Evidence for enhanced Bruton's tyrosine kinase activity in transitional and naïve B cells of patients with granulomatosis with polyangiitis

Abstract

Objectives: To determine Bruton's tyrosine kinase (BTK) protein and phosphorylation levels in B cell subsets of granulomatosis with polyangiitis (GPA) patients and to investigate the effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production.

Methods: BTK protein and phosphorylation levels were determined by flow cytometry in peripheral blood B cells of 29 untreated GPA patients [9 active and 20 remission GPA patients (10 ANCA- and 10 ANCA+)], 9 age- and sex-matched healthy controls (HCs) and 9 untreated active RA patients. The effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production was determined in the same donors in peripheral blood mononuclear cell cultures.

Results: BTK protein levels were significantly increased in transitional and naïve B cells of active GPA and RA patients compared with remission GPA patients and HCs. Both B cell subsets of active patients were more sensitive to B cell receptor stimulation, as BTK and phospholipase Cγ2 phosphorylation were increased in these patients. In vitro BTK blockade had profound effects on B cell cytokine production, plasma cell formation and (auto)antibody production in both GPA patients and HCs. Interestingly, the effect of BTK blockade was less pronounced in active GPA patients, possibly due to increased activation of B cells.

Conclusion: We show that BTK protein and phosphorylation levels are most profoundly increased in newly emerging B cells of active GPA patients compared with remission patients. BTK blockade greatly inhibits in vitro B cell effector functions in GPA patients and HCs. These promising data identify BTK as an interesting novel therapeutic target in the treatment of GPA.

Keywords: B cells; BTK blocker; Bruton’s tyrosine kinase; vasculitis.

Fig. 1

BTK protein levels are increased in B cells from active GPA patients compared with HCs and remission patients (A) BTK protein levels in total peripheral B cells from HCs, active GPA, remission GPA and RA patients, analysed by intracellular flow cytometry. Depicted ratio is BTK MFI in B cells/BTK MFI in T cells. Histogram shows an overlay of a representative HC and active GPA patient. (B–F) BTK protein levels in naïve B cells (B), transitional B cells (C), IgD⁺ non-switched memory B cells (D), switched memory B cells (E) and plasmablasts (F). *P < 0.05; **P < 0.01; ***P < 0.001. act: active; BTK: Bruton’s tyrosine kinase; GPA: granulomatosis with polyangiitis; HCs: healthy controls; MFI: mean fluorescence intensity.

Fig. 2

Increased BCR activity in transitional and naïve B cells of GPA patients ex vivo (A) Correlations between BTK protein and ex vivo pBTK in total B cells of HCs (open circles) and active GPA patients (closed circles). Histogram overlay shows ex vivo pBTK expression in total B cells. (B) Ex vivo pBTK expression in transitional and naïve B cells of HCs, active and remission GPA patients. (C) Correlations between BTK protein and pPLCγ2 in total B cells of HCs (open circles) and active GPA patients (closed circles). Histogram overlay shows pPLCγ2 expression in total B cells. (D) Ex vivo pPLCγ2 expression in transitional and naïve B cells of HCs, active and remission GPA patients. **P < 0.01. B: B cells; BCR: B cell receptor; BTK: Bruton’s tyrosine kinase; pBTK: phosphorylated BTK; GPA: granulomatosis with polyangiitis; HCs: healthy controls; MFI: mean fluorescence intensity; pPLCγ2: phosphorylated phospholipase Cγ2.

Fig. 3

Increased in vitro BCR responsiveness in transitional and naïve B cells of active GPA patients pBTK expression on transitional B cells (A) and naïve B cells (B) in vitro, unstimulated (left) and upon stimulation with anti-IgM Fab₂ fragments for 5 min (aIgM; right). Histogram shows overlays of pBTK MFI of a representative HC and active GPA patient. (C) Ratio of pBTK MFI on different B cell subsets of anti-IgM stimulated sample/unstimulated samples. (D, E) pPLCγ2 expression on transitional B cells (D) and naïve B cells (E) in vitro, unstimulated (left) and upon stimulation with anti-IgM F(ab)₂ fragments for 5 min (right). Histogram shows overlays of pPLCγ2 MFI of a representative HC and active GPA patient. (F) Ratio of pPLCγ2 MFI on different B cell subsets of anti-IgM stimulated sample/unstimulated sample. *P < 0.05; **P < 0.01; ***P < 0.001. act: active; aIgM: anti-IgM; B: B cells; BCR: B cell receptor; GPA: granulomatosis with polyangiitis; HC: healthy control; MFI: mean fluorescence intensity; pBTK: phosphorylated Bruton’s tyrosine kinase; pPLCγ2: phosphorylated phospholipase Cγ2; stim: stimulated; unstim: unstimulated.

Fig. 4

BTK protein levels of B cells of GPA patients correlate with B cell activation and decreased pro-inflammatory T cell subsets in the circulation (A) CD86 expression on total peripheral B cells of HCs, active and remission GPA patients. Histogram shows an overlay of a representative HC and active GPA patient. (B–D) Correlation of BTK protein with CD86 expression on total B cells of patients and HCs (B), proportions of circulating pro-inflammatory (C) or regulatory (D) CD4⁺ T cell subsets in active GPA patients (circles) and remission GPA patients (squares). act: active; B: B cells; BCR: B cell receptor; BTK: Bruton’s tyrosine kinase; GPA: granulomatosis with polyangiitis; HC: healthy control; MFI: mean fluorescence intensity; T: T cells; Tfh-like: T follicular helper-like cell; Tfr-like: T follicular helper regulatory cell.

Fig. 5

BTK blockade inhibits in vitro B cell cytokine production, plasma cell formation and (auto)antibody production (A) The frequencies of IFNγ⁺ (top left), TNFα⁺ (top right), IL-10⁺ (bottom left) and IL-6⁺ (bottom right) B cells are given in samples stimulated with anti-IgM only (open circle) and samples stimulated with anti-IgM + BTK blocker (open squares) for HCs, active and remission GPA patients. (B) For HCs, active and remission GPA patients, the frequencies of memory B cells (left) and plasma cells (right) within CD19⁺CD22⁺ B cells are depicted. Open circles represent samples stimulated with anti-IgM/BAFF, open squares represent samples stimulated with anti-IgM/BAFF in the presence of the BTK blocker. (C) PR3–ANCA levels (left) for anti–IgM/BAFF/IL–21 and anti–IgM/BAFF/IL–21 + BTK blocker stimulated samples for active (open circles), ANCA– (open squares), and ANCA+ (open diamonds) remission GPA patients, and total IgG concentrations (right) in anti–IgM/BAFF/IL–21 (open circles) and anti–IgM/BAFF/IL–21 + BTK blocker (open squares) stimulated samples for HCs, active and remission GPA patients. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. BAFF: B cell activating factor; BTK: Bruton’s tyrosine kinase; GPA: granulomatosis with polyangiitis; HCs: healthy controls; RU: respone units.