Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

Experimental Hematology 2011;39:1064–1071

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression Esther Mogaa, Elisabet Cant
oa, Silvia Vidala, C
andido Juareza, Jorge Sierrab, and Javier Brionesb a

Department of Immunology; bHematology, Hospital Santa Creu i Sant Pau, Barcelona, Spain (Received 13 April 2011; revised 12 July 2011; accepted 5 August 2011)

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 FcgRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcgRIIIA 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. Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is characterized by the progressive accumulation of CD5þ neoplastic B cells, which results from a dynamic balance between cell death and proliferation [1,2]. 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 [3]. Despite these advances, CLL remains an incurable disease [4]. 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 [5]. This prompted us to investigate ways of improving the antitumoral effect of rituximab against CLL.

Offprint requests to: Javier Briones, M.D., Ph.D., Service of Hematology, Hospital Santa Creu i Sant Pau, 08041 Barcelona, Spain; E-mail: [email protected]

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 FcgR on effector cells such as monocyte/macrophages and natural killer (NK) cells [6–11]. In patients with non-Hodgkin lymphoma and CLL, NK cell-mediated ADCC represents one of the most important mechanisms of action of rituximab [9,12,13]. Although many cytokines have been studied to induce durable NK cell activation and expansion, most reports have shown a rather modest effect and the requirement for additional stimuli or cell expansion [10,14]. 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 [15]. Interleukin-15 has many activating and homeostatic functions on lymphocytes, and acts at different stages of

0301-472X/$ - see front matter. Copyright Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2011.08.006

E. Moga et al./ Experimental Hematology 2011;39:1064–1071

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 [16-19]. It has recently shown that IL-15 trans-presentation promotes human NK cell development and differentiation in vivo [20]. Accordingly, an IL-15 receptor agonist may provide a therapeutic tool to increase the pool of IL-15responsive cells during immunotherapy strategies. On the other hand, transforming growth factor-beta (TGF-b) is a pleiotropic cytokine with potent immunosuppressive effects that is highly expressed by CLL cells [21,22]. The TGF-b can suppress NK cell spontaneous killing and cytokine production, as well as the expression of activating NK receptors such as NKp30 and NKG2D [23]. In addition, TGF-b inhibits CD16-mediated IFN-g production and ADCC in human NK cells [24,25]. 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-b in regulating rituximab-mediated ADCC against CLL cells. Materials and methods Patients, cells, and culture conditions 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 [26], and all were treatment na€ıve 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. 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

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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 mg/mL (Invitrogen, Paisley, Scotland), human holo-transferrin 50 mg/mL (Sigma-Aldrich), insulin from bovine pancreas 5 mg/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 [27]. 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 mg/mL) [28], or CpG ODN control (ODN 2216 control; sequence 5’-ggGGGAGCATGCTGcggggg-3’; 5 mg/mL; both ODN synthesized and purified by MWG-Biotech AG, Ebersberg, Germany), or IL-15 (PeproTech; 10 ng/mL). Depletion of natural killer cells 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. Antibody-dependent cellular cytotoxicity Cytotoxicity was tested using a standard 51Cr release assay. Briefly, target cells were labeled with 50 mCi 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

Table 1. Patient’s characteristics Patient number CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL

1 2 3 4 5 6 7 8 9 10

Rai stage

Cromosomal abnormalitiesa

CD20 MFI

ZAP70 statusb

CD38 statusc

IgVH mutational statusd

ADCC PBMC medium þ rituximabe

ADCC PBMC IL-15 þ rituximabe

0 II I III 0 II I II 0 III

None Del13q None None Del13q Del13q Del17p None None Tris 12

142 251 130 95 44 137 108 102 150 172

Neg Pos Neg Neg Neg Neg Neg Neg Neg Neg

Neg Pos Pos Neg Neg Neg Neg Neg Neg Neg

Mut Unmut ND Unmut Mut Mut Mut Unmut ND ND

11.4 2.6 6.5 35.6 11.3 46.6 2.6 6.4 25.5 37.7

29.4 2.2 44 51.7 36.6 50 8.1 12.8 35.9 47.2

MFI 5 Mean fluorescence intensity; IgVH 5 immunoglobulin variable heavy-chain; PBMC 5 peripheral blood mononuclear cell; IL-15 5 interleukin-15; CLL 5 chronic lymphocytic leukemia; Neg 5 negative; Pos 5 positive; Mut 5 mutated; Unmut 5 unmutated; FISH 5 fluorescense in situ hybridization; ND 5 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.

