Interleukin-21 and rituximab enhance NK cell functionality in patients with B-cell chronic lymphocytic leukaemia

Leukemia Research 35 (2011) 914–920

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Interleukin-21 and rituximab enhance NK cell functionality in patients with B-cell chronic lymphocytic leukaemia Christian W. Eskelund a,∗ , Line Nederby a,b , Anna H. Thysen a , Anni Skovbo a , Anne S. Roug b , Marianne E. Hokland a a b

Institute of Medical Microbiology and Immunology, Aarhus University, Aarhus, Denmark Department of Haematology, Aarhus University Hospital, Aarhus, Denmark

a r t i c l e

i n f o

Article history: Received 10 June 2010 Received in revised form 24 January 2011 Accepted 4 February 2011 Available online 26 February 2011 Keywords: B-cell chronic lymphocytic leukaemia NK cells IL-21 Rituximab Immunotherapy Cancer immunology

a b s t r a c t We have examined natural killer (NK) cell functionality of 54 B-CLL patients upon in vitro stimulation with interleukin-21 (IL-21), together with the anti-CD20 antibody, rituximab. Upon stimulation with rituximab-coated target cells IFN-␥ production was reduced in patients’ NK cells compared to healthy donors’, while both natural- and antibody-dependent cytotoxicity (ADCC) was normal. Following additional stimulation with IL-21, IFN-␥ production, natural cytotoxicity and ADCC were significantly augmented in patients. A complete restoration of IFN-␥ production, however, required the depletion of malignant cells prior to stimulation. Collectively, our data show that NK cells of B-CLL patients are reversibly inhibited, but that their functionality can be normalized by stimulation with IL-21 and when inhibitory effects of the malignant B-CLL cells are eliminated by depletion. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction B-cell chronic lymphocytic leukaemia (B-CLL) is the most prevalent type of leukaemia in the Western world. It is characterized by clonal proliferation of mature B cells in the bone marrow, secondary lymphoid organs and in the circulation, ultimately leading to pancytopenia due to massive bone marrow infiltration. The course of the disease can be indolent, requiring no need for therapy, or aggressive and hence medical intervention is required [1–3]. B-CLL patients with B symptoms or bone marrow failure are most often treated with cytoreductive drugs and monoclonal antibodies (mAbs) such as anti-CD20 or anti-CD52 as single agents or in combination. Such medical intervention may, to differing degree, inhibit the immune system, thus increasing the risk of opportunistic infections [1,2]. The introduction of mAbs has substantially improved the efficacy of B-CLL therapy [4]. They exert direct proapoptotic effects on the target cell [5] as well as indirect effects such as activation of the complement system and antibody-dependent cellular cytotoxicity (ADCC) mediated by cytotoxic effector cells expressing Fc receptors

∗ Corresponding author at: Institute of Medical Microbiology and Immunology, Aarhus University, The Bartholin Building, Wilhelm Meyers Allé 4, 8000 Aarhus, Denmark. Tel.: +45 8942 1741; fax: +45 8619 6128. E-mail address: [email protected] (C.W. Eskelund). 0145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2011.02.006

for IgG, e.g. natural killer (NK) cells and monocytes/macrophages [6,7]. NK cells are large granular lymphocytes with a CD56+ /CD3− phenotype, representing 8–15% of peripheral blood lymphocytes in healthy individuals [8,9]. The NK cells are divided into two subsets: one expressing large amounts of CD56 (CD56bright ) and one expressing small amounts (CD56dim ). The CD56bright cells, constituting approximately 10% of the NK cells in the peripheral blood, are considered to have a primarily regulatory function, producing cytokines such as interferon (IFN)-␥. The CD56dim cells comprise the cytotoxic subset containing cytoplasmic granules of perforin and granzymes and expressing large amounts of the Fc-receptor, CD16 (Fc␥RIII) [8,9]. Activated NK cells express the ␣-subunit of the interleukin (IL)-2 receptor, CD25 [10], and CD69 is expressed on NK cells during early activation [11]. NK cells play a key role in the immune response against transformed and virus-infected cells [12]. They exert natural cytotoxicity as a consequence of receptor–ligand interactions, while ADCC is carried out when CD16 binds to the Fc-regions of antibody–antigen complexes on the target [13–15]. Of relevance to this study, NK cells have been shown to exert ADCC in vitro when the Burkitt’s lymphoma Daudi cell line is coated with the anti-CD20 antibody rituximab [15]. Even from the time of diagnosis most B-CLL patients are immune incompetent due to neutropenia, hypogammaglobulinaemia and impaired normal B-, T- and NK-functions, and immunosuppression

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Table 1 Demographic data on B-CLL patients and healthy donors. Healthy donors (n = 10)

Untreated patients (n = 16)

Treated patients (n = 38)

Gender (% females) Infections experienced within < 1 yeara , median (range)

64 (54–74) 40 –

Time to treatment, month, median (range)

–

74 (36–84) 44 0 (0–3) –

Bone marrow infiltration at time of diagnosis (%), median (range)

–

68 (33–89) 47 1 (0–6) 6 (0–216) 90 (10–97)

