Ikaros Degradation By CC-220 (Iberdomide) Correlates with Normalization of SLE CD4 T Cell Phenotype

Introduction: CC-220 is a second-generation IMiD drug that degrades members of the IKZF family and is being explored for treating Systemic Lupus Erythematosus (SLE). SLE is driven by an abnormal immune response, and adaptive immune cells such as B and T cells are known to show distinct phenotypes in SLE patients. In vitro analysis has shown that CC-220 effectively inhibits autoantibody production and B cell proliferation in SLE patient samples; however, its effect on SLE T helper (Th) cells remains unexplored. In this study, we aimed to determine the phenotypic changes in SLE CD4 T cells induced by CC-220 treatment.

Methods: For evaluation of Th cell phenotypes that could mimic those in the germinal center, PBMCs from healthy control (HC) and SLE patients were treated with CytoStem (a reagent that couples MHC with TCR) in the presence of B cell proliferation media to create pseudo germinal center. After 5 days of proliferation, the media were changed, and cells were treated with CC-220 for an additional 5 days. Anti-dsDNA antibodies were profiled, and B cells were analyzed for the CD27+CD38+ precursor of plasma cell phenotype. Subsequently, CD4 cells were analyzed for CXCR5+PD1+ Tfh cells, as well as ICOS, Ikaros, and Aiolos levels, and profiled for intracellular cytokines (ICS). To assess the phenotype of SLE Th cells observed in co-culture is independent of media and if influenced by other cells, CD4+ T cells were isolated using a magnetic beads-based negative selection method from healthy control (HC) and SLE PBMCs. The cells were stimulated with a soluble CD3/CD28 cocktail in the presence of 100nM CC-220 for 3 days. After removal of the media, cells were stimulated with PMA for 6 hours. Multiplex cytokine analysis was performed on the supernatant post-stimulation.

Results: We identified an abnormal Th cell phenotype in SLE patients using multiplex profiling of both secreted and intracellular cytokines. The pseudo in-vitro germinal center setup produced significantly higher levels of anti-dsDNA antibodies in SLE samples compared to HC. Additionally, there was hyperproliferation of B cells with significantly elevated levels of plasma cells in SLE samples. Treatment with CC-220 significantly reduced the levels of anti-dsDNA antibodies and inhibited B cell proliferation.

The Th cell profile showed that these cells were hyperproliferative in SLE samples compared to HC. Treatment with CC-220 significantly reduced this proliferation; however, it increased the levels of PD1+CXCR5+ Tfh cells. Despite the increase in Tfh cells, the co-stimulation receptor ICOS was significantly reduced with CC-220 treatment. ICS analysis revealed that the majority of SLE Th cells were Th2 type, with significantly elevated levels of IL-4. SLE samples also exhibited a higher Th9 phenotype with significantly elevated levels of IL-9. CC-220 treatment normalized these cytokine levels and significantly increased the proportion of IL-10 and TNF-α positive Th cells. CC-220 treatment also restored the IL-2 levels in SLE Th cells to the levels observed in HC. The assessment of SLE Th cells without influence of other cells and media condition, also showed Th2 dominant phenotype compared to HC. SLE Th cells showed significantly lower secretion of IL-2 and TNF-α, whereas they exhibited significantly higher secretion of Th2 cytokines – IL-4, IL-5, and IL-13 compared to HC. Treatment with CC-220 shifted this abnormal phenotype in SLE Th cells, with secretions returning to levels observed in HC.

Analysis of Ikaros and Aiolos levels showed that these were significantly higher in SLE Th cells, and treatment with CC-220 significantly reduced their levels. We observed substantial variability in Ikaros levels among donors, and the fold change in several cytokines directly correlated with the fold change in Ikaros. In correlation with Ikaros change, positive correlations was observed for Th2 cytokines – IL-4, IL-6, IL-9, IL-5, and IL-13, whereas negative correlations was observed for IL-2, TNF-α, and IL-10. Similar analysis for Aiolos did not show any correlation.

Conclusion: SLE Th cells show significant distinct phenotype and are skewed towards Th2. CC-220 treatment normalizes the SLE Th cell phenotype, and the normalization correlates with the fold change in Ikaros after degradation.