Background Polymorphisms in IKZF1 (Ikaros) and IKZF3 (Aiolos), which are transcriptions factors essential for B cell activation, maturation and differentiation, have been associated with Systemic Lupus Erythematosus (SLE). However the functional role of IKZF1 and IKZF3 in the context of failed B cell checkpoints and aberrant plasmablast development in SLE has not been previously characterised. Iberdomide (a cereblon modulator) known to induce the degradation of transcription factors IKZF1 and IKZF3 is being explored as a therapeutic target for SLE. The aim of this study was to utilise iberdomide to evaluate the effect of inhibition of IKZF1 and IKZF3 on transcriptional programmes underlying B cell differentiation, gene expression and immunoglobulin production in SLE B cells.
Methods CD19 +B cells were isolated from peripheral blood of 25 SLE patients and stimulated with IL-2, IL-10, IL-15, CD40L and TLR7 ligand Resiquimod for 5 days to induce plasmablast differentiation. In separate studies, B cells were treated from the outset with iberdomide (10 nM) or vehicle and subsequently differentiated, or differentiated plasmablasts (day 4) were treated with iberdomide or vehicle for 18 hour. Treated plasmablasts underwent fluorescence-activated cell sorting (FACS), and IgG/IgM secretion analysed with ELISA. FACS-sorted CD27-IgD+naïve B cells and CD20lowCD27+CD38+plasmablasts were subjected to bulk ultra-low input RNA-seq along with matched baseline B cells. Unsupervised clustering, differential gene expression and pathway analysis were performed on transcriptome data.
Results Day 0 iberdomide (n=9), but not day 4 iberdomide (n=16), significantly reduced the CD20lowCD27+CD38+plasmablast numbers following cell culture (p=0.03). Similarly, Day 0 iberdomide significantly decreased supernatant IgG/IgM concentrations (p=0.050 and 0.017, respectively), but not day 4 iberdomide. RNA-seq of sorted naïve B cells and plasmablasts cultured with day 4 iberdomide demonstrated significant differential gene expression in both populations (400 and 461 differentially modulated genes in naïve B cells and plasmablasts, FDR-adjusted p<0.05). Pathway analysis showed that IKZF1/IKZF3 inhibition resulted in downregulation of JAK-STAT signalling downstream of IL12 (FDR=7.92E-04), IL12 signalling (FDR=0.0014), and p53 signalling regulation of cell death (FDR=0.0043) and showed a trend to upregulation of RUNX1 signalling and Rho GTPase cycle.
Conclusions Iberdomide exposure significantly blocked SLE B cell differentiation into plasmablasts, but did not alter fully differentiated plasmablast viability, confirming the role of IKZF1 and IKZF3 in the process of B cell differentiation into aberrant plasmablasts in SLE. Our study demonstrates that IKZF1 and IKZF3 inhibition results in differential expression of key B cell development transcriptional gene modules in both SLE naïve B cells and plasmablasts.
Funding Source(s): This study was supported by research funding from Celgene Corporation.
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