Age, median (range) (years)

a

70 (19–90)

Infections requiring treatment.

is aggravated during treatment with standard regimens. Moreover, resistance to treatment regimens develops with progression of the disease [16]. The introduction of new therapeutic strategies based on immunostimulating drugs is of highest importance in antileukaemia treatment. Previous studies have shown impaired, but reversible NK cell cytotoxicity in patients with B-CLL [17–19], whereas others have described the prognostic importance of NK cells in B-CLL [20]. This makes these cells an obvious target for immunostimulation. It has been demonstrated that interleukin-21 (IL-21) cytokine has a pro-apoptotic effect on B-CLL cells and it continues to enjoy a favourable safety profile in the clinic [21,22]. Reportedly, IL-21 enhances both the natural cytotoxicity of NK cells and ADCC mediated by rituximab [22–24]. Additionally, in combination with IL-15, it has been demonstrated to increase IFN-␥ production by NK cells [25]. Considering the natural response of NK cells against malignant cells and their presumed involvement in mAb based therapy, this cell type is probably important for an effective immune response against malignant B cells in B-CLL. We have therefore analyzed the NK cell system of 54 B-CLL patients with regard to NK cell number, activation status, cytotoxicity and IFN-␥-producing potential. 2. Materials and methods 2.1. Patients and healthy donors Peripheral blood samples from B-CLL patients (n = 54) were collected in EDTA vacuum tubes during routine health checks or prior to scheduled treatment at the Department of Haematology, Aarhus University Hospital, Denmark. The male:female ratio of the patients was 30 to 24 and the median age was 69 (range 33–89). Sixteen patients were untreated and 38 were, or had previously been, enrolled in treatment protocols (basic clinical data on each patient in Supplementary Material, Table 1). Blood samples from healthy donors were used as a control (n = 10). Both patient groups and the apparently healthy donors were matched in terms of gender and age (Table 1). Since classification of patients in Rai and Binet’s staging systems is not part of the general practice at the Department of Haematology, this information is not presented. The study was approved by the local ethical committee, and all patients provided informed consent before inclusion.

lymphoblastic B cell, was used as stimulating agent in cytokine producing assays as well as a target cell in the cytotoxicity tests [26]. The cells were cultured in RPMI1640 containing NaHCO3 , 10% FCS, 0.8 mg/ml penicillin/streptomycin, and 2 mM l-glutamine (culture medium). 2.4. NK cell analysis by flow cytometry The PBMCs were resuspended in PBS with 0.5% BSA and 0.09% NaN3 (staining buffer). Cells were stained with titrated concentrations of the appropriate antibodies for 15 min, washed twice in staining buffer and analyzed on a FC500 flow cytometer (Beckman Coulter, CA). Viability of the lymphocyte population was measured with a ViaProbe, 7-AAD (BD Biosciences, San Jose, CA) and was >99% for all samples from patients and healthy donors. The following antibodies were purchased from BD Biosciences: anti-CD3APC, anti-CD56PE, anti-CD25FITC, anti-CD69FITC and anti-CD25PC7. Anti-CD19FITC was purchased from Dako (Glostrup, DK). From all antibodies were titrated to give optimum antibody concentrations for the staining procedures. FlowJoTM for Macintosh, version 8.8.3 (Tree Star Inc., OR) was used for data analysis. Lymphocytes were gated based on the forward/side-scatter plot and the NK cell subset was defined as CD56+ /CD3− . 2.5. Detection of NK functionality markers by flow cytometry IFN-␥ production by the NK cells was measured by intracellular staining. The PBMCs were resuspended in either culture medium alone or in culture medium with added IL-21 (25 ng/ml) (kindly donated by Novo Nordisk A/S, DK), and incubated overnight at 37 ◦ C in 5% CO2 . Daudi cells were coated with rituximab (Mabthera® ) (Roche, Basel, Switzerland) and mixed with PBMCs in a ratio of 1:2. After 3 h of incubation, 10 ␮g/ml Brefeldin A (Sigma–Aldrich) was added. One hour later, the cells were washed in staining buffer and the samples were incubated with anti-CD3APC (BD Biosciences) and anti-CD56FITC (BD Biosciences) for 15 min. Cells were then fixed with FACS Lysing solution (BD Biosciences), permeabilized with FACS Permeabilizing solution 2 (BD Biosciences) and stained with anti-IFN-␥PE (BD Biosciences) for 30 min. Subsequently, cells were fixed in 0.9% (v/v) formaldehyde (Merck, Darmstadt, Germany) and analyzed by flow cytometry collecting at least 8 × 102 events in the CD56+ /CD3− gate. As a control PBMCs were stimulated with 25 ng/ml IL-2 (Chiron, Amsterdam, Holland) and 5 ng/ml IL-12 (Peprotech, London, UK) for 4 h. In an autologous set-up NK cell functionality was measured by CD107a expression on purified (>96%) CD56+ /CD3− NK cells stimulated with CD19+ /CD5+ (>97%) autologous B-CLL cells using fluorescence activated cell sorting (FACSAriaIII, BD Biosciences). Anti CD107a antibody purchased from Ebioscience (San Diego, CA). 2.6. CD19 depletion of the PBMC

