ILC subsets and changes in ILCs after pomalidomide. (A-C) Student t distribution analysis of the single-cell RNA sequencing data showing the NK and (A-B) ILC1 cluster and the (C) key genes (LTβ, IL7-R, GNLY, GZMB) that distinguish ILC1 and NK cells in human marrow. (D) Expression of Ikaros (Ikzf1) and Aiolos (Ikzf3) in ILC subsets (ILC1, ILC2, and ILC3). (E) Depletion of Ikzf1 and Ikzf3 in ILCs following exposure to pomalidomide in vitro. (i) Depletion of IKZF1: representative plot showing the level of Ikaros (Ikzf1) in ILC1, ILC2, and ILC3 in control cells and cells treated with pomalidomide (100 ng/mL). Bar graph shows data from 3 experiments. (ii) Depletion of IKZF3: representative FACS plots showing the level of Aiolos (Ikzf3) in ILC1, ILC2, and ILC3 in control cells and cells treated with pomalidomide (100 ng/mL). Bar graph shows data from 3 experiments. (F) Fold change in IFN-γ–producing ILC1 after culture with pomalidomide. Bar graph shows the IFN-γ production by ILC1 in HD PBMCs (n = 5) after overnight culture with IL-12 (10 ng/mL) alone (control) or with pomalidomide (100 ng/mL). (G) Changes in cytokine production by ILCs after a single dose of pomalidomide. PBMCs from MM patients (n = 3) were isolated before therapy and 4 hours after a single dose of pomalidomide and analyzed for cytokine production by ILCs after stimulation with PMA and ionomycin. Representative FACS plot showing the cytokine production by ILCs. Bar graph shows cumulative data from 3 patients. (H) Change in Ikaros levels in ILCs as well as CD3 in vivo after pomalidomide therapy.