CD137 (4-1BB) Engagement Fine-Tunes Synergistic IL-15– and IL-21–Driven NK Cell Proliferation

Laurent Vidard, Christine Dureuil, Jérémy Baudhuin, Lionel Vescovi, Laurence Durand, Véronique Sierra and Eric Parmantier
J Immunol August 1, 2019, 203 (3) 676-685; DOI: https://doi.org/10.4049/jimmunol.1801137

Key Points

  • Three signals are required to trigger NK cell proliferation.

  • 4-1BBL fine-tunes NK cell proliferation but not NK cell cytolytic activity.

Abstract

To understand and dissect the mechanisms driving human NK cell proliferation, we exploited the methodology used in cell therapy to numerically expand NK cells in the presence of K562-derived artificial APC (aAPCs) and cytokines. For four consecutive weeks, high expression of CD137L by a K562-derived aAPC cell line could sustain NK cell expansion by 3 × 105–fold, whereas low expression of CD137L by the parental K562 cell line only supported the expansion by 2 × 103–fold. The level of expression of CD137L, however, did not modulate the sensitivity of K562 cells to the intrinsic cytotoxicity of NK cells. Similarly, the low NK cell proliferation in the presence of the parental K562 cell line and cytokines was increased by adding agonistic anti-CD137 Abs to levels similar to CD137L-expressing K562-derived aAPCs. Finally, synergy between IL-15 and IL-21 was observed only upon CD137 engagement and the presence of aAPCs. Therefore, we conclude that NK cell proliferation requires cell-to-cell contact, activation of the CD137 axis, and presence of IL-15 (or its membranous form) and IL-21. By analogy with the three-signal model required to activate T cells, we speculate that the cell-to-cell contact represents “signal 1,” CD137 represents “signal 2,” and cytokines represent “signal 3.” The precise nature of signal 1 remains to be defined.

This article is featured in In This Issue, p.583

Introduction

Since the approval of ipilimumab, an anti–CTLA-4 Ab, by the U.S. Food and Drug Administration (FDA) in 2011 for the treatment of metastatic melanoma, immunotherapy has generated a considerable interest in cancer therapy. Anti-PD1 (nivolumab and pembrolizumab) and/or anti–PD-L1 (atezolizumab) Abs targeting the PD-1/PD-L1 checkpoint signaling pathway have now been approved for the treatment of melanoma, non–small cell lung cancer, Hodgkin lymphoma, metastatic head and neck squamous cell carcinoma, kidney carcinoma, and bladder cancer. The engagement of tumor cells and T cells by anti-CD3/CD19 bispecific T cell engager (blinatumomab) is another approach approved by the FDA to treat B cell acute lymphoblastic leukemia (ALL). More recently, and for the first time, the FDA has granted an accelerated approval to pembrolizumab based on a tumor’s biomarkers (microsatellite instability–high and mismatch repair–deficient cancers) without regard to the tumor’s original location. In addition to Abs and biologics, small molecules, vaccines, cytokines, and adoptive cell therapies represent future directions in cancer immunotherapy.

With the recent approval by the FDA of chimeric Ag receptor (CAR) T cell therapy (Kymriah [tisagenlecleucel]) for some pediatric and young adult patients with a specific form of ALL, cancer therapy is entering a new era. However, the ongoing challenges for adoptive cell therapy lie in the capacity to manufacture sufficient numbers of clinical-grade immune cells as well as other factors such as sterility, engraftment, proliferation, persistence of the effector functions of the engrafted cells, and histocompatibility with the host. Many acellular synthetic and cell-based artificial Ag-presenting systems have been developed to overcome the fastidious and time-consuming preparation of patient-derived autologous APCs (1, 2). Because NK cells are very potent effector cells, are associated with a lower risk of inducing graft-versus-host disease and, in contrast to T cells, do not require MHC restriction to display graft-versus-tumor activity, we decided to focus on the numerical expansion of these cells.