2.2. Peripheral blood mononuclear cells Compared to heparinized blood samples, the functional and phenotypic behaviours of the cells were not influenced by the use of EDTA as an anti-coagulant (data not shown). PBMCs were isolated from whole blood by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Bio-sciences AB, Sweden) according to the manufacturer’s instructions. The cells were suspended in 70% RPMI-1640 with NaHCO3 (Gibco Invitrogen, Merelbeke, Belgium) 20% heat-inactivated foetal calf serum (FCS) (Gibco, Paisley, UK) and 10% dimethyl sulfoxide (Sigma–Aldrich, St. Louis, MO) before storage at −135 ◦ C. The PBMCs were thawed in phosphate-buffered saline (PBS) with 20% FCS and washed in PBS with 0.5% bovine serum albumin (BSA) before being processed for further investigation. 2.3. Cell line The human Burkitt’s Lymphoma Daudi cell line (ATCC, Rockville, MD), which is CD20+ and epidermal growth factor receptor negative (EGFR)− , NK cell-sensitive,

To remove malignant cells, PBMCs were depleted of CD19 positive cells (i.e. the B-CLL clone and B cells) using Dynabeads CD19 Pan B (Invitrogen Dynal A/S, Norway) according to the manufacturer’s instructions. Purity was tested by surface staining using anti-CD3ECD (Beckman Coulter), anti-CD14PC7 (BD Biosciences), anti-CD56FITC (BD Biosciences), anti-CD19PE (Dako) and anti-CD45APC (Dako). 2.7. Chromium release assay A standard 4-h chromium-release assay with Daudi cells as target was used to measure the NK cells’ cytotoxic potential. ADCC was analyzed using Daudi coated with rituximab as target cell. Daudi incubated with the anti-EGFR antibody cetuximab (Erbitux® ) (Merck, Darmstadt, Germany) served as an isotype-matched control in the ADCC assay and measured the natural cytotoxicity of the NK cells. Daudi cells were incubated with the respective antibodies (1 ␮g/ml) for 20 min and washed twice. The CD19-depleted PBMCs, incubated overnight in culture medium at 37 ◦ C and 5% CO2 either with or without 25 ng/ml IL-21, were used as effector cells. Effector and target cells were mixed in the ratios 1.25:1, 5:1, and 20:1, and the assay was performed in triplicate. After a 4-h incubation period at 37 ◦ C in a 5% CO2 atmosphere,

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Table 2 Peripheral blood cell counts in treated and non-treated patient samples (medians with 25–75 percentiles).

Leukocytes (×106 /ml) Lymphocytes (×106 /ml) NK cells (×106 /ml) % NK cells of lymphocytes % NK cells of CD19− lymphocytes

Patients (n = 54)

Untreated patients (n = 16)

Treated patients (n = 38)

27.40 (5.97–62.00) 22.95 (1.94–52.40) 0.20 (0.10–0.52) 1.58 (0.45–5.21) 12.19 (4.87–17.79)

49.90 (39.63–110.15) 40.50 (32.70–104.98) 0.60 (0.33–0.76) 1.13 (0.40–2.37) 12.01 (6.57–15.21)

9.08 (5.32–38.50) 4.50 (1.24–34.10) 0.13 (0.08–0.26) 1.73 (0.55–11.35) 12.19 (4.87–22.18)

the supernatants were analyzed on a ␥-counter (Cobra auto-gamma, Packard, Canberra, Australia). Based on the percentage of CD56+ /CD3− NK cells obtained by flow cytometry, the NK:Daudi ratios were calculated. Cytotoxic activity (percent lysis) as a function of NK:Daudi ratio was plotted as a conventional logarithmic dose–response curve as previously described [27]. 2.8. Statistical analysis Statistical analyses were performed using the Mann–Whitney’s U test. Wilcoxon signed rank test was used to compare paired data. p-Values less than 0.05 were considered significant. Unless otherwise indicated, values are expressed as medians with 25–75 percentiles in parentheses. The figures show the medians and error-bars indicate the 25–75 percentiles. Statistical analyses were performed using GraphPad Prism version 5 software (La Jolla, CA).

3. Results 3.1. Quantitation of peripheral blood NK cells in B-CLL patients The distribution of leucocytes and NK cells in the peripheral blood of B-CLL patients and healthy donors was quantified by flow cytometry. Patient data are listed in Table 2. Since the malignant pool of cells comprised a dominant part of the total lymphocyte population, the CD19+ cells were excluded from the lymphocyte population during the analyses of flow cytometric data. The percentages of NK cells in the residual lymphocytic pool were comparable between patients and control donors (12.19% (4.87–17.79) and 11.94% (10.16–13.39), respectively, p = 0.87) (Fig. 1a). To distinguish the treatment-mediated effects from diseaseinduced alterations we isolated the more homogeneous group of untreated patients (n = 16) to compare with the more heterogeneous group of currently or previously treated patients (n = 38). Apart from treatment status, the basic clinical data were similar in the two groups (Table 1 and Table 1 of Supplementary Material). Likewise, there was no difference in the percentage of CD56+ /CD3− NK cells in the CD19− lymphocyte population between the two groups (12.01% (6.57–15.21) and 12.19% (4.87–22.18)) (Table 2). As expected, the absolute count of NK cells in treated patients was significantly lower than that in untreated patients (0.13 × 106 /ml

