Depletion of conventional mature B cells and compromised specific antibody response in bovine immunoglobulin μ heavy-chain transgenic mice

In this study,we introduced the bovine immunoglobulinμheavy-chain gene(the orphaned gene on BTA11)into mouse germline cells.Bovine IgM was highly expressed in selected transgenic lines,and it largely inhibited rearrangements of the endogenous immunoglobulin heavy chain(IgH)genes in these lines.The forced expression of bovine IgM resulted in reduced numbers of pro-and pre-B cells but increased the number of immature B cells in the transgenic mice.Bovine IgM-expressing B cells can migrate from the bone marrow to the spleen,but most of the cells are arrested at the T1 transitional B cell stage,leading to a significantly lower number of T2 transitional and mature B cells in the spleen.Although the serum concentrations of endogenous IgM and IgG in the transgenic mice were significantly decreased,the IgA levels were slightly increased compared to the WT mice.The bovine IgM level in the serum was only one-tenth to one-fifth of that of endogenous mouse IgM,suggesting that most of the serum immunoglobulin were contributed by endogenous IgH gene-expressing B cells.These transgenic mice also exhibited a lower frequency of unique complementarity determining region 3(CDR3)sequences in their VH repertoire and Vκrepertoire but exhibited an increased frequency of unique CDR3 in their Vλrepertoire.Compared to the WT mice,the transgenic mice had a significantly higher percentage of mouse IgMexpressing B cells that expressedλchains.Finally,we showed that the transgenic mice were deficient in a specific antibody response to antigen stimulation.

Development of the expressed immunoglobulinμchain repertoire during maturation of mice B cells

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 Igμchain repertoire analysis of pre-B,immature B and spleen B cell populations.We found that the majority of VH gene segments,VH families,JH 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 VH,D,and JH 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 Igμchain repertoire,especially the repertoire of CDRH3,was fine-tuned when B cells passed through several checkpoints of selection during the process of maturation.