Lenalidomide promotes CD154 gene therapy phenotype. (A) Whole-cell extracts from 48-hour–treated CLL B cells were analyzed by immunoblot for p73 and BID. Actin was used as internal loading control. (B) Representative histograms show expression of DR5 measured by flow cytometry in CLL B cells cultured without (filled gray) or with (filled black) lenalidomide. The gray- and black-lined open histograms correspond to CLL B cells cultured, respectively, with vehicle or lenalidomide and stained with an isotype monoclonal antibody of irrelevant specificity. (C) Peripheral blood mononuclear cells from before and day 8 of therapy were cultured in the presence of ActD and 107 cells were collected over a 4-hour time course as in Figure 3B (n = 5, P < .05). (D) Whole-cell extracts from patients' peripheral blood mononuclear cells before the dose and on day 8 were analyzed by immunoblot for p73 and BID (representative findings in 6 of 9 patients). (E) Purified recombinant human-ROR1 was run in each lane of an SDS-PAGE gel and then transferred to nitrocellulose membrane. Ponceau red staining was performed to assess both purity and equal loading. The membrane was cut into pieces and probed with rabbit anti–human ROR1 (Cell Signaling), goat anti–human ROR1 (R&D), healthy donor serum (ND), or serum from the index case immediately before lenalidomide (before) and on month 6 of therapy (after). The pieces were incubated with HRP-conjugated anti–rabbit, anti–goat, or anti–human IgG.