(0.08–0.26) and 0.60 × 106 /ml (0.33–0.76), respectively, p = 0.0001) (Table 2). No statistically significant difference was found in the distribution of the CD56dim and CD56bright NK cell subsets, neither between the two patient groups (data not shown) nor when comparing the patients with the healthy donors (Fig. 1b). 3.2. NK cells of B-CLL patients show increased CD25 expression The activation status of the peripheral blood NK cells of B-CLL patients was evaluated by their expression of CD25 and CD69. Compared to the healthy donors a significantly higher proportion of the NK cells from patients expressed CD25 (2.38% (1.54–4.21) and 0.86% (0.57–1.20), p = 0.0001) (Fig. 1c). The expression of CD25 was similar in the two patient groups (p = 0.45) (data not shown). No statistically significant difference in CD69 expression was seen between the patients and the healthy donors (p = 0.10). Likewise, the treated and non-treated patients displayed comparable levels of CD69 (p = 0.78) (data not shown). 3.3. NK cells from B-CLL patients show a decreased capacity to produce IFN- To examine the baseline capacity of the NK cells to produce IFN-␥, the NK cells were stimulated using the two standard NK cell activating cytokines: IL-2 and IL-12 [28]. The percentage of NK cells producing IFN-␥ in response to the stimulation was largely the same in patients and healthy donors (Fig. 2a). Knowing that NK cells of B-CLL patients had the potential to produce IFN-␥, their response to rituximab-coated Daudi cells (RDC) was tested. The percentage of IFN-␥ producing NK cells was measured after 4 h of stimulation with RDC in 34 randomly selected patients and compared to the percentage of such cells in 10 healthy donors. Both groups showed a significant increase in the percentage of IFN-␥ producing NK cells compared with un-stimulated samples (p < 0.0001 in patients, p = 0.002 in healthy controls) (Fig. 2b). How-

Fig. 1. NK cells of patients (Pt) and healthy donors (HD). Figures show median values and error-bars indicate 25–75 percentiles. (a) NK cells expressed as percentage of CD19− lymphocytes. Differences in NK cell distribution were not statistically significant (Pt, n = 54 and HD, n = 10). (b) The distribution of CD56dim NK cells. There was no statistically significant difference in the percentage of CD56dim NK cells (Pt, n = 54 and HD, n = 10). (c) Expression of CD25 on NK cells of patients and healthy donors. The patients demonstrated significantly higher expression of CD25 (Pt, n = 37 and HD, n = 10).

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no specific treatment regimen was associated with either high- or low IFN-␥ production (data not shown). Cetuximab-treated Daudi cells included as an isotype control showed no statistically significant activation of NK cells compared to no stimulation (data not shown). 3.4. Identical cytotoxic potential of NK cells from B-CLL patients and healthy donors The cytotoxic potential of the NK cells was tested in a standard 4 h 51 Cr release assay using cells from untreated patients (n = 8) and healthy donors (n = 5). Only untreated patients were tested since they constituted a homogenous group presenting a more precise picture of the disease-related implications on the NK cells. Prior to the assay, depletion of CD19 positive cells was performed on the PBMCs. The healthy donors were efficiently depleted of CD19+ cells (<0.5% CD19+ cells after depletion). However, in B-CLL patients the depletion was less efficient, leaving a number of CD19dim expressing cells in the samples, median 16.05% (7.19–27.7). The highest NK:Daudi ratio was used for statistical comparison of the cytotoxicity of patients and healthy donors (Fig. 2c). The natural cytotoxicity assay demonstrated a median cytotoxic activity of 17.02% (11.97–27.54) for the patients and 22.87% (13.25–34.45) for the healthy donors (p = 0.62). In the ADCC assay, the cytotoxic activity was 50.59% (43.86–55.20) and 44.11% (40.52–54.50), respectively (p = 0.83) (Fig. 2c). 3.5. IL-21 stimulation increases IFN- production and cytotoxicity of B-CLL NK cells

Fig. 2. Functional activity of NK cells of patients and healthy donors. Figures show medians and error-bars indicate 25–75 percentiles. (a) Dot plots showing IFN-␥ production of NK cells induced by either IL-2 and IL-12 or no stimulation. Diagrams display the results for one representative patient and a healthy donor. (b) Percentage of IFN-␥+ NK cells either unstimulated or stimulated with RDCs (Pt, n = 34 and HD, n = 10). (c) Cytotoxicity (% lysis) of NK cells measured by 51 Cr-release assay. Daudi cells were either uncoated (natural cytotoxicity, NC) or coated with rituximab (ADCC). PBMCs were depleted of CD19+ cells prior to assay (Pt, n = 8 and HD, n = 5). NK:Daudi ratios at 2.82:1 for patients and 2.96:1 for healthy donors were used for statistical comparisons of NK cytotoxicity (represented by dotted lines).

