sign in sign up
<<
Home , CD28, CD80, Natural killer cell

ANTICANCER RESEARCH 31: 475-480 (2011)

Natural Killer Cell Line YT Exerts Against CD86+ Myeloma Cells

ULF HARNACK*, HEIKO JOHNEN* and GABRIELE PECHER

Medical Clinic of Oncology and Hematology, Charité-Universitätsmedizin Berlin, Campus Mitte, 10117 Berlin, Germany

Abstract. Background: NK cell lines providing an The susceptibility of myeloma cells to natural killer (NK) unlimited source of effector cells might be suitable for use in cell-mediated has been shown in vitro and in vivo (4- adoptive . This study determined the cytolytic 6). However, autologous or allogeneic NK cells are difficult activity of the human NK-like cell line YT against myeloma to maintain and to expand ex vivo on the large scale cell lines and primary myeloma cells. Materials and necessary for adoptive immunotherapy (AIT) (7). Methods: Lysis of the myeloma cell lines MM1S and U266 Furthermore, the high doses of systemically administered and of primary human myeloma cells by YT was measured (IL)-2 that are required for the activation and using a flow-cytometric cytotoxicity assay. Furthermore, it expansion of NK cells ex vivo and after transfer in vivo was investigated whether the cytotoxicity correlates with the expand also regulatory T (Treg) cells which are able to expression of CD86 on myeloma cells and the effect of suppress NK cell activity (8-10). Instead, human NK cell different doses of IL-2 on cytolysis was tested. Results: YT lines, which provide an unlimited source of NK effector cells showed killing of myeloma cell lines and primary myeloma with stable phenotype, may be suitable for use in AIT (11- cells. The extent of cytolysis correlated with the expression 15). The human NK-like leukaemia cell line YT was of CD86 on myeloma cells and was not augmented by pre- established from cells in the pericardial fluid of a patient incubation of YT with high dose of IL-2. Conclusion: The with acute lymphoblastic (ALL) and thymoma human NK-like cell line YT could be useful in (16). YT cells were described as having their T-cell immunotherapy of patients with CD86+ multiple myeloma. (TCR) genes in germline configuration being also CD3–, CD16–, CD56+ CD57– and to exert cytotoxicity against Multiple myeloma (MM) is a B-cell neoplasia in which CD80/CD86-expressing T and B lymphoblastic cell lines via malignant plasma cells accumulate in the and a CD28-mediated mechanism (16-19). Myeloma cells and secrete large amounts of a monoclonal . Despite plasma cells from healthy donors lack expression of CD80 significant progress in the treatment of MM due to improved (20). However, malignant plasma cells show strong variation efficacy of autologous and allogeneic stem cell in the expression of CD86, which is associated with poor transplantation and introduction of the inhibitor prognosis and is absent from or weakly expressed on non- bortezomib and the immunomodulatory drugs , malignant plasma cells (20-22). Malignant and healthy and dexamethasone, a large percentage of MM plasma cells of the bone marrow express syndecan-1 patients unfortunately experience relapse (1-3). Thus, (CD138) on their surface but typically differ by the absence treatment of MM awaits the development of additional or presence of CD19, respectively (23). This study tested the therapeutic options including immunotherapeutic strategies. cytotoxic activity of the NK-like killer cell line YT against human primary myeloma cells and myeloma cell lines in vitro and determined whether cytolysis correlated with the expression of CD86 on the tumor cells. *Both Authors contributed equally to this work. Materials and Methods Correspondence to: PD Dr. Gabriele Pecher, Charité- Universitätsmedizin Berlin, Medical Clinic of Oncology and Primary myeloma cells and cell lines. Primary myeloma cells were Hematology, Campus Mitte, Charitéplatz 1, 10117 Berlin, Germany. isolated from the bone marrow of a patient with MM after informed Tel: +49 30450513131, Fax: +4930450514906, e-mail: gabriele. consent was given. The myeloma cell lines MM1S and U266 were [email protected] kindly provided by Dr. Sezer, Charité Universitätsmedizin Berlin, Germany and the cell line YT was a kind gift from Junji Yodoi, Key Words: Myeloma, YT, CD86, interleukin-2, cytotoxicity. Kyoto University, Kyoto, Japan. All cell lines and primary cells

