Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression
2011; Elsevier BV; Volume: 39; Issue: 11 Linguagem: Inglês
10.1016/j.exphem.2011.08.006
ISSN1873-2399
AutoresEsther Moga, Elisabet Cantó, Sílvia Vidal, C. Juárez, Jorge Sierra, Javier Briones,
Tópico(s)Lymphoma Diagnosis and Treatment
ResumoChemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a 51chromium release assay. The IL-15 significantly enhanced in vitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). In addition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL. Chemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a 51chromium release assay. The IL-15 significantly enhanced in vitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). In addition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL. Chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is characterized by the progressive accumulation of CD5+ neoplastic B cells, which results from a dynamic balance between cell death and proliferation [1Zenz T. Mertens D. Küppers R. Döhner H. Stilgenbauer S. From pathogenesis to treatment of chronic lymphocytic leukaemia.Nat Rev Cancer. 2010; 10: 37-50PubMed Google Scholar, 2Chiorazzi N. Rai K.R. Ferrarini M. Chronic lymphocytic leukemia.N Eng J Med. 2005; 352: 804-815Crossref PubMed Scopus (1305) Google Scholar]. Advances in the therapy for CLL, particularly “chemoimmunotherapy” regimens combining cytotoxic agents such as alkylating agents and purine nucleoside analogs with monoclonal antibodies such as rituximab, have improved complete response rates and survival [3Hallek M. Fischer K. Fingerle-Rowson G. et al.Lancet. 2010; 376: 1164-1174Abstract Full Text Full Text PDF PubMed Scopus (1510) Google Scholar]. Despite these advances, CLL remains an incurable disease [4Delgado J. Briones J. Sierra J. Emerging therapies for patients with advanced chronic lymphocytic leukaemia.Blood Rev. 2009; 23: 217-224Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar]. Rituximab, a chimeric anti-CD20 monoclonal antibody, represents one of the most important advances in the treatment of lymphoproliferative disorders in the last 30 years. In contrast to other indolent lymphomas, the clinical activity of rituximab as a single agent in CLL is modest with very rare complete remissions even at very high doses [5O’Brien S.M. Kantarjian H. Thomas D.A. et al.Rituximab dose-escalation trial in chronic lymphocytic leukemia.J Clin Oncol. 2001; 19: 2165-2170Crossref PubMed Scopus (559) Google Scholar]. This prompted us to investigate ways of improving the antitumoral effect of rituximab against CLL. Considerable evidence, based on in vitro studies, mouse models, and clinical data, reveals that rituximab can mediate cytotoxicity of both normal and malignant B cells by harnessing effector functions of the immune system. These include complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC) and/or phagocytosis; these latter two processes are promoted by activating FcγR on effector cells such as monocyte/macrophages and natural killer (NK) cells [6Golay J. Zaffaroni L. Vaccari T. et al.Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis.Blood. 2000; 95: 3900-3908PubMed Google Scholar, 7Clynes R.A. Towers T.L. Presta L.G. Ravetch J.V. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets.Nat Med. 2000; 6: 443-446Crossref PubMed Scopus (2264) Google Scholar, 8Cartron G. Dacheux L. Salles G. et al.Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene.Blood. 2002; 99: 754-758Crossref PubMed Scopus (1638) Google Scholar, 9Manches O. Lui G. Chaperot L. et al.In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas.Blood. 2003; 101: 949-954Crossref PubMed Scopus (323) Google Scholar, 10Golay J. Manganini M. Facchinetti V. et al.Rituximab-mediated antibody-dependent cellular cytotoxicity against neoplastic B cells is stimulated strongly by interleukin-2.Haematologica. 2003; 88: 1002-1012PubMed Google Scholar, 11Dall’Ozzo S. Tartas S. Paintaud G. et al.Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship.Cancer Res. 2004; 64: 4664-4669Crossref PubMed Scopus (367) Google Scholar]. In patients with non-Hodgkin lymphoma and CLL, NK cell-mediated ADCC represents one of the most important mechanisms of action of rituximab [9Manches O. Lui G. Chaperot L. et al.In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas.Blood. 