Skip to main content
NCBI home page
Cancer Immunology, Immunotherapy : CII logoLink to Cancer Immunology, Immunotherapy : CII
. 2011 Jun 26;60(12):1683–1695. doi: 10.1007/s00262-011-1067-6

Effect of the simultaneous administration of glucocorticoids and IL-15 on human NK cell phenotype, proliferation and function

Ardiana Moustaki 1, Kimon V Argyropoulos 1, Constantin N Baxevanis 1, Michael Papamichail 1, Sonia A Perez 1,✉
PMCID: PMC11029608  PMID: 21706285

Abstract

We have previously reported a synergistic effect between hydrocortisone (HC) and IL-15 on promoting natural killer (NK) cell expansion and function. In the present study, we extend our findings to methylprednisolone (MeP) and dexamethasone (Dex), thus ascribing to glucocorticoids (GCs) a general feature as positive regulators of IL-15-mediated effects on NK cells. We demonstrate that each GC when combined with IL-15 in cultures of peripheral blood (PB)-derived CD56+ cells induces increased expansion of CD56+CD3− cells displaying high cytolytic activity, IFN-γ production potential and activating receptor expression, including NKp30, NKp44, NKp46, 2B4, NKG2D and DNAM-1. Furthermore, GCs protected NK cells from IL-15-induced cell death. The combination of IL-15 with GCs favored the expansion of a relatively more immature CD16low/neg NK cell population, with high expression of NKG2A and CD94, and significantly lower expression of KIR (CD158a and CD158b) and CD57, compared to IL-15 alone. IL-15-expanded NK cells, in the presence or absence of GCs, did not express CD62L, CXCR1 or CCR7. However, the presence of GCs significantly increased the density of CXCR3 and induced strong CXCR4 expression on the surface of NK cells. Our data indicate that IL-15/GC-expanded NK cells, apart from their increased proliferation rate, retain their functional integrity and exhibit a migratory potential rendering them useful for adoptive transfer in NK cell-based cancer immunotherapy.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-011-1067-6) contains supplementary material, which is available to authorized users.

Keywords: Natural killer cells, Glucocorticoids, IL-15, Proliferation, Apoptosis, Chemokine receptors

Introduction

Natural killer (NK) cells are effector lymphocytes of the innate immune system that play an important role on controlling viral infections and tumor development [1]. The antiviral and antitumor effect of NK cells is not only attributable to their ability to kill target cells, but also to their regulatory function in initiation and modulation of immune responses, mainly through production of cytokines and chemokines [2, 3]. Furthermore, recent studies have shown that mouse NK cells demonstrate immune memory, a feature of adaptive immune system cell populations, e.g., T and B lymphocytes [4].

NK cells are a heterogeneous population in terms of phenotype, function and proliferative potential. In humans, two main subsets have been characterized based on surface CD56 intensity and CD16 expression: the more cytolytic CD56dimCD16bright population that represents the ~90% of circulating NK cells and the CD56brightCD16dim/neg with high proliferative capacity in response to cytokine activation and high cytokine production potential [5]. CD56dimCD16bright are more mature cells and are considered to differentiate from the CD56brightCD16dim/neg subset [6]. Further surface marker analysis reveals several phenotypically and functionally distinct subpopulations within the above two main NK cell subsets [7, 8].

Recently, purified NK cells have emerged as a powerful tool in cancer immunotherapy [9, 10]. However, acquisition of efficient numbers of activated NK cells remains a limiting factor in NK-cell-based immunotherapies. Furthermore, the use of immunosuppressive regimens, such us glucocorticoids (GCs), is a possible hurdle when applied along with adoptive immune cell transfer.

GCs represent a family of steroid hormones that participate in modifying the immune response [11]. The most prominent effect of GCs, which is the basis of their pharmacological use as anti-inflammatory agents, is immunosuppression [12]. GCs exert their anti-inflammatory properties mainly through induction of apoptosis [13–15], modulation of cytokine production [16], downregulation of cell surface adhesion molecules [17], inhibition of upregulation of CD40-ligand on activated CD4+ T cells [18] and direct inhibition of early TCR signaling events [19].

GCs are frequently used in conjunction with other treatments for patients with cancer. The potential of GCs to downregulate immune responses is considered a drawback for NK cell-based immunotherapy. Although GCs are primarily considered to deliver apoptotic/suppressive signals, their effect on immune responses is most likely dual. Therefore, characterization of factors that modulate the effect of GCs on compartments of the immune system is of great importance for cancer immunotherapy when combined with GC administration.

The dual role of GCs is demonstrated by the effect of dexamethasone (Dex, a synthetic GC) on hybridoma and normal T cells. Although Dex induces apoptosis in both types of cells, it can also antagonize apoptosis induced by other stimuli, like CD3 [20]. The strong upregulation of the IL-7Rα transcript by GCs on T cells is another example of the positive effect of GCs on the immune system, given the capacity of IL-7 to regulate the proliferation, survival and differentiation of T cells [21]. It has also been observed that GCs can accelerate anti-TCR-induced lymphocyte mitogenesis [22]. Moreover, Hinrichs et al. [23] have reported that Dex has no impact on the proliferation and function of activated pmel-1 CD8+ cells, and these cells can successfully/effectively be transferred to B16 melanoma bearing mice receiving Dex. Finally, a synergism between active immunization and treatment with GCs has been shown in patients with prostate cancer vaccinated with a personalized peptide vaccine and treated with Dex [24].

We have previously shown that hydrocortisone (HC), in synergy with IL-15, induces a remarkable increase in the proliferation of peripheral blood NK cells (CD56+CD3− cells), without any significant effect on their functional activity [25]. We have exploited this feature of HC when combined with IL-15 to obtain large numbers of NK cells for adoptive transfer in patients with advanced non-small cell lung cancer [26] and in a preclinical mouse model of metastatic lung cancer [27]. In both cases, these IL-15/HC-expanded NK cells were found to have significant anti-tumor potential.