0250-7005/2011 $2.00+.40 475 ANTICANCER RESEARCH 31: 475-480 (2011) were maintained in RPMI-1640 medium supplemented with 10% expressed CD86 at very low density and lacked expression fetal bovine serum (both from Biochrom AG, Berlin, Germany) and of CDSO (Figure 2A, B). MM1S showed moderate 100 U/ml penicillin and 100 μg/ml streptomycin (Biochrom AG). expression of CD80, whereas U266 exhibited low expression Maintenance of YT cells required addition of 5-10 U/ml human of this surface molecule. Both myeloma cell lines were recombinant IL-2 (Proleukin, Chiron) to the medium. negative for expression of CD80 (Figures 2C-F). Isolation of primary myeloma cells. Mononuclear cells were isolated from freshly prepared bone marrow of a patient with MM by ficoll Discussion density gradient centrifugation followed by enrichment of CD138+ cells using antibody-conjugated magnetic microbeads in accordance Recent studies revealed that bone-marrow-derived myeloma to the manufacturer’s instructions (Miltenyi, Bergisch-Gladbach, cells are sensitive to NK cell-mediated cytolysis (4-6). Germany). The purity of primary myeloma cells was determined by Therefore this study tested whether the human NK cell-like flow cytometry using a PE-labeled anti-CD138 antibody (Miltenyi Biotec) and a FITC-labeled anti-CD19-antibody (Jackson cell line YT shows cytolytic activity against myeloma cell Immunoresearch, Baltimore, USA). lines and primary myeloma cells in vitro. Indeed, YT cells exerted moderate killing of MM1S cells and lower cytotoxicity Phenotypical analysis of myeloma cells. The expression of - against U266 cells and CD19–CD138+ plasma cells freshly molecules on myeloma cells and on YT cells was determined by isolated from the bone marrow of a patient with MM. flow cytometry using a FACScan (Becton-Dickinson, Heidelberg, Myeloma cells lack expression of CD80 but show different Germany). FITC-labeled anti-human CD80 (clone 37711.111) and amounts of CD86 on their surface as described by others and CD86 (37301.111) were obtained from R&D Systems and control antibodies were obtained from eBioscience observed in this study (20-22). Furthermore, the extent of (Frankfurt, Germany). YT cells were confirmed to express CD28 as CD86 surface expression was recently shown to be associated described by others (18) using a biotin-conjugated anti-human with the pathophysiology of myeloma, including enhanced CD28 (CD28-2) antibody from eBioscience (data not shown). The growth rate, induction of immune-suppressive CD4+ T-cells biotinylated antibody was counterstained with DTAF-labeled and shorter survival time of patients (21, 22). In accordance streptavidin (Jackson Immunoresearch). to the described MHC-non-restricted cytotoxicity via interaction of CD28 on YT cells with CD80/86 on T and B Flow cytometric cytotoxicity assay. The cytotoxicity of YT cells pre- incubated with a low dose (10 U/ml) or high dose (1000 U/ml) of IL- lymphoma and cells, the cytolysis correlated with 2 over one week was determined by a flow-cytometric based assay. the extent of the expression of CD86 on the surface of the In brief, 1×106 target cells were stained with 0.1 μM myeloma cells (18, 19). Similarly, the myeloma cell line carboxyfluorescein diacetate succinimidylester (CFDA-SE) in PBS RPMI-8226, which expresses low levels of CD86, was shown over 5-10 min; 5×103 labeled target cells were incubated with YT to be almost resistant to YT-mediated lysis (18), whereas the effector cells for four hours in 200 μl complete medium in triplicates CD80+CD86++ ARH-77 cell line which was reportedly and at E:T ratios as indicated. Subsequently, the percentage of dead strongly killed by YT was meanwhile re-classified as a B- target cells was determined by flow cytometry using a FACscan after staining the cells with 2 μg/ml propidium iodide (PI). lymphoblastoid and not as a myeloma cell line (16, 24-26). NK cell cytotoxicity is significantly increased after Statistics. Data are presented as mean±the standard error of the treatment with larger amounts of IL-2 (27, 28). Thus, this mean (SEM). Analysis of flow cytometric data was performed using study determined the effect of a pre-treatment of YT with a the CellQuest software version 4.0.2 (BD Biosciences, Heidelberg, high dose of IL-2 on the lysis of myeloma cell lines. Germany). However, YT cell cytotoxicity was not increased by IL-2, which could be due to the CD28-mediated killer mechanism Results shown to be executed in the absence of IL-2 (18, 19). Moreover, YT cells reportedly express the IL-2 receptor (IL- YT cell-mediated cytolysis of primary myeloma cells and 2R) α-chain (CD25) only weakly or lack its expression and, myeloma cell lines. YT cells treated with a low dose of IL-2 in contrast to primary NK cells, do not up-regulate CD25 in showed moderate cytotoxicity against the myeloma cell line response to IL-2 which obviates the generation of the MM1S and exerted less cytolytic activity against U266 trimeric high affinity IL-2R (16, 29, 30). Similarly, the (Figure 1A). Pre-treatment of YT cells with a high dose of growth rate of YT cells was shown not to be increased after IL-2 did not increase their cytotoxicity against the myeloma incubation with high doses of IL-2 (30). Nevertheless, the cell lines (Figure 1B). Primary myeloma cells which were independence of YT cell killer activity from significant enriched from bone marrow cells to a purity of 73% were amounts of exogenous IL-2 might be advantageous lysed by YT to a similar degree as U266 (Figure 1 C, D). compared with the need for high doses of the for activation and expansion of autologous or allogeneic NK Expression of CD80/CD86 on myeloma cells. The bone- cells before and after adoptive transfer, which is associated marrow-derived CD138+ plasma cells of the patient with the expansion of NK cell inhibitory Treg cells and

