Assessing B lymphocyte clonal diversity and expansion by high-throughput sequencing of rearranged IgH loci from naïve and memory repertoires (IRM8P.708)

Abstract Diversity in B cell antigen specificity arises from structural diversity of the B cell receptor generated by somatic rearrangement of the immunoglobulin heavy (IgH) and light chain loci during B cell development. Antigen-experienced (memory) B cells are stimulated to proliferate upon successful binding of antigen, whereas the extent to which antigen-inexperienced (naïve) cells proliferate, and therefore the total diversity of circulating naïve B cells, is unclear. High-throughput sequencing of rearranged IgH CDR3 regions was performed for sorted memory and naïve B cell samples from each of 3 adults. For each sample, approximately 5 million B cells were split among 188 libraries for multiplex PCR amplification of IgH CDR3 segments, allowing us to estimate clone abundances using the number of libraries occupied by each distinct CDR3 segment. Modeling sample clone abundances as a superposition of homogeneous Poisson processes, memory repertoire clone abundance was characterized. Data from naïve samples suggest extreme diversity with more than 98% of clones occupying only one library (i.e., almost surely present in only one cell in the sample). A measure-theoretic upper bound on mean clone abundance in the naive repertoire was computed based on Monte Carlo simulation, indicating that naïve B cells typically undergo little, if any, expansion. We estimate the diversity of circulating naive B cells to be on the order of 100 million in healthy adults.