2003; 101: 949-954Crossref PubMed Scopus (323) Google Scholar, 12Glennie M.J. French R.R. Cragg M.S. Taylor R.P. Mechanisms of killing by anti-CD20 monoclonal antibodies.Mol Immunol. 2007; 44: 3823-3837Crossref PubMed Scopus (435) Google Scholar, 13Fischer L. Penack O. 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This warrants further investigation of innate immune system activating agents to improve rituximab efficacy against CLL. In a recent National Cancer Institute sponsored workshop to search for immunotherapy drugs with high potential for the treatment of cancer, interleukin-15 (IL-15) ranked top within a list of 20 agents from more than 100 analyzed [15Cheever M.A. Twelve immunotherapy drugs that could cure cancers.Immunol Rev. 2008; 222: 357-368Crossref PubMed Scopus (256) Google Scholar]. Interleukin-15 has many activating and homeostatic functions on lymphocytes, and acts at different stages of the immune response by expanding and activating NK cells. It plays a pivotal role in NK-cell proliferation and cytotoxicity, and regulates NK-cell macrophage interaction [16Lucas M. Schachterle W. Oberle K. Aichele P. Diefenbach A. Dendritic cells prime natural killer cells by trans-presenting interleukin 15.Immunity. 2007; 26: 503-517Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 17Fehniger T.A. Caligiuri M.A. Interleukin 15: biology and relevance to human disease.Blood. 2001; 97: 14-32Crossref PubMed Scopus (793) Google Scholar, 18Mrozek E. Anderson P. Caligiuri M.A. Role of interleukin-15 in the development of human CD56+ natural killer cells from CD34+ hematopoietic progenitor cells.Blood. 1996; 87: 2632-2640PubMed Google Scholar, 19Carson W.E. Fehniger T.A. Haldar S. et al.A potential role for interleukin-15 in the regulation of human natural killer cell survival.J Clin Invest. 1997; 99: 937-943Crossref PubMed Scopus (347) Google Scholar]. It has recently shown that IL-15 trans-presentation promotes human NK cell development and differentiation in vivo [20Huntington N.D. Legrand N. Alves N.L. et al.IL-15 trans-presentation promotes human NK cell development and differentiation in vivo.J Exp Med. 2009; 206: 25-34Crossref PubMed Scopus (384) Google Scholar]. Accordingly, an IL-15 receptor agonist may provide a therapeutic tool to increase the pool of IL-15-responsive cells during immunotherapy strategies. On the other hand, transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with potent immunosuppressive effects that is highly expressed by CLL cells [21Li M.O. Wan Y.Y. Sanjabi S. Robertson A.K. Flavell R.A. Transforming growth factor-beta regulation of immune responses.Annu Rev Immunol. 2006; 24: 99-146Crossref PubMed Scopus (1690) Google Scholar, 22Lotz M. Ranheim E. Kipps T.J. Transforming growth factor beta as endogenous growth inhibitor of chronic lymphocytic leukemia B cells.J Exp Med. 1994; 179: 999-1004Crossref PubMed Scopus (116) Google Scholar]. The TGF-β can suppress NK cell spontaneous killing and cytokine production, as well as the expression of activating NK receptors such as NKp30 and NKG2D [23Castriconi R. Cantoni C. Della Chiesa M. et al.Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells.Proc Natl Acad Sci U S A. 2003; 100: 4120-4125Crossref PubMed Scopus (493) Google Scholar]. In addition, TGF-β inhibits CD16-mediated IFN-γ production and ADCC in human NK cells [24Meadows S.K. Eriksson M. Barber A. Sentman C.L. Human NK cell IFN-gamma production is regulated by endogenous TGF-beta.Int Immunopharmacol. 2006; 6: 1020-1028Crossref PubMed Scopus (36) Google Scholar, 25Bellone G. Aste-Amezaga M. Trinchieri G. Rodeck U. Regulation of NK cell functions by TGF-beta 1.J Immunol. 1995; 155: 1066-1073PubMed Google Scholar]. In an attempt to improve rituximab activity against CLL cells, we investigated the ability of IL-15 to enhance in vitro rituximab-mediated ADCC on primary CLL cells. In addition, we evaluated the role of TGF-β in regulating rituximab-mediated ADCC against CLL cells. Heparinized peripheral blood was obtained after informed consent from patients with CLL. Patients were diagnosed according to the National Cancer Institute Working Group 1996 criteria [26Cheson B.D. Bennett J.M. Grever M. et al.National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment.Blood. 