ever, the healthy donors had a significantly higher percentage of IFN-␥ producing NK cells than did the patients (3.70% (2.09–4.50) and 0.79% (0.50–0.94), respectively, p < 0.0001). Without stimulation there was no significant difference between the two groups (p = 0.47) (Fig. 2b). Moreover, we found no statistically significant difference between treated and untreated patients (p = 0.17), and

The effect of IL-21 as a supplementary stimulation of the NK cells was tested. The addition of IL-21 caused a significant increase in IFN-␥+ NK cells in both patients and healthy donors (p < 0.0001 and p = 0.002, respectively). Nevertheless, the healthy donors showed a significantly higher percentage of IFN-␥+ NK cells than the patients (7.96% (4.80–8.59) and 1.38% (1.15–2.23), p < 0.0001) (Fig. 3a). We found no difference between treated and untreated patients (p = 57), and again there was no tendency for any particular treatment regimen to affect the IFN-␥ production differently from the other treatment regimens (data not shown). The relative increase in IFN-␥-producing NK cells mediated by supplementary IL-21 stimulation was 1.78 (1.40–2.68) in the patients and 2.16 (1.3–2.29) in the healthy donors. The difference was not statistically significant (p = 0.50) (Fig. 3b). Importantly, upon stimulation with IL-21, the patients and the healthy donors showed similar cytotoxic activity (Fig. 3c). At the highest NK:Daudi ratio, the natural cytotoxicity was 39.55% (22.15–49.08) in the patients and 41.86% (40.5–48.36) in the healthy donors (p = 0.52). ADCC was 63.85% (54.75–71.76) for the patients and 58.60% (56.96–61.63) for the healthy donors (p = 0.83). In patients the natural cytotoxicity increased from 17.02% (11.97–27.54) to 39.55% (22.15–49.08) (p = 0.0078) and ADCC from 50.69% (43.86–55.20) to 63.85% (54.75–71.76) (p = 0.023) when stimulated with IL-21 (Fig. 3d). 3.6. B-CLL cells inhibit NK cell function The proportion of NK cells producing IFN-␥ in CD19-depletedand non-depleted PBMCs was measured after RDC and IL-21 stimulation in 8 untreated patients. Without stimulation, no difference was found in the percentage of IFN-␥ producing NK cells (0.43% (0.29–0.58) in depleted samples and 0.34% (0.34–0.35) in non-depleted samples, p = 0.63). Interestingly, however, upon stimulation with IL-21 and RDC, the depleted PBMCs showed a significantly higher percentage of IFN-␥+ NK cells than the

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Fig. 3. IFN-␥ production and cytotoxicity of NK cells upon stimulation with IL-21 in addition to RDCs. Figures show median values and error-bars indicate 25–75 percentiles. (a) Percent IFN-␥+ NK cells of patients and healthy donors after RDC stimulation. PBMCs are either unstimulated or stimulated with IL-21 (Pt, n = 34 and HD, n = 10). (b) Relative increase in IFN-␥+ NK cells mediated by IL-21 activation prior to RDC stimulation, ratio = (IFN-␥+ NKRDC+IL-21 )/(IFN-␥+ NKRDC ) (Pt, n = 34 and HD, n = 10). (c) Cytotoxic activity (% lysis) of NK cells against Daudi cells either uncoated (natural cytotoxicity, NC) or coated with rituximab (ADCC) in a 4-h 51-chromium release assay. PBMCs were depleted of CD19+ cells and stimulated with IL-21 prior to assay (Pt, n = 8 and HD, n = 5). NK:Daudi ratios at 2.82:1 for patients and 2.96:1 for healthy donors were used for statistical comparisons of NK cytotoxicity (represented by dotted lines). (d) Increase in cytotoxic activity (% lysis) caused by IL-21 stimulation. Results are those of highest NK:Daudi ratio in patients only (NC and ADCC, respectively) (n = 8).

non-depleted PBMCs (4.14% (3.20–5.22) and 2.22% (1.05–3.34), respectively, p = 0.039) (Fig. 4a and b). We tested the experimental setup using cells from two healthy donors. In both we found a similar expression of IFN-␥ in NK cells before and after CD-19 depletion. This was also the case when using control magnetic beads or merely the addition of depletionprocedure washing medium (data not shown). In two patients the response of NK cells to autologous BCLL cells was examined in the same experimental setting as above. Cell populations were sorted by a fluorescence activated cell sorter achieving >95% purity of CD56+ /CD3− NK cells and CD19+ /CD5+ B-CLL cells. Importantly, we showed that autologous rituximab-coated B-CLL cells were able to induce activation to a level comparable to normal donors (as measured by the expression of CD107a) of autologous and IL-21 stimulated NK cells (data not shown). 4. Discussion IL-21 has been shown to enhance both cytotoxicity and IFN-␥ production of healthy donors’ NK cells [15,24]. Furthermore, previous studies of B-CLL patients have demonstrated a decreased NK cell cytotoxicity, which could be restored to normal levels by IL-2 [18,22]. In the present study we provide evidence that both cytotoxicity and IFN-␥ production of NK cells from B-CLL patients can be enhanced by stimulation with IL-21 and rituximab, however, full reinstatement of NK cell function requires a substantial depletion of the malignant cells.

