Assessing B Lymphocyte Clonal Diversity, Expansion, and Convergent Evolution By High-Throughput Sequencing Of Rearranged IGH Segments From Naïve and Memory Repertoires

Diversity in B lymphocyte antigen specificity, necessary for effective adaptive immunity, arises from structural diversity of the B cell receptor generated by random somatic rearrangement of the immunoglobin heavy (IgH) and light chain loci during lymphocyte development. Antigen-experienced (memory) B cells are stimulated to proliferate upon successful binding of antigen, whereas the extent to which unexperienced (naïve) cells proliferate, if it all, is unclear. During memory clonal expansion, daughter cells undergo somatic hypermutation, which introduces random single nucleotide variants to the rearranged gene segments, selectively optimizing antigen affinity. Among memory B cells that express identical receptor proteins, the extent to which the cells are identical by descent, or rather have convergently evolved from different naïve clones to the same specificity, is also unknown.

We performed high-throughput sequencing of rearranged IgH gene segments for B cells sorted into memory (CD19+, CD27+) and naïve (CD19+, CD27-,IgM+, IgD+) samples (∼5 million cells each) from each of three adults. Each sample was split among 188 libraries for multiplex PCR amplification of IgH sequences at ∼10x coverage, allowing us to estimate clonal abundance by measuring the number of libraries occupied by each sequence.

Using a maximum entropy inverse model, all three memory samples evince three distinct subpopulations, corresponding to memory cell types of differing abundance. Data from naïve samples suggest extreme diversity with more than 98% of clones occupying one library (i.e., almost surely present in only one cell in the starting material). 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. Additionally, somatic hypermutation in the memory samples was investigated to detect convergent evolution of distinct naïve B cell clones toward common amino acid sequence (hence common antigen specificity) in the memory B cell compartment.