In the present work, we sought first to investigate whether MeP and Dex, two very commonly used GCs in the clinical practice, could act similarly to HC on NK cells. After having established that all tested GCs enhanced NK cell proliferation and protected them from cytokine-induced cell death without compromising their functional potential, we investigated how GCs affect the phenotype of NK cells in terms of NK cell activating receptors, maturation markers and chemokine receptors, in order to obtain information on the differential effects of GCs on different subpopulations of circulating NK cells and their possible impact on their clinical use in cancer treatment.

Materials and methods

Isolation of peripheral blood CD56+ cells

The present study was approved by the Scientific Committee of Saint Savas Cancer Hospital. Heparinized blood samples were acquired from healthy volunteers (median age 31 years; range 26–56). PB mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density centrifugation using standard procedures. CD56+ cells were isolated from PBMC using anti-CD56-coated magnetic microbeads (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer’s instructions. Whenever pure NK cells were used, depletion of CD3+ cells using anti-CD3 magnetic beads and LD columns (Miltenyi) was performed prior to CD56+ cell selection. The purity of the isolated populations was always more than 97%.

Cell lines

The human cell lines K562 and Daudi were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 medium (Life Technologies, Paisley, Scotland), supplemented with 10% fetal bovine serum (FBS; Biosera, East Sussex, United Kingdom), 2 mM l-glutamine (Life Technologies) and 50 μg/ml gentamicin, in a 37°C humidified incubator with 5% CO2.

Cell cultures

Freshly isolated CD56+ or CD56+CD3− cells were plated in 24-well plates at 1 × 106 cells/ml in 1 ml α-MEM (Life Technologies) with 20% FBS, 2 mM l-glutamine and 50 μg/ml gentamicin, supplemented with 30 ng/ml recombinant human IL-15 (specific activity 1.7 × 107 IU/mg, CellGenix, Freiburg, Germany) in the absence or presence of HC (Pfizer, Belgium NV), MeP or Dex (Vianex, Athens, Greece) at the indicated concentrations. The approximate equivalent concentrations of these three GCs were determined according to their glucocorticoid potency (Table S1). Medium was replaced every 3–4 days, and cell density was adjusted to 0.5–1.0 × 106 cells/ml. Cell number was evaluated using a standard hemocytometer, and dead cells were excluded by trypan blue dye. For NK cell number determination, cell number counts were adjusted by the percentage of CD56+CD3− in each sample, as determined by flow cytometry analysis.

Monoclonal antibodies and immunophenotyping

The following anti-human mAb and reagents were purchased from BD Biosciences (San Jose, CA): FITC–conjugated anti -CD244 (clone 2-69), -CD62L (clone Dreg 56), -Granzyme B (clone ICRF44), -CD107a (clone H4A3), CD107b (clone H4B4); PE–conjugated Annexin V, anti-CD56 (clone B159), -NKp46 (clone 9E2/NKp46), -NKG2D (clone 1D11), DNAM-1 (clone DX11), -CD94 (clone HP-3D9), -CD57 (clone NK-1), -CD158a (clone HP-3E4), -CD158b (clone CH-L), -CCR7 (clone 3D12), -CXCR1 (clone 5A12). -CXCR3 (clone 1C6/CXCR3), -CXCR4 (clone 12G5), -perforin (clone δG9); PerCP–conjugated anti-CD3 (clone SK7); 7-AAD (Amino-actinomycin D). PE–conjugated anti-NKG2A (clone Z199),-NKp30 (clone Z25), -NKp44 (clone Z231), PE-Cy5-conjugated anti-CD56 (clone NKH-1) and -CD16 (clone 3G8) were from Beckman-Coulter (Immunotech, France). FITC-conjugated anti-CD16 (clone CB16), PE-conjugated CD16 (clone CB16) and allophycocyanin (APC)-conjugated anti-CD3 (clone UCHT1) were purchased from eBioscience (San Diego, CA).

For intracellular detection of perforin and granzyme B, CD56+ cells were fixed and permeabilized with the Cytofix/Cytoperm Kit (BD Biosciences).

NK cell apoptosis was determined by Annexin V and 7-AAD uptake on gated CD56+CD3− cells.

Data were collected on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FCS3 Express (De Novo Software, Los Angeles, CA). Histograms were analyzed by calculating the specific fluorescence intensity (SFI) [obtained by subtracting geometric mean of the isotype control antibody fluorescence from geometric mean of the specific antibody fluorescence].

CFSE-based proliferation assay

Freshly isolated CD56+ cells were labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Leiden, The Netherlands) in PBS containing 5% (v/v) FCS, for 5 min at room temperature, as previously described [28]. After extensive wash in PBS/5% FBS, labeled cells were cultured under the indicated conditions. Proliferation of NK cell subsets, assessed by CFSE dilution, was monitored up to 10 days by multiparametric flow cytometry.

CFSE-based cytotoxicity assay

Cytotoxic activity of cultured CD56+ cells was determined by a fluorescent-based flow cytometric assay against the NK-sensitive cell line K562, as previously described [29]. Briefly, target cells were labeled with 1 μM CFSE for 5 min at 37°C and incubated with effector NK cells at the indicated ratios for 4 h in a 37°C humidified incubator with 5% CO2. As controls, targets or NK cells were incubated alone to measure spontaneous cell death. Following incubation, cells were stained with 7-AAD, washed and fixed with 1% PFA in PBS. Flow cytometric analysis was performed within 1 h. Dead target cells were defined as CFSE+7-AAD+ labeled cells. Percent cytotoxicity was calculated by the formula:

graphic file with name M1.gif

CD107 degranulation/mobilization assay

CD107 was used as an indicator of effector NK cell degranulation. Ten-day-expanded CD56+ cells were co-incubated with K562 or the LAK-sensitive cell line Daudi at a 1/1 ratio for 4 h at 37°C in the presence of anti-CD107a/b mAbs. Monensin (eBioscience) was added during the last 3 h of incubation at a 2-μM final concentration. Cells were stained with APC-conjugated anti-CD3 and PE-conjugated anti-CD56 prior to flow cytometric analysis.

