IgD shapes the pre-immune naïve B cell compartment in humans

Abstract

B cell maturation and immunoglobulin (Ig) repertoire selection are governed by expression of a functional B cell receptor (BCR). Naïve B cells co-express their BCR as IgM and IgD isotype. However, the role of the additionally expressed IgD on naïve B cells is not known. Here we assessed the impact of IgD on naïve B cell maturation and Ig repertoire selection in 8 individuals from 3 different families with heterozygous loss-of-function or loss-of expression mutations in IGHD. Although naïve B cells from these individuals expressed IgM on their surface, the IGHD variant in heterozygous state entailed a chimeric situation by allelic exclusion with almost half of the naïve B cell population lacking surface IgD expression. Flow cytometric analyses revealed a distinct phenotype of IgD-negative naïve B cells with decreased expression of CD19, CD20 and CD21 as well as lower BAFF-R and integrin-β7 expression. IgD-negative B cells were less responsive in vitro after engaging the IgM-BCR, TLR7/9 or CD40 pathway. Additionally, a selective disadvantage of IgD-negative B cells within the T2 transitional and mature naïve B cell compartment as well as reduced frequencies of IgM^lo/- B cells within the mature naïve B cell compartment lacking IgD were evident. RNA-Ig-seq of bulk sorted B cell populations showed an altered selection of distinct V_H segments in the IgD-negative mature naïve B cell population. We conclude that IgD expression on human naïve B cells is redundant for generation of naïve B cells in general, but further shapes the naive B cell compartment starting from T2 transitional B cells. Our observations suggest an unexpected role of IgD expression to be critical for selection of distinct Ig V_H segments into the pre-immune Ig repertoire and for the survival of IgM^lo/- naïve B cells known to be enriched in poly-/autoreactive B cell clones.

Keywords: B cell; B cell maturation; B cell receptor; IgD; IgM; immunoglobulin repertoire.

Figure 1

Lack of surface IgD expression in a fraction of naïve B cells in individuals carrying heterozygous IGHD variants. (A) Pedigree of individuals with heterozygous IGHD variants. Individuals affected by a disease are shown in black. Wild type (wt) and mutant (mut) describes the presence of respective IGHD alleles in each individual. (B) Representative dot plot showing IgD expression on CD19⁺CD27^-IgM⁺ naïve B cells from a control individual as well as an individual carrying a heterozygous IGHD variant. (C) Representative sequencing chromatograms (IGHD, genomic DNA) of 3 individuals with different heterozygous IGHD variants and controls. (D) Compiled flow cytometric data showing the frequency of IgD-negative B cells within CD19⁺CD27^-IgM⁺ naïve B cells in control individuals and family members without (IGHD-wt) or with (IGHD-het.) a heterozygous IGHD variant (One-way ANOVA with Turkey`s multiple comparison). n.s., not significant.

Figure 2

Characterization of IGHD variants reveal loss-of-expression or loss-of-function (A) Flow cytometric analysis of surface IgD expression on CD19⁺CD27^-IgM⁺ naïve B cells from 3 individuals carrying different heterozygous IGHD variants (upper row). Intracellular IgD expression within surface IgD+ (blue line) or surface IgD- (red line) CD19⁺CD27^-IgM⁺ naïve B cells from the same individuals (lower row). Dashed lines represent isotype controls. (B) Proportion of unique IgM or IgD sequence reads assessed by RNA-based IgH high-throughput sequencing within sorted CD19⁺CD27^-IgM⁺CD10^- IgD⁺ or IgD^- mature naïve B cells from 3 individuals carrying different heterozygous IGHD variants (C) Immunoblot analysis of IgD Expression in 293T cells not transfected (n.t.) or transfected with an empty vector or an IgD-FLAG expression vector with wild type IgD sequence or the p.P6L variant. (D) Histograms showing flow cytometric analysis of CD69 expression on sorted IgD+ or IgD- CD19+CD27-IgM+ naïve B cells from an individual carrying the p.P6L IGHD variant stimulated in vitro with anti-IgM or anti-IgD for 48 hours. Fold induction (stimulated versus non-stimulated) of CD69 expression is shown on the right.

