Biology of Blood and Marrow Transplantation 10:40-48 (2004) ᮊ 2004 American Society for Blood and Marrow Transplantation 1083-8791/04/1001-0004$30.00/0 doi:10.1016/j.bbmt.2003.09.013 Allogeneic versus Syngeneic Killer Splenocytes as Effector Cells for the Induction of Graft-versus- Tumor Effect Shoshana Morecki, Elena Yacovlev, Yael Gelfand, Anna Vilensky, Shimon Slavin Department of Bone Marrow Transplantation & Cancer Immunotherapy, Cell Therapy & Transplantation Research Center, Hadassah University Hospital, Jerusalem, Israel Correspondence and reprint requests: Shoshana Morecki, PhD, Department of BMT, Hadassah University Hospital, Jerusalem 91120, Israel (e-mail: [email protected]). Received August 8, 2003; accepted September 22, 2003 ABSTRACT The effect of allogeneic versus syngeneic killer cells derived from normal or severe combined immunodefi- ciency disease (SCID) mice was evaluated for induction of antitumor reaction in a murine model of mammary carcinoma. Tumor cells of H-2d origin were injected intravenously into H-2d/b mice 24 hours after total body irradiation (4 Gy). On the following day, lymphokine-activated killer (LAK) splenocytes, derived from either minor (H-2d) or major (H-2b) histocompatibility complex (MHC)–mismatched parental normal mice or MHC (H-2b)–mismatched SCID mice, were given intravenously. LAK cells of H-2d normal or SCID mice, syngeneic to the tumor, were inoculated in parallel. The results show that LAK cells derived from minor histocompat- ibility complex–mismatched or MHC-mismatched parental normal mice improved the probability of tumor- free survival as compared with LAK cells syngeneic to the tumor cells, but they aggravated the severity of graft-versus-host disease. SCID splenocytes serving as a source of natural killer (NK) cells were expanded and activated in vitro by rIL-2 to obtain a sufficient number of DX5؉ CD3؊ CD8؊ NK cells (SCID-LAK). H-2b SCID-LAK cells did not cause graft-versus-host disease and significantly delayed tumor growth compared with syngeneic H-2d SCID-LAK cells, as indicated by tumor colony assays in vitro and adoptive transfer experi- ments. However, the graft-versus-tumor effect was not long lasting, and treated mice finally died of tumor. Our results show an advantage of allogeneic over syngeneic cell therapy for achieving a graft-versus-tumor effect by rIL-2–activated T cells and NK cells. Periodic repetition of NK treatments may be required to achieve more durable antitumor effects. © 2004 American Society for Blood and Marrow Transplantation KEY WORDS Allogeneic killer cells ● Syngeneic killer cells ● SCID mice ● Graft-versus-tumor effect INTRODUCTION disease (GVHD) [12]. Furthermore, donor lympho- cyte infusion given after HSCT was shown to play a Immunotherapy strategies that aim to amplify an key role in eradicating malignant cells and preventing immune response and break tolerance to the tumor relapse [3-6,13-15]. The antitumor effects of alloreac- cells are mostly based on the use of tumor-syngeneic/ tive donor lymphocytes, designated as GVL and graft- autologous effector cells in both experimental models versus-tumor (GVT) effects, were ascribed mainly to and cancer patients [1-11]. During the last decade, an allogeneic T lymphocytes that recognize malignant increasing number of protocols based on an allogeneic cells in a major histocompatibility complex (MHC)– reaction between immunocompetent effector cells and restricted fashion and to MHC-nonrestricted natural tumor target cells were developed. This strategy killer (NK) and lymphokine-activated killer (LAK) emerged from clinical observations after allogeneic cells [16-20]. Unlike T cells, which are activated hematopoietic stem cell transplantation (HSCT), through recognition of foreign epitopes, it is sug- where there seemed to be a correlation between graft- gested that NK-cell activation is mediated via MHC versus-leukemia (GVL) effects and graft-versus-host class I missing-self epitopes when the negative signals 40 Allogeneic Killer Cells for Cancer of killing-inhibitory receptors (KIR) on the NK cells lands) in culture medium of 4T1 supplemented with Ϫ cannot be neutralized [21-23]. Allogeneic cell therapy 2-mercaptoethanol (5 ϫ 10 5 mol/L) and sodium (alloCT) has pronounced antitumor effects but is of- pyruvate (1 mmol/L; complete LAK medium). After 4 ten complicated by the development of GVHD, which days in a CO2 incubator at 37°C, cells were washed ϩ is ascribed to immunocompetent donor CD4 and and tested for their ability to exert an antitumor re- ϩ CD8 T cells that target organs such as skin, gut, sponse in vivo. liver, and lung [24-27]. In contrast to T cells, alloge- neic NK cells can mediate antitumor responses with- Propagation of LAK SCID Cells out causing GVHD when administered to immuno- Single-cell suspensions of splenocytes and bone compromised mice or humans [17-20], thus marrow (BM) cells