Fig. 2. CD21lo cells are clonally related to plasmablasts and memory B cells but form distinct clades.

454 sequencing was performed on cDNA libraries generated from PCR amplified antibody genes from plasmablasts, memory B cells, and CD21lo cells isolated from 3 subjects that received the 2010–2011 seasonal influenza vaccine. (A) Number of clonal families of each size (n=3). Clone numbers were normalized for sequencing sample size by rarefaction. (B) Pie charts showing the proportion of Day 14 CD21lo containing clones that also contain PB or Day 14 memory sequences. (C) Mean number of mutations in the V region for Day 14 CD21lo and plasmablast sequences for each influenza specific clone (n=13). (D) Difference in V region nucleotide mutation number between the most and least mutated sequences within each influenza specific clone (n=13). (E–G) Representative maximum likelihood trees. Trees are rooted on the germline VH-JH sequence of the clone. Empirical probability of a Day 14 CD21lo sequence will have another Day 14 CD21lo sequence as its nearest neighbor in the phylogenetic tree or vice versa for plasmablast (n=219) (H) or Day 14 memory B cell (n=87) (I) or sequences in experimentally verified flu specific clones (n=12) (J). Significance was determined with a two-tailed paired t- test. Each dot represents the probability calculated for an individual clone. The line denotes the mean. (K) Percentage of clones that contain Day 14 CD21lo sequences that also contain sequences from Day 90 CD21lo, Day 90 memory, or both.