NK cells belong to the family of innate lymphoid cells and are important components of the innate immunity to kill stressed, infected, and transformed cells in the body. Normal, stressed, diseased, and tumor cells display a repertoire of ligands that interact with either activating or inhibitory receptors expressed at the surface of NK cells. The delicate integration of activating and inhibitory signals in NK cells determines the outcome: elimination of the stressed/diseased cells or tolerance toward the normal cells. The list of activating receptors includes CD16, the low-affinity Fc receptor; NKG2D; members of the natural cytotoxic receptor (NCR) family, such as NKp30, NKp44, and NKp46; and members of the killer-cell Ig-like receptors (KIRs) characterized by a short cytoplasmic tail. The inhibitory receptors include CD94/NKG2A/C/E, KLRG1, and KIRs with a typical long cytoplasmic tail.

Human NK cells are characterized by the expression of CD56 and lack of CD3 and can be divided into two subpopulations based on the expression of CD56 and CD16. The CD56brightCD16− subset is most abundant in tissues, and the cells are phenotypically less mature and more prone to cytokine secretion. In contrast, the CD56dimCD16+ subset represents ∼90% of NK cells in the blood. In this case, the cells are phenotypically more mature and are licensed to kill stressed and diseased cells (natural cytotoxicity and/or Ab-dependent cellular cytotoxicity [ADCC]). NK cells are widely distributed throughout the body, including the blood, lymph nodes, bone marrow, skin, gut, uterus, kidney, joints, breast, liver, lung, and placenta (3). NK cells rapidly migrate to inflamed tissues or secondary lymphoid organs, where they play a defensive role against pathogens.

One of the most efficient methods used to numerically expand NK cells is based on a K562 tumor cell line platform (1). The K562 cell line was originally derived from a 53-y-old woman with chronic myelogenous leukemia in terminal blast crises and phenotypically resembles an erythroleukemia line. K562 cells are easily killed by NK cells because they lack the MHC required to inhibit the effector function of NK cells and are the prototype human NK–sensitive target. K562 cells grow in suspension, with a doubling time of 30–40 h and can be easily genetically manipulated for the stable inclusion of new genes. The ectopic expression of costimulatory receptor ligands with or without MHC genes turns K562 cells into artificial APC (aAPC).

aAPC-derived K562 cells have been developed to sustain the proliferation and numerical expansion of NK (4, 5) and CAR-engineered NK cells (6). We exploited the methodology to gain more insight into the mechanisms driving NK cell proliferation and their effector functions. Surprisingly, we found that NK cells have a tremendous replicative capacity that is, to some extent, comparable to T cells. In the presence of cytokines such as IL-15 (or IL-2) and IL-21, NK cell proliferation relies on the presence of aAPCs, and the fold expansion was strictly correlated with the level of CD137 (4-1BB) engagement. CD137 activation controls not only the level of cytokine-driven NK cell proliferation but also the NK cell viability during the culture period. Therefore, we conclude that the optimal culture condition to sustain the proliferation of NK cells requires cell–cell contact, activation of the CD137 axis, and presence of IL-15 (or its membranous form) and IL-21 in the culture medium.

Materials and Methods

Cell lines

The chronic myelogenous leukemia cell line K562 (CLL-243) as well as the Burkitt lymphoma cell lines RAMOS (CRL-1596) and Daudi (CLL-213) were obtained from the American Type Culture Collection. The ALL cell line NALM-6 was available in our laboratory. All cell lines were maintained in RPMI 1640 (31870-025; Life Technologies) supplemented with 10% heat-inactivated FBS (10500056; Life Technologies) and 2 mM l-glutamine (25030-024; Life Technologies). When NK cells were cocultured with the K562 tumor cell line or its aAPC derivatives, the previous medium was also supplemented with 50 μM β2-ME (31350-010; Life Technologies), 50 μg/ml of gentamicin (G1522; Sigma), and, unless otherwise mentioned, with 100 U/ml (100 ng/ml) of IL-2 (589108; BioLegend), 50 U/ml (150 ng/ml) of IL-15 (570308; BioLegend), and 100 U/ml (50 ng/ml) of IL-21 (571208; BioLegend). For the maintenance of CD86/CD137L– and IL-15RA/IL-15–engineered aAPCs, the previous medium was supplemented with 0.8 μg/ml of puromycin (P9620; Sigma) and 10 μg/ml of blasticidin (A11139-03; Life Technologies).