1996; 87: 4990-4997PubMed Google Scholar], and all were treatment naïve at the time of obtaining the cell sample (Table 1). All patients provided informed consent, and the study was approved by the Ethics Committee of our institution.Table 1Patient’s characteristicsPatient numberRai stageCromosomal abnormalitiesaAs determined by FISH with probes for 11q22.3, 13q14, 17p13.1, and cr.12.CD20 MFIZAP70 statusbPositivity denotes ≥20% cells expressing ZAP70.CD38 statuscPositivity denotes ≥30% cells expressing CD38.IgVH mutational statusdMutated IgVH gene denotes more than 2% mutations compared with germline sequence.ADCC PBMC medium + rituximabePercentage of cytotoxicity at ratio 13.3.ADCC PBMC IL-15 + rituximabePercentage of cytotoxicity at ratio 13.3.CLL 10None142NegNegMut11.429.4CLL 2IIDel13q251PosPosUnmut2.62.2CLL 3INone130NegPosND6.544CLL 4IIINone95NegNegUnmut35.651.7CLL 50Del13q44NegNegMut11.336.6CLL 6IIDel13q137NegNegMut46.650CLL 7IDel17p108NegNegMut2.68.1CLL 8IINone102NegNegUnmut6.412.8CLL 90None150NegNegND25.535.9CLL 10IIITris 12172NegNegND37.747.2MFI = Mean fluorescence intensity; IgVH = immunoglobulin variable heavy-chain; PBMC = peripheral blood mononuclear cell; IL-15 = interleukin-15; CLL = chronic lymphocytic leukemia; Neg = negative; Pos = positive; Mut = mutated; Unmut = unmutated; FISH = fluorescense in situ hybridization; ND = not done.a As determined by FISH with probes for 11q22.3, 13q14, 17p13.1, and cr.12.b Positivity denotes ≥20% cells expressing ZAP70.c Positivity denotes ≥30% cells expressing CD38.d Mutated IgVH gene denotes more than 2% mutations compared with germline sequence.e Percentage of cytotoxicity at ratio 13.3. Open table in a new tab MFI = Mean fluorescence intensity; IgVH = immunoglobulin variable heavy-chain; PBMC = peripheral blood mononuclear cell; IL-15 = interleukin-15; CLL = chronic lymphocytic leukemia; Neg = negative; Pos = positive; Mut = mutated; Unmut = unmutated; FISH = fluorescense in situ hybridization; ND = not done. The peripheral blood mononuclear cell (PBMC) fraction was isolated by Ficoll-Hypaque gradient centrifugation and aliquots of cells were frozen in 10% dimethylsulfoxide according to standard procedures, and stored in liquid nitrogen before use. Before the cytotoxicity assay, CLL cells were cultured overnight in Iscove’s Dulbecco Medium (Sigma-Aldrich, Madrid, Spain) and supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories, Linz, Austria), albumin bovine serum 0.5% (Calbiochem, Darmstadt, Germany), gentamycin 15 μg/mL (Invitrogen, Paisley, Scotland), human holo-transferrin 50 μg/mL (Sigma-Aldrich), insulin from bovine pancreas 5 μg/mL (Sigma-Aldrich), and IL-4 2 ng/mL (PeproTech, London, United Kingdom), hereafter called B-Medium, at 37°C, in a humidified 5% CO2 atmosphere. The PBMCs were obtained from healthy volunteers, after obtaining informed consent, and were processed and analyzed as previously reported [27Moga E. Alvarez E. Cantó E. et al.NK cells stimulated with IL-15 or CpG ODN enhance rituximab-dependent cellular cytotoxicity against B-cell lymphoma.Exp Hematol. 2008; 36: 69-77Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. Briefly, to induce immune responses in vitro, PBMC or purified NK cells were cultured in RPMI complete medium at a density of 1 × 106/mL/well for 20 hours with either CpG oligodeoxynucleotides (ODN) A (ODN 2216; sequence 5’-ggGGGACGATCGTCgggggg-3’; 5 μg/mL) [28Krug A. Rothenfusser S. Hornung V. et al.Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells.Eur J Immunol. 