Immunology and inflammation Lupus Sci Med: first published as 10.1136/lupus-2020-000445 on 16 March 2021. Downloaded from Effects of targeting the transcription factors Ikaros and Aiolos on B cell activation and differentiation in systemic lupus erythematosus Felice Rivellese ,1 Sotiria Manou- Stathopoulou,1 Daniele Mauro,1 Katriona Goldmann,1 Debasish Pyne,2 Ravindra Rajakariar,3 Patrick Gordon,4 Peter Schafer,5 Michele Bombardieri,1 Costantino Pitzalis,1 Myles J Lewis 1 To cite: Rivellese F, ABSTRACT Manou- Stathopoulou S, Objective To evaluate the effects of targeting Ikaros and Key messages Mauro D, et al. Effects of Aiolos by cereblon modulator iberdomide on the activation What is already known about this subject? targeting the transcription and differentiation of B- cells from patients with systemic factors Ikaros and Aiolos The transcription factors Ikaros and Aiolos, which lupus erythematosus (SLE). ► on B cell activation and are critical for B cell differentiation, are implicated in Methods CD19+ B- cells isolated from the peripheral differentiation in systemic systemic lupus erythematosus (SLE) pathogenesis. blood of patients with SLE (n=41) were cultured with lupus erythematosus. Targeting Ikaros and Aiolos using the cereblon mod- TLR7 ligand resiquimod ±IFNα together with iberdomide ► Lupus Science & Medicine ulator iberdomide has been proposed as a promising 2021;8:e000445. doi:10.1136/ or control from day 0 (n=16). Additionally, in vitro B- cell therapeutic agent. lupus-2020-000445 differentiation was induced by stimulation with IL-2/IL-10/ IL-15/CD40L/resiquimod with iberdomide or control, given What does this study add? at day 0 or at day 4. At day 5, immunoglobulins were ► Additional material is ► Targeting Ikaros and Aiolos with iberdomide inhibits published online only. To view, measured by ELISA and cells analysed by flow cytometry. the TLR7- mediated differentiation of plasmablasts in please visit the journal online RNA- Seq was performed on fluorescence- activated cell- human SLE. - + low + + (http:// dx. doi. org/ 10. 1136/ sorted CD27 IgD naïve- B- cells and CD20 CD27 CD38 ► Iberdomide inhibits TLR7 and interferon driven au- lupus- 2020- 000445). plasmablasts to investigate the transcriptional toantibody production by SLE autoreactive B cells. consequences of iberdomide. ► RNA- sequencing was used to detect differentially Results Iberdomide significantly inhibited theTLR7 and expressed genes in iberdomide- treated differentiat- http://lupus.bmj.com/ FR, SM- S and DM contributed IFNα-mediated production of immunoglobulins from SLE ed SLE plasmablasts, thus identifying genes which equally. B- cells and the production of antinuclear antibodies as are most directly under the control of Ikaros and well as significantly reducing the number of CD27+CD38+ Received 16 September 2020 Aiolos. plasmablasts (0.3±0.18, vehicle 1.01±0.56, p=0.011) Revised 22 December 2020 + How might this impact on clinical practice or future Accepted 28 January 2021 and CD138 plasma cells (0.12±0.06, vehicle 0.28±0.02, p=0.03). Additionally, treatment with iberdomide from developments? day 0 significantly inhibited the differentiation of SLE B- ► This study provides strong evidence that therapeu- cells into plasmablasts (6.4±13.5 vs vehicle 34.9±20.1, tic targeting of Ikaros and Aiolos can ameliorate key on September 25, 2021 by guest. Protected copyright. p=0.013) and antibody production. When given at later pathogenic processes in human SLE. stages of differentiation, iberdomide did not affect the numbers of plasmablasts or the production of antibodies; however, it induced a significant modulation of gene cell lineages and their maturation into effector expression involving IKZF1 and IKZF3 transcriptional cells.1 IKZF1 (Ikaros) is an essential regulator programmes in both naïve B-cells and plasmablasts (400 of common lymphoid progenitor (CLP) stem and 461 differentially modulated genes, respectively, false cells. Ikzf1- deficient mice lack CLP and fail to discovery rate<0.05). generate mature B/T lymphocytes, natural Conclusion These results demonstrate the relevance of 2 3 Ikaros and Aiolos as therapeutic targets in SLE due to their killer and dendritic cells. IKZF1 also acts ability to modulate B cell activation and differentiation as a checkpoint at the pro- B cell to pre- B cell © Author(s) (or their 4 5 employer(s)) 2021. Re- use downstream of TLR7. transition stage. In contrast, IKZF3 (Aiolos) permitted under CC BY. is necessary for memory B cell and plasma cell Published by BMJ. formation and Ikzf3 deficient mice lack both For numbered affiliations see of these mature B cell populations.6 7 end of article. INTRODUCTION IKZF1, IKZF3 and more recently IKZF2 Correspondence to The Ikaros family of zinc- fingers (IKZF) (Helios) have been identified as suscepti- Dr Myles J Lewis; myles. lewis@ proteins are transcription factors critical for bility loci in systemic lupus erythematosus qmul. ac. uk the development of key haemopoietic stem (SLE) in large- scale genome- wide associated Rivellese F, et al. Lupus Science & Medicine 