Supplementary Figure 1: Optimize the dose and schedule of lenalidomide-combined vaccine therapy. Ten Balb/c mice per group were immunized intramuscularly with 50 g plasmid DNA encoding MCP3 chemokine-fused A20 lymphoma-derived idiotype antigen (MCP3-sFv) on days 0, 14 and 28. Lenalidomide was given i.p at 5 mg/kg or 50 mg/kg using either a continuous schedule for 35 consecutive days (a) or an intermittent schedule (b). Control mice received the plasmid DNA alone or PBS. Two weeks after final vaccination, all mice were challenged with a lethal dose of 2×105 A20 lymphoma cells by intraperitoneal injection and followed for tumor development.
Supplementary Figure 2: Pharmacokinetics of plasma lenalidomide level in mice.Fifteen Balb/c mice were injected i.p with a daily dose of 5 mg/kg lenalidomide for 35 consecutive days. Blood samples were collected 0.5 (n=3), 1 (n=3), 2 (n=3), 4 (n=2), 8 (n=2) and 24 (n=2) hours after the final dose of lenalidomide on Day 35. Plasma was analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Composite pharmacokinetic parameters were calculated using Watson LIMS™ (version 7.4, Thermo Fisher, Philadelphia, PA).
Supplementary Figure 3. Effects of lenalidomide on MDSC, Treg and NK cells in mice with established tumors. Five Balb/c mice per group were injected with 2×105 A20 lymphoma cells intraperitoneally on day 0. Mice were then treated either with 5 mg/kg lenalidomide for consecutive 21 days starting on day 1, or with 100 mg/kg cyclophosphamide on days 13 and 14 or nothing (controls). All mice were sacrificed three weeks after tumor challenge. Splenocyte single cell suspensions were prepared and individual mice were analyzed by specific immunostaining for MDSC, Treg and NK cells. The data are representative of 2 independent experiments showing percentage of splenic MDSC, Treg in CD4 T-cell population and NK cells in CD3- lymphocytes (Mean SEM). Differences between groups were analyzed by using student t test.