Continuous Germinal Center Invasion Contributes to the Diversity of the Immune Response

Antibody responses are characterized by increasing affinity and diversity over time. Affinity maturation occurs in germinal centers by a mechanism that involves repeated cycles of somatic mutation and selection. How antibody responses diversify while also undergoing affinity maturation is not as well understood. Here, we examined germinal center (GC) dynamics by tracking B cell entry, division, somatic mutation and specificity. Our experiments show that naïve B cells continuously enter GCs where they compete for T cell help and undergo clonal expansion. Consistent with late entry, invaders carry fewer mutations but can contribute up to 30 % or more of the cells in late-stage germinal centers. Notably, cells entering the germinal center at later stages of the reaction diversify the immune response by expressing receptors that show low affinity to the immunogen. Paradoxically, the affinity threshold for late GC entry is lowered in the presence of high affinity antibodies. RNA-seq profiling of Follicular B cells and Germinal Center B cells. Cells were sorted directly into a solution of 1% 2-β-mercaptoethanol (Sigma) in TCL Buffer (Qiagen, 1031576) and RNA was isolated using RNAClean XP Beads (Beckman Coulter, A63987). Near full-length mRNA was reverse transcribed as previously described (Islam et al., 2014) (Trombetta et al., 2014). Multiplexed libraries were prepared for sequencing using a Nextera XT DNA Library Preparation Kit (Illumina). Libraries were sequenced on an Illumina instrument at the Rockefeller University Genomics Core.