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Detecting selection in B cell Ig sequences is critical to understanding affinity maturation and can provide insights into Ag-driven selection in normal and pathologic immune responses. The most common sequence-based methods for detecting selection analyze the ratio of replacement and silent mutations using a binomial statistical analysis. However, these approaches have been criticized for low sensitivity. An alternative method is based on the analysis of lineage trees constructed from sets of clonally related Ig sequences. Several tree shape measures have been proposed as indicators of selection that can be statistically compared across cohorts. However, we show that tree shape analysis is confounded by underlying experimental factors that are difficult to control for in practice, including the sequencing depth and number of generations in each clone. Thus, although lineage tree shapes may reflect selection, their analysis alone is an unreliable measure of in vivo selection. To usefully capture the information provided by lineage trees, we propose a new method that applies the binomial statistical framework to mutations identified based on lineage tree structure. This hybrid method is able to detect selection with increased sensitivity in both simulated and experimental data sets. We anticipate that this approach will be especially useful in the analysis of large-scale Ig sequencing data sets generated by high-throughput sequencing technologies.

B lymphocytes producing high-affinity Abs are critical for protection from extracellular pathogens, such as bacteria and parasites. The process by which high-affinity B cells are selected during the immune response has never been elucidated. Although it has been shown that high-affinity cells directly outcompete low-affinity cells in the germinal center (GC), whether there are also intrinsic differences between these cells has not been addressed. It could be that higher affinity cells proliferate more rapidly or are more likely to enter cell cycle, thereby outgrowing lower affinity cells. Alternatively, higher affinity cells could be relatively more resistant to cell death in the GC. By comparing high- and low-affinity B cells for the same Ag, we show here that low-affinity cells have an intrinsically higher death rate than do cells of higher affinity, even in the absence of competition. This suggests that selection in the GC reaction is due at least in part to the control of survival of higher affinity B cells and not by a proliferative advantage conferred upon these cells compared with lower affinity B cells. Control over survival rather than proliferation of low- and high-affinity B cells in the GC allows greater diversity not only in the primary response but also in the memory response.