Scientific Report 2007

Immunology

Control of V(D)J Recombination and Formation of the Antibody Repertoire in Normal and Autoimmune Mice

A.J. Feeney, C.R. Espinoza, J. Lamoureux, M. Cherrier, C.R. Xu, J. Carey, S. Salerno, T. Wong

A main focus of our laboratory is the molecular analysis of factors that influence the composition of the antibody repertoire and elucidation of the mechanisms that control the V(D)J rearrangement process. In each precursor B lymphocyte, a different set of V, D, and J genes recombine to form exons for the light and heavy chains of the antibody molecule. Each locus has many V, D, and J genes, but the gene segments are not used equally. One of our goals is to understand the basis of this nonrandom use of gene segments.

We previously showed that much of this bias occurs because V genes undergo recombination with different intrinsic frequencies due to differences in the sequence of the binding site for the recombinase flanking each gene segment. However, other factors clearly influence recombination frequencies; currently, we are focusing on the role of transcription factors and chromatin modifications in controlling accessibility to V(D)J recombination and recombination frequency.

Genes in loci that are undergoing V(D)J recombination are often associated with histones that are acetylated. We hypothesized that the extent of histone modification affects the frequency of recombination of individual genes, and indeed we observed a positive correlation between the relative rearrangement frequency of individual genes in vivo and the extent of acetylation of histones associated with those genes, as assessed by chromatin immunoprecipitation. We are now identifying other histone modifications that likely play a role in regulating gene use in the antibody repertoire.

We have also uncovered a novel role for the transcription factor Pax5 in promoting V(D)J rearrangement. Pax5 is essential for B-cell development. In the absence of Pax5, V_H-to-DJ_H rearrangement is severely impaired. We found Pax5 binding sites in the coding regions of many V_H genes. Furthermore, in collaboration with Z. Zhang and M. Cooper, University of Alabama, Birmingham, we showed that Pax5 binds to the recombinase proteins RAG1 and RAG2. Hence, we propose that Pax5 may recruit RAG1 or RAG2 to the recombination signal sequence or may stabilize the interaction of the RAG complex with its binding site. We are investigating other roles of Pax5 in shaping the antibody repertoire.

B cells in the spleen are divided into functionally distinct subsets. We are investigating differences in the antibody repertoires between B cells in the marginal zone, which respond to blood-borne pathogens, and B cells in the follicle, the largest population of splenic B cells. We previously showed that B cells made during fetal and neonatal life lack an enzyme, terminal deoxynucleotidyl transferase; this enzyme greatly diversifies the antibody repertoire in adults. We now have evidence that B cells generated early in ontogeny are preferentially selected into the marginal zone compartment, suggesting that the fetal/neonatal repertoire of antibodies, which is quite different from that generated in adults, may be particularly useful against blood-borne pathogens.

In other studies, we are examining the breakdown of B-cell tolerance in autoimmunity. When precursor B cells successfully recombine both heavy- and light-chain gene segments, they express a B-cell receptor for the first time. If the receptor is autoreactive, then the immature B cell normally continues to undergo light-chain V-J rearrangement until an innocuous receptor is made. This process is termed receptor editing and is an important checkpoint in B-cell tolerance. We have evidence that this process is not functioning as efficiently in lupus-prone mice as in nonautoimmune mice, and we are investigating why this difference occurs. Such misregulation of this key checkpoint could lead to the release of autoreactive B cells into the periphery, where they can become activated to secrete autoantibodies and cause autoimmune disease.

Publications

Espinoza, C.R., Feeney, A.J. Chromatin accessibility and epigenetic modifications differ between frequently and infrequently rearranging V_H genes. Mol. Immunol. 44:2675, 2007.

Feeney, A.J. Genetic and epigenetic control of V gene rearrangement frequency. In: V(D)J Recombination. Ferrier, P. (Ed.). Landes Bioscience, Austin, TX, in press.

Lamoureux, J.L., Watson, L.C., Cherrier, M., Skog, P., Nemazee, D., Feeney, A.J. Reduced receptor editing in lupus-prone MRL/lpr mice. J. Exp. Med., in press.