Systems Immunology Approach for Antibody Discovery and B Cell Repertoire Analysis

Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells1,2 or recombinant antibody libraries3–6. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between ~1% and >10% of the total repertoire. We paired the most abundant variable heavy (VH) and variable light (VL) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities. 

In an ongoing study, we performed a systems immunology analysis of the humoral response in mice, focusing on the molecular, cellular, and physiological diversity of plasma cell responses. Specifically, we used high-throughput repertoire analysis to quantify the immunoglobulin diversity in plasma cells located in the various lymphoid organs of mice following immunization with a protein antigen. For example, we were able to quantify junctional clonal diversity and frequency across multiple lymphoid organs (based on V_H complementarity determining region 3 (CDRH3)). Moreover, we used clustering analysis to identify clonal correlations, therefore gaining insight into the overlap of repertoires in different lymphoid compartments. Importantly, we discovered that mice with high serum antigen-specific antibody titers converged to the same highly abundant plasma cell IgG V_H clones, suggesting immunoglobulin equilibration across lymphoid organs may be correlated to the strength of the immune response.