The NK cell population of untreated B-CLL patients tended to be larger compared with the NK cell population of healthy individuals. However, patients receiving therapy usually had significantly lower numbers of NK cells, presumably due to therapeutic toxicity. In support of these findings, expansion of NK cells in untreated BCLL patients has previously been reported [29]. Importantly, the percentage of NK cells in the non-malignant cell pool was similar in treated and untreated patients (Table 2). The NK cells of both treated and untreated patients exhibited an increased expression of CD25 compared to the healthy donors (Fig. 1c), whereas expression of the early activation marker CD69 on the same cells was not, as expected, significantly elevated. The elevation of CD25 and unresponsiveness of CD69 might suggest a more chronic activation of the NK cells, which could possibly be an effect of the malignant clone. Upon stimulation with the Daudi cell line coated with rituximab, we observed an increase in IFN-␥ producing NK cells among both healthy donors and patients, however, the latter were significantly poorer responders, with no relation to either treatment or therapeutic regimen (Fig. 2b). Stimulation with cetuximab-treated Daudi cells only produced a minimal IFN-␥ response not statistically different from no stimulation. This suggests that the IFN-␥ production observed in this experiment is triggered primarily through CD16–rituximab interaction. Of particular interest was our finding that supplemental IL-21 stimulation induced a similar relative increase in IFN-␥+ NK cells in patients as well as in healthy donors (Fig. 3b). Similar results have previously been noted by Roda et al. [24], who investigated IFN-␥

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Fig. 4. CD19+ cells affected IFN-␥ production by NK cells stimulated with RDC and IL21 (n = 8). Figures show median values and error-bars indicate 25–75 percentiles. (a) Dot plots of a representative patient showing IFN-␥ production in NK cells from samples with and without depletion of CD19+ cells prior to IL-21 and RDC stimulation. (b) Percentage of IFN-␥+ NK cells in samples with and without depletion of CD19+ cells (n = 8). Grey area indicates the interquartile range and dotted line indicates the median percentage of IFN-␥+ NK cells of healthy donors (non-depleted).

secretion of NK cells after co-culture with a mAb-coated human breast cancer cell line. They showed a significantly elevated IFN␥ secretion upon pre-stimulation of NK cells with IL-21, however only in healthy donors. Interestingly, we now demonstrate that the same can be achieved in NK cells from B-CLL patients. After depletion of CD19+ cells from the PBMCs, the cytotoxic activity of NK cells was measured by a conventional 4-h chromiumrelease assay. This showed that activity levels in patients and healthy donors were comparable (Figs. 2c and 3c). Moreover, we demonstrated that both natural cytotoxicity and ADCC were significantly augmented by IL-21 stimulation (Fig. 3d). These results were somewhat surprising as other studies have reported lower cytotoxic capacities for B-CLL NK cells than for NK cells from healthy donors [18,19]. However, this discrepancy may be assigned to different experimental approaches, e.g. the use of distinct techniques for malignant clone depletion. In these studies the depletion was accomplished either by lysis of malignant cells by a combination of a monoclonal B cell antibody and complement, or by positively selecting rosette-forming cells from the PBMCs. Nevertheless, one of these studies found that the cytotoxic activity of NK cells obtained from B-CLL patients could be fully restored by stimulation with IL-2 [18]. Importantly, we show the same effect of IL-21. To examine the clinical aspect of the study we performed an autologous setup using sorted B-CLL cells as targets for sorted autologous NK cells. Importantly, we showed that rituximab-coated B-CLL cells were able to induce activation of autologous NK cells,

