News and Publications

Checkpoints in the Development of B cells in Young and Aged Mice

N.R. Klinman, G.H. Kline, N.B. Leaf, S.N. Zaharevitz, T.A. Hayden, M. Klyusner

Development of B cells depends on a highly regulated progression of molecular and cellular events. The most crucial of these events is the rearrangement of segments of the gene that encodes the variable (V) region of the immunoglobulin heavy (H) chain. Because recombination is not precise, nucleotides are lost or gained at the junctions of the segments. This gain or loss of nucleotides, in combination with the assortment of 10²--10³ structurally distinct V_H gene segments, 13 distinct D gene segments, and 4 J_H segments, creates an enormously diverse set of H chains. Thus, the development and expression of B cells depend on the expression of a protein (H chain) whose fine structure is unpredictable and whose function is therefore highly variable. Because of this situation, developmental checkpoints exist to ensure that the only pre-B cells that clonally expand and mature are those with H chains that are likely to be able to assemble with light (L) chains and encode antibodies that do not recognize self-antigens.

One of these checkpoints requires that the nascent H chain be able to associate with surrogate light chain, the assembled product of 2 pro-B cell polypeptides, V-pre B and lambda 5, to form a functional pre-B cell receptor. Because H chains that cannot assemble with surrogate light chain also do not associate with L chains, cells with H chains that would not generate H-L heterodimers are not permitted to mature. A second checkpoint represents either the elimination of newly generated B cells with receptors that recognize self-antigens or the initiation of new L-chain expression in such cells and thus the expression of new receptors (receptor editing).

Evidence of the impact of the failure to pass through these checkpoints has been obtained by analyzing the sequence of the genes that encode H-chain V regions in genomic DNA from various developmental B-cell subsets. The failure of cells expressing nascent H chains encoded by any of several of the 10²--10³ V_H gene segments is measured as a decrease in the ratio of productive vs nonproductive rearrangements of these V_H gene segments as cells progress from 1 maturational stage to another. Because clonal maturation and expansion depend on the expression of a functional H chain, all maturing pre-B cells and B cells express a productive H-chain rearrangement, whereas less than half express a nonproductively rearranged H-chain V gene. Therefore, for most V_H gene segments, more than 70% of rearrangements in pre-B cells and B cells are productive. However, evidence indicates that the amino acid sequence of the H chain also has a role in determining the functionality and ability of the chain to mediate B-cell development. For example, for certain V_H gene segments and rearrangement motifs, the proportion of nonproductive rearrangements is increased. The most dramatic example of this increase is H chains encoded by the V_H81X gene segment. For these chains, the proportion of productive rearrangements decreases at each of the checkpoints, that is, as cells mature from pro-B cells to pre-B cells, and as B cells mature.

To investigate the mechanisms that regulate these developmental checkpoints, we are analyzing the sequence motifs of a library of productively rearranged H-chain V regions obtained from cells that did not accomplish the transition from pro-B cell to pre-B cell. In collaboration with H.M. Jäck, University of Erlangen-Nürnberg, Erlangen, Germany, we have constructed transgenic mice that express a V_H81X-encoded H chain that does not assemble with surrogate light chains and L chains. By showing that the H chains encoded by this transgene do not mediate clonal maturation, we have verified the need for H chains to associate with surrogate light chains as a checkpoint in B-cell maturation. As sequence motifs are identified that favor such "dysfunctionality" in H chains encoded by V_H81X or other V_H gene segments, we will attempt to correlate amino acid sequence and functionality by expressing selected H chains in cell lines and transgenic mice.

Interestingly, analysis indicated that although B-cell longevity in aged mice is increased, B-cell generation is markedly decreased. Furthermore, studies have shown that B-cell development is blocked at both of the checkpoints described earlier.

PUBLICATIONS

Kline, G.H., Hartwell, L., Beck-Engeser, G.B., Keyna, U., Zaharevitz, S., Klinman, N.R., Jäck, H.-M. Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional µ heavy chains. J. Immunol. 161:1609, 1998.

Klinman, N.R. The cellular origin of memory B cells. Semin. Immunol. 9:241, 1997.

Klinman, N.R. Repertoire diversification of primary vs memory B-cell subsets. Curr. Top. Microbiol. Immunol. 229:133, 1998.

Klinman, N.R., Kline, G.H. The B-cell biology of aging. Immunol. Rev. 160:103, 1997.