Interleukin-21 Potently Induces Direct and Indirect Cytotoxicity of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphomacharacterized by overexpression of cyclin D1 (CCND1) in 95% of patients due to the t(11;14)(q13;q32) chromosomal translocation. This incurable lymphoma is highly chemoresistant, with short response duration, frequent relapses and eventual death, even with the most aggressive chemotherapeutic regimens. Interleukin 21 (IL-21), a member of the IL-2 cytokine family, possesses potent anti-tumor activity against a variety of cancers not expressing IL-21 receptor (IL-21R) through activation of the immune system. Previously, we established that apart from its immuno-stimulatory effects, IL-21 exerts direct cytotoxicity on IL-21R-expressing diffuse large B cell lymphoma (DLBCL) cells (Sarosiek KA et al.Blood, 2010). Herein we carried out a comprehensive study to delineate the effects of IL-21 on MCL cell lines and primary tumors. Flow-cytometric analysis revealed that all MCL cell lines (Mino, HBL2, Jeko1, G519, IRM2, SP53, Z138, UPN1 and L128) as well as primary tumors expressed surface IL-21R at variable levels. Treatment of Mino, HBL2 and SP53 cells with IL-21 (100ng/mL) led to a marked time-dependent decrease in cell proliferation and increased cell death. In contrast, Jeko1, IRM2, L128, Z138, UPN1 and G519 cells exhibited resistance to IL-21 treatment. Similarly, primary MCL tumors treated with IL-21 in vitro exhibited significant cell death in 4 of 5 cases expressing IL-21R. To decipher the mechanism of IL-21-induced direct cytotoxicity, responsive and resistant cell lines as well as primary tumors were utilized. Similarly to our previous study in DLBCL, IL-21 stimulation resulted in dramatic phosphorylation of STAT1 and STAT3 in IL-21 responsive cell lines (Mino, HBL-2, SP53) and a primary tumor, while minimal STAT5 phosphorylation was observed only in Mino. We have previously demonstrated that IL-21-induced cell death in DLBCL is mediated by STAT3-induced upregulation of c-Myc expression. Correspondingly, IL-21 treatment led to c-Myc upregulation only in IL-21-sensitive MCL cell lines and primary tumors but not in the resistant cell lines and primary tumors, independent of the IL-21R expression levels. Knockdown of c-Myc prevented IL-21-induced Mino cell death, whereas c-Myc overexpression in resistant MCL cell lines facilitated IL-21-induced cytotoxicity. Furthermore, IL-21 resulted in upregulation of the pro-apoptotic protein Bax and downregulation of the anti-apoptotic proteins Bcl-XL and Bcl-2, as previously observed in DLBCL. Knockdown of STAT3 or Bax using specific siRNAs in Mino cells resulted in abrogation of the IL-21-induced cell death. In contrast to a previous report (Gelebart P et al. Leukemia, 2009), knockdown of STAT1 or overexpression of dominant negative STAT1 failed to prevent IL-21-induced Mino cell death. We also discovered that apart from its direct cytotoxic effects, IL-21 also leads to NK-cell dependent lysis of MCL cell lines resistant to direct cytotoxicity. In vivo treatment with IL-21 resulted in complete FC-muMCL1 tumor regression in syngeneic mice (p<0.0001). To understand the contribution of immune system components to the IL-21 induced anti-tumor activity, we carried out cell subset depletion studies. In vivo depletion of NK cells together with CD4+ T cells abrogated IL-21 induced tumor regression of the FC-muMCL1 in xenograft mice, suggesting that both NK and CD4+ cells contribute to the indirect immune mediated cytotoxicity of MCL. Collectively, our data indicate that IL-21 has potent anti-tumor activity against MCL cells via direct (IL-21R mediated) and indirect (immune cell mediated) effects, and should be evaluated in clinic for treatment of patients with MCL.