Characterization of the B-cell receptor repertoires in peanut allergic subjects undergoing oral immunotherapy

Abstract

B-cell receptors (BCRs) play a critical role in adaptive immunity as they generate highly diverse immunoglobulin repertoires to recognize a wide variety of antigens. To better understand immune responses, it is critically important to establish a quantitative and rapid method to analyze BCR repertoire comprehensively. Here, we developed "Bcrip", a novel approach to characterize BCR repertoire by sequencing millions of BCR cDNA using next-generation sequencer. Using this method and quantitative real-time PCR, we analyzed expression levels and repertoires of BCRs in a total of 17 peanut allergic subjects' peripheral blood samples before and after receiving oral immunotherapy (OIT) or placebo. By our methods, we successfully identified all of variable (V), joining (J), and constant (C) regions, in an average of 79.1% of total reads and 99.6% of these VJC-mapped reads contained the C region corresponding to the isotypes that we aimed to analyze. In the 17 peanut allergic subjects' peripheral blood samples, we observed an oligoclonal enrichment of certain immunoglobulin heavy chain alpha (IGHA) and IGH gamma (IGHG) clones (P = 0.034 and P = 0.027, respectively) in peanut allergic subjects after OIT. This newly developed BCR sequencing and analysis method can be applied to investigate B-cell repertoires in various research areas, including food allergies as well as autoimmune and infectious diseases.

High-affinity allergen-specific human antibodies cloned from single IgE B cell transcriptomes

“…Convergent antibody evolution is believed to occur in response to a number of pathogens such as influenza (26) and HIV (22). Although our results offer a single additional example, another study of peanut-allergic individuals (27) reported IgE VH sequences that used identical V and J genes and shared at least 70% CDR3 identity with one or more of the six convergent antibodies in our dataset ( fig. S9).…”

Human IgE producing B cells have a unique transcriptional program and generate high affinity, allergen-specific antibodies

“…A third individual had Ara h 2-specific antibodies that utilized a similar heavy V gene and the same light chain V gene. Although our study was limited by sample size, there is evidence supporting the importance of these genes within the peanut-allergic population more broadly: one independent study of IgE heavy chain sequences from peanut allergic individuals (64) reported IgE heavy chains that utilized identical V and J genes and shared at least 70% CDR3 identity with one or more of the six convergent antibodies in our dataset (fig. S12); another study (9) reported Ara h 2 specific IgG and IgM antibodies that utilized similar IGHV3-30 genes.…”

“…Similar IgE heavy chain CDR3 sequences in an independent dataset . Pairwise CDR3 sequence identity of the six convergent heavy chain CDR3 sequences from CF1 of the present study and three IgE heavy chain CDR3 sequences derived from multiple patients in a separate peanut allergy immune repertoire sequencing study (64). Each CDR3 sequence from this separate study shares at least 70% identity with one or more CDR3 sequences from the present study.…”

Recent developments and highlights in immune monitoring of allergen immunotherapy

“…Furthermore, targeted sequencing of T-cell receptor or Ig genes can reveal T-cell and B-cell signatures associated with disease or immune tolerance. [58][59][60] Proteomic approaches allow for the large-scale study of key proteins in solution, as well as those derived from cell fractions. The latter can also be examined in the context of single cells through advances in multi-parameter flow cytometry and mass cytometry.…”

“…58 In addition, the repertoire of IGH transcripts has been shown to undergo changes following AIT with more oligoclonality in IGHA and IGHG transcripts. 59 Furthermore, paired analysis of IgG transcripts in ryegrass pollen-allergic patients before and after 3 years of SLIT showed that usage of IGHG2 and IGHG4 was increased within the total pool of unique IGHG transcripts. 33 Together, these studies demonstrate the potential of measuring changes in the IGH transcript repertoire that correlate with allergic disease and effects of treatment, and might be utilized as biomarkers.…”