2001; 31: 2154-2163Crossref PubMed Scopus (753) Google Scholar], or CpG ODN control (ODN 2216 control; sequence 5’-ggGGGAGCATGCTGcggggg-3’; 5 μg/mL; both ODN synthesized and purified by MWG-Biotech AG, Ebersberg, Germany), or IL-15 (PeproTech; 10 ng/mL). The NK-depleted PBMC cells were obtained by negative selection from PBMCs using the EasySep Human CD56 Positive Selection Kit with an EasySep magnet (Stem Cell Technologies, Paris, France). Magnetically labeled cells were then separated from unlabeled cells using the EasySep procedure, following manufacturer’s instructions. The supernatant fraction containing T cells, granulocytes, and monocytes, but not NK cells, were used for the ADCC experiments. Cytotoxicity was tested using a standard 51Cr release assay. Briefly, target cells were labeled with 50 μCi of chromium-51 (Amersham Biosciences, Little Chalfont Buckinghamshire, United Kingdom) for every 1 × 106 CLL cells for 2 hours. Previously, CLL cells were cultured in B-Medium overnight at a density of 1 × 106 per mL/well. Cells were then washed and incubated in the presence of rituximab (MabThera, Roche, Italy) or polyclonal human immunoglobulin G (IgG; containing 68.7% IgG1; Flebogamma, Grifols, Spain), both at 10 μg/mL, for 45 minutes at 37°C in the presence of RPMI-5 which is composed of RPMI 1640 medium HEPES modification 40 mL (Sigma-Aldrich), RPMI 1640 60 mL (PAA Laboratories), 5% human serum (Sigma-Aldrich), L-glutamine 1% (Invitrogen), and gentamycin 15 μg/mL (Invitrogen). Previous experiments using rituximab at 0.01 to 100 μg/mL demonstrated that 10 μg/mL was a saturating dose (data not shown). Decreasing concentrations of effector cells (PBMC or purified NK cells) were added to 3 × 104 target cells in round-bottom 96-well plates (Nunc, Roskilde, Denmark) in a final volume of 200 μL. The plates were centrifuged for 1 minute at 1,500 revolutions per minute, and were incubated for 4 hours at 37°C. One hundred μL of supernatant were collected from each well and counted in a gamma-counter (Cobra auto-gamma Packard, Perkin Elmer, Madrid, Spain). The percentage of cytotoxicity was the mean of triplicate wells and was calculated using the following equation: (E − S)/(M − S) × 100, where E = experimental counts per minute (cpm), S = spontaneous cpm, and M = maximum cpm. Spontaneous release and maximum release were determined from wells containing target cells incubated in medium alone or in 1% Triton X-100, respectively. Genotyping of FcγRIIIA-158V/F polymorphism was performed as shown by Koene et al. [29Koene H.R. Kleijer M. Algra J. Roos D. von dem Borne A.E. de Haas M. Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype.Blood. 1997; 90: 1109-1114Crossref PubMed Google Scholar], using a nested polymerase chain reaction followed by allele-specific restriction enzyme digestion as described elsewhere [27Moga E. Alvarez E. Cantó E. et al.NK cells stimulated with IL-15 or CpG ODN enhance rituximab-dependent cellular cytotoxicity against B-cell lymphoma.Exp Hematol. 2008; 36: 69-77Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. Supernatants from cell cultures were tested for IFN-γ by FlowCytomix, in accordance with the manufacturer’s instructions (Bender MedSystem, Vienna, Austria). A paired t test was used to compare rituximab-mediated ADCC with unstimulated versus stimulated effector cells. The level of significance was p < 0.05. The SPSS software (version 14.0; SPSS Inc., Chicago, IL, USA) was used for the analysis. We previously reported that both IL-15 and CpG ODN could enhance in vitro rituximab-ADCC against a lymphoma B-cell line [27Moga E. Alvarez E. Cantó E. et al.NK cells stimulated with IL-15 or CpG ODN enhance rituximab-dependent cellular cytotoxicity against B-cell lymphoma.Exp Hematol. 2008; 36: 69-77Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. Preliminary assays showed that 10 ng/mL IL-15 was the minimal concentration with a detectable effect on cytotoxicity assays against CLL cells, and 2.5 μg/mL CpG ODN A was a cytotoxic saturating dose (data not shown). Therefore, PBMCs were activated with IL-15 (10 ng/mL), or CpG ODN A (2.5 μg/mL), or CpG ODN control (C, 2.5 μg/mL). The cytotoxicity of PBMC stimulated with IL-15 was 33.8% ± 3.9% lysis, higher than PBMC stimulated with an active CpG ODN A (25.6% ± 4.9% lysis, at a ratio of 13.3; p = 0.01; Fig. 1A). Subsequently, we analyzed the rituximab-mediated ADCC. In the presence of rituximab (10 μg/mL), PBMC stimulated with CpG ODN A showed a significant ADCC activity against CLL cells (35.9% ± 6.3% lysis, at a ratio of 13.3), in comparison with CpG ODN C (18% ± 4.1% lysis, at a ratio of 13.3; p = 0.006). However, when PBMCs were activated with IL-15, rituximab-mediated ADCC was greatly enhanced (43.5% ± 4.8% vs. 35.9% ± 6.3% lysis; IL-15-activated PBMC