Bone Marrow Transplantation (2004) 33, 1137–1141 & 2004 Nature Publishing Group All rights reserved 0268-3369/04 $25.00
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Immunotherapy Murine B-cell leukemia lymphoma (BCL1) cells as a target for NK cell-mediated immunotherapy L Weiss1, S Reich1, O Mandelboim2 and S Slavin1 1 Department of Bone Marrow Transplantation and Cancer Immunotherapy, Cell Therapy and Transplantation Immunobiology Research Center, Hadassah University Hospital, Jerusalem, Israel; and 2The Lautenberg Center for General and Tumor Immunology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel
Summary: Natural killer (NK) cells are important for their ability to recognize and lyse tumor cells and virus infected cells. NK cells express triggering receptors that are specific for nonMHC ligands. This article describes the 35S release cytotoxic assay, which measures the ability of NK cells derived from spleen cells taken from polyIC-treated mice to lyse B-cell leukemia (BCL1) cells. BCL1 cells express ligands for NKp46 on the cell surface membrane and they are sensitive to allogeneic but not syngeneic IL-2 activated natural killer cells. Bone Marrow Transplantation (2004) 33, 1137–1141. doi:10.1038/sj.bmt.1704475 Published online 12 April 2004 Keywords: B-cell leukemia (BCL1); murine; lymphokine activated killer cells (LAK cells); immunity
Immunocompetent T lymphocytes, natural killer (NK) cells, natural killer NK T cells (NKT) and recombinant interleukin 2 (rIL-2) activated lymphocytes derived from an allograft may play a major role in induction of graft– versus-leukemia or lymphoma (GVL) effects, following allogeneic bone marrow or blood stem cell transplantation (BMT). Although there seems to be no question on the role of alloreactive T cells in induction and maintenance of GVL effects in experimental animals and man,1–4 thus explaining the inseparable effects of GVL and graft-versushost disease (GVHD), the therapeutic role of NK cells, especially against lymphoid target cells, remains unclear. Recently, Ruggeri et al5 demonstrated the pivotal role of NK cells in GVL. Patients with acute myeloid leukemia treated with haploidentical stem cell allografts with alloreactive NK cells in the direction of GVHD responded much better and featured fewer relapses, however, no benefit from alloreactive NK cells could be documented in patients with acute lymphoblastic leukemia.5 Since NK
Correspondence: Dr S Slavin, Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah University Hospital, Jerusalem 91120, Israel; E-mail:
[email protected] Received 13 August 2003; accepted 29 December 2003 Published online 12 April 2004
cells do not induce GVHD, the possible use of NK or NKT cells for induction of GVL effects while avoiding GVHD appears most attractive. It was already documented that lymphokine (rIL-2) activated killer lymphocytes (LAK cells), generated in vitro or in vivo, are capable of killing a wide variety of malignant cells in an MHC nonrestricted manner.6,7 NK cell activity can be suppressed in vivo by treatment with anti-asialo GM1 antibodies (AGM1).8 We have previously documented that allogeneic, but not syngeneic LAK, cells are capable of inducing GVL effects in vivo in (BALB/cXC57BL/6)F1 (F1) recipients inoculated with a large BCL1 inoculum following cell therapy with parental (C57BL/6) spleen cells.9 Furthermore, rIL-2 activation improved dramatically the GVL capacity of allogeneic lymphocytes following BMT against both lymphoid10–14 and myeloid tumor cells.15 On the other hand, in vivo treatment with AGM1, for 10 days post BMT in C57BL/6-F1 chimeras, abrogated the GVL effect and resulted in the development of leukemia relapse and death of all recipients.16 Since AGM1 administered in vivo can also kill T cells in addition to NK cells, it was essential to determine if allogeneic NK cells alone could induce GVL effects. Here we show that B-cell leukemia (BCL1) cells express ligands for NKp46 on the cell surface membrane and, consequently, they are sensitive to NK cell activity as was determined by the 35S release cytotoxic test. Allogeneic, but not syngeneic LAK, cells are capable of cytolysis of BCL1 cells.