IFN-gamma secretion assay

IFN-γ secretion by NK cells was assessed on 10-day-expanded CD56+ cells after an additional 24-h incubation in fresh medium containing IL-15 (30 ng/ml), IL-12 (2 ng/ml; R&D Systems) and IL-18 (100 ng/ml; MBL International, Woburn, MA) [30], in the absence or presence of GCs. An IFN-γ secretion assay kit (Miltenyi) was used according to the manufacturer’s instructions. Cells were also labeled with FITC-conjugated anti-CD3 for NK cell discrimination and analyzed by flow cytometry.

Statistical analysis

Data were analyzed by GraphPad Prism version 4 (Graphpad Software, Inc., San Diego, CA). One-way ANOVA for repeated measures (RM) test was utilized for total NK cell comparisons and two-way RM ANOVA test for subpopulation evaluations. P < 0.05 was considered significant. Asterisks on the graphs indicate: *P < 0.05, **P < 0.01 and ***P < 0.001.

Results

Enhanced expansion of activated NK cells in the presence of GCs

We have previously shown enhanced proliferation of NK cells when activated with IL-15 (or IL-2) in the presence of HC [25]. In contrast, it has been reported that MeP inhibits the IL-2- or IL-15-induced proliferation of NK cells [31, 32]. To investigate whether GCs differentially regulate NK cell activation, we examined the effect of MeP and Dex (the most commonly used GCs in clinical practice), in comparison to HC, on NK cells under the conditions described in our previous work [25].

As shown in Fig. 1, both MeP (Fig. 1a-i) and Dex (Fig. 1a-ii) enhanced the proliferation of IL-15-activated NK cells, similarly to HC. The effective range for MeP is between 2 × 10−5 and 2 × 10−7 M, and for Dex between 4 × 10−6 and 4 × 10−8 M, although donor-dependent variability was observed. In all further experiments, MeP was used at 2 × 10−6 M and Dex at 4 × 10−7 M concentrations that approximately correspond to 1 × 10−5 M HC.

Fig. 1.

Fig. 1

Open in a new tab

GCs enhance the expansion of IL-15-stimulated NK cells. CD56+ cells from healthy donors were cultured with IL-15 in the absence or presence of GCs, and NK cell counts (a) or CFSE-based proliferation analyses (b) were performed. NK cell-enumeration was performed by total viable CD56+ cell counts at 5-day intervals, along with immunophenotyping in order to estimate the number of NK (CD56+CD3−) cells depicted on the plots. a-i, -ii Growth curves of IL-15 expanded NK cells from one representative donor in the absence or presence of different concentrations of MeP (M-5,-6,-7: 2 × 10−5,−6,−7 M) and Dex (D-6,-7,-8: 4 × 10−6,−7,−8 M), respectively. An IL-15/HC (10−5 M) -treated group was also included. a-iii, -iv NK cell fold expansion from 8 different donors cultured at the indicated conditions for 5 or 20 days, respectively. Concentrations of GCs used in iii and iv: HC: 1 × 10−5 M; MeP: 2 × 10−6 M; Dex: 4 × 10−7 M. b Histograms of CFSE dilution on gated live NK cells from one representative donor show the progressive proliferation of NK cells cultured under the indicated conditions. Cumulative data from 3 independent experiments are presented as mean (±SEM) percent of highly proliferating NK cells (>5 divisions) on day 8. Statistical significance of IL-15/GC-treated samples compared to IL-15 treated cells is indicated above respective bars. **P < 0.01, ***P < 0.001

A more detailed analysis of the cell growth kinetics revealed a tendency for reduced NK cell proliferation rates in response to IL-15/GC at culture initiation, although not statistically significant (Fig. 1a-iii, day 5). However, after 10 days in culture, the expansion rate was accelerated (Fig. 1a-i, -ii) resulting to a 10-fold mean increase in cell number by day 20 (Fig. 1a-iv), when compared to NK cell stimulated with IL-15 alone.

These data were further confirmed by CFSE-labeling experiments (Fig. 1b), which clearly showed that NK cells activated with IL-15 in the presence of GCs exhibited accelerated proliferation. Despite the temporary cell-cycle entry delay of IL-15/GC activated NK cells (Fig. 1b, day 4 and day 6 for the donor presented), by day 8 more than 50% of proliferating cells had completed more than 5 divisions. In contrast, only about 20% of IL-15 stimulated cells could accomplish more than 5 divisions. These findings indicate that either the IL-15/GC-induced reprogramming of NK cells requires a prolonged time or that NK stimulation in the presence of GCs results in the selective expansion of a small cell subset, initially masked by the dominant population.

Glucocorticoids protect NK cells from IL-15-induced apoptosis

We have previously shown that HC protects NK cells from activation-induced apoptosis. Similarly, MeP and Dex also protected NK cells from IL-15-induced apoptosis: the percentage of Annexin V+ (apoptotic) NK cells was reduced by almost 50% in 10 day cultures with IL-15/GC compared to IL-15 alone (Fig. 2a, b). Furthermore, the presence of GC mostly protected from apoptosis the CD16− NK cell population (Fig. 2c).

Fig. 2.

Fig. 2

Open in a new tab

IL-15/GC-expanded NK cells are resistant to activation-induced apoptosis. Apoptosis of 10-day-expanded NK cells was assessed at the single-cell level by labeling cells with Annexin V/7-AAD. a Percentages of apoptotic (Annexin V+) cells expressed as mean values (± SEM) from 3 independent experiments. Statistical significance of IL-15/GC-treated samples compared to IL-15 treated cells is indicated above respective bars. b Representative dot plots of 7AAD versus Annexin V labeled NK cells cultured at the indicated conditions. The percentages of early (Annexin V+7AAD−) and late apoptotic (Annexin V+7AAD+) cells are depicted in the respective quadrants. Concentrations of GCs used: HC: 1 × 10−5 M; MeP: 2 × 10−6 M; Dex: 4 × 10−7 M. c Percentages of apoptotic cells among CD16+ and CD16− gated NK subpopulations cultured with IL-15 alone or in the presence of HC (10−5 M). Bars represent mean values (±SEM) from 3 independent experiments. ns nonsignificant, *P < 0.05, ***P < 0.001

IL-15/GC-activated and -expanded NK cells remain fully functional

We have previously reported that HC did not affect the function of IL-15 activated CD56+ cells [25]. Thus, in the following series of experiments, we focused on the functional potential of NK cells activated in the presence of MeP and Dex.

