High throughput profiling of the B cell repertoire identifies systematic changes in the repertoire of individuals with Crohn's disease

Abstract

The B cell repertoire contains the recombined sequences that encode the entire antibody repertoire of an individual. The repertoire is made from three antigenic binding chains, namely the immunoglobulin heavy chain (IGH) and two immunoglobulin light chains, κ (IGK) and λ (IGL). Compared to the T cell repertoire, the B cell repertoire is understudied in inflammatory bowel diseases (IBD) even though different antibodies such as ASCA (Anti-Saccharomyces cerevisiae) and ANCA (Anti-Neutrophil Cytoplasmic Antibodies) have been shown to be elevated in individuals with IBD. To address this limitation, we profiled the B cell repertoire of peripheral blood from 27 treatment-naive individuals with CD and 21 age-matched symptomatic controls using bulk B cell receptor sequencing. The repertoire of individuals with CD showed a reduction in diversity and an increase in clonality. Furthermore, we observed a significant reduction in the expansion of IgM and IgD and an expansion of IgA2, and IgG2 clonotypes in individuals with CD relative to controls, suggesting an antigen-driven expansion. This was also supported by higher levels of somatic hypermutations, particularly in the complementary determining region 2 (CDR2) of immunoglobulin heavy chain, in individuals with CD relative to the control group. Thus, despite the small sample size, we identified multiple alterations in the B cell repertoire of individuals with CD, highlighting the potential of the B cell repertoire in identifying antigenic exposures implicated in the disease, demanding now larger international studies, ideally including also treatment-naive and pre-clinical cases.

Keywords: B cell; B cell repertoire sequencing; IBD; Ig isotype; somatic hypermutation; treatment-naïve.

Figure 1

Repertoire diversity and inequality across BCR loci (productive clonotypes). (A) Schematic overview of the IBSEN-III peripheral blood BCR-seq workflow. Treatment-naive individuals (Crohn’s disease (CD) and symptomatic controls (SC)) were sampled; peripheral blood was collected into PAXgene tubes, RNA was extracted, and the BCR repertoire was amplified using multiplex PCR targeting IGH, IGK, and IGL rearrangements prior to sequencing and downstream analysis. Per-sample diversity metrics were calculated from productive clonotypes for the heavy chain (IGH) and light chains (IGK, IGL) and compared between Crohn’s disease (CD; orange) and control subjects (SC; grey). (B–D) Shannon diversity index (UMI-weighted clonotype abundance) for IGH, IGK, and IGL, respectively. (E–G) Chao1 richness estimator for IGH, IGK, and IGL, respectively. (H–J) Gini coefficient of the clonotype abundance distribution (higher values indicate greater clonal inequality/expansion) for IGH, IGK, and IGL, respectively. Each dot represents one sample; horizontal black lines indicate the median. P-values are from two-sided Mann-Whitney U tests (NS, not significant). Panel (A) was Created in BioRender. ElAbd, H. (2026) https://BioRender.com/cj89mj7.

Figure 2

Differences in IGH isotype usage between Crohn’s disease (CD) and symptomatic controls (SC) using MiXCR-calculated unique-molecule fractions. (A) UMI-weighted isotype fractions per sample, computed by summing MiXCR calculated UMI fractions across clonotypes assigned to each IGH isotype/subclass (IgM, IgD, IgG1-4, IgA1–2 and IgE). (B) Clonotype-count (unweighted) isotype fractions per sample, computed as the fraction of unique clonotypes assigned to each isotype/subclass. Each dot represents one sample; horizontal black lines indicate group medians. CD samples are shown in orange and SC samples in grey. Statistical comparisons were performed per isotype using two-sided Mann-Whitney U tests with Benjamini-Hochberg (BH/FDR) correction across isotypes within each panel; adjusted p-values are shown above each isotype. Because axes are log-scaled, samples with missing or zero values were displayed at a small constant (1×10^-6) for plotting only; raw values (including zeros) were retained for statistical testing.

Figure 3

Region-resolved somatic hypermutation (SHM) in IGH differs between CD and SC. Per-sample, region-specific SHM rates were quantified from MiXCR allele-reassigned IGH clonotypes by comparing observed nucleotide sequences to their inferred germline counterparts for CDR1 (A), CDR2 (B), FR2 (C), FR3 (D), and FR4 (E). For each sample and region, we computed a UMI-weighted mean SHM rate (mismatches/compared bases), where compared bases include only positions with unambiguous A/C/G/T in both observed and germline sequences. Each dot represents one sample (CD vs SC), with boxplots summarizing the distribution across samples (median and interquartile range). Statistical differences between CD and SC were assessed using a two-sided Mann-Whitney U test per region and corrected for multiple testing across regions using Benjamini-Hochberg (BH).