Materials and methods Murine B-cell leukemia/lymphoma BCL1, a spontaneous B-cell leukemia/lymphoma of BALB/c origin, originally described by Slavin and Strober,17 was maintained in vivo by passages in BALB/c mice. Inoculation of more than 10 cells results in typical B-cell leukemia/ lymphoma characterized by marked splenomegaly, extreme peripheral blood lymphocytosis (up to 200 000/mm3) and death of all tumor-bearing mice.
Detection of ligand for the Nkp46 receptor on BCL1 cells The generation, production and staining procedures of the Ig fusion proteins were previously described.18,19 Briefly, the PCR-generated fragments were cloned into a
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mammalian expression vector containing the Fc portion of human IgG1 (a kind gift from B Seed). Sequencing of the constructs revealed that all cDNAs were in frame with the human Fc genomic DNA and were identical to the reported sequences. COS-7 cells were transiently transfected with the plasmids containing cDNAs using Fugene6 reagent (Boehringer, Mannheim, Germany) according to the manufacturer’s instructions, and supernatants were collected and purified on a protein G column. SDS/PAGE analysis revealed that all Ig fusion proteins were approximately 95% pure and of the proper molecular mass. To assay for the binding various cells were incubated with 50 mg/ml of fusion protein for 2 h on ice. The cells were washed and incubated with Fc-fragment-specific (minimal cross-reaction to bovine, horse, and mouse serum proteins), phycoerythrin (PE)-conjugated affinity-purified F(ab0 )2 fragment of goat anti-human IgG (Jackson Immuno Research) for 1 h and analyzed by flow cytometry with a FACScan (Becton Dickinson).
Mice BALB/c (H-2d/d), C57BL/6 (H-2b/b), (BALB/c C57BL/6) F1 (H-2d/b) (F1) mice (2–4 months old) were purchased from Harlan Breeding Facility (Jerusalem, Israel). Mice were kept under clean conditions with autoclaved cages, sawdust, food and acidified water supplied ad libitum. All animal protocols were approved by the Institutional Committee for Animal Experimentation.
Immunization of mice C57BL/6 mice serving as donors of immune lymphocytes were immunized with 30 106 spleen cells obtained from BCL1-bearing mice (containing 490% BCL1), on days 20 and 10 before testing in the 35S release cytotoxic test.
PolyIC PolyIC (Sigma, St Louis, USA; Catalog Number P-0913) was injected intraperitoneally (200 mg/mouse) to C57BL/6 mice 20 h before using their spleen cells for cytotoxic test.
were incubated overnight with 5 ml 35S methionine (NEG709A 185.00 MBq (5.00 mCi) easytagt methionine, L-[35S] –43.48 TBq mmol (1175.0Ci/mmol), Perkin-Elmer Life Science, Boston, MA, USA) in RPMI medium without methionine. Spleen cells from normal BALB/c and F, and C57BL/6 normal immunized and polyIC treated, were washed twice and suspended in RPMI 1640 medium supplemented by 10% fetal calf serum. LAK cells were collected as mentioned above. The cytotoxic activity of the effectors cells from different sources was measured in 5-h 35 S-release assay as previously described.20
Results Effect of PolyIC treatment on NK activity against BCL1 cells As shown in Figure 1, in vivo treatment of mice with PolyIC augmented the NK activity in all three mice tested. Spleen cells from PolyIC treated mice showed cytotoxic activity of up to 14.0% at 50 : 1 effectors to target cell ratio as compared with 4.1% in normal spleen cells. Higher cytotoxic activity was also noticed at 25 : 1 and 12.5 : 1 effectors to target cell ratio compared with normal cells, one out of two experiments.