Reagents

The following reagents were used: 4-1BB (CD137) IgG1 Fc (838-4B-100; R&D Systems), anti–IL-15 Ab (clone 34559, MAB2471; R&D systems), anti–IL-15RA Ab (clone JM7A4, ab91270; Abcam), anti-CD86 Ab (clone IT2.2, 305410; BioLegend), anti-CD137L Ab (clone 5F4, 311502; BioLegend), anti-CD137 Ab (clone 4B4-1, 309811, BioLegend), and anti-CD137 Ab (clone BBK-2, LS-C88297; LifeSpan BioSciences).

Engineering of aAPCs

An attR1-attR2 cassette encoding ccdB and the chloramphenicol resistance gene (Cm[R]) was synthesized by GeneArt. Two BamHI sites and one BglII site were mutated with silent mutations before synthesis. The BglII 1.7-Kd fragment was cloned into the PiggyBac transposon vector pCMV3470 linker previously digested with BamHI.

CD137L (TNFSF9, NM_003811), CD86 (NM_006889), IL-15RA (IL15RA, NM_002189), and IL-15 (IL15, NM_172175) were synthesized by GeneArt as transcription units flanked with one CMV promoter and one polyadenylation recognition motif. When necessary, silent mutations were introduced to remove unwanted restriction sites before synthesis. CD86 and IL-15RA transcription units were flanked with attL1 and attL4 recombination sites, and CD137L and IL-15 transcription units were flanked with attR4 and attR3 recombination sites. Puromycin and blasticidin resistance genes were synthesized by GeneArt as transcription units flanked by one SV40 promoter and one polyadenylation recognition motif. All antibiotic resistance-encoding transcription units were flanked with attL3 and attL2 recombination sites. The three-fragment vectors were generated by LR recombination with the MultiSite Gateway PRO 3.0 (12537-103; Life Technologies) according to the manufacturer’s instructions. A schematic representation of the construct is depicted in Supplemental Fig. 1.

K562 cells were nucleofected according to the manufacturer’s instructions. Briefly, 106 K562 cells were resuspended in 100 μl of Nucleofector Solution (VCA-1003; Lonza) at room temperature (RT) and were mixed with 2 μg of pCM3323 (Transposase) and 4 μg of PiggyBac expression vector (encoding the genes of interest). The cells were then transferred to a nucleofection cuvette, placed in the Nucleofector device, and nucleofected using the optimized program for K562 cells (T-01). Immediately after nucleofection, 500 μl of preequilibrated culture medium was added to the cuvette before the transfer of the nucleofected cells into one well of a 12-well plate containing 1.5 ml of prewarmed complete culture. Cells were then incubated for 3 d at 37°C in an incubator (5% CO2 and 95% relative humidity). The respective antibiotics were added to the culture medium, and cells were kept under selection for 3–4 wk. When necessary, the enrichment of cells expressing the transgenes was performed either by cell sorting or panning.

Staining procedure and flow cytometry

In a 96-well plate, cells (3 × 105 per well) were resuspended in 50–100 μl of cold PBS containing 2% BSA and were incubated for 10 min at RT with 5 μl of human Fc block reagent (564219; Becton Dickinson). When necessary, 50 μl of brilliant stain buffer (563794; BD Biosciences) previously equilibrated at 4°C was added, and the plates were placed on ice. Abs were added, and the plates were incubated in the dark for 30 min at 4°C. The cells were washed twice with 200 μl of cold PBS containing 2% BSA, and the pellets were resuspended in 200 μl of a 1/1000 dilution of fixable Viability Dye eFluor 780 (65-0865; eBioscience). The cells were incubated for 30 min in the dark and then were washed twice. The pellets were resuspended in 200 μl of Fix-and-Lyse solution (VersaLyse and IOTest 3 Fixative Solution, A09777 and A07800, respectively; Beckman Coulter), according to the manufacturer’s instructions, for 10 min at RT. Finally, the cells were washed twice before fluorescence acquisition on a BD LSRFortessa ×20 cell analyzer. The cytometer settings were calibrated and adjusted each day after running cytometer setup and tracking beads and using FACS Diva application settings (BD). OneComp eBeads (01-1111-42; eBiosciences) were used for compensation controls. In all experiments, lymphocytes were defined based on a forward scatter × side scatter gate. To identify viable and single cells, forward scatter–area by allophycocyanin-Cy7-A and forward scatter–area by forward scatter–height gates were used, respectively. Data were analyzed using BD FACSDiva v8.0.1 software. The following Abs were used following the manufacturer’s instructions: anti-CD3 BV510 (clone UCHT-12, 563109; BD), anti-CD56 BV421 (clone NCAM16, 562751; BD), anti-CD16 BV711 (clone 3G8, 302044; BioLegend), anti-CD69 BUV737 (clone FN50, 564439; BD), anti-NKG2D PE (clone 1D11, 320806; BioLegend), anti–IL-15 FITC (clone IC2471F, 34559; BD), anti–IL-15RA PE (clone JM7A4, 330208; BioLegend), anti-CD86 PE-Cy7 (clone IT2.2, 305422; BioLegend), anti-CD137L allophycocyanin (clone 5F4, 311506; BioLegend), and anti-CD137L PE (clone 5F4, 311504; BioLegend).

