News and Publications

Formation of the B-Cell Repertoire

A.J. Feeney, B. Nadel, P. Goebel, G. Escuro, A. Tang, G. Lugo, V. Love

Much of the diversity of antibodies is due to the many V, D, and J gene segments in the genome. In each precursor lymphocyte, a unique combination of V_H, D_H, and J_H gene segments and V_L and J_L gene segments recombine at the DNA level to form exons to encode the 2 chains for that cell's receptor. In addition to this combinatorial diversity, marked diversity is created at the junctions of the V, D, and J segments. The main focus of our laboratory is the analysis of factors that influence the composition of the initially formed repertoires. We are particularly interested in revealing biases imposed by the V(D)J recombination mechanism itself on nonrandom use of genes and junctional diversity.

Previously, we found that the antibody repertoire shows nonrandom use of V genes. This unequal use could be due to frequent antigenic selection and expansion of B cells whose receptors use the commonly observed V genes, or the V genes could rearrange with different frequencies. To determine the initial relative frequency of rearrangement of the individual V genes, we purified DNA from pre-B cells isolated from human bone marrow and determined that the initial rearrangement was biased.

The next aim was to determine why the genes rearranged nonrandomly. Our hypothesis is that a major reason for unequal V rearrangement is the naturally occurring variation in the recombination signal sequences (RSSs) that flank each V, D, and J gene segment. The RSS is the site where the recombinase enzyme complex binds to combine 2 gene segments. The RSS is composed of a conserved heptamer sequence and a conserved nonamer sequence separated by a 12- or a 23-bp spacer. Each change from the consensus heptamer or nonamer reduces the efficiency of recombination of the adjacent gene segment to a different degree. We constructed plasmid-based recombination substrates to analyze the relative efficiency of recombination of various natural RSSs. In these substrates, 2 V genes are placed in competition for rearrangement to a single J gene. Thus, small differences in the rearrangement potential of each RSS can be accurately measured.

All VIII genes have a consensus RSS, and these genes are common in the peripheral repertoire. All VII genes have a characteristic change in the nonamer of the RSS. To test whether VIII genes were overused because they contained consensus nonamers, we compared a VIII gene with a VII gene in the competition substrate. To our surprise, the VIII gene recombined less well than did the VII. We made several constructs to dissect the region responsible for these findings. We found that the change in the nonamer was deleterious, as predicted. We determined that the main reason for the difference in recombination frequency between the 2 V genes was the sequence of the spacer of the RSS. When we made an artificial substrate in which the only difference between the 2 V segments was the spacer, we reconstructed most of the bias in rearrangement observed with the intact V genes.

We then tested the 2 other functional VIII genes to determine the relative frequency of rearrangement within the VIII family. We observed up to a 2.7-fold difference in rearrangement of the 3 V genes, and this difference correlated with the relative frequency of rearrangement of these 3 VIII genes in the bone marrow B-cell precursors. Thus, although previously it was thought that only the length of the spacer of the RSS was important, these experiments revealed that the sequence of the spacer also influences the composition of the antibody repertoire.

PUBLICATIONS

Nadel, B., Tang, A., Escuro, G., Lugo, G., Feeney, A.J. Sequence of the spacer in the RSS affects V(D)J rearrangement frequency and correlates with nonrandom V usage in vivo. J. Exp. Med 187:1495, 1998.

Nadel, B., Tang, A., Feeney, A.J. V_H replacement is unlikely to contribute significantly to receptor editing due to an ineffectual embedded recombination signal sequence. Mol. Immunol., in press.

Nadel, B., Tang, A., Lugo, G., Love, V., Escuro, G., Feeney, A.J. Decreased frequency of rearrangement due to synergistic effect of nucleotide changes in the heptamer and nonamer of the recombination signal sequence of the V gene A2b, which is associated with increased susceptibility of Navajos to Haemophilus influenzae type b disease. J. Immunol., in press.

O'Rourke, A.M., Escuro, G., Feeney, A.J. Cloning and sequencing of the cDNA for mouse cytohesin-1. Immunogenetics, in press.