476 Harnack et al: YT Cell Cytotoxicity Towards Myeloma

Figure 2. Expression of CD86 and CD80 on human myeloma cells. The expression of B-7 molecules on CD138+ plasma cells of a patient with MM (A, B) and on myeloma cell lines (C-F) determined by flow cytometry.

killer immunoglobulin-like receptors which regulate recognition and lysis of tumor cells by NK cells on YT cells may enable therapeutic usage of the NK-like cell line independently from the MHC class-I repertoire of the recipient (36-38). Taken together, this study provides evidence that human myeloma cells are sensitive to YT cell-mediated cytolysis dependent on the expression of CD86 on tumor cells. Furthermore, the cytotoxicity of YT cells is not improved by Figure 1. Cytotoxicity of YT cells against human myeloma cells. A, B: YT treatment with a large dose of IL-2. effector cells cultured before in medium containing 10 or 1000 U/ml IL- 2 were incubated with myeloma cell lines for 4 hours at the indicated References E:T-ratios and cytolysis of target cells was measured using a flow + cytometric NK-cell assay. C, D: Purity of CD138 myeloma cells and 1 Ludwig H, Bolejack V, Crowley J, Bladé J, Miguel JS, Kyle RA, cytolysis by YT. Myeloma cells were enriched from bone marrow of a Rajkumar SV, Shimizu K, Turesson I, Westin J, Sonneveld P, patient with MM by magnetic bead sorting and incubated with YT cells Cavo M, Boccadoro M, Palumbo A, Tosi P, Harousseau JL, Attal for 4 hours at the indicated E:T ratios to determine their sensitivity to M, Barlogie B, Stewart AK and Durie B: Survival and years of YT-mediated cytolysis. Data shown are representative of two or three independently performed experiments with similar results (cell lines) or life lost in different age cohorts of patients with multiple of one experiment (patient cells) all performed in triplicates. myeloma J Clin Oncol 28: 1599-1605, 2010. 2 Richardson PG, Weller E, Lonial S, Jakubowiak AJ, Jagannath S, Raje NS, Avigan DE, Xie W, Ghobrial IM, Schlossmann RL, Mazumder A, Munshi NC, Vesole DH, Joyce R, Kaufman JL, Doss D, Warren DL, Lunde LE, Kaster S, Delaney C, Hideshima induction of a vascular leak syndrome (8-10, 28, 31-34). In T, Mitsiades CS, Knight R, Esseltine DL and Anderson KC: fact, YT cells were shown to provide a constitutively Lenalidomide, bortezomib, and dexamethasone combination therapy in patients with newly diagnosed multiple myeloma. activated killer phenotype due to production of significant 116: 679-686, 2010. amounts of cytotoxic molecules, such as perforins and 3 Palumbo A and Rajkumar SV: Multiple myeloma: chemotherapy , and expression of the -inducing Fas- or transplantation in the era of new drugs. Eur J Haematol 84: ligand (35). Moreover, the absence of surface-expressed 379-390, 2010.

