Lenalidomide modulates MM expression of NK cell–activating ligands and in combination with IPH2101 enhances in vivo tumor-cell rejection. (A) Incubation of U266 MM cells in lenalidomide (5μM) for 5 days led to alteration of NK cell–ligand expression, representative results shown for each ligand (open curves are isotype and control, shaded curve is lenalidomide treated). The average Ab-binding capacity of MICA, DR4, ULBP2, and CD112 increased by 73.6% ± 26.6%, 95.6% ± 9.5%, 86.1% ± 16.1%, and 114% ± 6.9%, respectively, compared with control cells (P < .05 for all comparisons, left). The expression of ULBP-1, CD155, ULBP-3, and ULBP-4 was not affected (right). (B) A similar effect was observed in primary MM cells (n = 7) all of which expressed ULBP-1 at baseline; a representative result is shown. Lenalidomide (5nM) for 24 hours increased the expression of ULBP-1 by 153% ± 24% (P = .016), but did not reproducibly alter expression of any of the other ligands shown in all cases examined. (C) NK-cell lysis of U266 cells was augmented 1.22 ± 0.05-fold relative to control (P < .05) when targets were pretreated with lenalidomide to increase activating ligand expression as shown in panel A. However, this effect was lost when NK cells were preincubated in blocking Abs against NKG2D, DNAM-1, and TRAIL, suggesting functional relevance to lenalidomide's modulation of activating ligands on MM target cells. A similar effect was observed in patient-derived NK cells against autologous MM-cell targets (right; n = 2 independent experiments). (D) A single 5-μg dose of 5E6 led to a slight reduction of observed RMA cells, and pretreatment of animals with lenalidomide for 21 days enhanced this effect (control mean 16.5 ± 5.9 vs 5E6 plus lenalidomide, 7.8 ± 3.5, P < .01). Similar results were observed with 5E6 (10 μg) with lenalidomide at 14 or 28 days (E). The combination of 5E6 and lenalidomide significantly reduced tumor burden (11.8 ± 5.1 for control vs 3.4 ± 1.6 for 5E6 plus lenalidomide, P < .005).