Calcein release assay

Four million target cells were resuspended in 4 ml of culture medium and were incubated with 10 μl of 2 mM calcein AM (C3100MP; Invitrogen) for 30 min at 37°C (5% CO2) in the dark. Cells were then washed three times and resuspended at 4 × 105 per ml in culture medium containing 10 mM probenecib (P36400; Invitrogen). Effector cells were washed and resuspended at 2 × 106 per ml in culture medium. Two-fold serial dilutions of effector cells were performed in 96-well flat-bottom plates in duplicate. A total of 20,000 prelabeled target cells were added in each well. Volumes were adjusted to 200 μl per well by adding either 50 μl of medium or Ab solution in RPMI complete medium.

After 4 h at 37°C, 5% CO2, the wells were gently mixed to evenly distribute the released calcein in the supernatant, and the plates were centrifuged for 2 min at 300 × g. A total of 100 μl of supernatants were then collected and transferred into black 96-well plates (655076; Greiner). Fluorescence was recorded on a Victor ×5 Light Plate Reader (2030-0050; PerkinElmer) with excitation and emission at 485 and 535 nm, respectively. Maximum and spontaneous release controls were set up in duplicate using 1% Triton X-100 and RPMI complete medium, respectively. Specific lysis was calculated using the formula ([test release−spontaneous release]/[maximum release−spontaneous release]) × 100.

NK cell expansion

Buffy coats from healthy donors were provided by the Etablissement Français du Sang after obtaining written informed consent. NK cells were purified using the human NK cell isolation kit (130-092-657; Miltenyi Biotec) according to the manufacturer’s instructions. The purity of NK cells was evaluated by flow cytometry and ranged from 85 to 95%, and their viability was ≥95%. A total of 400,000 freshly purified NK cells were cocultured with 8 × 105 50-Gy x-irradiated aAPCs in 3 ml of culture medium and, unless otherwise mentioned, with 100 U/ml of IL-2, 50 U/ml of IL-15, and 100 U/ml of IL-21. When necessary, the cells were further diluted with cytokine-containing medium. Generally, after 1 wk, 30–60 ml of NK cells were obtained. The cell numbers and viabilities were monitored every week. Every other week, the cells were either allowed to rest in cytokine-containing medium (107 NK cells in 30 ml of medium) or were restimulated by the addition of aAPCs and cytokines (5 × 106 NK cells + 107 aAPCs in 30 ml of medium).

Cell proliferation

Proliferation was monitored by cell count and trypan blue dye exclusion using a Vi-Cell XR cell counter (Beckman Coulter) or with a WST-1 colorimetric assay (11644807001; Roche) using a Victor ×5 Light Plate Reader (2030-0050; PerkinElmer) with a filter of 450 nM or flow cytometry.

For the WST-1 assay, freshly purified NK cells were seeded at 4000 cells per well in 96-well flat-bottom plates in duplicate in complete RPMI medium. A total of 8,000 x-irradiated (50-Gy) aAPCs were added, and, unless otherwise stated, Abs, fusion protein cytokines, or medium were added in a 100 μl volume to adjust the final volume of the culture to 200 μl. Cultures were incubated for 7 d at 37°C, 5% CO2. One week later, 20 μl of WST-1 reagent was added to each well, and the plates were incubated for an additional 4 h at 37°C, 5% CO2. The absorbance was measured at 450 nm in a Victor ×5 Light Plate Reader. The percentage of proliferation was calculated using the formula (test OD 450 nm/maximum OD 450 nm) × 100.