477 ANTICANCER RESEARCH 31: 475-480 (2011)

4 Frohn C, Höppner M, Schlenke P, Kirchner H, Koritke P and 19 Teng JM, Liu XR, Mills GB and Dupont B: CD28-mediated Luhm J: Anti-myeloma activity of natural killer . cytotoxicity by the human leukemia NK cell line YT involves Brit J Haematol 119: 660-664, 2002. phosporylation, activation of phosphatidylinositol-3- 5 Carbone E, Neri P, Mesuraca M, Fulciniti MT, Otsuki T, Pende kinase, and kinase C. J Immunol 156: 3222-3232, 1996. D, Groh V, Spies T, Pollio G, Cosman D, Catalano L,Tassone P, 20 Brown RD, Pope B, Yuen E, Gibson J and Joshua DE: The Rotoli B and Venuta S: HLA class I, NKG2D, and natural expression of T-cell-related co-stimulatory molecules in multiple cytotoxicity receptors regulate multiple myeloma cell myeloma. Leuk Lymphoma 31: 379-384, 1998. recognition by natural killer cells. Blood 105: 251-258, 2005. 21 Pope B, Brown RD, Gibson J, Yuen E and Joshua D: B7-2- 6 Soriani A, Zingoni A, Cerboni C, Iannitto ML, Ricciardi MR, Di positive myeloma: incidence, clinical characteristics, prognostic Gialleornado V, Cippitelli M, Fionda C, Petrucci MT, Guarini A, significance, and implications for tumor immunotherapy. Blood Foà R and Santoni A: ATM-ATR-dependent up-regulation of 96: 1274-1279, 2000. DNAM-1 and NKG2D ligands on multiple myeloma cells by 22 Yamashita T, Tamura H, Satoh C, Shinya E, Takahashi H, Chen therapeutic agents results in enhanced NK cell susceptibility and L, Kondo A, Tsuji T, Dan K and Ogata K: Functional B7.2 and is associated with a senescent phenotype. Blood 113: 3503-3511, B7-H2 molecules on myeloma cells are associated with a growth 2009. advantage. Clin Res 15: 770-777, 2009. 7 Klingemann HG and Martinson J: Ex vivo expansion of natural 23 Bataille R, Jégo G, Robillard N, Barillé-Nion S, Harousseau JL, killer cells for clinical applications. Cytotherapy 6: 15-22, 2004. Moreau P, Amiot M and Pellat-Deceunynck C: The phenotype 8 Brandenburg S, Takahashi T, de la Rosa M, Janke M, Karsten G, of normal, reactive and malignant plasma cells. Identification of Muzzulini T, Orinska Z, Bulfone-Paus S and Scheffold A: IL-2 “many and multiple myelomas” and of new targets for myeloma induces in vivo suppression by CD4(+)CD25(+)Foxp3(+) therapy. Haematologica 91: 1234-1240, 2006. regulatory T-cells. Eur J Immunol 38: 1643-1653, 2008. 24 Chiorini JA, Wendtner CM, Urcelay E, Safer B, Hallek M and 9 Lundqvist A, Yokoyama H, Smith A, Berg M and Chields R: Kotin RM: High-efficiency transfer of the T-cell co-stimulatory Bortezomib treatment and regulatory T-cell depletion enhance molecule B7-2 to lymphoid cells using high-titer recombinant the antitumor effects of adoptively infused NK cells. Blood 113: adeno-associated vectors. Human Gene Ther 6: 1531-1541, 6120-6127, 2009. 1995. 10 Smyth MJ, Teng MW, Swann J, Kyparissoudis K, Godfrey DI 25 Drexler HG, Dirks WG and MacLeod RA: False human and Hawakaya J: CD4+CD25+ T-regulatory cells suppress NK hematopoietic cell lines: cross-contaminations and cell-mediated immunotherapy of cancer. J Immunol 176: 1582- misinterpretations. Leukemia 13: 1601-1607, 1999. 1587, 2006. 26 Pellet-Deceunynk C, Amiot M, Bataille R, van Riet I, van Camp 11 Tam YK, Martinson JA, Doligosa K and Klingemann HG: Ex B, Omede P and Boccadoro M: Human myeloma cell lines as a vivo expansion of the highly cytotoxic human natural killer-92 tool for studying the biology of multiple myeloma: a reappraisal cell line under current good manufacturing practice conditions 18 years after. Blood 86: 4001-4002, 1995. for clinical adoptive cellular immunotherapy. Cytotherapy 5: 27 DeBlaker-Hohe DF, Yamauchi A, Yu CR, Horvath-Arcidiacono 259-272, 2003. JA and Bloom ET: IL-12 synergizes with IL-2 to induce 12 Arai S, Meagher R, Swearingen M, Myint H, Rich E, Martinson lymphokine-activated cytotoxicity and and J and Klingemann H: Infusion of the allogeneic cell line NK-92 in fresh human NK cells. Cell Immunol 165: in patients with advanced renal cell cancer or melanoma: a phase 33-43, 1995. I trial. Cytotherapy 10: 625-632, 2008. 28 Miller JS, Tessmer-Tuck J, Pierson BA, Weisdorf D, McGlave P, 13 Suck G, Branch DR and Keating A: Irradiated KHYG-1 retains Blazar BR, Katsanis E, Verfaillie C, Lebkowski J, Radford J Jr. cytotoxicity: potential for adoptive immunotherapy with a and Burns LJ: Low-dose subcutaneous interleukin-2 after line. Int J Radiat Biol 82: 355-361, 2006. autologous transplantation generates sustained in vivo natural 14 Schirrmann T and Pecher G: Tumor-specific targeting of a cell killer cell activity. Biol Blood Marrow Transplant 3: 34-44, 1997. line with natural killer cell activity by asialoglycoprotein receptor 29 Alileche A, Goldman CK and Waldmann TA: Differential effects gene transfer. Cancer Immunol Immunother 50: 549-556, 2001. of IL-2 and IL-15 on expression of IL-2 receptor alpha. Biochem 15 Schirrmann T and Pecher G: Human natural killer cell line Biophys Res Commun 285: 1302-1308, 2001. modified with a chimeric immunoglobulin T-cell receptor gene 30 Nagashima S, Mailliard R, Kashii J, Reichert TE, Herbermann leads to tumor growth inhibition in vivo. Cancer Gene Ther 9: RB, Robbins P and Whiteside TL: Stable transduction of the 390-398, 2002. interleukine-2 gene into human natural killer cell lines and their 16 Yodoi J, Teshigawara K, Nikaido T, Fukui K, Noma T, Honjo T, phenotypic and functional characterization in vitro and in vivo. Takigawa M, Sasaki M, Minato N, Tsudo M, Uchiyama T and Blood 91: 3850-3861, 1998. Maeda M: TCGF (IL 2)-receptor inducing factor(s). I. 31 Bachanova V, McKenna D, Curtsinger J, Panoskaltsis-Mortari A, Regulation of IL 2 receptor on a natural killer-like cell line (YT Lindgren BR, Cooley S, Weisdorf D and Miller JS: Allogeneic cells). J Immunol 134: 1623-1630, 1985. natural killer cells for refractory lymphoma. Cancer Immunol 17 Yoneda N, Tatsumi E, Kawano S, Teshigawara K, Oka T, Fukuda Immunother 59: 1739-1744, 2010. M and Yamaguchi N: Detection of Epstein-Barr virus genome in 32 Siegler U, Meyer-Monard S, Jörger S, Stern M, Tichelli A, natural killer-like cell line YT. Leukemia 6: 136-141, 1992. Gratwohl A, Wodnar-Filipowicz A and Kalberer CP: Good 18 Azuma M, Cayabyab M, Buck D, Phillips JH and Lanier LL: manufacturing practice-compliant cell sorting and large-scale Involvement of CD28 in MHC-unrestricted cytotoxicity mediated expansion of single KIR-positive alloreactive human natural by a human natural killer leukemia cell line. J Immunol 149: killer cells for multiple infusions to leukemia patients. 1115-1123, 1992. Cytotherapy 12: 750-763, 2010.

