Cancer Immunol Immunother (2013) 62:1637–1648 DOI 10.1007/s00262-013-1466-y
ORIGINAL ARTICLE
Lenalidomide overcomes suppression of human natural killer cell anti‑tumor functions by neuroblastoma microenvironment‑associated IL‑6 and TGFβ1 Yibing Xu · Jianping Sun · Michael A. Sheard · Hung C. Tran · Zesheng Wan · Wei Yao Liu · Shahab Asgharzadeh · Richard Sposto · Hong Wei Wu · Robert C. Seeger
Received: 20 May 2013 / Accepted: 12 August 2013 / Published online: 27 August 2013 © The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Background Treatment for children with high-risk neuroblastoma with anti-disialoganglioside mAb ch14.18, IL-2, and GM-CSF plus 13-cis-retinoic acid after myeloablative chemotherapy improves survival, but 40 % of patients still relapse during or after this therapy. The microenvironment of high-risk neuroblastoma tumors includes macrophages, IL-6, and TGFβ1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression. Methods Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFβ1, and lenalidomide in various combinations and then characterized using cytotoxicity (direct and antibody-dependent cell-mediated cytotoxicity), cytokine, flow cytometry, and Western blotting assays. Anti-tumor activity of NK cells with lenalidomide, ch14.18, or both was evaluated with a xenograft model of neuroblastoma. Results CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFβ1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFβ1 Electronic supplementary material The online version of this article (doi:10.1007/s00262-013-1466-y) contains supplementary material, which is available to authorized users. Y. Xu · J. Sun · M. A. Sheard · H. C. Tran · Z. Wan · W. Y. Liu · S. Asgharzadeh · R. Sposto · H. W. Wu · R. C. Seeger (*) Division of Hematology/Oncology, Children’s Hospital Los Angeles, 4650 Sunset Boulevard, Mailstop #57, Los Angeles, CA 90027, USA e-mail:
[email protected] H. C. Tran · S. Asgharzadeh · R. Sposto · R. C. Seeger Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
activate the STAT3 and SMAD2/3 pathways in NK cells and suppress IL-2 induction of cytotoxicity, granzymes A and B release, perforin expression, and IFNγ secretion. Lenalidomide blocks IL-6 and TGFβ1 activation of these signaling pathways and inhibits their suppression of NK cells. Neuroblastoma cells in NOD/SCID mice exhibit activated STAT3 and SMAD2/3 pathways. Their growth is most effectively inhibited by co-injected peripheral blood mononuclear cells (PBMC) containing NK cells when mice are treated with both ch14.18 and lenalidomide. Conclusion Immunotherapy with anti-tumor cell antibodies may be improved by lenalidomide, which enhances activation of NK cells and inhibits their suppression by IL-6 and TGFβ1. Keywords Neuroblastoma · NK cells · ADCC · Lenalidomide · IL-6 · TGFβ1
Introduction Addition of anti-disialoganglioside (GD2) chimeric mAb ch14.18, IL-2, and GM-CSF immunotherapy to 13-cis-retinoic acid improved event-free survival (EFS) for patients with high-risk neuroblastoma, but approximately 40 % still relapsed during or after this therapy [1]. An immunosuppressive tumor microenvironment could negatively impact the anti-tumor cell activity of antibody therapy that is largely reliant upon antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Our gene expression analyses of high-risk, metastatic neuroblastomas without MYCN gene amplification using Affymetrix® microarrays and TaqMan® low-density arrays revealed that high expression of genes related to inflammation, including IL-6, IL-6R, and TGFβ1, was associated
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with a poor 5-year EFS [2–4]. Furthermore, we found that high-risk primary neuroblastoma tumors contain CD68+ tumor-associated monocytes/macrophages (TAMs) producing IL-6 and that bone marrows with neuroblastoma metastases include CD33+ CD14+ monocytic cells, which also express IL-6 [3]. Experimentally, we have shown that neuroblastoma cells stimulate blood monocytes to secrete IL-6 and that TAMs stimulate the growth of neuroblastoma cells in NOD/SCID mice, at least partially via IL-6 secretion [3]. IL-6, which induces activation of the STAT3 signaling pathway, has not been definitively shown to affect the function of human NK cells. Treatment for patients with recombinant human IL-6 has been reported to suppress the ability of their peripheral blood mononuclear cells (PBMC) to kill K562 cells in vitro [5]. Although K562 cells are sensitive to NK cell