derived from SCID mice were suggesting that NK alloreactivity warrants further prepared in complete LAK medium supplemented study as a potentially safer antitumor modality. with 10 ␮g/mL indomethacin (Sigma, St. Louis, MO). Encouraged by the efficient antitumor effects of Cell suspensions at a concentration of 1 ϫ 106/mL alloCT in previous studies, yet aware of the associated were plated in 6-well tissue culture plates (Corning, complications, we compared the efficacy of allogeneic New York, NY) or tissue culture flasks containing versus syngeneic cell therapy (synCT) by using various 3000 IU/mL rIL-2, according to the total number of sources of lymphokine-activated T and NK cells in an cells obtained. Cell and rIL-2 concentrations were attempt to induce GVT effects in a murine model of adjusted with fresh complete medium to 1 ϫ 106/mL mammary carcinoma. and 3000 IU/mL, respectively, every 3 to 4 days. Flow Cytometry Analysis MATERIALS AND METHODS Peripheral blood mononuclear cells (5 ϫ 105) Mice were stained directly with phycoerythrin anti-mouse ϩ BALB/c (H-2d) (BALB), C57BL/6 (H-2b) (C57) Pan NK cells (DX5) to detect NK1.1 cells, fluores- d d/b DBA/2 (H-2 ) (BALB/cXC57BL/6) F1 (H-2 )(F1), cein isothiocyanate (FITC) anti-mouse CD3 (17A2), and C.B-17/IcrHsd-scid (H-2d; SCID, severe com- and FITC anti-mouse CD8a (53-6.7). Phycoerythrin bined immunodeficiency disease) (C.B-17) mice, aged rat immunoglobulin M isotype standard (R4-22) and 9 to 12 weeks, were obtained from Harlan Sprague FITC rat immunoglobulin G2b isotype standard Dawley (Indianapolis, IN). C57BL/6J-PrkdcϽscidϾ/ (A95-1) were used as isotype controls. All reagents SzJ (H-2b) (C57 SCID) mice were obtained from were purchased from BD PharMingen (San Diego, Jackson Laboratory (Bar Harbor, ME). All mice were CA). All samples were preincubated for 10 min on ice maintained in the Hadassah University Hospital in with unlabeled rat anti-mouse CD32/16 antibodies accordance with the national laws and regulations for (Southern Biotechnology Associates, Inc., Birming- the protection of animal welfare. ham, AL) to prevent nonspecific staining through the Fc fraction of the labeled antibodies. Staining was Tumor Cells performed as described previously [31]. 4T1 is a tumor cell line established from a cell 4T1 Clonogenic Assay subpopulation isolated from a single spontaneously arising mammary tumor in a BALB/c (H-2d) mouse Lung metastases were measured as already de- [28]. 4T1 cells were maintained in 10% fetal bovine scribed [32]. Briefly, lungs were harvested from 3 mice serum, 2 mmol/L glutamine streptomycin 100 ␮g/ of each experimental group 13 to 14 days after tumor mL, penicillin 100 U/mL, and 1% nonessential amino inoculation. Single-cell suspensions were prepared in acid (culture medium of 4TI) and prepared for intra- serial dilutions in culture medium of 4T1 including 60 venous (IV; 0.25 mL per mouse) and intradermal (0.1 ␮mol/L 2-amino-6 mercaptopurine (Sigma) and mL per mouse) inoculation or were prepared for in plated in 100 ϫ 20 mm tissue culture dishes (Becton vitro assays as described previously [29]. Yac-1 (H-2a) Dickinson Labware, Franklin Lakes, NJ). Tumor col- (Yac) is a cell line sensitive to the cytotoxic activity of onies were fixed and stained with 0.03% methylene NK cells [30] and acts as target cells in chromium 51 blue 10 to 14 days after plating. The number of tumor release assays. Cells were kept at 37°C in a humidified colonies was calculated per mouse lung for each ex- 5% CO2/air incubator. perimental group relative to 100% 4T1 colonies in the control group of untreated mice. Preparation of LAK Normal Splenocytes Measurement of Cytotoxic Activity Spleen cells (1 ϫ 106/mL) were plated in 225-cm2 tissue culture flasks (Costar, Cambridge, MA) con- Cells harvested from normal LAK and SCID- taining 6000 IU/mL recombinant interleukin (rIL)-2 LAK cultures were washed, diluted to 5 ϫ 106/mL, (Proleukin; Chiron BV, Amsterdam, The Nether- and tested in a standard 4-hour chromium 51 release BB&MT 41 S. Morecki et al. Table 1. Effects of Syngeneic versus Allogeneic Cell Therapy on Survival of Tumor-Inoculated Mice Cause of Death No. Disease- Survival,* Free Effector Cells Median (Range) No. Mice Tumor-Related GVHD-Related Survivors Tumor only 33 (20-137) 31 31 0 0 F1 splenocytes 33 (22-205)‡ 26 25 0 1 BALB splenocytes 28 (25-50) 24 24 0 0 C57 splenocytes 17 (13-20) 15 0 15 0 BALB LAK cells 33 (27-50) 18 18 0 0 DBA LAK cells† 41 (28-180)‡ 18 14 2 2 F1 LAK cells 38 (30-138)‡ 10 9 0 1 C57 LAK cells 18 (14-20) 10 0 10 0 ϫ ϫ 4 (BALB/c C57BL/6) F1 mice were inoculated with 5 10 4T1 cells 24 hours after receiving 4 Gy of total body irradiation. One day later, 20 to 25 ϫ 106 effector cells derived from normal mice were given to them intravenously, and rIL-2 was administered intraperitoneally for 3 consecutive days at 5 ϫ 104 IU/d, starting from the day of cell therapy.