Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy

Abstract

Background: Specific immunotherapy (SIT) is the only treatment with proved long-term curative potential in patients with allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B-cell repertoires are not well understood.

Objective: We sought to characterize the IgE sequences expressed by allergen-specific B cells and track the fate of these B-cell clones during SIT.

Methods: We used high-throughput antibody gene sequencing and identification of allergen-specific IgE with combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and the nasal mucosa from aeroallergen-sensitized subjects before and during the first year of subcutaneous SIT.

Results: Of 52 distinct allergen-specific IgE heavy chains from 8 allergic donors, 37 were also detected by using high-throughput antibody gene sequencing of blood samples, nasal mucosal samples, or both. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships.

Conclusion: In the future, combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies might aid assessment of SIT mechanisms and efficacy.

Keywords: Aeroallergens; IgE; allergen-specific antibodies; clonotype evolution; immunoglobulin class-switch; local immunity; repertoire; specific immunotherapy.

Figure 1

Timing of SIT and sample collection. Blood samples at 2 months and 1 year timepoints were obtained approximately 1 week after the preceding SIT vaccine injection. Samples were not obtained from the non-vaccinated group at 2 months.

Figure 2

Detection of sequences related to the isolated allergen-specific scFv over the time course of SIT, using HTS of antibody heavy chain rearrangements. Filled boxes indicate the time points at which such related sequences were also detected using HTS in the blood (red) or nasal biopsy (blue) samples. Sequences only identified after selection of scFv from antibody libraries but not by HTS are not shown.

Figure 3

Analysis of isotype expression, tissue localization, and clonal persistence of B cells belonging to clonal lineages containing IgE-expressing members in SIT patients. (A) Isotype expression by allergen-specific B cell clones containing IgE members for which the allergen specificity was determined, compared to clones from SIT patients where the IgE specificity is not known. The clones of known allergen specificity are more likely to contain B cells expressing other IgA-switched antibodies. Significance was determined by Fisher’s exact test (two-tailed p-values * = 0.01 to 0.05, ** = 0.001 to 0.01, *** = 0.0001 to 0.001, **** = < 0.0001) Clones containing only IgE members are not shown on graph, but the difference was not significant with a two-tailed p-value of 0.077. Numbers above bars indicate the total number of clusters representing the given category. (B) Tissue and blood distribution of allergen-specific B cell clones containing IgE-expressing members. Clones of known allergen specificity were detected in both nasal biopsy and blood samples more frequently than clones whose allergen specificity was not identified. Data from all time points were included in this analysis. Clones in the ‘blood’ or ‘biopsy’ categories are also counted in the ‘blood and biopsy’ category. (C) Persistent detection of B cell from known allergen-specific clones versus clones of unknown specificity, during SIT. The clones whose allergen-specificity was identified were more likely to be identified in more than one time point during the SIT course. (D) Time points at which allergen-specific IgE clones were detected. Numbers on the x-axis indicate the time point in days.

Figure 4

Antibody heavy chain somatic mutation trees describing putative clonal evolution relationships between members of allergen-specific B cell clones. Clone sets related to scFv IT5-rD13 (A), IT6-nD16 (B), IT6-rD128 (C), IT6-nD17 (D), IT6-rD111 (E) are shown. IgE sequences identified as allergen-specific in phage display experiments are represented by diamonds, while clone members identified by deep sequencing of nasal biopsies are shown as rectangles, and clone members identified by deep sequencing of peripheral blood mononuclear cell specimens are displayed as circles. The colors indicate the time point at which sequences were identified (red nodes = 0 months, blue = 2 months, green = 1 year of SIT). The symbols indicate the isotype expressed by the clone member. Nodes with no internal symbol represent IgE clone members, nodes with triangles represent IgG1 and nodes with a circled dot represent IgA members. Additional examples of trees are shown in Supplementary Figure E8 in the Online Repository.