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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 mg/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 mg/mL (Invitrogen). Previous experiments using rituximab at 0.01 to 100 mg/mL demonstrated that 10 mg/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 mL. The plates were centrifuged for 1 minute at 1,500 revolutions per minute, and were incubated for 4 hours at 37 C. One hundred mL 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 5 experimental counts per minute (cpm), S 5 spontaneous cpm, and M 5 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. Analysis of FcgRIIIA polymorphism Genotyping of FcgRIIIA-158V/F polymorphism was performed as shown by Koene et al. [29], using a nested polymerase chain reaction followed by allele-specific restriction enzyme digestion as described elsewhere [27]. Measurement of IFN-g concentration Supernatants from cell cultures were tested for IFN-g by FlowCytomix, in accordance with the manufacturer’s instructions (Bender MedSystem, Vienna, Austria). Statistical analysis 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.

Results Interleukin-15-activated PBMC significantly enhance rituximab-dependent cellular cytotoxicity against CLL cells We previously reported that both IL-15 and CpG ODN could enhance in vitro rituximab-ADCC against a lymphoma B-cell line [27]. 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 mg/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 mg/mL), or CpG ODN control (C, 2.5 mg/mL). The cytotoxicity of PBMC stimulated with IL-15 was 33.8% 6 3.9% lysis, higher than PBMC stimulated with an active CpG ODN A (25.6% 6 4.9% lysis, at a ratio

of 13.3; p 5 0.01; Fig. 1A). Subsequently, we analyzed the rituximab-mediated ADCC. In the presence of rituximab (10 mg/mL), PBMC stimulated with CpG ODN A showed a significant ADCC activity against CLL cells (35.9% 6 6.3% lysis, at a ratio of 13.3), in comparison with CpG ODN C (18% 6 4.1% lysis, at a ratio of 13.3; p 5 0.006). However, when PBMCs were activated with IL-15, rituximab-mediated ADCC was greatly enhanced (43.5% 6 4.8% vs. 35.9% 6 6.3% lysis; IL-15-activated PBMC vs. CpG ODN A activated PBMC, respectively, at a ratio of 13.3; p 5 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). Both CLL and the effector cells influence the rituximab-mediated ADCC against CLL cells Next, we analyzed rituximab-mediated ADCC in an extended number of CLL samples (total number, n 5 10). Collectively, we observed that IL-15-activated PBMC showed a significant higher cytotoxicity compared with unstimulated PBMC (17.6% 6 4.4% vs. 2.7% 6 1.5% lysis, respectively, at a ratio of 13.3; p 5 0.002; Fig. 2A). Moreover, rituximab-mediated ADCC was enhanced when PBMCs were activated with IL15 compared with unstimulated PBMCs (33.2% 6 4.9% vs. 13.4% 6 2.5% lysis, respectively, at a ratio of 13.3; p 5 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-15activated PBMCs showed an enhanced ADCC against CLL cells in all pairs tested (Fig. 3B). Natural killer cells are the main effector cells involved in rituximab-mediated ADCC against CLL cells 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% 6 2.5% versus 1.5% 6 2.3% lysis, at a ratio of 13.3; IL-15-activated PBMCs versus IL-15-activated PBMCs (-NK) was 47.2% 6 2.8% versus 8.8% 6 4.5% lysis, at a ratio of 13.3. Interleukin-15 overcomes the negative effect of TGF-b on the rituximab-mediated ADCC response and IFN-g production of PBMC To evaluate the influence of TGF-b, a potent immunoregulatory molecule involved in the suppression of NK cell

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Figure 1. Interleukin-15 (IL-15) enhances rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) of chronic lymphocytic leukemia (CLL) greater than CpG oligodeoxynucleotides (ODN) A. (A) Cytotoxicity assay of peripheral blood mononuclear cell (PBMC) incubated for 24 hours in medium supplemented with CpG ODN C 2.5 mg/mL (control), or with CpG ODN A 2.5 mg/mL, or with IL-15 10 ng/mL. The CpG ODN C 6.6% 6 2.1% versus CpG ODN A 25.6% 6 4.9% lysis (*p 5 0.04). The CpG ODN A 25.6% 6 4.9% versus IL-15 33.8% 6 3.9% lysis (**p 5 0.01). Results shown are representative of three independent experiments performed with three PBMC and three CLL samples (mean 6 SEM). (B) The ADCC assay of PBMC incubated for 24 hours in medium supplemented with CpG ODN C 2.5 mg/mL, or with CpG ODN A 2.5 mg/mL, or with IL-15 10 ng/mL. The CpG ODN C 18% 6 4.1% versus CpG ODN A 35.9% 6 6.3% lysis (*p 5 0.006). The CpG ODN A 35.9% 6 6.3% versus IL-15 43.5% 6 4.8% lysis (**p 5 0.02). Results shown are representative of five independent experiments performed with five PBMC and five CLL samples (mean 6 SEM). (C) The ADCC assay of PBMCs incubated for 24 hours in medium supplemented with CpG ODN A 2.5 mg/mL plus IL-15 10 ng/mL. The CpG ODN A 38% 6 6.7% versus IL-15 47% 6 4.7% lysis (*p 5 0.05). The IL-15 47% 6 4.7% versus CpG ODN A þ IL-15 52% 6 1.7% lysis. Results shown are representative of three independent experiments performed with three PBMC and three CLL samples (mean 6 SEM) The Effector:Target ratio is 13.3. The lytic activity was then assessed in a standard 4-hour chromium release assay using CLL cells as target.

