The landscape of the immunoglobulin repertoire in endemic pemphigus foliaceus

Abstract

Introduction: Primarily driven by autoreactive B cells, pemphigus foliaceus (PF) is an uncommon autoimmune blistering skin disease of sporadic occurrence worldwide. However, PF reaches a prevalence of 3% in the endemic areas of Brazil, the highest ever registered for any autoimmune disease, which indicates environmental factors influencing the immune response in susceptible individuals. We aimed to provide insights into the immune repertoire of patients with PF living in the endemic region of the disease, compared to healthy individuals from the endemic region and a non-endemic area.

Methods: We characterized the B-cell repertoire in i) nontreated patients (n=5); ii) patients under immunosuppressive treatment (n=5); iii) patients in remission without treatment (n=6); and two control groups iv) from the endemic (n=6) and v) non-endemic areas in Brazil (n=4). We used total RNA extracted from peripheral blood mononuclear cells and performed a comprehensive characterization of the variable region of immunoglobulin heavy chain (IGH) in IgG and IgM using next-generation sequencing.

Results: Compared to individuals from a different area, we observed remarkably lower clonotype diversity in the B-cell immune repertoire of patients and controls from the endemic area (p < 0.02), suggesting that the immune repertoire in the endemic area is under geographically specific and intense environmental pressure. Moreover, we observed longer CDR3 sequences in patients, and we identified differential disease-specific usage of IGHV segments, including increased IGHV3-30 and decreased IGHV3-23 in patients with active disease (p < 0.04). Finally, our robust network analysis discovered clusters of CDR3 sequences uniquely observed in patients with PF.

Discussion: Our results indicate that environmental factors, in addition to disease state, impact the characteristics of the repertoire. Our findings can be applied to further investigation of the environmental factors that trigger pemphigus and expand the knowledge for identifying new targeted and more effective therapies.

Keywords: B cells; autoimmunity; environmental factors; immunoglobulin repertoire; pemphigus foliaceus; skin disease.

Figure 1

Pipeline for the immune repertoire characterization. MIG: molecular identifier group. CDR3: complementarity determining region 3.

Figure 2

Clonotype abundance among groups. The clonotype frequency proportion differs among groups of samples (Low, < 0.1%; Medium, between 0.1 and 1%; Hyperexpanded, > 1%).

Figure 3

Clonotype diversity of the IGHM and IGHG gene segments. Alpha diversity was assessed with PD (phylogenetic diversity index). Controls from the non-endemic region and controls from the endemic region showed significant differences in most comparisons for both isotypes. Comparisons between endemic samples were non-significant.

Figure 4

Principal coordinate analysis (PCoA) based on the beta diversity of the CDR3 amino acid sequence. For IGHM, we considered the complete repertoire, showing a tendency of clustering (endemic vs. non-endemic, p = 0.11). For IGHG, we only analyzed the clonotypes with medium to high frequencies, showing a significant clustering between regions (endemic vs. non-endemic, p = 0.003).

Figure 5

Clustering of samples according to the disease status. We performed a principal component analysis with the frequencies of the IGHV gene segments that were differentially used between groups (see Table 2 ). Shaded areas represent sample groups (pink: individuals with active disease; green: individuals without disease). The group with active disease includes non-treated and under-treatment patients. The group without disease includes controls from the endemic area and patients in remission. PF: Patients with pemphigus foliaceus.

Figure 6

CDR3 length distribution in the study population groups. The curves represent the distribution of the CDR3 region’s length. Control samples from the non-endemic region (represented as thick light-blue curves) were compared to samples from the endemic region. Deviations from the Gaussian distribution were only observed in patients (p < 0.05, Shapiro-Wilk normality test). The vertical dotted line represents the CDR3 median length. aa = amino acid.

Figure 7

Similarity networks among clonotypes of patients and controls from the endemic area. (A) Network consisting solely of clonotypes from patients (PF). (B-E) Networks consisting predominantly of clonotypes from patients with a few from controls (CT) from the endemic area (Details of the network composition and clonotype sequences are shown in Supplementary Table. S5 ). The networks were constructed based on CDR3 amino acid sequences of IGHG clonotypes. The nodes represent unique clonotype sequences and are labeled with the sample IDs. Each clonotype differs from the adjacent clonotype by the quantity of amino acid residues indicated by the numbers between nodes (Hamming distances). The amino acid consensus sequences indicate the similarity between clonotype sequences in each network. Letter heights indicate the conservation in each position, with the most frequent amino acid placed on top.