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thus stressing the clinical relevance of the study. However, a larger study group is needed to fully clarify this. Both cytotoxicity and IFN-␥ production could be augmented by supplemental stimulation with IL-21, but in contrast to cytotoxicity the level of IFN-␥ production was still significantly impaired in the patients. However, a critical difference between the two experimental setups is that of the effector cell suspension. IFN-␥ production was measured after culture of a full PBMC suspension with Daudi cells, while in the cytotoxicity assay the effector cells were depleted of CD19+ cells prior to the culture. This might suggest that the impaired IFN-␥ production was caused by the malignant cells directly or indirectly inhibiting NK cell functionality. Hence, in order to clarify this possible effect, we repeated the IFN-␥ assay on PBMCs depleted of CD19 expressing cells. Interestingly, as a result of depletion prior to stimulation with rituximab-coated Daudi cells and IL-21 the percentage of IFN-␥ producing NK cells became comparable to that of the healthy donors (Fig. 4b). This effect was apparent even when the depletion was only partial. These results demonstrate that the impaired IFN-␥ producing aptitude of the BCLL NK cells in this study was at least partly an outcome of inhibiting effects exerted by the malignant clone. One explanation could be competition for IL-21 due to binding to the B-CLL cells. To explore this we analyzed whether increased concentrations of IL-21 could overcome the NK inhibitory effect. Thus, analyzing the effect of increased IL-21 concentrations, we showed a rise in the number of IFN-␥+ NK cells in non-depleted samples, although this does not explain the entire effect. This suggests that the competition for IL-21 binding might be part of the explanation, however other inhibitory effects of B-CLL cells such as secretion of immunomodulating cytokines, direct inhibition by cell-to-cell contact and/or competitive inhibition in other aspects might add to the inhibitory effect of B-CLL cells. In this context, Buggins et al. [30] showed that supernatants from B-CLL cells had an inhibitory effect on T cells, presumably mediated by IL-6. Ongoing studies in our laboratory seek to unveil the mechanisms underlying the NK-inhibiting effects of B-CLL cells. To test that NK inhibition was not induced by the depletion procedure itself we evaluated the experimental setup of the depletion procedure using two healthy donors. In both we found similar expression of IFN-␥ in NK cells before and after CD-19 depletion, and the same was present when using control magnetic beads or simply adding depletion-procedure washing medium (data not shown). This demonstrated that the depletion procedure did not interfere with the IFN-␥ expression of the NK cells. Even so, a second aspect of the experiment need to be stressed, namely the efficacy of the depletion. Comparing the CD19 profile of the cell suspension before and after the depletion we discovered that a group of CD19dim cells remained, while practically all CD19bright cells were removed. There was a considerable inter-patient variation, which correlated with the number of CD19dim cells before the depletion. We did not find any relationship between the efficacy of the depletion and the IFN-␥ expression of the NK cells. This indicates that the suppressive effect of the B-CLL cells was either mainly exerted by the CD19bright population, or else it was simply created by the great surplus of malignant cells. Either way, the difference in the number of malignant cells did not interfere with the interaction of NK and Daudi cell, since the Daudi:PBMC ratio was not changed. Collectively, our data suggest that the functional capacity of the NK cells from B-CLL patients is comparable to that of cells from healthy donors, but that the peripheral blood environment in the patients somehow prevents the cells from fully exploiting their capacity. The fact that this is true for both the treated and untreated patients indicates that the inhibiting effect on NK cells is disease-induced and not merely treatment-related. As described in Section 1, immune-stimulation is highly desired in the treatment of B-CLL both due to the antitumor effect and to

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combat opportunistic infections in some patients. Particularly IL-21 is of great interest because of its high tolerability in clinical studies versus cytokines with similar effects such as IL-2 [21,31,32]. In this study the combination of IL-21 and rituximab has been shown to have a significant effect on both the IFN-␥ production by B-CLL NK cells and their cytotoxicity in vitro. This supports the need for further investigations in clinical trials. Additional beneficial effects of this treatment have previously been described. Reportedly, rituximab and IL-21 have direct proapoptotic effects on the B-CLL cells [5,22] and the combination of IL-21 and mAbs has shown promising effects in animal models [24]. In humans, a trial is presently being conducted which will test IL-21 and rituximab as a combination therapy for advanced stages of B lymphoproliferative malignancies (Timmerman et al. [33]). 5. Conclusion In conclusion, we have shown that NK cells of B-CLL patients are reversibly inhibited, but that their functionality can be normalized by stimulation with IL-21 and depletion of the malignant cells. Conflict of interest statement All authors have no conflict of interest to declare. Acknowledgements This study was supported with grants from the Danish Cancer Society and the Karen Elise Jensen Foundation. We thank Novo Nordisk A/S, Denmark, for providing the IL-21. We thank Professor Malcolm Turner for critical reading of the manuscript. Contributions. CWE, LN, and MEH contributed to study concept and design; CWE, AHT, AS, and ASR to acquisition of data; CWE, LN, AS, and MEH to analysis and interpretations of data; and CWE and LN to drafting of the manuscript. Critical revision of the manuscript for important intellectual content was done by AHT, AS, ASR, and MEH. CWE, LN, AHT, AS, ASR, and MEH contributed to the final approval of the version to be submitted. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.leukres.2011.02.006. References [1] Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med 2005;352:804–15. [2] Morra E, Nosari A, Montillo M. Infectious complications in chronic lymphocytic leukaemia. Hematol Cell Ther 1999;41:145–51. [3] Zent CS, Kyasa MJ, Evans R, Schichman SA. Chronic lymphocytic leukemia incidence is substantially higher than estimated from tumor registry data. Cancer 2001;92:1325–30. [4] Keating MJ, O’Brien S, Albitar M, Lerner S, Plunkett W, Giles F, et al. Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia. J Clin Oncol 2005;23:4079–88. [5] Shan D, Ledbetter JA, Press OW. Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 1998;91:1644–52. [6] Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 2000;6:443–6. [7] Mavromatis B, Cheson BD. Monoclonal antibody therapy of chronic lymphocytic leukemia. J Clin Oncol 2003;21:1874–81.