cytotoxicity, on rituximab-mediated ADCC, we analyzed the ADCC of IL-15-activated PBMC in the presence of TGF-b. As shown in Figure 4A TGF-b decreased the rituximab-mediated ADCC against CLL cells (PBMC medium vs. PBMC medium þ TGF-b was 23.3% 6 1.7% vs. 17.6% 6 1.9% lysis, at a ratio of 13.3; p 5 0.02). However, rituximab-mediated ADCC was completely restored when PBMCs were activated with IL-15 (40.7% 6 7.9% lysis of IL-15-activated PBMC þ TGF-b vs. 45.5% 6 5.1% lysis of IL-15-activated PBMC without TGF-b; p O 0.05; Fig. 4B). Next, we analyzed the impact of TGF-b on IFN-g secretion by PBMC. The TGF-b significantly decreases IFN-g secretion by PBMC (unstimulated PBMC in absence or presence of TGF-b; 424.5 6 90.6 pg/mL vs. 238.2 6 85.3, respectively; p 5 0.01). In contrast, IL-15 induced a dramatic secretion of IFN-g by PBMC (2625.4 6 450.3 pg/mL). However, in the presence of TGF-b þ IL-15, IFN-g levels were maintained significantly elevated compared to TGFb alone (IL-15-activated PBMC þ TGF-b vs. unstimulated PBMCþ TGF-b; 1488.5 6 262.3 pg/mL vs. 238.2 6 85.3 respectively; p 5 0.003).

Interleukin-15-activated PBMCs enhance rituximab-mediated ADCCs against CLL cells independently of FcgR polymorphism We analyzed the FcgRIIIA polymorphism of the PBMCs used in the ADCC assays. The FcgRIIIA 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% 6 7.8% vs. 19.9% 6 13% lysis, respectively; p 5 0.05). In contrast, when PBMCs were activated with IL-15, FF effector cells showed a rituximabmediated ADCC comparable to that of VF PBMCs (28.9% 6 17.6% vs. 39% 6 14.5% lysis, respectively; p 5 0.2).

Discussion Various strategies are being studied to enhance rituximabassociated 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

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A

60

% specific

% specific 51 Cr release

40 20 0

cytotoxicity/ADCC

60

p=0.002

Cr release

cytotoxicity

51

A

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13.3

6.6

3.3

PBMC medium

50 40 30

PBMC medium + rituximab

20

IL-15

10

IL-15 + rituximab

0

CLL#2 / CLL#6 / CLL#9 / CLL#4 / CLL#5 / CLL#7 / PBMC#2 PBMC#2 PBMC#9 PBMC#9 PBMC#5 PBMC#5

ratio E:T

ratio 13.3

PBMC medium

IL-15

B

cytotoxicity/ADCC

% specific

20

Cr release 51

p=0.001

40

50

30

PBMC medium + rituximab

20

IL-15

% specific

60

51

B

Cr release

60

ADCC

PBMC medium

40

10

IL-15 + rituximab

0

CLL#5 / CLL#5 / CLL#7 / CLL#7 / CLL#6 / CLL#6 / PBMC#2 PBMC#10 PBMC#7 PBMC#3 PBMC#6 PBMC#4

0 13.3

6.6 ratio E:T

3.3

PBMC medium + rituximab IL-15 + rituximab Figure 2. Interleukin-15 (IL-15) enhances rituximab-mediated antibodydependent cellular cytotoxicity (ADCC) against chronic lymphocytic leukemia (CLL) cells. (A) Cytotoxicity assay of peripheral blood mononuclear cell (PBMC) incubated for 24 hours in medium alone (dotted line) or supplemented with IL-15 (10 ng/mL; -) when target cells were incubated for 45 minutes with immunoglobulin G (IgG) control (10 mg/mL). (B) The ADCC assay of PBMC incubated for 24 hours in medium alone (line) or supplemented with IL-15 (10 ng/mL; :) when target cells were incubated for 45 minutes with rituximab (10 mg/mL). The lytic activity was then assessed in a standard 4-hour chromium release assay. The percentage of lysis was calculated as described in Methods. The results are representative of 10 independent experiments, performed with PBMC from 10 healthy donors and 10 CLL samples (mean 6 SEM).

