Highly sensitive and unbiased approach for elucidating Ig repertoires
S. Lin1, M.Tian1, Z. Ba1, Z. Du1, Y. Zhang1, Q. Pan-Hammarström2, J. Hu1*, and F. W. Alt1*
1Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115 Howard Hughes Medical Institute. 2Karolinska Institute, Sweden
Developing B cells undergo V(D)J recombination to assemble germline V, D, and J segments into exons that encode the antigen-binding variable region of immunoglobulin (Ig) heavy (H) and light (L) chains. IgH and IgL chains associate to form the B cell receptor (BCR), which upon antigen binding activates B cells to secrete BCR as an antibody. Each clonally independent B cell expresses a unique set of IgH and IgL variable regions. V(D)J recombination generates diverse primary Ig repertoires that result from a combinatorial assortment of many different Vs, Ds, and Js, coupled with V(D)J junction diversification to generate the complementary determining region 3 for antigen contact. Evaluation of primary Ig repertoires and how they are further molded by secondary mutation and affinity maturation processes are important to the B cell development, vaccine, and antibody fields. HTGTS repertoire sequencing (HTGTS-Rep-seq) is an unbiased, sensitive, and accessible assay to quantify Ig repertoires. The assay quantitatively identifies the vast majority of IgH and IgL V(D)J exons from progenitor and mature mouse B cells in their productive and non-productive configurations, and also quantifies DJH intermediates. HTGTS-Rep-seq can be used for human samples, following antibody maturation, or comparing repertoires pre- and post- B cell eliminating treatments (i.e. CAR-T cell therapy or BM transplants). We have used HTGTS-Rep-seq on mouse models that express an HIV broadly neutralizing antibody precursor and shown that these mice can generate a diverse set of HIV-neutralizing antibody lineages in response to specific immunogens. Additionally, preliminary studies in human peripheral B cells and cord blood-derived (CBD) CD19+ cells reveal a bias for proximal VH usage in CBD CD19+ cells over peripheral B cells—a developmental bias also observed in mice.
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