For flow cytometry, after 7 d of duplicate cultures, DAPI (R37606; Life Technologies) and beads at a final concentration of 50,000 beads/ml (Polybead Microspheres 4.5 μm, 17135-5) were added to each well of a 96-well flat-bottom plate according to the manufacturer’s instructions. A total of 100 μl of cell culture was analyzed by flow cytometry. NK cells and beads were gated based on their forward scatter versus side scatter profile. Live NK cells were gated based on their forward scatter versus BUV496 profile. The concentration of NK cells was calculated using the formula ([live NK cell events/beads events] × bead concentration). The fold expansion was determined by dividing the concentration of NK cells obtained at day 7 by the concentration of NK cells seeded at day 0.

ELISA

NK cells were mixed with target cells as described in the Calcein release assay section, with either a human IgG1 control (403102; BioLegend) or a human IgG1 anti-human CD20 Ab (hcd20-mab1; InvivoGen) at 5 μg/ml. Alternatively, NK cells were seeded in 96-well flat-bottom plates in duplicate previously coated with either 10 μg/ml of anti-CD16 Ab (clone 3G8, 302014; BioLegend) or 10 μg/ml of murine IgG1 control (clone MOPC-21, 400153; BioLegend). After 18 h at 37°C, 100 μl of supernatants was collected, and the production of IFN-γ was measured using the Human IFN-γ ELISA kit (550612; BD) according to the manufacturer’s instructions. Optical densities were determined using a TECAN Infinite M1000 at 450 and 570 nm. Optical imperfections in the plate were corrected by subtracting the reading at 570 nm from the reading at 450 nm.

Statistical analysis

For each experiment, the means of duplicate measurements were calculated and pooled. For the analysis of the dose or ratio, values equal to 0 were not considered in the analysis. If normality assumption was verified, two-way ANOVA with random effects (and repeated measures if needed) was performed with a heterogeneous variance structure across groups if necessary. In the case of normality assumption violation, two-way ANOVA with random effects (and repeated measures if needed) was performed on rank-transformed data. Depending on the objectives, this analysis was followed by contrast analyses to test the comparisons of interest and then by Bonferroni–Holm adjustments for multiplicity issues. Statistical data were computed using SAS version 9.4 for Windows 7. Differences were considered significant when p <0.05.

Results

CD137L (4-1BBL) supports the proliferation and sustains the viability of NK cells in the presence of cytokines

We identified ∼50 articles in the literature in which aAPCs based on the K562 platform have been successfully used to amplify Ag-specific CD4+ (7) and CD8+ T cells (8), tumor-infiltrating lymphocytes, (9, 10), regulatory T cells (11), γδ T cells (12, 13), NK cells (4, 5), and NKT cells (14). K562-derived aAPCs have also been useful for the numerical expansion of CAR-engineered CD8+ T (15, 16), γδ T (17), and NK cells (6). The most widely used costimulatory receptor ligands were CD86 and CD137L. To gain further insight into the role of aAPCs, costimulatory receptors and cytokines in sustaining the proliferation and survival of NK cells, we first tried to develop “membrane-bound” chimeric forms of IL-15 and IL-21 as described previously (6, 18) but could not obtain surface expression in K562 cells. Therefore, we decided to engineer a K562 cell line by transfecting it with a PiggyBac transposon encoding CD86 and CD137L (Supplemental Figs. 1, 2) and to supplement the culture medium with IL-2, IL-15, and IL-21, the most commonly used cytokines in systems using aAPCs.