vs. CpG ODN A activated PBMC, respectively, at a ratio of 13.3; p = 0.02; Fig. 1B). In these conditions, IL-15 seems to be a stronger stimulus to enhance rituximab-mediated ADCC against CLL cells. In addition, these two agents have neither additive nor a synergistic effect (Fig. 1C). Next, we analyzed rituximab-mediated ADCC in an extended number of CLL samples (total number, n = 10). Collectively, we observed that IL-15-activated PBMC showed a significant higher cytotoxicity compared with unstimulated PBMC (17.6% ± 4.4% vs. 2.7% ± 1.5% lysis, respectively, at a ratio of 13.3; p = 0.002; Fig. 2A). Moreover, rituximab-mediated ADCC was enhanced when PBMCs were activated with IL-15 compared with unstimulated PBMCs (33.2% ± 4.9% vs. 13.4% ± 2.5% lysis, respectively, at a ratio of 13.3; p = 0.001; Fig. 2B). To account for the variability of the PBMC capacity to mediate the ADCC effect against a CLL sample, rituximab-mediated ADCC was tested using three PBMCs as effector, each of them with two different CLL samples. In all pairs, except one, IL-15-activated PBMC showed superior rituximab-mediated ADCC (Fig. 3A). Alternatively, the sensitivity of a CLL sample to be lysed with IL-15-activated effector cells was also tested in an independent assay. Rituximab mediated-ADCC was tested with three CLL samples, each of them with two different PBMCs. The IL-15-activated PBMCs showed an enhanced ADCC against CLL cells in all pairs tested (Fig. 3B).Figure 3Chronic lymphocytic leukemia (CLL) cells and donor’s peripheral blood mononuclear cell (PBMC) influence the rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC). The PBMCs were incubated overnight in medium alone or supplemented with interleukin-15 (IL-15), 10 ng/mL. The lytic activity was then assessed in a standard 4-hour chromium release assay using immunoglobulin G (IgG)-coated CLL cells or rituximab-coated CLL cells as targets. Results shown are (A) the cytotoxicity and ADCC of three donor’s PBMC each one cultured with two different CLL samples, and (B) the cytotoxicity and ADCC of three CLL samples each one cultured with two different donor’s PBMCs. Data shown are the mean ± SD of triplicate wells from each donor PBMC/CLL sample.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We studied if NK cells were involved in rituximab-mediated ADCC against CLL cells. The NK-depleted PBMCs (-NK) did not show a significant rituximab-mediated ADCC against CLL cells: unstimulated PBMCs versus unstimulated PBMCs (-NK) was 25.3% ± 2.5% versus 1.5% ± 2.3% lysis, at a ratio of 13.3; IL-15-activated PBMCs versus IL-15-activated PBMCs (-NK) was 47.2% ± 2.8% versus 8.8% ± 4.5% lysis, at a ratio of 13.3. To evaluate the influence of TGF-β, a potent immunoregulatory molecule involved in the suppression of NK cell cytotoxicity, on rituximab-mediated ADCC, we analyzed the ADCC of IL-15-activated PBMC in the presence of TGF-β. As shown in Figure 4A TGF-β decreased the rituximab-mediated ADCC against CLL cells (PBMC medium vs. PBMC medium + TGF-β was 23.3% ± 1.7% vs. 17.6% ± 1.9% lysis, at a ratio of 13.3; p = 0.02). However, rituximab-mediated ADCC was completely restored when PBMCs were activated with IL-15 (40.7% ± 7.9% lysis of IL-15-activated PBMC + TGF-β vs. 45.5% ± 5.1% lysis of IL-15-activated PBMC without TGF-β; p > 0.05; Fig. 4B). Next, we analyzed the impact of TGF-β on IFN-γ secretion by PBMC. The TGF-β significantly decreases IFN-γ secretion by PBMC (unstimulated PBMC in absence or presence of TGF-β; 424.5 ± 90.6 pg/mL vs. 238.2 ± 85.3, respectively; p = 0.01). In contrast, IL-15 induced a dramatic secretion of IFN-γ by PBMC (2625.4 ± 450.3 pg/mL). However, in the presence of TGF-β + IL-15, IFN-γ levels were maintained significantly elevated compared to TGF-β alone (IL-15-activated PBMC + TGF-β vs. unstimulated PBMC+ TGF-β; 1488.5 ± 262.3 pg/mL vs. 238.2 ± 85.3 respectively; p = 0.003). We analyzed the FcγRIIIA polymorphism of the PBMCs used in the ADCC assays. The FcγRIIIA genotype of the 10 PBMCs analyzed was as follows: five were Valine (V)/Phenylalanine (F) heterozygous and five Phenylalanine (F)/Phenylalanine (F) homozygous. Rituximab-mediated ADCC was marginally decreased when effector cells were FF compared to VF (8.6% ± 7.8% vs. 19.9% ± 