cytotoxicity, this study did not demonstrate a direct effect of IL-6 upon NK cells or any other possible mechanism [5]. The potential importance of STAT3 pathway activation in suppressing NK cell cytotoxicity is supported by experiments showing that NK cells from tumor-bearing mice with induced ablation of STAT3 signaling are more cytotoxic against T cell lymphoma YAC-1 cells, which are NK cell-sensitive, than those from tumor-bearing mice with wild-type STAT3 [6]. TGFβ1 activates the SMAD2/3 signaling pathway in human NK cells, which results in the suppression of ADCC and secretion of IFNγ, TNFα, and GM-CSF in vitro [7–9]. Culture of blood NK cells with TGFβ1 decreases the expression of CD16 after 24 h [10, 11]. NK cells from patients with glioblastoma, lung, and colorectal cancer have decreased the expression of the activating immunoreceptor NKG2D, and TGFβ1 in serum and cerebrospinal fluid is responsible for this down-regulation [12, 13]. Based upon the above observations, we hypothesized that IL-6 and TGFβ1 in the neuroblastoma microenvironment may contribute to the failure of treatment with mAb ch14.18 by suppressing anti-tumor functions of NK cells. Using purified NK cells, we found that anti-tumor NK cell functions induced in vitro by IL-2, including direct cytotoxicity, ADCC, and IFNγ secretion, are suppressed by conditioned medium (CM) from neuroblastoma/monocyte co-cultures that contain IL-6 and TGFβ1 and that suppression is removed by the depletion of these cytokines from the CM. Additionally, these recombinant cytokines suppressed IL-2 activation of NK cells. Lenalidomide is an immune-modulating drug that activates T cells to secrete IL-2, which in turn activates NK cell cytotoxicity and ADCC [14, 15]. Clinical trials in children and adults demonstrated a greater number of NK cells and increased cytotoxicity, decreased T regulatory cells, and increased secretion of IL-2, IL-15, and GM-CSF after 21 days of lenalidomide treatment [16, 17]. Preclinical in vitro data have shown that lenalidomide can enhance
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ADCC mediated by human NK cells with rituximab (antiCD20) against lymphoma and chronic lymphocytic leukemia cells [18–20], with SGN-40 (anti-CD40) against multiple myeloma and chronic lymphocytic leukemia cells [21, 22], and with trastuzumab (anti-HER2/neu) and cetuximab (anti-EGFR) against solid tumor cell lines [23]. Because of these clinical and preclinical effects, we hypothesized that lenalidomide also could prevent the suppression of IL-2 activation of NK cells by neuroblastoma/monocyte CM, IL-6, and TGFβ1. Indeed, we show that lenalidomide blocks the activation of STAT3 and SMAD2/3 signaling pathways and prevents the suppression of NK cell activation in vitro. Further, NK cells among PBMC that are coinjected with neuroblastoma cells into NOD/SCID mice have the greatest anti-tumor effect when mice are treated with both lenalidomide and mAb ch14.18.
Materials and methods For additional details on “Materials and methods,” see Supplemental Methods. Reagents Details for reagents are provided in Supplemental Methods. Anti-GD2 chimeric mAb ch14.18 was provided by the National Cancer Institute-Frederick. Lenalidomide was provided by Celgene and was dissolved in dimethyl sulfoxide (final DMSO concentration for in vitro and in vivo experiments was 0.03 and 5 %, respectively). Neuroblastoma cell lines and tumors The CHLA-255 parent cell line, the firefly luciferase (Fluc) and humanized Renilla luciferase (hRluc) transfected CHLA-255 cell lines, and the LA–N-1 cell line were cultured in IMDM with 15 % FBS. Primary untreated tumors from patients with high-risk, stage 4 (metastatic) neuroblastoma were obtained from patients enrolled and consented in therapeutic and biology protocols of the Children’s Oncology Group (COG). NK cells, monocytes, and cell culture PBMC were isolated from blood of normal adult donors by density separation using Ficoll-Hypaque [24]. NK cells and monocytes were isolated from PBMC by negative selection using a NK Cell Isolation Kit or a Monocyte Cell Isolation Kit, an LS Column, and a MidiMACS™ Separator according to the manufacturer’s protocol (Miltenyi Biotec). Purified NK cells (CD56+ CD16+ CD3−) were >95 %, and purified monocytes (CD14+) were >95 %. NK cells were
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removed from PBMC by positive selection using CD56 MicroBeads and the column and separator mentioned above (CD56+ was