We first assessed the cytolytic potential of NK cells expanded in vitro with IL-15, in the absence or presence of GCs. MeP and Dex, similarly to HC, did not impair the K562- and Daudi-induced degranulation (Fig. 3a-i, -ii; Supplementary Fig. S1a, b), perforin and granzyme B content (Fig. 3a-iii, -iv; Supplementary Fig. S1c, d) or the cytotoxic activity of NK cells as shown by killing of K562 targets (Fig. 3a-v; Supplementary Fig. S1e). The capacity of NK cells to produce IFN-γ also remained unaffected in NK cells expanded with IL-15 plus GCs (Fig. 3a-vi; Supplementary Fig. S1f).

Fig. 3.

Fig. 3

Open in a new tab

Functional potential of IL-15/GC-treated NK cells. a Purified NK cells stimulated for 10 days with IL-15, in the absence or presence of GCs, were tested for: surface expression of the degranulation marker CD107a/b after 4 h incubation with K562 (i) or Daudi (ii) target cells at an effector-to-target ratio (E/T) of 1/1; intracellular expression of perforin (iii) and granzyme B (iv); (v) their cytotoxic capacity against K562 target cells in a 4 h CFSE- and 7-AAD-based assay. Histograms of 7-AAD-stained K562 target cells (CFSE+) are shown. Gray dotted line represents the spontaneous cytotoxicity of K562 in the absence of effector cells; and (vi) IFN-γ production after an additional 24 h incubation with IL-12 and IL-18. b Surface expression of NK-activating receptors on IL-15 or IL-15/GC-activated NK cells. Overlay histograms show results of one representative experiment out of 3 performed. Gray lines represent control samples stained with isotype-matched mAbs. Concentrations of GCs used: HC: 1 × 10−5 M; MeP: 2 × 10−6 M; Dex: 4 × 10−7 M

It is well known that the function of NK cells is regulated by a balance of negative and positive signals [33]. A number of NK-activating receptors have been identified, capable of positively regulating functional activities of NK cells [33, 34]. NK cells cultured for 10 days with IL-15 in the absence or presence of GCs expressed similar amounts of NKp30, NKp44, NKG2D, CD244 (2B4) and DNAM-1, whereas NKp46 expression was higher in the presence of GCs compared to IL-15 alone (Fig. 3b; Supplementary Fig. S2).

Phenotype of NK cells activated and expanded in the presence of GC

NK cells are a heterogeneous population comprising several phenotypically defined subsets which represent different maturation/activation stages [5, 7, 8, 35, 36]. Resting peripheral blood NK cells can be divided primarily into two main populations based on the CD56 intensity: the more mature CD56dim and the relatively immature CD56bright NK cell subsets, which are further characterized by the expression of CD16, NKG2A, CD94, KIR and CD57 surface antigens (Fig. 4a). Thus, we next evaluated the phenotypic alterations associated with the expansion of NK cells in the presence of GCs. Freshly isolated and ex vivo expanded NK cells were analyzed by flow cytometry for the expression of maturation-related cell surface markers. Analysis was based on the presence or absence of CD16 marker, because CD56-based NK cell subsets were no longer distinguishable upon cell activation.

Fig. 4.

Fig. 4

Open in a new tab

IL-15-expanded NK cells in the presence of GCs acquire a distinct immunophenotypic profile. a Flow cytometric analysis of freshly isolated PB NK cells. Cell surface expression of CD16, NKG2A, CD94, KIR and CD57 antigens is presented versus the expression of CD56. b Representative immunophetype of freshly isolated (first row) or 10-day-expanded NK cells with IL-15 in the absence of presence of GCs. Cytograms show the expression of the antigen of interest versus CD16 expression. The percentage of each subpopulation is indicated in the corresponding quadrant. Concentrations of GCs used: HC: 1 × 10−5 M; MeP: 2 × 10−6 M; Dex: 4 × 10−7 M

Immunophenotypic analysis revealed that the frequency of CD16− NK cells was greatly increased in the presence of IL-15 and GCs, representing approximately 50% (IL-15/HC: 51.97 ± 0.54, IL-15/MeP: 44.79 ± 0.76, IL-15/Dex: 43.09 ± 0.46) of 10-day-expanded total NK cells (Fig. 4b; Fig. 6b), while in the presence of IL-15 alone, the predominant NK subpopulation (85.39 ± 2.03; P < 0.001 when compared to each IL-15/GC-treated group) expressed CD16. As shown in Fig. 4b and Supplementary Fig. S3, IL-15/GC-expanded NK cells (both CD16+ and CD16− populations) expressed more NKG2A and CD94 and displayed considerably lower frequencies of KIR (CD158a and CD158b) and CD57 expressing cells, compared to their IL-15 stimulated counterparts. These immunophenotypic features of IL-15-expanded NK cells in the presence of GCs reflect a more immature phenotype, suggesting that GCs might selectively favor the expansion of the less differentiated CD56brightCD16− NK cells. Indeed, as shown in Fig. 5 by CFSE dilution, the percentage of highly proliferating cells (>5 divisions) within the CD16−/low population was substantially greater in the presence of IL-15/HC than with IL-15 alone. This imbalance in the proliferation rate of CD16+ and CD16− NK cells, due to differential effect of GC, could explain the relatively immature phenotype of IL-15/GC-expanded NK cells, since the CD16− NK cell subset lacks CD57 expression, exhibits less KIR molecules and higher expression levels of NKG2A and CD94, compared to the CD16+ cell population.

Fig. 6.