drugs) [30,31]. 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 [17]. Herein, we provide evidence of the importance of NK cell direct activation for the enhancement of rituximabmediated ADCC. When comparing the effect of IL-15

ratio 13.3

Figure 3. Chronic 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 6 SD of triplicate wells from each donor PBMC/ CLL sample.

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 [32]. 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) [33]. However, the CD20 expression of the CLL samples used

E. Moga et al./ Experimental Hematology 2011;39:1064–1071

B

cytotoxicity/ADCC PBMC medium

60 50 40

**

30 20 10

PBMC medium + TGF

rituximab

*

rituximab + TGF

0 ratio 13.3

cytotoxicity/ADCC IL-15

% specific 51 Cr release

% specific 51 Cr release

A

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60 IL-15 + TGF

50 40 30 20

IL-15 + rituximab

10

IL-15 + rituximab + TGF

0 ratio 13.3

Figure 4. Cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of peripheral blood mononuclear cell (PBMC) against chronic lymphocytic leukemia (CLL) cells in the presence of transforming growth factor-beta (TGF-b). (A) Effect of TGF-b (3 ng/mL) on cytotoxicity and ADCC assay of unstimulated PBMC. Cytotoxicity assay of PBMC medium (6.7% 6 0.6% lysis) versus PBMC medium þ TGF-b (3.6% 6 1% lysis; *p 5 0.01). The ADCC assay of PBMC medium (23.3% 6 1.7% lysis) versus PBMC medium þ TGF-b (17.6% 6 1.9% lysis; **p 5 0.02). (B) Effect of TGF-b (3 ng/mL) on cytotoxicity and ADCC assay of PBMC incubated for 24 hours in medium supplemented with interleukin-15 (IL-15; 10 ng/mL). Cytotoxicity assay of IL-15-activated PBMC (31.8% 6 3.5% lysis) versus IL-15-activated PBMC þ TGF-b (27.8% 6 4.6% lysis). The ADCC assay of IL-15-activated PBMC (45.5% 6 5.1% lysis) versus IL-15-activated PBMC þ TGF-b (40.7% 6 7.9% lysis). Results shown are representative of three independent experiments performed with three PBMC and three CLL samples (mean 6 SEM). The lytic activity was assessed in a standard 4-hour chromium release assay using CLL cells as targets.

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 patientderived 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 [34], 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 [35]. Another well-known factor that influences rituximab-mediated ADCC refers to the role of genetic polymorphisms at amino acid 158 in the FcgRIIIA 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 [8,11,29,36]. 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 [8,36,37]. However, although less well studied, no correlation between the FcgRIIIA genotype and treatment outcome with rituximab has been reported in a small study with patients with CLL treated with rituximab monotherapy [38]. 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) FcgRIIIA genotype [39]. 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 FcgRIIIA 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 [40]. In addition, CLL cells express high levels of immunosuppressive factors, such as TGF-b, that contributes to the immune dysfunction [22]. Recently, it has been shown that TGF-b inhibits CD16-mediated IFN-g production and ADCC in human NK cells [41]. With this background, we investigated if IL-15 could overcome the inhibitory effect of TGF-b 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-b. Remarkably, IL-15-activated PBMCs had preserved rituximab-mediated ADCC in the presence of TGF-b. The IL-15 is known to enhance NK cell cytotoxicity via

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upregulation of effector molecules such as IFN-g, perforin, and granzymes [42]. In our study, IFN-g secretion was greatly reduced after exposure of PBMC to TGF-b 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 wellknown 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 [43]. 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 [44, 45]. 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 [46] and, in this scenario, the administration of an NK cell-activating cytokine with rituximab could be of potential benefit to those patients. Support and financial disclosure declaration This work was supported by grants from the Instituto de Salud Carlos III (FIS 06/09, G03/179, and C03/010). Conflict of interest The authors declare no conflicts of interest to declare.

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