13% lysis, respectively; p = 0.05). In contrast, when PBMCs were activated with IL-15, FF effector cells showed a rituximab-mediated ADCC comparable to that of VF PBMCs (28.9% ± 17.6% vs. 39% ± 14.5% lysis, respectively; p = 0.2). Various strategies are being studied to enhance rituximab-associated cytotoxic mechanisms against lymphoma B-cells. One of these strategies focuses on the combination of rituximab with different biologic agents to stimulate the innate immune system (e.g., cytokines, immunomodulatory drugs) [30Khan K.D. Emmanouilides C. Benson Jr., D.M. et al.A phase 2 study of rituximab in combination with recombinant interleukin-2 for rituximab-refractory indolent non-Hodgkin’s lymphoma.Clin Cancer Res. 2006; 12: 7046-7053Crossref PubMed Scopus (94) Google Scholar, 31Poiré X. Kline J. Grinblatt D. et al.Phase II study of immunomodulation with granulocyte-macrophage colony-stimulating factor, interleukin-2, and rituximab following autologous stem cell transplant in patients with relapsed or refractory lymphomas.Leuk Lymphoma. 2010; 51: 1241-1250Crossref PubMed Scopus (12) Google Scholar]. To this purpose, we analyzed the impact of IL-15 on rituximab-mediated ADCC against CLL cells. Our results demonstrated that IL-15 is a potent enhancer of rituximab-mediated ADCC of PBMCs against CLL cells. Our data suggest that IL-15 acts directly on NK cells, and these cells are the main effector cells mediating rituximab-dependent ADCC, because IL-15-activated NK-depleted PBMCs have no cytolytic effect on CLL cells. This is in agreement with the fact that NK cells are one of the most important cells responding to IL-15, because they constitutively express the IL-15 receptor [17Fehniger T.A. Caligiuri M.A. Interleukin 15: biology and relevance to human disease.Blood. 2001; 97: 14-32Crossref PubMed Scopus (793) Google Scholar]. Herein, we provide evidence of the importance of NK cell direct activation for the enhancement of rituximab-mediated ADCC. When comparing the effect of IL-15 with CpG, a molecule that indirectly activates NK cells through their effect on dendritic cells, IL-15 showed a greater rituximab-mediated killing of CLL cells. Moreover, the addition of CpG did not significantly increase the cytolytic activity of IL-15-activated PBMC against CLL cells, in agreement with cytotoxic studies performed in other hematological malignancies [32Wysocka M. Benoit B.M. Newton S. Azzoni L. Montaner L.J. Rook A.H. Enhancement of the host immune responses in cutaneous T-cell lymphoma by CpG oligodeoxynucleotides and IL-15.Blood. 2004; 104: 4142-4149Crossref PubMed Scopus (75) Google Scholar]. As expected, some degree of variability has been detected when rituximab-mediated ADCC against a CLL sample was tested with PBMCs from different donors. Alternatively, for a particular PBMC donor sample, variability on the cytolytic effect was also detected between two different CLL samples. A number of factors may account for such differences. First, different CD20 expression on the CLL samples may explain the variability obtained from a single PBMC, which is partially associated to cytogenetic features (i.e., patients with trisomy 12 CLL showed strong leukemic cell CD20 expression and had a high rate of response to rituximab-based therapy) [33Tam C.S. Otero-Palacios J. Abruzzo L.V. et al.Chronic lymphocytic leukaemia CD20 expression is dependent on the genetic subtype: a study of quantitative flow cytometry and fluorescent in-situ hybridization in 510 patients.Br J Haematol. 2008; 141: 36-40Crossref PubMed Scopus (64) Google Scholar]. However, the CD20 expression of the CLL samples used in our study was uniformly low, with no significant differences between them, which make this option unlikely. Moreover, among the CLL samples tested, only one showed trisomy 12, and did not show a greater killing to any of the PBMCs used (data not shown). Second, the variability of the cytotoxic effect of the PBMC may be due to the allogeneic nature of the effector cells. In this regard, the use of patient-derived NK cells would be more clinically relevant, but is hampered by the very low numbers of NK cells in the peripheral blood of our patients with CLL. Because all CLL-PBMC pairs tested were allogeneic, differences in cytotoxicity may be explained, at least in part, due to differences in HLA molecules on CLL samples (i.e., HLA-C) and the killer immunoglobulin receptor expressed in NK cells within the PBMC [34Moretta L. Moretta A. Killer immunoglobulin-like receptors.Curr Opin Immunol. 