The role of Bruton's tyrosine kinase in the development and BCR/TLR-dependent activation of AM14 rheumatoid factor B cells

Abstract

The protein kinase Btk has been implicated in the development, differentiation, and activation of B cells through its role in the BCR and TLR signaling cascades. These receptors and in particular, the BCR and either TLR7 or TLR9 also play a critical role in the activation of autoreactive B cells by RNA- or DNA-associated autoantigens. To explore the role of Btk in the development of autoreactive B cells, as well as their responses to nucleic acid-associated autoantigens, we have now compared Btk-sufficient and Btk-deficient mice that express a prototypic RF BCR encoded by H- and L-chain sdTgs. These B cells bind autologous IgG2a with low affinity and only proliferate in response to IgG2a ICs that incorporate DNA or RNA. We found that Btk-sufficient RF(+) B cells mature into naïve FO B cells, all of which express the Tg BCR, despite circulating levels of IgG2a. By contrast, a significant proportion of Btk-deficient RF(+) B cells acquires a MZ or MZ precursor phenotype. Remarkably, despite the complete inability of RF(+) Xid/y B cells to respond to F(ab')2 anti-IgM, RF(+) Xid/y B cells could respond well to autoantigen-associated ICs. These data reveal unique features of the signaling cascades responsible for the activation of autoreactive B cells.

Keywords: Btk; Toll-like receptor; Xid; autoantibody; autoreactive B cells; marginal zone B cells.

Figure 1.. Serum isotype and clonotype levels in non-Tg and AM14 sdTg mice.

(A–E) Comparison of BALB/c WT (●; n=6) and BALB/c AM14 (□; n=8) serum isotype titers. Mean ± sem is shown. Statistical significance was analyzed by two-way ANOVA (**P ≤ 0.01; *P ≤ 0.05). (F) Sera were adsorbed on a 4G7 affinity column. Total Ig levels of the original sera (filled symbols) and eluates (open symbols) were determined by ELISA.

Figure 2.. Loss of MZ and B1 B cells in BALB/c AM14 B cells.

Splenocytes from BALB/c WT (left) and BALB/c AM14 (right) mice were analyzed by flow cytometry using the markers B220 and 4G7 to identify clonotype+ B cells (A) and B220 and AA4.1, to identify mature and immature B cells (B). B220⁺ B cells were analyzed further for MZ (CD21^hi CD23^lo) and FO (CD21^lo/− CD23^hi) compartments (C) or for MZ B cells and MZP (IgM^hi and CD21^hi; D). (E) Peritoneal cells were stained with B220 and CD5 to identify B1 cells. Representative plots of at least five independent experiments are shown.

Figure 3.. B cell subset distribution of (CBA/N×BALB/c) AM14 Xid/y mice resembles non-Tg Xid/y mice.

Splenocytes of (CBA/N×BALB/c) Xid/+, Xid/y, AM14 Xid/+, and AM14 Xid/y were analyzed for B cell subset distribution by flow cytometry. Representative plots of three independent experiments are shown. Splenocytes were stained with B220 and AA4.1 (A) or B220 and the anti-clonotype 4G7 (B) or IgM and IgD (C). B220-gated B cells were stained for MZ (CD21^hi CD23^lo) and FO (CD21^lo/− CD23^hi) B cell (D) or with IgM and CD21 (E). (F–I) Spleen sections were stained for IgM and MOMA-1 to visualize the localization of the MZ ring around the B cell follicle. Representative sections of two independent experiments are shown.

Figure 4.. Serum isotype and clonotype levels in AM14 Xid/+ and AM14 Xid/y mice.

(A–E) Comparison of (CBA/N×BALB/c) Xid/+ (●; n=3), Xid/y (○; n=4), AM14 Xid/+ (■; n=8), and AM14 Xid/y (□; n=8) serum isotype titers. Mean ± sem is shown. Statistical analysis was performed by two-way ANOVA (**P ≤ 0.01).

Figure 5.. Ca²⁺-dependent activation of AM14 Xid/y B cells is muted.

B220-purified B cells from the indicated strains were stimulated with of F(ab′)₂ anti-IgM (A), anti-RP105 (B), or anti-CD38 (C) for 30 h. Entry into cell cycle was measured by ³H-thymidine incorporation. Mean ± sem of four to 10 independent experiments is shown. One-way ANOVA was used to test for statistical significance (*P≤0.05; **P≤0.01; ***P≤0.005).

Figure 6.. AM14 Xid/y B cells respond to TLR activation.

Splenic B220⁺ B cells from (CBA/N×BALB/c) Xid/+ (●), Xid/y (○), AM14 Xid/+ (■), and AM14 Xid/y (□) were cultured with the indicated concentrations of LPS (A), CL097 (B) or CpG (C) for 30 h. Proliferation was ascertained by ³H-thymidine uptake. Mean ± sem of three independent experiments. Statistical significance was ascertained by two-way ANOVA (*P≤0.05).

Figure 7.. AM14 Xid/y B cells respond to nucleic acid containing ICs.

(A) B220-purified B cells from AM14 Xid/+ and AM14 Xid/y were stimulated with CpG or the mAb PL2-3 (n=14), PA4 (n=7), and BWR4 (n=7) for 24 h, and proliferation was assessed by ³H-thymidine incorporation. Mean ± sem is shown, and statistical significance was determined by Student's t-test (*P≤0.05; ***P≤0.005). (B) AM14 Xid/+ (■; n=3) and AM14 Xid/y (□; n=4) B cells were cultured in media alone (left), BLyS (middle), or BWR4 (right). Cell death was determined at the indicated time-points by flow cytometry using TO-PRO-3. (C) CFSE-labeled B220⁺ AM14 Xid/+ (upper) and AM14 Xid/y (lower) B cells were cultured for 72 h as described in B and then stained with TO-PRO-3 and analyzed by flow cytometry. Representative plots of five independent experiments are shown. (D) The cell-surface expression of CD69 and CD86 by AM14 Xid/+ (gray line) and AM14 Xid/y (black line) B cells stimulated for 3 days with BWR4 was determined by flow cytometry. Isotype control is shown by the solid curve. Representative plots of two independent experiments are shown.