Fig. 6

Open in a new tab

Discrete chemokine receptor expression on IL-15/GC-expanded NK cells. Expression of a repertoire of chemokine receptors was assessed on freshly isolated or in vitro expanded NK cells. a Flow cytometric analysis of freshly isolated PB NK cells. Cell surface expression of CD16, CD62L, CXCR1, CXCR3 and CXCR4 antigens is presented versus the expression of CD56. b Representative immunophenotype of freshly isolated (first row) or 10-day-expanded NK cells with IL-15 in the absence of presence of GCs. Cytograms show the expression of the antigen of interest versus CD16 expression. The percentage of each subpopulation is indicated in the corresponding quadrant. Concentrations of GCs used: HC: 1 × 10−5 M; MeP: 2 × 10−6 M; Dex: 4 × 10−7 M

Fig. 5.

Fig. 5

Open in a new tab

CD16 expression defines NK cell populations with different proliferative capacity. a CFSE-labeled NK cells from healthy donors were stimulated with IL-15 in the absence or presence of HC. Representative density plots of CFSE intensity versus CD16 expression were obtained on days 6 and 9. The percentages of rapidly proliferating (>5 successful divisions) cells within CD16+ (gate R1) and CD16− NK cell population (gate R2) are presented. b Cumulative results of the percentage of rapidly proliferating (>5 successful divisions) CD16+ and CD16− NK cells stimulated with IL-15 in the absence or presence of HC. Data represent the mean values (±SEM) from 3 independent experiments. ns nonsignificant, ***P < 0.001

Homing receptor expression on NK cells activated and expanded in the presence of GC

NK cells are further distinguished in different subpopulations according to their chemokine receptor expression profiles (Fig. 6a), which determine their trafficking capacity [37]. In the following series of experiments, we investigated the expression of several chemokine/homing receptors on NK cells activated and expanded with IL-15 in the presence or absence of GCs. As depicted in Fig. 6b and Supplementary Fig. S4, the expression of CD62L and CXCR1 significantly declined under both culture conditions (i.e., IL-15 alone or IL-15 plus GCs); however, the presence of GCs retarded this process (Fig. 6b; Supplementary Fig. S4, and day-5 data not shown). Of notice, although we were able to detect CCR7 on the majority of resting CD56bright NK cells, in line with previous reports, CCR7 was not detectable on activated NK cells independently of the culture conditions (data not shown). The expression of CXCR3 was significantly increased in IL-15/GC-expanded NK cells compared to IL-15-expanded cells (Fig. 6b; Supplementary Fig. S4). Surface expression of CXCL12 receptor, CXCR4, was highly induced by GCs in the presence of IL15, while only a small proportion (approx. 3%) of IL-15-expanded NK cells expresses this receptor.

Discussion

In the present study, we demonstrate that MeP and Dex act similarly to HC [25] on activated PB-derived human NK cells: NK cells cultured with IL-15 in the presence of MeP or Dex, even at pharmacological concentrations, exhibited a significantly enhanced growth compared to NK cells cultured with IL-15 alone, without losing their functional capacity, as demonstrated by their cytolytic activity, cytokine production and expression of activating receptors.

A more detailed analysis of the proliferation kinetics of NK cells cultured with IL-15/GC compared to IL-15 alone revealed that, in most donors, NK cells initially responded to GC combined with IL-15 by a transient (5–7 day) inhibition of cell proliferation, followed in all cases by enhanced proliferation. This transient growth inhibition could not be attributed to increased cell death, since GCs protected NK cells from IL-15-induced apoptosis. These findings are in line with previous data by Chiosone et al. [32] who reported an inhibitory effect of MeP combined with IL-15 on NK cell proliferation, viability and activating NK receptor expression in short-term (i.e., 5 day) cultures.

The transient delay observed during the first 5-7 days in cultures of NK cells with GC and IL-15 could be attributed either to an adaptation to the GC culture period or could imply that a minor subpopulation is positively responding and requires some time to expand. A possible candidate could be the CD56bright population, considered to be more immature [38, 39] compared to CD56dim cells which would possibly explain why immature NK cells have been found by us [40, 41] and others [42, 43] to be greatly induced by GCs. Indeed, our data indicate that the more immature CD16−/low population, which represents less than 5% of circulating NK cells, responded by significantly increased proliferation to activation with IL-15 in the presence of GCs.

Since CD16 mediates the antibody-dependent cellular cytotoxicity (ADCC), a key mechanism of tumor cell destruction during antibody-based therapy of several malignancies, it should be useful to improve our culture conditions in order to longer maintain this population. A combination of IL-15/CG with LCL as feeders [44] is currently under investigation.

Additional phenotypic analysis confirmed that NK cells, in response to IL-15/GC, exhibit a more immature phenotype compared to cells expanded with IL-15 alone. Apart from the CD16− NK cells, which represent the more immature cells [35], among the CD16+ NK cells those expressing high CD94 [36], high NKG2A, low KIR [7], and lack CD57 expression [7, 8] are considered as less differentiated NK cells compared to their CD16+CD94lowNKG2Alow/negKIRbrightCD57+ counterparts. The latter, terminally differentiated, population was significantly reduced in 10-day NK cell cultures with IL-15 in the presence of GCs compared to IL-15 alone. Furthermore, NKs expressing CD57 have been reported to represent highly mature and possibly terminally differentiated NK cells with no or very limited proliferative potential [8]. This is in agreement with the data presented herein showing that the percentage of the CD16+CD57+ NK cells is considerably reduced in IL-15/GC cultures. Further experiments with highly enriched phenotypically defined subpopulations of NK cells will confirm whether the presence of GCs during cytokine activation favors a particular subset of cells and will unravel the mechanisms underlying this phenomenon.

NK cells are further distinguished in different subpopulations according to their homing/chemokine receptor expression profiles, determining their trafficking capacity [37]. The adhesion molecule CD62L (L-Selectin) is highly expressed on the surface of the great majority of CD56bright NK cells and is moderately expressed on a subset of CD56dim NKs (our results and [45, 46]). Upon stimulation with IL-15, in the presence or absence of GCs, both CD16+ and CD16− NK cells progressively lose CD62L expression. Similar results have been reported for IL-2-activated and -expanded NK cells intended for clinical use [45]. Most of the CD56dimCD16+ NK cells express CXCR1. Activation of NK cells with IL-2 or IL-15 results in downregulation of CXCR1 expression [47] in accordance with our findings. The presence of GCs only delayed CXCR1 downregulation.

