CD23+IgG1+ memory B cells are poised to switch to pathogenic IgE production in food allergy

Abstract

Food allergy is caused by allergen-specific immunoglobulin E (IgE) antibodies, but little is known about the B cell memory of persistent IgE responses. Here, we describe, in human pediatric peanut allergy, a population of CD23⁺IgG1⁺ memory B cells arising in type 2 immune responses that contain high-affinity peanut-specific clones and generate IgE-producing cells upon activation. The frequency of CD23⁺IgG1⁺ memory B cells correlated with circulating concentrations of IgE in children with peanut allergy. A corresponding population of "type 2-marked" IgG1⁺ memory B cells was identified in single-cell RNA sequencing experiments. These cells differentially expressed interleukin-4 (IL-4)- and IL-13-regulated genes, such as FCER2/CD23⁺, IL4R, and germline IGHE, and carried highly mutated B cell receptors (BCRs). In children with high concentrations of serum peanut-specific IgE, high-affinity B cells that bind the main peanut allergen Ara h 2 mapped to the population of "type 2-marked" IgG1⁺ memory B cells and included clones with convergent BCRs across different individuals. Our findings indicate that CD23⁺IgG1⁺ memory B cells transcribing germline IGHE are a unique memory population containing precursors of high-affinity pathogenic IgE-producing cells that are likely to be involved in the long-term persistence of peanut allergy.

Fig. 1.. Frequency of IgG⁺CD23⁺ memory B cells correlates with total IgE concentrations in peanut allergy.

(A) Percentages of surface CD23⁺ B cells in total CD19⁺ B cells, in CD19⁺CD27⁻IgM/IgD⁺ B cells (Naïve), and in CD19⁺CD27⁺ B cells (CD27⁺ memory) were analyzed in peanut-allergic patients (n=58; red dotted bar) and non-allergic healthy children (n=13; blue solid bar). Data are presented as median ± 95 % CI. Data were analyzed by unpaired t-test. (B and C) Shown are correlations between the frequency of CD23⁺ memory B cells (B) or CD23⁻ memory B cells (C) with total plasma IgE concentrations. Data were analyzed by Spearman correlation. (D) CD23⁺ B cell populations were analyzed among peanut-allergic patients with high peanut-specific IgE (PA PsIgE>100, n=18; red solid bar) or with low peanut-specific IgE (PA PsIgE<5, n=16; red shaded bar), as well as in non-allergic individuals (n=13; blue solid bar). Data are presented as median ± 95 % CI. Data were analyzed by unpaired t-test for naïve B and switched CD27⁺ or Mann-Whitney test for total CD27⁺ and IgG⁺ CD27⁺.

Fig. 2.. scRNA-seq seq reveals CD23⁺ memory B cells transcribe IGHE, are enriched for IGHG BCR constant regions, and are highly mutated.

(A) Shown is a Uniform Manifold Approximation and Projection (UMAP) plot of cells obtained from peanut-allergic and non-allergic individuals. Each point represents a single cell. (B) Shown is scaled gene expression of cells in cluster 5. Tiles show scaled, normalized expression of cluster 5. Genes are positively differentially expressed genes (adjusted p < 0.05) for cluster 5. IL-4 and IL-13 regulated genes IGHE, FCER2, IL4R, and CD86 are shown in red. (C) Shown is the proportion of cells in each scRNA-seq defined cluster that uses each isotype constant region. Enrichment for IGHG-associated BCRs was analyzed by chi-square test (Cluster 5: 61.7% of cells, p < 2.2e-16). (D) Shown is the frequency of IGHV-gene SHM for cells in each cluster. Cells are colored by associated constant region. Data in (D) are presented as box and whiskers plots in which boxes represent the median as well as first and third quartiles, and whiskers extend to the largest and smallest values within 1.5 times the interquartile range from the boxes.

Fig. 3.. IGHE transcribing B cells primarily use IGHG1 and IGHG4 BCR constant regions and are associated with cluster 5.

(A) Distribution of normalized IGHE expression among all B cells that express at least one IGHE transcript. Dashed lines show the amount of IGHE transcription in the two observed IGHE-constant region B cells. “IGHE-transcribing” cells are defined as cells with normalized IGHE expression at least as high as one of these two observed cells using IGHE-constant regions.(B) IGHE-transcribing cells were associated with each cluster and constant region. Cells of the grid are labeled by the total number of IGHE-transcribing cells and colored by the percentage of cells in each cluster that are IGHE-transcribing.

Fig. 4.. IgG switched CD23⁺ memory B cells from peanut-allergic individuals with PsIgE>100 showed an increased frequency of B cells producing peanut-specific IgG.

(A) Two distinct populations of CD27⁺ memory B cells were sorted for memory B cell culture experiments. (B) Culture supernatants of IgG⁺ memory B cells from non-allergic individuals (N=7, left), peanut-allergic individuals with PsIgE<5 (N=7, middle), and peanut-allergic individuals with PsIgE>100 (N=7, right) were tested for the reactivity against peanut by ELISA. Percent of positive wells among the total wells (indicated in each circle) of each culture is shown. Shaded orange pie slice represents a percent positive among CD23⁻IL-4R⁻ memory B cell culture wells and solid orange pie slice represents a percent positive among CD23⁺/IL-4R⁺ memory B cell culture wells. Differences in the percentage of positive wells were determined by chi-square test. (C) Culture supernatants of IgG⁺ memory B cells from peanut-allergic individuals with PsIgE>100 (N=4) were tested for the reactivity of IgG against peanut (top) and Ara h 2 (bottom) by ELISA. Percent of positive wells among the total wells (indicated in each circle) of each culture is shown. Shaded red pie slice represents a percent positive among CD23⁻IL-4R⁺ memory B cell culture wells and the solid red pie slice represents a percent positive among CD23⁺ memory B cell culture wells. Differences in the percentage of positive wells were determined by chi-square test.

Fig. 5.. BCRs from Ara h 2-binding IgG1⁺ B cells are highly mutated, use IGHG1 constant regions, and express FCER2 and IGHE.

(A) Shown is the frequency of IGHV-gene somatic hypermutation for DT and Ara h 2-sorted B cells with either IGHG or IGHM constant regions from either peanut-allergic or non-allergic individuals. Data were analyzed by Mann-Whitney U test. Data are presented as box and whiskers plots in which boxes represent the median as well as first and third quartiles, and whiskers extend to the largest and smallest values within 1.5 times the interquartile range from the boxes. (B) Shown are the proportions of DT and Ara h 2-sorted IGHG B cells with associated IGHG sub-isotypes. (C) FCER2, GLT-IGHE, and total-IGHE gene expression was evaluated in single sorted IgG1⁺ B cells. mRNA was quantified in single IgG1⁺ B cells derived from Ara h 2 binders (n=34 cells) from 6 peanut-allergic individuals with PsIgE>100 and DT binders from 6 peanut-allergic individuals with PsIgE>100 (n=14 cells) and 3 non-allergic individuals (n=23 cells). Data are presented as mean±SD and were analyzed by Mann-Whitney U test.

Fig. 6.. Convergent Ara h 2-binding sequence clusters were observed across multiple donors.

(A) Amino acid alignment of heavy and light chain sequences is shown for two shared convergent sequence clusters. The upper sequence in each plot shows the predicted germline V and J gene sequences, followed by clonal members. Brackets indicate complementarity determining regions (CDRs) on sequence alignments. (B and C) ELISA curves are shown for monoclonal antibodies in the PA13P1E10 family (B) and the 4C5GF1 family (C) generated from V(D)J sequences of convergent clusters from (A).