[8] Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-cell subsets. Trends Immunol 2001;22:633–40. [9] Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, et al. Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset. Blood 2001;97:3146–51. [10] Nagler A, Lanier LL, Phillips JH. Constitutive expression of high affinity interleukin 2 receptors on human CD16-natural killer cells in vivo. J Exp Med 1990;171:1527–33. [11] Ziegler SF, Ramsdell F, Alderson MR. The activation antigen CD69. Stem Cells 1994;12:456–65. [12] Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol 2008;9:503–10. [13] Moretta A, Marcenaro E, Parolini S, Ferlazzo G, Moretta L. NK cells at the interface between innate and adaptive immunity. Cell Death Differ 2008;15:226–33. [14] Colucci F, Schweighoffer E, Tomasello E, Turner M, Ortaldo JR, Vivier E, et al. Natural cytotoxicity uncoupled from the Syk and ZAP-70 intracellular kinases. Nat Immunol 2002;3:288–94. [15] Dall’Ozzo S, Tartas S, Paintaud G, Cartron G, Colombat P, Bardos P, et al. Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration–effect relationship. Cancer Res 2004;64:4664–9. [16] Sturm I, Bosanquet AG, Hermann S, Guner D, Dorken B, Daniel PT. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy. Cell Death Differ 2003;10:477–84. [17] Guven H, Gilljam M, Chambers BJ, Ljunggren HG, Christensson B, Kimby E, et al. Expansion of natural killer (NK) and natural killer-like T (NKT)-cell populations derived from patients with B-chronic lymphocytic leukemia (B-CLL): a potential source for cellular immunotherapy. Leukemia 2003;17:1973–80. [18] Kay NE, Zarling J. Restoration of impaired natural killer cell activity of B-chronic lymphocytic leukemia patients by recombinant interleukin-2. Am J Hematol 1987;24:161–7. [19] Ziegler HW, Kay NE, Zarling JM. Deficiency of natural killer cell activity in patients with chronic lymphocytic leukemia. Int J Cancer 1981;27:321–7. [20] Palmer S, Hanson CA, Zent CS, Porrata LF, Laplant B, Geyer SM, et al. Prognostic importance of T and NK-cells in a consecutive series of newly diagnosed patients with chronic lymphocytic leukaemia. Br J Haematol 2008;141:607–14. [21] Thompson JA, Curti BD, Redman BG, Bhatia S, Weber JS, Agarwala SS, et al. Study of recombinant interleukin-21 in patients with metastatic melanoma and renal cell carcinoma. J Clin Oncol 2008;26:2034–9. [22] Gowda A, Roda J, Hussain SR, Ramanunni A, Joshi T, Schmidt S, et al. IL-21 mediates apoptosis through up-regulation of the BH3 family member BIM and enhances both direct and antibody-dependent cellular cytotoxicity in primary chronic lymphocytic leukemia cells in vitro. Blood 2008;111:4723–30. [23] Brady J, Hayakawa Y, Smyth MJ, Nutt SL. IL-21 induces the functional maturation of murine NK cells. J Immunol 2004;172:2048–58. [24] Roda JM, Parihar R, Lehman A, Mani A, Tridandapani S, Carson 3rd WE. Interleukin-21 enhances NK cell activation in response to antibody-coated targets. J Immunol 2006;177:120–9. [25] Strengell M, Matikainen S, Siren J, Lehtonen A, Foster D, Julkunen I, et al. IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells. J Immunol 2003;170:5464–9. [26] Klein E, Klein G, Nadkarni JS, Nadkarni JJ, Wigzell H, Clifford P. Surface IgMkappa specificity on a Burkitt lymphoma cell in vivo and in derived culture lines. Cancer Res 1968;28:1300–10. [27] Bryant J, Day R, Whiteside TL, Herberman RB. Calculation of lytic units for the expression of cell-mediated cytotoxicity. J Immunol Methods 1992;146:91–103. [28] Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan S, et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989;170: 827–45. [29] Lundin J, Porwit-MacDonald A, Rossmann ED, Karlsson C, Edman P, Rezvany MR, et al. Cellular immune reconstitution after subcutaneous alemtuzumab (antiCD52 monoclonal antibody, CAMPATH-1H) treatment as first-line therapy for B-cell chronic lymphocytic leukaemia. Leukemia 2004;18:484–90. [30] Buggins AG, Patten PE, Richards J, Thomas NS, Mufti GJ, Devereux S. Tumorderived IL-6 may contribute to the immunological defect in CLL. Leukemia 2008;22:1084–7. [31] Rosenberg SA, Yang JC, White DE, Steinberg SM. Durability of complete responses in patients with metastatic cancer treated with high-dose interleukin-2: identification of the antigens mediating response. Ann Surg 1998;228:307–19. [32] Atkins MB, Kunkel L, Sznol M, Rosenberg SA. High-dose recombinant interleukin-2 therapy in patients with metastatic melanoma: long-term survival update. Cancer J Sci Am 2000;6(Suppl. 1):S11–4. [33] Timmerman JM, Byrd JC, Andorsky DJ, Siadak MF, DeVries TA, Hausman DF, et al. Efficacy and safety of recombinant interleukin-21 (rIL-21) and rituximab in relapsed/refractory indolent lymphoma. J Clin Oncol 2008.