Resting PB NK cells exhibit low levels of the chemokine receptor CXCR3 and are CXCR4low/neg. Beider et al. [47] have reported that activation of NK cells, both primary and NK cell lines, with IL-2 resulted in downregulation of CXCR4 and significant upregulation of CXCR3, similarly to our data presented herein with IL-15-activated PB NK cells. The presence of GCs further enhanced CXCR3 levels and induced the strong expression of CXCR4. CXCR4 upregulation by Dex has been previously reported in T lymphocytes [48].

The effectiveness of adoptive NK cell-based therapy depends upon the ability of the transferred cells to traffic to and be maintained at the tumor site(s). Thus, the expression of homing receptors on the IL-15/GC-expanded NK cells is of great importance when these cells are intended for use in cancer immunotherapy.

CXCR3 expression on NK cells is considered to be a prerequisite for their infiltration into tumors [49]. Furthermore, several tumor types of hematopoietic or non-hematopoietic origin home to organs where SDF-1/CXCL12 is expressed, mostly in the bone marrow [50, 51]. Thus, the IL-15/GC-activated and -expanded NK cells, which express high levels of both CXCR3 and CXCR4, have the potential of trafficking to tumors and/or to the sites of micrometastasis.

In conclusion, PB-derived NK cells activated in vitro with IL-15 in the presence of GCs expand vigorously, remain functional and potentially possess improved migratory capacity, thus being valuable for use in NK cell-based cancer immunotherapy. The in vivo physiological role of GC synergy with IL-15 and/or other activation signals on NK cells remains to be elucidated and is currently under investigation. Finally, our data imply that combining in vivo GC treatment with NK cell adoptive transfer and concomitant administration of IL-15 (or IL-2) would permit the in vivo NK cell expansion, which is a prerequisite for their anti-tumor clinical efficacy.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 3964 kb) (3.9MB, pdf)

Acknowledgments

This study was supported in part by a donation from OPAP SA to M.P.

Conflict of interest

The authors declare no competing financial interests.

