Targeting BMI-1 to deplete antibody-secreting cells in autoimmunity

Abstract

Objectives: B cells drive the production of autoreactive antibody-secreting cells (ASCs) in autoimmune diseases such as Systemic Lupus Erythematosus (SLE) and Sjögren's syndrome, causing long-term organ damage. Current treatments for antibody-mediated autoimmune diseases target B cells or broadly suppress the immune system. However, pre-existing long-lived ASCs are often refractory to treatment, leaving a reservoir of autoreactive cells that continue to produce antibodies. Therefore, the development of novel treatment methods targeting ASCs is vital to improve patient outcomes. Our objective was to test whether targeting the epigenetic regulator BMI-1 could deplete ASCs in autoimmune conditions in vivo and in vitro.

Methods: Use of a BMI-1 inhibitor in both mouse and human autoimmune settings was investigated. Lyn ^-/- mice, a model of SLE, were treated with the BMI-1 small molecule inhibitor PTC-028, before assessment of ASCs, serum antibody and immune complexes. To examine human ASC survival, a novel human fibroblast-based assay was established, and the impact of PTC-028 on ASCs derived from Sjögren's syndrome patients was evaluated.

Results: BMI-1 inhibition significantly decreased splenic and bone marrow ASCs in Lyn ^-/- mice. The decline in ASCs was linked to aberrant cell cycle gene expression and led to a significant decrease in serum IgG3, immune complexes and anti-DNA IgG. PTC-028 was also efficacious in reducing ex vivo plasma cell survival from both Sjögren's syndrome patients and age-matched healthy donors.

Conclusion: These data provide evidence that inhibiting BMI-1 can deplete ASC in a variety of contexts and thus BMI-1 is a viable therapeutic target for antibody-mediated autoimmune diseases.

Keywords: B cells; BMI‐1; Sjögren's syndrome; Systemic Lupus Erythematosus; antibody; autoimmunity.

Figure 1

Depletion of ASCs in autoimmune‐prone Lyn ^−/− mice treated with the BMI‐1 small molecule inhibitor PTC‐028. (a) Schematic of the experimental design. Lyn ^−/− mice (≥6 months of age) were treated with PTC‐028 or vehicle control once daily on days 0–11 and then assessed on day 15. (b) Representative flow cytometric plots of splenic ASCs from Lyn ^−/− mice treated with vehicle control or PTC‐028. (c) Frequency of splenic ASC as a proportion of total lymphocytes and (d) total number of splenic ASCs in Lyn ^−/− mice treated with vehicle control or PTC‐028 on day 15. (e) Representative flow cytometric plots of bone marrow ASCs from Lyn ^−/− mice treated with vehicle control or PTC‐028. (f) Frequency of bone marrow ASC as a proportion of total lymphocytes and (g) total number of bone marrow ASCs in Lyn ^−/− mice treated with vehicle control or PTC‐028 on day 15. Data are presented as the mean ± SEM with individual data points representing one mouse. Data are combined from three independent experiments. n = 11 or 12 per group. Mann–Whitney non‐parametric two‐tailed tests were used for statistical analysis.

Figure 2

BMI‐1 inhibition reduced total IgG3 and anti‐DNA IgG in Lyn ^−/− mice. (a) Assessment of serum antibody by ELISA. Total IgG3 in Lyn ^−/− mice treated with vehicle control or PTC‐028 once daily on days 0–11 and then assessed on day 15. Data shown are representative of three independent experiments. n = 4 per group. (b) IgG3 immune complexes and (c) anti‐DNA IgG in Lyn ^−/− mice treated with vehicle control or PTC‐028 once daily on days 0–11 and then assessed on day 15. n = 11 or 12 per group. Data are combined from three independent experiments. Mann–Whitney non‐parametric two‐tailed tests were used for statistical analysis. *P ≤ 0.05.

Figure 3

BMI‐1 inhibition regulated cell cycle genes in ASCs isolated from Lyn ^−/− mice. RNA sequencing was performed on splenic ASCs (B220^lowCD138^hi) sort‐purified from Lyn ^−/− mice on day 15 following treatment with PTC‐028 or vehicle control from days 0–11. (a) Volcano plot comparing average gene expression in ASCs from Lyn ^−/− mice treated with either PTC‐028 or vehicle control. False discovery rate (FDR) (minimum cut‐off = 0.05) is compared to fold change (Minimum cut‐off ±1.5). Upregulated genes are displayed in red, downregulated genes are displayed in blue and grey representing non‐significant results. Labelled genes (in green) are genes associated with the cell cycle that are regulated by BMI‐1. (b) Gene ontology (GO) analysis on upregulated genes showing the top 15 most statistically enriched biological processes. The number of enriched genes (no. genes) is listed for each process. (c) Cell cycle enrichment analysis shows enrichment scores in our RNA sequencing data set for genes associated with the cell cycle (CC), G1/S phase and G2/M phase. n = 2 per group.

Figure 4

BMI‐1 regulated survival of ASCs isolated from Sjögren's Syndrome patients. (a) Schematic of experimental design. ASCs (CD20⁻CD3⁻CD14⁻CD16⁻CD19⁺CD27^hiCD38^hi) were sort‐purified from ex vivo PBMCs isolated from either Sjögren's Syndrome patients or aged‐matched healthy donors. ASCs were cultured in human tonsil fibroblast supernatant with IL‐6 and APRIL for 6 days in the presence of PTC‐028 or a DMSO vehicle control. The viability of ASCs was analysed using flow cytometry. Total number of live (FVS780⁻) ASCs from (b) healthy donors and (c) Sjögren's Syndrome patients treated with vehicle control or PTC‐028. Data are represented as the mean from each biological replicate with connected data points originating from the same donor. n = 4 per group. The ratio‐paired t‐test was used for statistical analysis.