Anti-lymphoma activity of lenalidomide against non-germinal center B-cell subtype of diffuse large B-cell lymphoma

Abstract

Purpose: Lenalidomide shows anti-tumor activity against B-cell malignancies. Although differential sensitivities to lenalidomide were observed between diffuse large B-cell lymphoma (DLBCL) subtypes, an in vitro efficacy of lenalidomide has not been verified in DLBCL cells. Here, we investigated the direct effect of lenalidomide on DLBCL cells as well as immunomodulatory properties using human NK cells. Experimental Design: Direct effect of lenalidomide against DLBCL cell lines was evaluated by modified MTT or trypan blue exclusion assay. Immunoblotting was used to identify mechanism of growth inhibition. Human peripheral blood mononuclear cells (PBMCs) were prepared from healthy donors with leukoreduction system chambers. Primary NK cells were isolated from the PBMCs using anti-CD3 and anti-CD56 antibodies labeled with magnetic microbeads (MACS® separation). The cytotoxicity of lenalidomide-treated effector cells (PBMCs and NK cells) was analyzed by CD107a assay. NK cell-mediated growth inhibition was evaluated by Annexin V analysis. Flow cytometry was used to measure the expression patterns of lenalidomide-treated effector cells. Results: We found that lenalidomide induced significant growth inhibition in non-GCB (non-germinal center B-cell) DLBCL subtype (0.738±0.070, mean±SD) compared with GCB DLBCL subtype (1.143±0.130, mean±SD) at 5μM of lenalidomide (p<0.001). Similar result obtained from trypan blue exclusion. After exposure to lenalidomide to non-GCB DLBCL cell lines for three days, IRF4 expression decreased as well as NFκB signaling decreased in nucleus, whereas precursor form of NFκB increased in nucleus. When lenalidomide-treated NK cells were co-cultured with DLBCL cells, early apoptosis was induced more in lenalidomide-treated NK cells than lenalidomide-untreated NK cells. Although the percentage of CD3-CD56+ cell was similar between lenalidomide treated group and untreated group, CD56 expression on lenalidomide-treated NK cell was increased as a timedependent manner (56.2±8.25 to 176.24±56.83, median ± SD at day0 to day7) and it means the number of CD56bright NK cells increased after exposure to lenalidomide. Although lenalidomide upregulated inhibitory KIR expressions (KIR2DL1, KIR2DL2/3, and KIR3DL1) on CD56bright NK cell subset, it has down-regulated inhibitory KIR expressions on CD56dim NK cell. Conclusions: Lenalidomide showed antitumor activity against non-GCB DLBCL cells via decreased IRF4 expression through NFκB signaling pathway as well as modulation of NK cells to induce early apoptosis of DLBCL cells.