LAK cell activity against BCL1 cells As shown in Figure 2 (representing one of three experiments with similar results), allogeneic (C57BL/6) and semiallogeneic (F1) LAK cells expressed high cytotoxic activity against BCL1 cells compared with syngeneic (BALB/c) LAK cells and normal spleen cells. LAK cells from C57BL/6 or F1 mice at a ratio of 100 : 1 effectors to target cells resulted in up to 41% 35S release compared to 5% in LAK BALB/c and to 0–1% when naive spleen cells were derived from either BALB/c, C57BL/6 or F1 mice (mean of two mice in each group). 16 14
Preparation of LAK cells
12 % Release 35S
Spleen lymphocytes were stimulated with rIL-2 (Chiron, Amsterdam, The Netherlands) by culturing the cells at 6000 IU/ml at a cell concentration of 2.5–5 106/ml in 75 ml flasks (Nunc, Denmark) in RPMI 1640 medium (Biological Industries, Beit Haemek, Israel). The medium was supplemented with glutamine 2 mM, penicillin 100 U/ ml, streptomycin 100 mg/ml and 10% inactivated fetal calf serum. The cells were cultured for 5 days in humidified incubator with 5% CO2 in air at 371C. The cells were harvested with a cell scraper and viability was determined by the trypan blue dye exclusion method.
10 8 Poly IC 6 4 2 Normal 0 50:1
Cytotoxicity assay BCL1 cells were collected from the blood of BCL1-bearing BALB/c mouse containing 495% BCL1, and 10 106 cells Bone Marrow Transplantation
25:1
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Ratio effector : target Figure 1 NK activity in C57BL/6 mice treated with PolyIC against BCL1 cells. Results of three individual mice treated by polyIC are shown in comparison with normal controls, representing one out of two experiments.
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Figure 4 Cytotoxic activity of immune cells against BCL1 cells. Cytotoxic activity of C57BL/6 spleen cells, 10 days after the last immunization in vivo with BCL1 cells. *Pool of normal spleen cells, all the others are individual immunized spleen cells.
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Ratio effector cells : target cells Figure 2 NK activity of LAK cells. NK activity mediated by IL-2 activated spleen cells (LAK cells) obtained from C57BL/6 BALB/c and (BALB/c C57BL/6) F1 (F1) were tested against BCL1 cells in comparison with normal spleen cells. One out of three experiments is shown.
100 NKp46-Ig
NK activity against BCL1 cells by immune allogeneic cells Immune C57BL/6 spleen cells showed cytotoxic activity against BCL1 cells in all six mice as shown in Figure 4. Cytotoxic activity ranged between 9.4 and 17.6% 35S release in 50 : 1 effectors to target ratio compared to 2.7% in normal C57BL/6 spleen cells. Possible role of immune T cells, rather than NK cells, cannot be ruled out.
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Figure 3 NKp46-Ig binding to BCL1 cells. BCL1 cells and control cells obtained from BALB/c were incubated with 50 mg/ml of fusion protein for 2 h on ice. The cells were washed and inoculated with Fc – fragment specific PE conjugated affinity – purified F (ab0 )2 fragment of goat anti-human IgG for 1 h and analyzed by flow cytometry with a FACS scan.
NKp46-Ig staining BCL1 cells express a ligand for the NKp46-Ig receptor. It was recently demonstrated that the NKp46-Ig is the main lysis receptor for NK cells of mice and man.21 As killing of BCL1 tumors was observed with allogeneic NK cells, we tested whether BCL1 expresses a ligand for the NKp46-Ig receptors. Efficient recognition of the BCL1 cells was observed when the human NKp46-Ig was used (Figure 3). Both mouse and human NKp46-Ig share a high degree of homology.21 These results suggest that a ligand for the NKp46-Ig receptors is expressed on the mouse BCL1 cells.
