of heavy chain mutations that occur in the CDR3, mutations that occur in overlapping motifs and comparative regional mutation analysis for Framework Regions (FR)1, 2 and 3 and Complementarity Determining Regions (CDR)1, 2 and 3. Recently a Monte Carlo simulation was performed to determine the frequency at which random mutation occurs in the CDR3. Future plans include analysis of complex mutation patterns that develop from insertions and deletions and the adaptation of the same analyses to light chains.? PROJECT 2) ? Repertoire and mutation analysis of individual B cells from patients with genetic defects in proteins that regulate B cell development and function. The individual analyses allow us to isolate the contribution of single elements of the multi-component regulation of somatic hypermutation (SHM), class switching, selection, and plasma cell vs memory cell development. To perform these studies we amplify and sequence genomic heavy and light chain gene rearrangements in individual B cells. The technique allows us to analyze molecular mechanisms that are revealed in nonproductive rearrangements as well as the impact of selection that is imposed on productive rearrangements. The following defective signaling elements that influence developmental pathways, response thresholds and germinal center reactions are under investigation: (1) Molecules that regulate germinal center reactions which are required for B cell memory development, such as (a) CD40 ligand mutation (X-HIgM) and (b) CD40 deficiency; (2) deficiency in AID (Activation induced cytidine deaminase), the master regulator of hypermutation; (3) Mutated Fas which regulates B cell homeostasis through activation induced cell death (ALPS--Autoimmune Lymphoproliferative Syndrome); and (4) Xeroderma Pigmentosa Variant (XPV) defect in the DNA polymerase eta which is an error-prone polymerase believed to contribute to SHM. We found that CD40 ligand deficiency results in an abnormally skewed repertoire enriched with B cells using VH4 rearrangements that have a propensity for autoreactivity. Whereas the role of AID-induced G/C mutations are well understood, the mechanism by which A/T mutations occur was not known. (1) Analysis of X-HIgM mutations has revealed how the SHM develops in normal B cells:(a) in X-HIgM the hypermutable C in WRCY (R=A/G, Y=C/T, W=A/T) was mutated whereas the complementary G in RGYW was not mutated, (b) WA motif mutations overlap RGYW motifs, but not WRCY motifs, in which the U:G mismatch has undergone repair, and (c) transitions abnormally outnumber transversions. We concluded that the A/T mutation spectrum is linked to AID-targeted G/C mutations via repair and repair involving POL eta activity occurs preferentially on the non-transcribed strand. (2) Other studies revealed that the Fas mutation results in over-utilization of VH4 genes, failure to develop CD27+ memory B cells and increased survival of B cells with mutations that jeopardize stable Ig structure. (3) Studies of the AID deficient B cells revealed that: (a) mutations occur at a frequency that significantly exceeds that of non-B cells suggesting that DNA repair mechanisms are down-regulated in germinal center B cells, (b) AID targeted mutation in RGYW/WRCY motifs and POL eta mutational targeting in WA motifs were lost. No new mutational targeting motif was identified. (c) replacement substitutions were reduced in the CDRs, and d) transitions occurred more frequently than in normal B cells. These data led to the conclusions that in the absence of AID, mutations occur 20-fold less frequently than normal B cells and the mutations that do occur are associated with AID-independent cytidine deamination that is an inefficient mechanism for influencing the antigen binding site. Furthermore, the data suggest AID is required to recruit or increase the accessibility of POL eta and UNG. ? PROJECT 3) Pseudo-hybrid formations in heavy chain variable segments as a mechanism of receptor editing. Receptor editing/revision is a mechanism that B cells can use to avoid autoreactivity. One form of editing that is uniquely associated with VH4 family genes involves the invasion of an upstream VH4 gene into the FR3 region of a previously rearranged VH4 gene. In contrast to other reports, our in vitro data demonstrates the mechanism involved may infrequently depend on recombination activation genes (RAG). Further studies will explore the role of AID-dependent strand breaks in the formation of hybrid joins. ? PROJECT 4) Repertoire and mutation analysis of anergic CD19lowIgD+ cells in SLE patients. Anergy is a mechanism by which self-reactive B cells may become unresponsive; thus, CD19lowIgD+ cells were analyzed to determine whether this population was oliogoclonal or otherwise enriched with autoreactive B cells. We found the CD19lowIgD+ cells were polyclonal and used many of the same genes utilized by the CD19highIgD+ cells isolated from the same SLE patient. However, a greater representation of B cells expressing VH4 genes were included in the CD19lowIgD+ subset. We concluded that in SLE this form of anergy may be used to generally downregulate B cell hyperactivity, as well as to regulate the activity of specific VH4 B cells. ? PROJECT 5) Characterization of Circulating Human CD5+ B cells. The goals of this project include the functional and phenotypic characterization of circulating human CD5+ B cells and discernment of the role of CD5+ B cell subsets in systemic autoimmune disease (SLE). Based on previous studies, CD5+ B cells have been shown to play an important role in autoimmunity and autoimmune diseases. However, most of the data on human CD5+ B cells have been obscured by the heterogeneous characteristics of CD5+ B cells that need to be further characterized. Preliminary data suggests that the CDR5+ subsets from normal donors are functionally & phenotypically distinct populations. ? Other ongoing projects:? 1. The development of murine B cells expressing rearranged human Ig receptors in a transgenic model? 2. Repertoire and mutation analysis of peripheral blood B cells containing a CD40 snp
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