References

  • 1.Papamichail M, Perez SA, Gritzapis AD, Baxevanis CN. Natural killer lymphocytes: biology, development, and function. Cancer Immunol Immunother. 2004;53:176–186. doi: 10.1007/s00262-003-0478-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol. 2008;9:503–510. doi: 10.1038/ni1582. [DOI] [PubMed] [Google Scholar]
  • 3.Farag SS, Caligiuri MA. Human natural killer cell development and biology. Blood Rev. 2006;20:123–137. doi: 10.1016/j.blre.2005.10.001. [DOI] [PubMed] [Google Scholar]
  • 4.Sun JC, Lopez-Verges S, Kim CC, Derisi JL, Lanier LL. NK cells and immune “memory”. J Immunol. 2011;186:1891–1897. doi: 10.4049/jimmunol.1003035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, Carson WE, Caligiuri MA. Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset. Blood. 2001;97:3146–3151. doi: 10.1182/blood.V97.10.3146. [DOI] [PubMed] [Google Scholar]
  • 6.Chan A, Hong DL, Atzberger A, Kollnberger S, Filer AD, Buckley CD, McMichael A, Enver T, Bowness P. CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts. J Immunol. 2007;179:89–94. doi: 10.4049/jimmunol.179.1.89. [DOI] [PubMed] [Google Scholar]
  • 7.Bjorkstrom NK, Riese P, Heuts F, Andersson S, Fauriat C, Ivarsson MA, Bjorklund AT, Flodstrom-Tullberg M, Michaelsson J, Rottenberg ME, Guzman CA, Ljunggren HG, Malmberg KJ. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood. 2010;116:3853–3864. doi: 10.1182/blood-2010-04-281675. [DOI] [PubMed] [Google Scholar]
  • 8.Lopez-Verges S, Milush JM, Pandey S, York VA, Arakawa-Hoyt J, Pircher H, Norris PJ, Nixon DF, Lanier LL. CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset. Blood. 2010;116:3865–3874. doi: 10.1182/blood-2010-04-282301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Arai S, Klingemann HG. Natural killer cells: can they be useful as adoptive immunotherapy for cancer? Expert Opin Biol Ther. 2005;5:163–172. doi: 10.1517/14712598.5.2.163. [DOI] [PubMed] [Google Scholar]
  • 10.Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA, Yun GH, Fautsch SK, McKenna D, Le C, Defor TE, Burns LJ, Orchard PJ, Blazar BR, Wagner JE, Slungaard A, Weisdorf DJ, Okazaki IJ, McGlave PB. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood. 2005;105:3051–3057. doi: 10.1182/blood-2004-07-2974. [DOI] [PubMed] [Google Scholar]
  • 11.Ashwell JD, Lu FW, Vacchio MS. Glucocorticoids in T cell development and function*. Annu Rev Immunol. 2000;18:309–345. doi: 10.1146/annurev.immunol.18.1.309. [DOI] [PubMed] [Google Scholar]
  • 12.Rook GA. Glucocorticoids and immune function. Baillieres Best Pract Res Clin Endocrinol Metab. 1999;13:567–581. doi: 10.1053/beem.1999.0044. [DOI] [PubMed] [Google Scholar]
  • 13.Cohen JJ, Duke RC. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol. 1984;132:38–42. [PubMed] [Google Scholar]
  • 14.Herold MJ, McPherson KG, Reichardt HM. Glucocorticoids in T cell apoptosis and function. Cell Mol Life Sci. 2006;63:60–72. doi: 10.1007/s00018-005-5390-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Perandones CE, Illera VA, Peckham D, Stunz LL, Ashman RF. Regulation of apoptosis in vitro in mature murine spleen T cells. J Immunol. 1993;151:3521–3529. [PubMed] [Google Scholar]
  • 16.Almawi WY, Beyhum HN, Rahme AA, Rieder MJ. Regulation of cytokine and cytokine receptor expression by glucocorticoids. J Leukoc Biol. 1996;60:563–572. doi: 10.1002/jlb.60.5.563. [DOI] [PubMed] [Google Scholar]
  • 17.Cronstein BN, Kimmel SC, Levin RI, Martiniuk F, Weissmann G. A mechanism for the antiinflammatory effects of corticosteroids: the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1. Proc Natl Acad Sci USA. 1992;89:9991–9995. doi: 10.1073/pnas.89.21.9991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bischof F, Melms A. Glucocorticoids inhibit CD40 ligand expression of peripheral CD4+ lymphocytes. Cell Immunol. 1998;187:38–44. doi: 10.1006/cimm.1998.1308. [DOI] [PubMed] [Google Scholar]
  • 19.Baus E, Andris F, Dubois PM, Urbain J, Leo O. Dexamethasone inhibits the early steps of antigen receptor signalling in activated T lymphocytes. J Immunol. 1996;156:4555–4561. [PubMed] [Google Scholar]
  • 20.Zacharchuk CM, Mercep M, Chakraborti PK, Simons SS, Jr, Ashwell JD. Programmed T lymphocyte death. Cell activation- and steroid-induced pathways are mutually antagonistic. J Immunol. 1990;145:4037–4045. [PubMed] [Google Scholar]
  • 21.Franchimont D, Galon J, Vacchio MS, Fan S, Visconti R, Frucht DM, Geenen V, Chrousos GP, Ashwell JD, O’Shea JJ. Positive effects of glucocorticoids on T cell function by up-regulation of IL-7 receptor alpha. J Immunol. 2002;168:2212–2218. doi: 10.4049/jimmunol.168.5.2212. [DOI] [PubMed] [Google Scholar]
  • 22.Wiegers GJ, Labeur MS, Stec IE, Klinkert WE, Holsboer F, Reul JM. Glucocorticoids accelerate anti-T cell receptor-induced T cell growth. J Immunol. 1995;155:1893–1902. [PubMed] [Google Scholar]
  • 23.Hinrichs CS, Palmer DC, Rosenberg SA, Restifo NP. Glucocorticoids do not inhibit antitumor activity of activated CD8+ T cells. J Immunother. 2005;28:517–524. doi: 10.1097/01.cji.0000177999.95831.7b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Naito M, Itoh K, Komatsu N, Yamashita Y, Shirakusa T, Yamada A, Moriya F, Ayatuka H, Mohamed ER, Matsuoka K, Noguchi M. Dexamethasone did not suppress immune boosting by personalized peptide vaccination for advanced prostate cancer patients. Prostate. 2008;68:1753–1762. doi: 10.1002/pros.20847. [DOI] [PubMed] [Google Scholar]
  • 25.Perez SA, Mahaira LG, Demirtzoglou FJ, Sotiropoulou PA, Ioannidis P, Iliopoulou EG, Gritzapis AD, Sotiriadou NN, Baxevanis CN, Papamichail M. A potential role for hydrocortisone in the positive regulation of IL-15-activated NK-cell proliferation and survival. Blood. 2005;106:158–166. doi: 10.1182/blood-2004-08-3232. [DOI] [PubMed] [Google Scholar]
  • 26.Iliopoulou EG, Kountourakis P, Karamouzis MV, Doufexis D, Ardavanis A, Baxevanis CN, Rigatos G, Papamichail M, Perez SA. A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer. Cancer Immunol Immunother. 2010;59:1781–1789. doi: 10.1007/s00262-010-0904-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Salagianni M, Lekka E, Moustaki A, Iliopoulou EG, Baxevanis CN, Papamichail M, Perez SA. NK Cell adoptive transfer combined with ontak-mediated regulatory T cell elimination induces effective adaptive antitumor immune responses. J Immunol. 2011;186:3327–3335. doi: 10.4049/jimmunol.1000652. [DOI] [PubMed] [Google Scholar]
  • 28.Quah BJ, Warren HS, Parish CR. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc. 2007;2:2049–2056. doi: 10.1038/nprot.2007.296. [DOI] [PubMed] [Google Scholar]
