Effects of targeting the transcription factors Ikaros and Aiolos on B cell activation and differentiation in systemic lupus erythematosus

Abstract

Objective: To evaluate the effects of targeting Ikaros and Aiolos by cereblon modulator iberdomide on the activation and differentiation of B-cells from patients with systemic lupus erythematosus (SLE).

Methods: CD19⁺ B-cells isolated from the peripheral blood of patients with SLE (n=41) were cultured with TLR7 ligand resiquimod ±IFNα together with iberdomide or control from day 0 (n=16). Additionally, in vitro B-cell differentiation was induced by stimulation with IL-2/IL-10/IL-15/CD40L/resiquimod with iberdomide or control, given at day 0 or at day 4. At day 5, immunoglobulins were measured by ELISA and cells analysed by flow cytometry. RNA-Seq was performed on fluorescence-activated cell-sorted CD27^-IgD⁺ naïve-B-cells and CD20^lowCD27⁺CD38⁺ plasmablasts to investigate the transcriptional consequences of iberdomide.

Results: Iberdomide significantly inhibited the TLR7 and IFNα-mediated production of immunoglobulins from SLE B-cells and the production of antinuclear antibodies as well as significantly reducing the number of CD27⁺CD38⁺ plasmablasts (0.3±0.18, vehicle 1.01±0.56, p=0.011) and CD138⁺ plasma cells (0.12±0.06, vehicle 0.28±0.02, p=0.03). Additionally, treatment with iberdomide from day 0 significantly inhibited the differentiation of SLE B-cells into plasmablasts (6.4±13.5 vs vehicle 34.9±20.1, p=0.013) and antibody production. When given at later stages of differentiation, iberdomide did not affect the numbers of plasmablasts or the production of antibodies; however, it induced a significant modulation of gene expression involving IKZF1 and IKZF3 transcriptional programmes in both naïve B-cells and plasmablasts (400 and 461 differentially modulated genes, respectively, false discovery rate<0.05).

Conclusion: These results demonstrate the relevance of Ikaros and Aiolos as therapeutic targets in SLE due to their ability to modulate B cell activation and differentiation downstream of TLR7.

Keywords: B-lymphocytes; autoimmune diseases; lupus erythematosus; systemic.

Figure 1

TLR7-induced and IFN-induced activation of SLE B cells. (A) Overview of experimental setup: B cells from SLE patients were triggered with TLR7 ligand R848 (Resiquimod) with iberdomide (1–10–100 nM) or vehicle as shown. After 5 days, cells were harvested and underwent flow-cytometry, while supernatants were used to measure ELISA. (B, C). IgG and IgM measured in the supernatants of cells stimulated as above with iberdomide at 100 nM (B) or 1–10–100 nM (C). (D) Representative dot-plots of flow cytometry after gating as shown and (E) cumulative results. (F) ANA measurement by IF in the supernatants of cells treated as in (A). n=16 patients with SLE in (B), 6 in (C), 7 in (E), with representative results in (D) and (F), p<0.05 Mann-Whitney in (B) and (E). ANA, antinuclear antibodies; SLE, systemic lupus erythematosus.

Figure 2

Differentiation of SLE B cells into plasmablasts. (A) B cells from patients with SLE were triggered with HA-sCD40L (50 ng/mL) cross-linked with anti-HA IgG (1 µg/mL), IL-2 (20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL) and TLR7 ligand R848 (3 uM), plus Iberdomide (10 nM) or control vehicle from day 0. (B) Representative dot-plots of flow cytometry of cells harvested after 5 days of culture. Gating on live CD19+. (C) Cumulative results of flow cytometry (D) representative histogram of CD27 expression by B cells treated with iberdomide or vehicle. (E) Absolute numbers of plasmablasts (CD27+CD38+) and naïve B cells sorted after 5 days of culture as above. (F) IgG and IgM measured in the supernatants of cells treated as above. n=8 patients with SLE in (C), (E) and (F), with representative results in (B) and (D). P<0.05 Mann-Whitney in C-E-F. SLE, systemic lupus erythematosus.

Figure 3

Modulation of SLE B cell differentiation. (A) B cells from patients with SLE were triggered with HA-sCD40L (50 ng/mL) cross-linked with anti-HA IgG (1 µg/mL), IL-2 (20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL) and TLR7 ligand R848 (3 uM) for 4 days, then Iberdomide (10 nM) or control vehicle was added at day 4 for 18 hours. (B) Representative dot-plots of flow cytometry experiments with cells harvested after 5 days of culture. Gating on live CD19+. (C) Cumulative results of flow cytometry (D) representative histogram of CD27 expression by B cells treated with iberdomide or vehicle. (E) Absolute numbers of plasmablasts (CD27+CD38+) and naïve B cells (IgD+CD20 low) sorted after 5 days of culture as above. (F) IgG and IgM measured in the supernatants of cells treated as above. n=16 patients with SLE in C-F-G, with representative results in (B) and (D). P<0.05 Mann-Whitney test in C-E-F. SLE, systemic lupus erythematosus.

Figure 4

Modulation of gene expression by iberdomide. (A, B) Hierarchical clustering of differentially modulated genes from RNA sequencing of sorted naïve B cells (A) and plasmablasts (B) after treatment as shown in figure 3(A). (C, D) Volcano plots showing genes that are significantly modulated by iberdomide in naïve B cells (C) and plasmablasts (D). Labelling is showing genes known to be modulated by Ikaros and Aiolos. (E, F) percentage of the total genes modulated in naïve B cells (E) and plasmablasts (F) that are known to be targets of Ikaros and Aiolos or both. n=3 patients with SLE from three independent experiments. SLE, systemic lupus erythematosus.