Freshly purified NK cells were cocultured at a ratio of 1 NK cell per 2 aAPCs, with either the parental K562 cell line or K562 cells engineered to express CD86 and CD137L in the presence of IL-2, IL-15, and IL-21. When necessary, cells were diluted further with cytokine-containing medium. Every week, NK cells were counted and viabilities were recorded. Every other week, cells were either allowed to rest in cytokine-containing medium or alternately restimulated by the addition of aAPCs and cytokines. The rates of NK cell proliferation were higher after the first cycle of activation regardless of the aAPC used and were decreased for the remaining weeks (Fig. 1A). The parental K562 tumor cell line was significantly less potent in sustaining NK cell proliferation than the CD86/CD137L–expressing K562 cell line: a 30-fold versus 120-fold increase after 1 wk of coculture, respectively (p < 0.01 the first 2 wk and p < 0.001 the last 2 wk) (Fig. 1A). Although the viability of NK cells was not different for the first 2 wk (ranging from 70 to 80%) regardless of which aAPC was used, it significantly decreased to 40–50% in the last week when the parental K562 cell line was used instead of the CD86/CD137L–expressing K562 (p < 0.01 the third week and p < 0.0001 the fourth week) (Fig. 1B). This result shows that, when used as an aAPC, the K562 cell line forced to express CD86 and CD137L can support the proliferation of NK cells (3 × 105–fold expansion in 4 wk) (Fig. 1C) and sustain their viability for at least 4 wk (Fig. 1B).

FIGURE 1.
FIGURE 1.

CD137L (4-1BBL) supports the proliferation and sustains the viability of NK cells in the presence of cytokines. Freshly purified NK cells were mixed with 50-Gy x-irradiated aAPCs (either parental K562 [blue symbols] or K562 expressing CD86 and CD137L [red symbols]) in the presence of IL-2, IL-15, and IL-21 in duplicate cultures and serially restimulated every other week with aAPCs and cytokines for a total of 4 wk. Every week, cell counts (A) and viabilities (B) were monitored. The cumulative fold expansion after each week is displayed in graph (C). Shown are the means ± SEM from nine donors, and statistical analysis was performed as described in the Materials and Methods section. **p < 0.01, ***p < 0.001.

In the presence of cytokines, CD86- and CD137L-expressing aAPCs increase NK cells’ natural cytotoxicity but not ADCC

To evaluate the impact of CD86/CD137L–expressing aAPCs and cytokines (IL-2, IL-15, and IL-21) on the activity of NK cells, the natural cytotoxicity and ADCC were monitored after the purification of NK cells (i.e., day 0) and after 1 wk of coculture (day 7). The RAMOS tumor cell line, which was derived from a patient with Burkitt lymphoma and expresses CD20, was used as a target cell line in this assay. The natural cytotoxicity of the resting NK cells was low (Fig. 2B, blue squares), but their ability to perform ADCC was stimulated by the presence of a human anti-CD20 Ab (p < 0.0001 at ratios of 2.5:1 to 10:1) (Fig. 2B, blue squares versus blue circles). By contrast, culturing NK cells with CD86/CD137L–expressing aAPCs strongly activated the natural cytotoxicity and allowed the NK cells to efficiently lyse the RAMOS cells, even at a low effector/target ratio (p < 0.0001 at all ratios) (Fig. 2B, blue squares versus red squares). The addition of a human anti-CD20 Ab did not improve the natural cytotoxicity (p = 1.0 at all ratios) (Fig. 2B, red squares versus red circles). When freshly purified, the CD56dimCD16+ and CD56brightCD16− NK cell populations could be clearly differentiated (Fig. 2A). However, after 1 wk of activation, only one population could be observed, namely a mixture of the CD56dimCD16+ and CD56brightCD16− populations. In this case, the number of CD16+ cells and CD16 level of expression remained high even if we could observe a very slight decrease in the percentage of CD16-positive cells compared with the freshly purified population (67% versus 76%). Therefore, the difference between resting and activated NK cells with respect to ADCC does not seem to rely on the expression of CD16, suggesting that once natural cytotoxicity has reached its maximal capacity, it cannot be further augmented in vitro by the presence of Ab-coated target cells.

FIGURE 2.
FIGURE 2.

