Bruton's tyrosine kinase is essential for human B cell tolerance

Abstract

Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance, we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk, peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless, XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance.

Figure 1.

Peripheral XLA B cell are new emigrant B cells. Dot plots show CD10, IgM (top), CD10, and CD27 (bottom) expression on gated CD19⁺ B cells in a control (left) and XLA patient (right). Most peripheral blood B cells from XLA patients were CD19⁺ CD10⁺ IgM⁺ CD27⁻ new emigrant B cells that fail to develop into CD19⁺ CD10⁻ IgM⁺ CD27⁻ mature naive B cells.

Figure 2.

XLA B cells are a highly selected B cell population with a unique V_H and D gene usage. Proportions of V_H1 (A), V_H4 (B), D family (C) gene, and RF (D) usage in control new emigrant (open bars) and XLA B cells (closed bars). Control new emigrant B cell sequences included sequences shown in Table S1 and as reported previously (reference 1). D RF usage for combined D2, D5, and D6 family gene segments represented in panel D was assigned according to Corbett et al. (reference 27). *, statistically significant difference. P-values for differences between fractions are stated in the text and were calculated by the Fisher Exact Test.

Figure 3.

Extensive Igκ secondary recombination in XLA B cells. Jκ (A) and Vκ (B) usage in control new emigrant and XLA B cells. Control new emigrant B cell sequences included sequences shown in Table S1 and as reported previously (reference 1). Pie charts show proportion of different Jκ genes. The number of sequences analyzed in each fraction is indicated below the pie charts. The proximal Vκ locus (B) involved in 95% of Vκ–Jκ rearrangements is shown clustered into groups of V genes. The percent of each Vκ group is indicated on the y axis. *, statistically significant difference. P-values for differences between fractions are stated in the text and were calculated by the Fisher Exact Test.

Figure 4.

Extensive Igλ secondary recombination in XLA B cells. Jλ (A) and Vλ (B) usage in control new emigrant and XLA B cells. Control new emigrant B cell sequences included sequences shown in Table S1 and as reported previously (reference 1). Pie charts show proportion of different Jλ genes with the number of sequences analyzed indicated below. The Vλ locus (B) is shown clustered into groups of V genes. *, statistically significant difference. P-values for differences between fractions are stated in the text and were calculated by the Fisher Exact Test.

Figure 5.

A majority of XLA B cells express self-reactive antibodies. (A) Data shown are from ELISAs for anti–HEp-2 cell reactivity using recombinant antibodies from 29 control new emigrant (left), 29 XLA-Ins (middle), and 36 XLA-T117P B cells (right). Reactive antibodies are indicated by clone number. Dotted lines show ED38 positive control (reference 20). The percentage of autoreactive clones for each fraction is indicated. P-values for differences between fractions are shown and were calculated by the Fisher Exact Test. The p-value increases up to 0.013 when control new emigrant antibodies reported previously were included (reference 1). (B) XLA B cells express ANAs. Antibodies from XLA B cells show various patterns of ANA including nucleolar (X2-20) and nuclear and cytoplasmic patterns (X1-30, X2-53, X3-40, X3-69, and X-4-13).

Figure 6.

XLA B cells express polyreactive antibodies. Data shown are from ELISAs for reactivity with single-stranded DNA, double-stranded DNA, insulin, and LPS. Percentages represent frequency of polyreactive antibodies. Dotted lines show ED38 positive control (reference 20). Reactive antibodies are indicated by clone number. The frequency of polyreactive antibodies is significantly increased in XLA B cells (P ≤ 0.0001). *, control new emigrant clones reported previously (reference 1).