Site-specific labeling uncovers differences in levels and distribution of B-cell receptors of different isotypes on primary B cells.

Expression of the BCR is essential for survival, development, and effector functions of B cells. Naive B cells express surface IgM and IgD, while surface IgG1 is expressed by class-switched (memory) B cells. Despite similar overall structures, the different BCR isotypes show differences in distribution and expression levels. The dynamics of BCR behavior have been difficult to explore owing to a lack of appropriate tools that can track the BCR without causing concomitant activation. Using CRISPR-Cas9, we inserted a sortase recognition motif (LPETG [LeuProGluThrGly]) at the C-terminus of the OB1 transnuclear ovalbumin-specific Cκ chain (Igκ-LPETG mice). The surface BCR from Igκ-LPETG mice is fully functional and can be labeled site-specifically with biotin or fluorophores. Igκ-LPETG mice show near-normal B-cell development, with an increase in Igλ-producing cells, presumably due to massive contraction of the κ locus V-region cluster upon V-J recombination to generate the OB1 light chain. Using the Igκ-LPETG mice, we compared organization and density of BCRs on the surface of IgM/IgD+ B cells bearing a wild-type (WT) heavy chain locus and IgG1 B cells in the OB1 model. The density of IgG1 BCRs is much reduced compared to IgM/IgD BCRs on primary B cells. Upon activation, IgM/IgD BCRs are found in detergent-insoluble domains, whereas IgG1 BCRs are not. The isotype of the Ig heavy chain thus contributes to surface expression and nanoscale organization of the BCR.