BCL1, a spontaneously arising murine lymphocyte leukemia and lymphoma, which in addition to massive involvement of the spleen, bone marrow and blood, also infiltrates lymph nodes and liver, has served as a preclinical model for studying basic tumor biology and immunotherapy of leukemia/lymphoma since the late 1970s.22–25 Considering the fact that in clinical practice, lymphoid malignancies are hardly eradicated even when myeloablative doses of chemoradiotherapy at maximally tolerated doses are used for conditioning pretransplantation, relapse continues to be a major problem. BCL1 has served as a useful tool for studying innovative immunotherapy procedures, including cytokine therapy10 and particularly adoptive allogeneic T-cell mediated immunotherapy.1,9,11–15,25 Thus, it has been previously shown that GVL effects mediated by donor immunocompetent lymphocytes can result in complete eradication of all tumor cells following establishment of host-versus-graft unresponsiveness by induction of mixed chimerism following engraftment of donor stem cells.1,14,26 The efficacy of GVL effects induced by the allograft could be maximized by stimulation of donor lymphocytes with rIL-2.12–14 More recently, we have confirmed that maximal GVL effects can be induced by using specifically immune donor lymphocytes (submitted for publication). Unfortunately, in murine studies, similarly to clinical observations in patients receiving donor lymphocyte infusion (DLI) following allogeneic BMT,27 acute and chronic GVHD appear to be significant barriers, causing major morbidity Bone Marrow Transplantation
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and occasional mortality. NK cells, particularly IL-2activated NK cells, and more recently NK T cells, have been shown to induce remarkable antitumor effects in experimental systems and in clinical trials.5,27 The potential therapeutic benefits of alloreactive NK cells were recently published by Ruggeri et al,5 who elegantly demonstrated that alloreactive NK cells in the direction of host-versusgraft could result in allograft rejection, whereas alloreactive NK cells in the direction of graft-versus-host, could result in better engraftment, most likely by eradicating residual hematopoietic cells, alloreactive host T cells included, on the one hand, and host dendritic cells on the other, thus resulting in reduced incidence of a relapse. Unfortunately, rIL-2 activated lymphocytes may induce much more potent GVHD as compared with naı¨ ve lymphocytes, due to activation of T cells in parallel with NK cells.28 In attempting to analyze the role of NK cells, which may be much safer than T cells due to the fact that they cause no GVHD, we have previously documented that treatment of donor lymphocytes post-BMT with AGM1 can eradicate the therapeutic benefits induced by donor lymphocytes, thus suggesting that NK cells may also play an important role in inducing GVL post-transplantation across MHC in C57BL/6-F1 chimeras.16 Unfortunately, it has been previously shown that asialo GM1 may also affect T lymphocytes,29 and therefore it was never completely clear whether the negative effects of AGM1 were due solely to elimination of NK cells, or also to elimination or downregulation of T cells, particularly CD8-positive T cells.16 The present investigations were carried out in order to document directly the potential role of allogeneic NK cells isolated from lymphocytes undergoing alloreactivity in vivo, against labeled BCL1 cells. We have documented that in vivo, NK cells, in addition to T cells, play a role in induction of GVL effects against BCL1. Our previous investigations documented the role of NK cells by documenting increased incidence of relapse following induction of GVL effects induced by C57BL/6 in BALB/c mice and F1 hybrids following treatment of recipients with anti-asialo GM1.16 Indeed, the present investigations documented cell surface determinants of the ligand for the NKp46 receptor. This became evident by staining tumor cells with human NKp46-Ig fusion protein. The human and mouse NKp46 receptors share more than 80% degree of homology,30 thus explaining why the human NKp46-Ig was able to recognize mouse tumors. Recent work by Ruggeri et al5 demonstrated the essential role of allogeneic NK cells in induction of GVL effect, and the specificity of killing mediated by these cells could be explained by downregulation of class I on tumor cells. Interestingly, although GVL effects mediated by alloreactive NK cells were most effective in patients with AML, patients with lymphoid malignancies did not seem to benefit from alloreactive NK cells.5 It was suggested that lack of expression of ICAM-1 may be responsible for relative resistance of malignant lymphocytes to NK cell mediated lysis. In this work, we demonstrated the theoretical background that may explain the role of allogeneic NK cells in the induction of antitumor effects via the specific recognition of tumor cells by the NKp46 receptor, which may be upregulated by activation of Bone Marrow Transplantation
antitumor effector cells with rIL-2, yet, the role of other possible targets cannot be ruled out. Taken together, it seems that allogeneic rIL-2-activated lymphocytes, including activated T cells and alloreactive NK cells, can induce antitumor effects against malignant B lymphocytes. The likely possibility that antitumor cytotoxicity mediated by allogeneic NK cells may play a beneficial role on antitumor effects in vivo can explain the potential beneficial role of the NK cell family in induction of GVL effects in vivo, as well as justify the potential use of activated NK cells for induction of GVL effects independently of GVHD. Clinical application of alloreactive and cytokine activated NK cells for immunotherapy of hematologic malignancies, lymphoid malignancies included, may offer a unique advantage because it may be applied to induce antitumor effects while avoiding or minimizing GVHD.
