Inhibition of PI3K-δ prevents CLL cell immune activation induced by lenalidomide. (A) CD19⁺ cells from CLL patients (N = 25) were treated with or without 0.5μM lenalidomide and/or 10μM CAL-101 for 48 hours. Surface expression of CD20, CD40, or CD86 was evaluated by flow cytometry using CD20-phycoerythrin, CD40-phycoerythrin, or CD86-phycoerythrin antibodies and IgG1-phycoerythrin isotype control. (B) CD19⁺ cells from CLL patients (N = 15) were treated with or without 0.5μM lenalidomide and/or 10μM CAL-101 for 48 hours. RNA was extracted and converted to cDNA, and RT-PCR analysis was done to determine quantities of CD40, CD86, and CD154 mRNA. (C) CD19⁺ cells from CLL patients (N = 6) were treated with or without 0.5μM lenalidomide and/or CAL-101 for 48 hours. CLL cells were irradiated (20 Gy) and placed in culture with purified B cells, in the absence or presence of 5 μg/mL pokeweed mitogen. Quantification of IgM was determined by enzyme-linked immunosorbent assay. (D) CD19⁺ cells from CLL patients (N = 15) were treated with or without 0.5μM lenalidomide and/or 10μM CAL-101 for 48 hours. RNA was extracted and converted to cDNA, and RT-PCR analysis was done to determine quantities of bFGF. (E) CD19⁺ cells from CLL patients (N = 13) were treated with or without 0.5μM lenalidomide and/or 10μM CAL-101 for 48 hours. RNA was extracted and converted to cDNA, and RT-PCR analysis was done to determine quantities of VEGF.