Development of the expressed immunoglobulin &Mgr; chain repertoire during maturation of mice B cells

Abstract

In the bone marrow and spleen, the developing B cell populations undergo both negative and positive selections to shape their B cell receptor repertoire. To gain insight into the shift of the immunoglobulin heavy (IgH) chain repertoire during B cell development, we undertook large scale Ig μ chain repertoire analysis of pre-B, immature B and spleen B cell populations. We found that the majority of V H gene segments, V H families, J H and D gene segments, were observed to have significantly different usage frequencies when three B cell populations were compared, but the usage profile of the V H, D, and J H genes between different B cell populations showed high correlations. In both productive and nonproductive rearrangements, the length of CDRH3 shortened significantly on average when B cells entered the periphery. However, the CDRH3 length distribution of nonproductive rearrangements did not follow a Gaussian distribution, but decreased successively in the order 3n -2, 3n -1 and 3n, suggesting a direct correlation between mRNA stability and CDRH3 length patterns of nonproductive rearrangements. Further analysis of the individual components comprising CDRH3 of productive rearrangements indicated that the decrease in CDRH3 length was largely due to the reduction of N addition at the 5′ and 3′ junctions. Moreover, with development, the amino acid content of CDRH3 progressed toward fewer positively charged and nonpolar residues but more polar residues. All these data indicated that the expressed Ig μ chain repertoire, especially the repertoire of CDRH3, was fine-tuned when B cells passed through several checkpoints of selection during the process of maturation.

Sequencing and cloning of antigen-specific antibodies from mouse memory B cells

“…Multiple strategies have been designed to amplify the variable regions of antibody heavy chains from genomic DNA or mRNA. These technologies are well established for the human repertoire, but they have rarely been used for mouse repertoire analysis 19,20 . The major limitations of these methods are loss of heavy-and light-chain pairing at the cellular level, short read lengths and incompatibility for further cloning of antibodies.…”