Acknowledgements We thank the Danny Cunniff Leukemia Research Laboratory; the Gabrielle Rich Leukemia Research Foundation; the Cancer Treatment Research Foundation; the Novotny Trust and the Fig Tree Foundation for their continuous support of our ongoing basic and clinical research.
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20 Porgador A, Mandelboim O, Restifo NP, Strominger JL. Natural killer cell lines kill autologous beta2-microglobulindeficient melanoma cells: implications for cancer immunotherapy. Proc Natl Acad Sci USA 1997; 94: 13140–13145. 21 Moretta L, Biassoni R, Bottino C et al. Surface receptors that regulate the NK cell function: beyond the NK cell scope. Curr Top Microbiol Immunol 2002; 266: 11–22. 22 Vitetta ES, Yuan D, Krollick K et al. Characterization of the spontaneous murine B cell leukemia (BCL1). III. Evidence for monoclonality using an anti-idiotype antibody. J Immunol 1979; 122: 1649–1654. 23 Yuan D, Uhr JW, Knapp MR et al. Structural differences between m chain of cell associated and secreted immunoglobulin. In: Cooper M, Mosier D, Scher I, Vitetta E (eds.). B Lymphocytes in the Immune Response. Elsevier: Amsterdam, 1979; pp 23–31. 24 Knapp MR, Jones PP, Black SJ et al. Characterization of a spontaneous murine B cell leukemia (BCL1). 1. Cell surface expression of IgM, IgD, Ia and FcR. J Immunol 1979; 123: 992–999. 25 Weiss L, Morecki S, Vitetta ES, Slavin S. Suppression and elimination of BCL1 leukemia by allogeneic bone marrow transplantation. J Immunol 1983; 130: 2452–2455. 26 Prigozhina T, Gurevitch O, Morecki S et al. Non-myeloablative allogeneic bone marrow transplantation as immunotherapy for hematologic malignancies and metastatic solid tumors in pre-clinical models. Exp Hematol 2002; 30: 89–96. 27 Slavin S, Naparstek E, Nagler A et al. Allogeneic cell therapy with donor peripheral blood cells and recombinant human interleukin-2 to treat leukemia relapse post allogeneic bone marrow transplantation. Blood 1996; 87: 2195–2204. 28 Leshem B, Vourka Karussis U, Slavin S. Correlation between enhancement of graft vs leukemia effects following allogeneic bone marrow transplantation by rIL-2 and increased frequency of cytotoxic T-lymphocyte precursors in murine myeloid leukemia. Cytokines, Cell Mol Ther 2000; 6: 141–147. 29 Weiss L, Reich S, Slavin S. The role of antibodies to IL-2 receptor and anti-Asialo GMI antibodies on GVL effects induced by BMT in murine B cell leukemia. Bone Marrow Transplant 1995; 16: 457–461. 30 Moretta L, Bottino C, Pende D et al. Human natural killer cells: their origin, receptors and function. Eur J Immunol 2002; 32: 1205–1211 (Review).
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