478 Harnack et al: YT Cell Cytotoxicity Towards Myeloma

33 Yates J, Rovis F, Mitchell P, Afzali B, Tsang JY, Garin M, 37 Tarazona R, Borrego F, Galiani MD, Aguado E, Pena J, Coligan Lechler RI, Lombardi G and Garden OA: The maintenance of JE and Solana R: Inhibition of CD28-mediated natural human CD4+CD25+ regulatory T-cell function: IL-2, IL-4, IL-7 cytotoxicity by KIR2DL2 does not requie p56lck in the NK cell and IL-15 preserve optimal suppressive potency in vitro. Int line YT-Indy. Molecular 38: 495-503, 2001. Immunol 19: 785-799, 2007. 38 Bari R, Bell T, Leung WH, Vong QP, Chan WK, Das Gupta N, 34 Assier E, Jullien V, Lefort J, Moreau JL, Di Santo JP, BB Holladay M, Rooney B and Leung W: Significant functional Vargaftig, JR Lapa e Silva and J Thèze: NK cells and heterogeneity among KIR2DL1 alleles and a pivotal role of polymorphonuclear are both critical for IL-2 induced arginine 245. Blood 114: 5182-5190, 2009. pulmonary vascular leak syndrome. J Immunol 172: 7661-7668, 2004. 35 Montel AH, Bochan MR, Goebel WS and Brahmi Z: Fas- mediated cytotoxicity remains intact in perforin and antisense transfectants of a human NK-like cell line. Cell Immunol 165: 312-317, 1995. 36 Yu J, Heller G, Chewning J, Kim S, Yokohama WM and Hsu KC: Hierarchy of the human natural killer cell response is Received December 10, 2010 determined by class and quantity of inhibitory receptors for self- Revised January 19, 2011 HLA-B and HLA-C ligands. J Immunol 179: 5977-5989, 2007. Accepted January 20, 2011

479