2004; 16: 165-174Crossref Scopus (286) Google Scholar], which makes the ADCC of allogeneic NK cells higher compared to autologous (e.g., the patients’ own) NK cells. Nevertheless, although this may hold true in our study, IL-15-activated NK cells consistently showed higher rituximab-mediated ADCC in all CLL samples tested, compared to unstimulated NK cells, which validates our findings on the enhanced effect of IL-15 on rituximab-mediated ADCC. Importantly, it has been demonstrated that peripheral NK cells from patients with CLL are fully functional in terms of degranulation and ADCC, as compared with those from healthy donors. Moreover, the NK cell repertoire remains remarkably stable even in those patients with disease progression [35Le Garff-Tavernier M. Decocq J. de Romeuf C. et al.Analysis of CD16+CD56dim NK cells from CLL patients: evidence supporting a therapeutic strategy with optimized anti-CD20 monoclonal antibodies.Leukemia. 2011; 25: 101-109Crossref PubMed Scopus (41) Google Scholar]. Another well-known factor that influences rituximab-mediated ADCC refers to the role of genetic polymorphisms at amino acid 158 in the FcγRIIIA gene, either a phenylalanine or a valine (V). Thus, differences in rituximab responses have been correlated with the binding affinity of IgG1, related to V isoform, and the absolute number of CD16 receptors per effector cell, that ultimately influences the ability of the effector cells to perform ADCC [8Cartron G. Dacheux L. Salles G. et al.Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene.Blood. 2002; 99: 754-758Crossref PubMed Scopus (1638) Google Scholar, 11Dall’Ozzo S. Tartas S. Paintaud G. et al.Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship.Cancer Res. 2004; 64: 4664-4669Crossref PubMed Scopus (367) Google Scholar, 29Koene H.R. Kleijer M. Algra J. Roos D. von dem Borne A.E. de Haas M. Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype.Blood. 1997; 90: 1109-1114Crossref PubMed Google Scholar, 36Weng W.K. Levy R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma.J Clin Oncol. 2003; 21: 3940-3947Crossref PubMed Scopus (1093) Google Scholar]. Several clinical studies in patients with indolent non-Hodgkin lymphoma have supported this finding, showing a greater clinical response to rituximab when effector cells have the Valine (V)/Valine (V) polymorphism [8Cartron G. Dacheux L. Salles G. et al.Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene.Blood. 2002; 99: 754-758Crossref PubMed Scopus (1638) Google Scholar, 36Weng W.K. Levy R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma.J Clin Oncol. 2003; 21: 3940-3947Crossref PubMed Scopus (1093) Google Scholar, 37Treon S.P. Hansen M. Branagan A.R. et al.Polymorphisms in FcgammaRIIIA (CD16) receptor expression are associated with clinical response to rituximab in Waldenström’s macroglobulinemia.J Clin Oncol. 2005; 23: 474-481Crossref PubMed Scopus (222) Google Scholar]. However, although less well studied, no correlation between the FcγRIIIA genotype and treatment outcome with rituximab has been reported in a small study with patients with CLL treated with rituximab monotherapy [38Farag S.S. Flinn I.W. Modali R. Lehman T.A. Young D. Byrd J.C. Fc gamma RIIIa and Fc gamma RIIa polymorphisms do not predict response to rituximab in B-cell chronic lymphocytic leukemia.Blood. 2004; 103: 1472-1474Crossref PubMed Scopus (183) Google Scholar]. In a recent, retrospective analysis of patients with relapsed CLL treated with chemotherapy, addition of rituximab to the treatment seemed to improve survival of those patients with a low (FF) or intermediate (VF) FcγRIIIA genotype [39Dornan D. Spleiss O. Yeh R.F. et al.Effect of FCGR2A and FCGR3A variants on CLL outcome.Blood. 