2021;8:e000445. doi:10.1136/lupus-2020-000445 1 Lupus Science & Medicine Lupus Sci Med: first published as 10.1136/lupus-2020-000445 on 16 March 2021. Downloaded from Table 1 Summary of patient characteristics (n=41) peripheral blood B cells from patients with SLE, specifi- cally on (i) TLR7 and IFN mediated B cell activation and Age, years mean (SD), range 47 (13) 21–72 differentiation, (ii) plasmablast/plasma cell differentia- Gender (female) 81% tion and (iii) transcriptional programmes underlying B ANA+ 86% cell differentiation. dsDNA+ 62% APS+ 19% MATERIALS AND METHODS Sm+ 14% Patient recruitment Skin involvement* 60% Peripheral blood and clinical demographics were collected from patients with a diagnosis of SLE from Barts Arthritis 60% Health NHS Trust and Kings College Hospital NHS Trust. Serositis 15% All study participants provided written informed consent Renal disease 10% at time of sample collection and fulfilled the 2012 updated Leucopaenia 25% American College of Rheumatology classification criteria 29 Thrombocytopaenia 5% ACR criteria for SLE. Patient characteristics are summa- rised in table 1. Sample collection from patients and Hydroxychloroquine 86% subsequent analysis were approved by UK local ethical Steroids 37.5% committee (REC reference 17/WS/0172). Patients or the *Including malar rash, photosensitivity and discoid lupus. public were not involved in the design, conduct, reporting or dissemination plans of the research. studies.8–13 IKZF1 polymorphism rs4917014 was identi- Blood processing and B cell isolation fied as a trans- expression quantitative trait locus, driving Peripheral blood mononuclear cells were isolated from upregulation of type 1 IFN genes and downregulation of peripheral blood using gradient separation with Lymph- complement genes.14 Ikaros has also been shown to influ- oprep (Alere), with RBC lysis via incubation in ammo- ence TLR7 signalling,15 representing another link with nium chloride (RBC-lysis buffer, Stemcell) for 5 min on SLE pathogenesis.16 ice. CD19+ cells were isolated via negative magnetic selec- Ikaros and Aiolos have emerged as the therapeutic tion with EasySep Human B Cell Isolation Kit (Stemcell). targets of the immunomodulatory drugs thalidomide and Purity (>95%) of isolated B cells was verified by flow cytom- analogues such as lenalidomide, which act as agonists for etry. For selected experiments, 1–2×105 B cells at baseline the ubiquitination E3 ligase complex cereblon,17 thus were stored in RNAprotect Cell Reagent (QIAGEN) and inducing ubiquitination and proteasomal degradation frozen at −80°C for subsequent RNA extraction. 18–20 of Ikaros and Aiolos. Thalidomide and its analogues http://lupus.bmj.com/ are well established as treatment options for multiple B cell culture and TLR7/IFN stimulation 21 Human B cells freshly isolated from patients with SLE myeloma. Lenalidomide inhibits plasma cells differen- 6 tiation from healthy donors in vitro22 and has been found were resuspended at concentration 0.5×10 cells/mL in to be effective in cases of treatment-refractor y cutaneous Iscove’s modified Dulbecco’s medium (IMDM) with 10% manifestations of SLE.23–25 More recently, a novel cereblon fetal calf serum (FCS), 1% antibiotic- antimycotic (Gibco) ligand has been developed, iberdomide, which binds with and 3 µM synthetic TLR7 agonist Resiquimod (R848, Invi- vogen) with/without 1000 IU IFNα (Enzo), together with higher affinity than thalidomide and other analogues, on September 25, 2021 by guest. Protected copyright. resulting in greater Ikaros/Aiolos degradation.26 iberdomide 1, 10 or 100 nM or vehicle control. At 5 days, In preliminary studies, iberdomide reduced Ikaros and cells were harvested for fluorescence- activated cell sorting Aiolos expression in SLE B cells, while inhibiting antibody (FACS) analysis, and supernatants stored for ELISA. production and differentiation of B cells into plasmab- 27 B cell culture and plasmablast differentiation lasts. Iberdomide has undergone a phase I study, which Human B cells freshly isolated from SLE patients were explored the effects of oral administration in healthy resuspended and cultured using a modified plasmablast volunteers and confirmed the reduction of Ikaros and 30 28 differentiation protocol with HA- sCD40L (R&D) at 50 Aiolos at protein level in B cells, T cells and monocytes. ng/mL cross-linked with 1 µg/mL anti- HA IgG (R&D), Iberdomide also inhibited anti-dsDNA and antiphospho- IL-2 at 20 U/mL, IL-10 at 50 ng/mL (Peprotech), IL-15 lipid antibody production ex vivo from SLE mononu- at 10 ng/mL (Peprotech) and TLR7 ligand R848 at 3 µM clear cells. Iberdomide reduced absolute B cell counts, (Invivogen). Iberdomide (10 nM) or control vehicle was augmented IL-2 production from T cells and inhibited added at two timepoints, at either day 0 or 18 hours prior IL-1β production in response to proinflammatory stimuli, to harvest on day 4. After 5 days, cells were harvested for underpinning its further development for the treatment FACS, and supernatants stored for ELISA. of SLE, with an ongoing phase II trial ( Clinical-­​Trials.