Figure 3

Loss of IgD expression is not compensated by upregulation of IgM expression in IgD-negative B cells (A) Representative histograms of IgM and IgD surface expression in different peripheral blood B cell subsets analyzed by flow cytometry (CD19⁺CD27^-CD10⁺CD21^- transitional 1 (T1) B cells, CD19⁺CD27^-CD10⁺CD21⁺ transitional 2 (T2) B cells, and CD19⁺CD27^-CD10^-CD21⁺ mature naive (MN) B cells, (B) Compiled flow cytometric data of IgM and IgD expression levels (mean fluorescence intensity, MFI) on different peripheral blood B cell subsets derived from 9 individuals. (**, p < 0.01; ***, p < 0.001; n.s., not significant; One-Way-ANOVA with Turkey`s multiple comparison test). (C) Representative dot blot showing surface expression of kappa and lambda light chain on IgD-positive (blue) or IgD-negative (red) CD19<sup>+</sup>CD27<sup>-</sup>CD10<sup>-</sup>CD21<sup>+</sup> mature naïve B cells. Compiled data on expression levels are shown on the right (paired Student`s t-test). (D) Representative histograms obtained from flow cytometric analysis showing IgM expression levels on CD19⁺CD27^-CD10⁺CD21^- transitional 1 B cells as well as IgD⁺ or IgD^- CD19⁺CD27^-CD10^-CD21⁺ mature naïve B cells. Compiled data from 6 individuals carrying different IGHD variants is shown in the lower panel (mean fluorescence intensity, MFI; One-way ANOVA with Turkey`s multiple comparison). (E) Representative dot blot showing the frequency of IgM<sup>lo/-</sup> B cells within IgD<sup>+</sup> (blue) or IgD<sup>-</sup> (red) CD27<sup>-</sup>CD10<sup>-</sup>CD21<sup>+</sup> mature naïve B cells from an individual carrying a heterozygous IGHD-variant. Compiled data from individuals with different IGHD-variants showing frequencies (left) and absolute counts (right) of IgM<sup>lo/-</sup> B cells (paired Student`s t-test).

Figure 4

Reduced expression of co-stimulatory molecules on IgD-negative naïve B cells (A) Representative histograms obtained from flow cytometric analysis of IgD⁺ (blue line) or IgD^- (red line) CD19⁺CD27^-IgM⁺ naïve B cells from an individual carrying a heterozygous IGHD variant showing expression of different markers. Dashed lines represent isotype control. (B) Relative expression levels of each marker within 5 different individuals carrying heterozygous IGHD variants is shown as IgD⁺/IgD^- ratio (mean fluorescence intensity, MFI; *, p < 0.05; **, p < 0.01; ***, p < 0.001 one sample t-test with hypothesized mean of 1).

Figure 5

Impaired activation of IgD-negative naïve B cells after stimulation by IgM-BCR-, TLR7/9- or CD40 ligands (A) Representative dot blots showing expression of CD69 and CD86 on CD19⁺CD27^-IgM⁺ IgD⁺ or IgD^- naive B cells stimulated or not with F(ab)₂ anti-IgM, Gardiquimod (TLR7), CpG (TLR9) or CD40L for 48 hours from a control individual (upper row) or an individual carrying a heterozygous IGHD variant. (B) Compiled data from 4 control individuals as well as 5 individuals carrying different IGHD variants (paired Student`s t-test; **, p < 0.01; *, p < 0.05).

Figure 6

IgD-positive B cells outcompete IgD-negative B cells within the naïve B cell compartment (A) Representative dot blot obtained from flow cytometric analysis of CD19⁺ peripheral blood B cells from a control individual and an individual carrying a heterozygous IGHD variant showing the distribution of IgD⁺ and IgD^- cells within CD27^-IgM⁺CD10⁺CD21^- transitional 1 B cells, CD27^-IgM⁺CD10⁺CD21⁺ transitional 2 B cells and CD27^-IgM⁺CD10^-CD21⁺ mature naive B cells. (B) Compiled flow cytometric data of IgM and IgD surface expression levels in IgD⁺ transitional 1 (T1), transitional 2 (T2) and mature naïve IgD+ B cells in 8 individuals carrying different IGHD variants. The relative mean fluorescence intensity (MFI) was calculated as IgD or IgM MFI on different B cell subsets divided through the MFI on CD19^- non-B cells (background). (C) The distribution of IgD- B cells within each B cell subset was analyzed as ratio IgD-/IgD⁺ cells. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant; (B) One-Way-ANOVA with Turkey`s multiple comparison test; (C) one sample t-test with hypothesized mean of 1).

Figure 7

Altered distribution of V_H segments in the IgD-negative mature naïve B cell population Distribution of different V_H (A) and J_H (B) segments used in CD19⁺CD27^-CD21⁺CD10^-IgM⁺IgD^- mature naïve B cells is shown as relative difference compared to the matched IgD⁺ counterpart. Symbols represent the Δ frequency IgD^-/mutant versus IgD⁺/wild-type from individual samples and bars the mean ± SEM. V_H and J_H segments are ordered on the x-axis according to their mean relative difference (*, p < 0.05; **, p < 0.01; one sample t-test with hypothesized mean of 0). (C) Representation of species richness (q=0), Shannon diversity index (q=1) and Simpson diversity index (q=2) is shown as difference between IgD^- and IgD⁺ B cell subsets. Symbols represent the Δ value IgD^-/mutant versus IgD⁺/wild-type of each parameter from individuals samples and bars the mean ± SEM. (D) Distribution of CDR3 IgH length in IgD⁺ (blue) and IgD^- (red) mature naïve B cells. Each symbol represents the mean frequency ± SEM of a distinct CDR3 length.