Human B-cell isotype switching origins of IgE

Abstract

Background: B cells expressing IgE contribute to immunity against parasites and venoms and are the source of antigen specificity in allergic patients, yet the developmental pathways producing these B cells in human subjects remain a subject of debate. Much of our knowledge of IgE lineage development derives from model studies in mice rather than from human subjects.

Objective: We evaluate models for isotype switching to IgE in human subjects using immunoglobulin heavy chain (IGH) mutational lineage data.

Methods: We analyzed IGH repertoires in 9 allergic and 24 healthy adults using high-throughput DNA sequencing of 15,843,270 IGH rearrangements to identify clonal lineages of B cells containing members expressing IgE. Somatic mutations in IGH inherited from common ancestors within the clonal lineage are used to infer the relationships between B cells.

Results: Data from 613,641 multi-isotype B-cell clonal lineages, of which 592 include an IgE member, are consistent with indirect switching to IgE from IgG- or IgA-expressing lineage members in human subjects. We also find that these inferred isotype switching frequencies are similar in healthy and allergic subjects.

Conclusions: We found evidence that secondary isotype switching of mutated IgG1-expressing B cells is the primary source of IgE in human subjects, with lesser contributions from precursors expressing other switched isotypes and rarely IgM or IgD, suggesting that IgE is derived from previously antigen-experienced B cells rather than naive B cells that typically express low-affinity unmutated antibodies. These data provide a basis from which to evaluate allergen-specific human antibody repertoires in healthy and diseased subjects.

Keywords: B cell; IgE; antibody; direct; high-throughput DNA sequencing; indirect; isotype switching; repertoire.

Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

(A) Sequence alignment of clonally-related antibody heavy chains identified in B cells expressing different isotypes. Highlighted bases indicate positions of somatic mutation in V and J gene segments. The CDR3 region encoded by the VDJ junctions is also highlighted. (B) Tree representation of the clonal lineage. Sequences are labeled according to isotype. Tree building and visualization was performed using the ape package in R and the distance matrix obtained by comparing sequences using the ape “raw” method.

Figure 2

(A) Number of IgE nearest isotype neighbor events with each non-IgE isotype, observed in multi-isotype B cell clonal lineages. Numbers in the heat map grid indicate the number of times a given nearest-neighbor relationship was observed in the data. Coloring in the heat map indicates the proportion of nearest neighbor relationships to IgE for each isotype, with blue indicating low proportion and red indicating high proportion. Each row represents a different individual, IgG1 is usually the isotype with the closest mutational relationship to IgE. Patient identifier codes from allergic individuals are highlighted in red. (B) Nearest isotype neighbors for IgE. Probabilities indicate the relative frequencies for each other isotype to be the nearest neighbor to IgE within multiple-isotype lineages. (C) A model for frequencies of class switching to IgE in healthy individuals based on the data in Figure 2B. The width of each arc indicates the probability of a nearest mutational neighbor between each isotype pair.

Figure 3

(A) Average IGHV gene mutation frequency for members of IgE nearest neighbor relationships. Panels indicate the average mutation level for members of the indicated nearest-neighbor pair. (B) Average variable gene mutation frequency for sequences that are nearest neighbors to IgM. There are no significant differences between allergic and healthy individuals for these metrics after accounting for multiple hypothesis testing.

Figure 4

(A) Average IGHV gene mutation frequency per isotype. (B) Proportion of isotype sequences having less than 1% mutation in IGHV.