Treatment of MCL Cells with Combined Rituximab and Lenalidomide Enhances NK-Mediated Synapse Formation and Cell Killing.

Mantle cell lymphoma (MCL) is a subtype of aggressive B-cell non-Hodgkin's Lymphoma (NHL) characterized by poor prognosis and very few therapeutic options that improve survival. Lenalidomide is an immunomodulatory agent that has demonstrated significant clinical activity in the treatment of patients with MCL. A mechanism attributed to lenalidomide, which has also been demonstrated in B-CLL models, is the restoration of impaired T-cell activity to form immunological synapses, thus enhancing T-cell effector functions thereby eliminating aberrant tumor B cells. Natural killer (NK) cells are another cell type that eliminates aberrant cell types via a mechanism dependent on active formation of immunological synapses. By engaging in both direct killing and ADCC, NK cells are an important component of innate immunity eliminating transformed cell types. Here we assess the effect of lenalidomide on the ability of NK cells to form immune synapses with MCL cell lines and cell samples from MCL patients. We also evaluate whether lenalidomide-mediated immunologic activity is altered when lenalidomide is combined with rituximab, an anti-CD20 antibody shown to eliminate MCL cells through the ADCC mechanism.

To measure immune synapse formation, NK and JeKo-1 cells were pre-treated with DMSO or 1μM lenalidomide for 24 or 48 hrs. JeKo-1 cells (labeled with red PKH26) were incubated for 30 min with or without 10 μg/ml rituximab, and cell conjugates were fixed and stained with Phalloidin-FITC to measure mean fluorescent intensity and F-actin formation. Adenylate kinase (AK) release and 7AAD staining were used to measure NK-mediated cytotoxicity after NK and MCL cells were co-cultured at different target-to-effector ratios, and with and without rituximab. Flow cytometry was used to measure relative expression of cell surface markers CD56 (N-CAM), CD54 (I-CAM1), and NKG2D in both JeKo-1 cells and in B-cell samples from MCL patients.

Addition of lenalidomide enhanced the formation of immunological synapses between JeKo-1 cells and NK cells, increasing the number of synapses approximately 3-fold after 24 hrs of treatment. When lenalidomide was combined with rituximab the number of synapses increased 3.5-fold. A significant increase in the number of synapses also occurred when NK cells co-cultured with MCL patient samples were treated with lenalidomide (2.5-fold increase) and lenalidomide plus rituximab (3-fold increase). Lenalidomide also augmented the intensity of F-actin at the synaptic site, which indicates more matured synapses rich with F-actin. The increased synapse-forming activity resulting from treatment with rituximab plus lenalidomide also translated into enhanced NK-mediated cytotoxicity. After 48 hours of concurrent treatment with lenalidomide and rituximab, approximately 80% of JeKo-1 cells released AK compared with approximately 45% after treatment with lenalidomide alone. Comparable cell killing data upon treatment with lenalidomide plus rituximab was obtained using a 7AAD assay. Finally, to understand the mechanism by which lenalidomide enhances the formation of immune synapses and augments the rituximab-dependent NK cell-mediated cytotoxicity in MCL cells, we studied the effect of lenalidomide on effector and target cells separately. In the NK cells, lenalidomide treatment induced F-actin polymerization and polarization, and the accumulation of perforin, which is evidence for effector cell activation. Previously we showed that lenalidomide treatment of tumor cells induces changes in actin structure and increases expression of cell surface markers, including CD54 and co-stimulatory molecules, which correlated with increased antigen presentation properties of tumor cells. Here we show that lenalidomide and rituximab induced F-actin polymerization and polarization in the JeKo-1 cells when administered individually and in combination, and resulted in slightly increased cytotoxicity.

Our results suggest that in MCL, combined use of lenalidomide and rituximab enhances NK-mediated immune synapse formation and the resultant cytotoxicity. Combining lenalidomide with rituximab may also enhance the anti-tumor immune response mediated through enhanced activity of NK cells. These studies suggest that lenalidomide plus rituximab may have clinical utility in the treatment of patients with MCL.

Gaidarova:Celgene: Employment, Equity Ownership. Corral:Celgene: Employment. Glezer:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment. Lopez-Girona:Celgene: Employment.