The mutation patterns in B-cell immunoglobulin receptors reflect the influence of selection acting at multiple time-scales

Abstract

During the several-week course of an immune response, B cells undergo a process of clonal expansion, somatic hypermutation of the immunoglobulin (Ig) genes and affinity-dependent selection. Over a lifetime, each B cell may participate in multiple rounds of affinity maturation as part of different immune responses. These two time-scales for selection are apparent in the structure of B-cell lineage trees, which often contain a 'trunk' consisting of mutations that are shared across all members of a clone, and several branches that form a 'canopy' consisting of mutations that are shared by a subset of clone members. The influence of affinity maturation on the B-cell population can be inferred by analysing the pattern of somatic mutations in the Ig. While global analysis of mutation patterns has shown evidence of strong selection pressures shaping the B-cell population, the effect of different time-scales of selection and diversification has not yet been studied. Analysis of B cells from blood samples of three healthy individuals identifies a range of clone sizes with lineage trees that can contain long trunks and canopies indicating the significant diversity introduced by the affinity maturation process. We here show that observed mutation patterns in the framework regions (FWRs) are determined by an almost purely purifying selection on both short and long time-scales. By contrast, complementarity determining regions (CDRs) are affected by a combination of purifying and antigen-driven positive selection on the short term, which leads to a net positive selection in the long term. In both the FWRs and CDRs, long-term selection is strongly dependent on the heavy chain variable gene family.

Keywords: B-cell receptor; affinity maturation; antigen-driven selection; lineage trees; mutations; next generation sequencing.

Figure 1.

B-cell lineages divide the affinity maturation process into long (trunk) and short (canopy) time-scales. (a) Antibodies are composed of two identical heavy chains and two identical light chains. The mRNA coding for each chain is divided into FWRs and CDRs, which are interlaced in the linear genetic code. To identify the somatic mutations that are accumulated during affinity maturation, antibody sequences are first aligned with the corresponding inferred germline sequence. (b) After clustering the repertoire into clonally related sequences (clones) and building lineage trees, the affinity maturation process was divided into two non-overlapping intervals: trunk and canopy, which were separated by the MRCA in the lineage, and correspond to long and short time-scales, respectively. The number of mutations is indicated along each branch (assumed to be unity if no number). (Online version in colour.)

Figure 2.

Trunk mutations dominate B-cell repertoires from blood samples of healthy donors. (a) Clone size distribution for each of the sequenced isotypes from subject 420IV. Axes are logarithmic and bins are ‘logarithmic’ sized (each bin is double the size of the previous one). (b) The number of mutations associated with the trunk or canopy was determined for IgG sequences of subject 420IV. Mutations from different regions (FWRs, CDRs or both) are shown in the three panels.

Figure 3.

Mutations in the FWRs are negatively selected in both trunk and canopy. (a,c) The fraction of clones with significant negative (filled bars) or positive (hatched bars) selection as determined by (a) BASELINe and (c) MBSM applied to the FWRs. In each panel, the upper part refers to the trunk, while the lower part refers to the canopy. (b,d) The average selection strength across all sequences as determined by (b) BASELINe and (d) MBSM applied to the FWRs. For BASELINe (b), 95% CIs are shown for each of the selection estimations.

Figure 4.

Mutations in the CDRs are positively selected in the trunk. (a,c) The fraction of clones with significant negative (filled bars) or positive (hatched bars) selection as determined by (a) BASELINe and (c) MBSM applied to the CDRs. In each panel, the upper part refers to the trunk, while the lower part refers to the canopy. (b,d) The average selection strength across all sequences as determined by (b) BASELINe and (d) MBSM applied to the CDRs. For BASELINe (b), 95% CIs are shown for each of the selection estimations.

Figure 5.

Correlation of the V gene selection estimations between subjects. (a) Pearson's correlations are shown for the selection estimations calculated for each V gene using MBMS. Only V genes that were used in more than 1% of the sampled repertoire were included. (b) Scatter plot for the V gene selection estimations in the CDRs and canopy data of subject PGP1 versus subject hu420143. Colours represent different V genes as indicated in the colour key to the right. (Online version in colour.)