2010; 116: 4212-4222Crossref PubMed Scopus (48) Google Scholar]. Our in vitro results showed that expression of at least one V allele on NK cells (i.e., VF genotype) provided more cytotoxic activity than the FF genotype. Thus, rituximab-mediated ADCC against CLL cells is higher with VF than with FF effector cells. However, the correlation between the FcγRIIIA gene polymorphism and the increased cytotoxic effect of rituximab against CLL seems to be lost when PBMCs are activated with IL-15, suggesting that the reduced cytotoxicity of the effector cells expressing the FF genotype could be overcome by the treatment with IL-15. Several cellular immune defects have been described in patients with CLL that prevent the development of an immune response against tumor cells [40Ramsay A.G. Johnson A.J. Lee A.M. et al.Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug.J Clin Invest. 2008; 118: 2427-2437PubMed Google Scholar]. In addition, CLL cells express high levels of immunosuppressive factors, such as TGF-β, that contributes to the immune dysfunction [22Lotz M. Ranheim E. Kipps T.J. Transforming growth factor beta as endogenous growth inhibitor of chronic lymphocytic leukemia B cells.J Exp Med. 1994; 179: 999-1004Crossref PubMed Scopus (116) Google Scholar]. Recently, it has been shown that TGF-β inhibits CD16-mediated IFN-γ production and ADCC in human NK cells [41Trotta R. Col J.D. Yu J. et al.TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells.J Immunol. 2008; 181: 3784-3792PubMed Google Scholar]. With this background, we investigated if IL-15 could overcome the inhibitory effect of TGF-β on rituximab-mediated ADCC against CLL cells. In our study, we observed a reduction of both natural cytotoxicity and rituximab-mediated ADCC of PBMC after exposure to TGF-β. Remarkably, IL-15-activated PBMCs had preserved rituximab-mediated ADCC in the presence of TGF-β. The IL-15 is known to enhance NK cell cytotoxicity via upregulation of effector molecules such as IFN-γ, perforin, and granzymes [42Fehniger T.A. Cai S.F. Cao X. et al.Acquisition of murine NK cell cytotoxicity requires the translation of a pre-existing pool of granzyme B and perforin mRNAs.Immunity. 2007; 26: 798-811Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar]. In our study, IFN-γ secretion was greatly reduced after exposure of PBMC to TGF-β while it was restored after IL-15 treatment of PBMC. Our observations are consistent with the concept that IL-15 plays an important role in NK cell antitumor activity and can be useful to optimize the response to rituximab in patients with CLL. More important, and clinically relevant, IL-15 may have additional advantages over other well-known NK-activating cytokines such as IL-2, IL-18, and IL-21, because, contrary to IL-15, these cytokines have been associated to promotion of immunosuppressive regulatory T and NK cells [43Gowda A. Ramanunni A. Cheney C. et al.Differential effects of IL-2 and IL-21 on expansion of the CD4+ CD25+ Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia.mAbs. 2010; 2: 35-41Crossref PubMed Scopus (25) Google Scholar]. While very limited clinical data is available for IL-15, recent studies in nonhuman primates have shown that IL-15 effectively expands T and NK cells with a very favorable toxicity profile [44Berger C. Berger M. Hackman R.C. et al.Safety and immunologic effects of IL-15 administration in nonhuman primates.Blood. 2009; 114: 2417-2426Crossref PubMed Scopus (184) Google Scholar, 45Lugli E. Goldman C.K. Perera L.P. et al.Transient and persistent effects of IL-15 on lymphocyte homeostasis in nonhuman primates.Blood. 2010; 116: 3238-3248Crossref PubMed Scopus (86) Google Scholar]. This is of special interest because recent clinical studies suggest that rituximab maintenance after chemotherapy may prolong the duration of response in patients with CLL [46Del Poeta G. Del Principe M.I. Buccisano F. et al.Consolidation and maintenance immunotherapy with rituximab improve clinical outcome in patients with B-cell chronic lymphocytic leukemia.Cancer. 2008; 112: 119-128Crossref PubMed Scopus (89) Google Scholar] and, in this scenario, the administration of an NK cell-activating cytokine with rituximab could be of potential benefit to those patients. This work was supported by grants from the Instituto de Salud Carlos III ( FIS 06/09, G03/179, and C03/010 ). The authors declare no conflicts of interest to declare.
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