  • 29.Lecoeur H, Fevrier M, Garcia S, Riviere Y, Gougeon ML. A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity. J Immunol Methods. 2001;253:177–187. doi: 10.1016/S0022-1759(01)00359-3. [DOI] [PubMed] [Google Scholar]
  • 30.Fehniger TA, Shah MH, Turner MJ, VanDeusen JB, Whitman SP, Cooper MA, Suzuki K, Wechser M, Goodsaid F, Caligiuri MA. Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response. J Immunol. 1999;162:4511–4520. [PubMed] [Google Scholar]
  • 31.Vitale C, Chiossone L, Cantoni C, Morreale G, Cottalasso F, Moretti S, Pistorio A, Haupt R, Lanino E, Dini G, Moretta L, Mingari MC. The corticosteroid-induced inhibitory effect on NK cell function reflects down-regulation and/or dysfunction of triggering receptors involved in natural cytotoxicity. Eur J Immunol. 2004;34:3028–3038. doi: 10.1002/eji.200425418. [DOI] [PubMed] [Google Scholar]
  • 32.Chiossone L, Vitale C, Cottalasso F, Moretti S, Azzarone B, Moretta L, Mingari MC. Molecular analysis of the methylprednisolone-mediated inhibition of NK-cell function: evidence for different susceptibility of IL-2- versus IL-15-activated NK cells. Blood. 2007;109:3767–3775. doi: 10.1182/blood-2006-07-037846. [DOI] [PubMed] [Google Scholar]
  • 33.Lanier LL. Up on the tightrope: natural killer cell activation and inhibition. Nat Immunol. 2008;9:495–502. doi: 10.1038/ni1581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, Biassoni R, Moretta L. Activating receptors and co receptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol. 2001;19:197–223. doi: 10.1146/annurev.immunol.19.1.197. [DOI] [PubMed] [Google Scholar]
  • 35.Romagnani C, Juelke K, Falco M, Morandi B, D’Agostino A, Costa R, Ratto G, Forte G, Carrega P, Lui G, Conte R, Strowig T, Moretta A, Munz C, Thiel A, Moretta L, Ferlazzo G. CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. J Immunol. 2007;178:4947–4955. doi: 10.4049/jimmunol.178.8.4947. [DOI] [PubMed] [Google Scholar]
  • 36.Yu J, Mao HC, Wei M, Hughes T, Zhang J, Park IK, Liu S, McClory S, Marcucci G, Trotta R, Caligiuri MA. CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets. Blood. 2010;115:274–281. doi: 10.1182/blood-2009-04-215491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Berahovich RD, Lai NL, Wei Z, Lanier LL, Schall TJ. Evidence for NK cell subsets based on chemokine receptor expression. J Immunol. 2006;177:7833–7840. doi: 10.4049/jimmunol.177.11.7833. [DOI] [PubMed] [Google Scholar]
  • 38.Carson WE, Fehniger TA, Caligiuri MA. CD56bright natural killer cell subsets: characterization of distinct functional responses to interleukin-2 and the c-kit ligand. Eur J Immunol. 1997;27:354–360. doi: 10.1002/eji.1830270203. [DOI] [PubMed] [Google Scholar]
  • 39.Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-cell subsets. Trends Immunol. 2001;22:633–640. doi: 10.1016/S1471-4906(01)02060-9. [DOI] [PubMed] [Google Scholar]
  • 40.Perez SA, Sotiropoulou PA, Gkika DG, Mahaira LG, Niarchos DK, Gritzapis AD, Kavalakis YG, Antsaklis AI, Baxevanis CN, Papamichail M. A novel myeloid-like NK cell progenitor in human umbilical cord blood. Blood. 2003;101:3444–3450. doi: 10.1182/blood-2002-05-1501. [DOI] [PubMed] [Google Scholar]
  • 41.Perez SA, Mahaira LG, Sotiropoulou PA, Gritzapis AD, Iliopoulou EG, Niarchos DK, Cacoullos NT, Kavalakis YG, Antsaklis AI, Sotiriadou NN, Baxevanis CN, Papamichail M. Effect of IL-21 on NK cells derived from different umbilical cord blood populations. Int Immunol. 2006;18:49–58. doi: 10.1093/intimm/dxh348. [DOI] [PubMed] [Google Scholar]
  • 42.Vitale C, Cottalasso F, Montaldo E, Moretta L, Mingari MC. Methylprednisolone induces preferential and rapid differentiation of CD34+ cord blood precursors toward NK cells. Int Immunol. 2008;20:565–575. doi: 10.1093/intimm/dxn014. [DOI] [PubMed] [Google Scholar]
  • 43.Grzywacz B, Kataria N, Blazar BR, Miller JS, Verneris MR. Natural killer cell differentiation by myeloid progenitors. Blood. 2011;117:3548–3558. doi: 10.1182/blood-2010-04-281394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Berg M, Lundqvist A, McCoy P, Jr, Samsel L, Fan Y, Tawab A, Childs R. Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells. Cytotherapy. 2009;11:341–355. doi: 10.1080/14653240902807034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Huenecke S, Zimmermann SY, Kloess S, Esser R, Brinkmann A, Tramsen L, Koenig M, Erben S, Seidl C, Tonn T, Eggert A, Schramm A, Bader P, Klingebiel T, Lehrnbecher T, Passweg JR, Soerensen J, Schwabe D, Koehl U. IL-2-driven regulation of NK cell receptors with regard to the distribution of CD16+ and CD16- subpopulations and in vivo influence after haploidentical NK cell infusion. J Immunother. 2010;33:200–210. doi: 10.1097/CJI.0b013e3181bb46f7. [DOI] [PubMed] [Google Scholar]
  • 46.Juelke K, Killig M, Luetke-Eversloh M, Parente E, Gruen J, Morandi B, Ferlazzo G, Thiel A, Schmitt-Knosalla I, Romagnani C. CD62L expression identifies a unique subset of polyfunctional CD56dim NK cells. Blood. 2010;116:1299–1307. doi: 10.1182/blood-2009-11-253286. [DOI] [PubMed] [Google Scholar]
  • 47.Beider K, Nagler A, Wald O, Franitza S, Dagan-Berger M, Wald H, Giladi H, Brocke S, Hanna J, Mandelboim O, Darash-Yahana M, Galun E, Peled A. Involvement of CXCR4 and IL-2 in the homing and retention of human NK and NK T cells to the bone marrow and spleen of NOD/SCID mice. Blood. 2003;102:1951–1958. doi: 10.1182/blood-2002-10-3293. [DOI] [PubMed] [Google Scholar]
  • 48.Curnow SJ, Wloka K, Faint JM, Amft N, Cheung CM, Savant V, Lord J, Akbar AN, Buckley CD, Murray PI, Salmon M. Topical glucocorticoid therapy directly induces up-regulation of functional CXCR4 on primed T lymphocytes in the aqueous humor of patients with uveitis. J Immunol. 2004;172:7154–7161. doi: 10.4049/jimmunol.172.11.7154. [DOI] [PubMed] [Google Scholar]
  • 49.Wendel M, Galani IE, Suri-Payer E, Cerwenka A. Natural killer cell accumulation in tumors is dependent on IFN-gamma and CXCR3 ligands. Cancer Res. 2008;68:8437–8445. doi: 10.1158/0008-5472.CAN-08-1440. [DOI] [PubMed] [Google Scholar]
  • 50.Burger JA, Kipps TJ. CXCR4: a key receptor in the crosstalk between tumour cells and their microenvironment. Blood. 2006;107:1761–1767. doi: 10.1182/blood-2005-08-3182. [DOI] [PubMed] [Google Scholar]
  • 51.Muller V, Alix-Panabieres C, Pantel K. Insights into minimal residual disease in cancer patients: implications for anti-cancer therapies. Eur J Cancer. 2010;46:1189–1197. doi: 10.1016/j.ejca.2010.02.038. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1 (PDF 3964 kb) (3.9MB, pdf)

Articles from Cancer Immunology, Immunotherapy : CII are provided here courtesy of Springer

RESOURCES