In the presence of cytokines, CD86- and CD137L-expressing aAPCs increase NK cell natural cytotoxicity but not ADCC. (A) Percentage of NK cells and expression of CD16, CD69, and NKG2D before (left panel) and after purification (middle panel) as well as after a 1-wk cycle of activation (right panel). Shown are representative results from one of three donors tested. (B) Cytotoxicity of resting (blue symbols) and 1-wk–activated (red symbols) NK cells from the same donor against RAMOS cells in the presence (circles) or absence (squares) of anti-CD20 Ab (in a 4 h assay) at different E:T ratios in 96-well flat-bottom plates in duplicate. Shown are the means ± SEM from three donors, and statistical analysis was performed as described in the Materials and Methods section.

Cytokine starvation dampens NK cell natural cytotoxicity and IFN-γ production but restores their capacity to perform ADCC in the presence of Ab-coated tumor cells

Because ADCC shifts natural cytotoxicity from a minimal to a maximal intensity, we decided to decipher the respective roles of costimulatory receptor and cytokines in this process as well as their respective roles in IFN-γ production by NK cells. For this purpose, after 1 wk of culture in the presence of aAPCs and cytokines, amplified NK cells were extensively washed and seeded for one additional week under two different conditions: 1) with cytokines and 2) in the absence of both aAPCs and cytokines. Surprisingly, in the absence of both aAPCs and cytokines, a 3–7-fold amplification of NK cells was still observed, with viabilities only 10% below those seen in the presence of cytokines (data not shown). This finding suggests that contact with the aAPCs in the presence of cytokines within the first days might determine the fate of NK cells for the subsequent days. Each group of cells was then tested for the ability to lyse RAMOS cells in the presence or absence of an anti-CD20 Ab as well as their capacity to produce IFN-γ. NK cells precultured in the presence of cytokines retained their ability to kill RAMOS cells and to produce IFN-γ (Fig. 3A–C, red curves). NK cells precultured in the absence of both aAPCs and cytokines could still perform ADCC in the presence of the anti-CD20 Ab (p < 0.001 at ratios of 10:1 and 5:1) (Fig. 3A, blue squares versus blue circles) compared with NK cells precultured in the presence of cytokines (p = 1.0 at all ratios) (Fig. 3A, red squares versus red circles). IFN-γ production was only observed with cytokine-activated NK cells in the presence of RAMOS cells (p < 0.0001 at all ratios) (Fig. 3B, blue squares versus red squares and blue circles versus red circles) regardless of the presence of the anti-CD20 Ab and was improved by CD16 engagement (p < 0.0001 at all NK cells concentrations) (Fig. 3C, blue squares versus red squares) regardless of the presence of target cells. Altogether, these data suggest that cytokines are necessary for the optimal cytolytic effector functions of NK cells in the absence of aAPCs, and the triggering of CD16 partially compensates for the lack of cytokines. Furthermore, the production of IFN-γ following CD16 engagement is supported only when NK cells were previously grown in the presence of cytokines.

FIGURE 3.
FIGURE 3.

Cytokine starvation dampens NK cell natural cytotoxicity and IFN-γ production but restores their capacity to perform ADCC in the presence of Ab-coated tumor cells. NK cells were activated for 1 wk in the presence of 50-Gy x-irradiated CD86- and CD137L-expressing K562 and cytokines. The cells were extensively washed and cultured for one additional week in the presence of either cytokines (red symbols) or complete medium (blue symbols). (A) Natural cytotoxicity in the presence of a human IgG1 isotype control (squares) and ADCC in the presence of an anti-CD20 Ab (circles) were compared in a 4-h cytotoxicity assay against RAMOS cells at different E:T ratios in 96-well flat-bottom plates in duplicate. To assess the presence of IFN-γ in the supernatants, cells were cultured for 18 h at 37°C. (B) IFN-γ production by NK cells stimulated by the RAMOS cell line in the presence of either a human IgG1 isotype control (squares) or an anti-CD20 Ab (circles) in 96-well flat-bottom plates in duplicate. (C) IFN-γ production by NK cells stimulated by plate-bound anti-CD16 Ab in 96-well flat-bottom plates in duplicate. Shown are the means ± SEM from five donors, and statistical analysis was performed as described in the Materials and Methods section.

Cell-to-cell interaction, activation of the CD137 axis, and the presence of cytokines are required for optimal proliferation of human NK cells

To gain further insight into the mechanisms driving NK cell expansion, a proliferation assay in 96-well plates was developed in which K562 cells with or without CD86 and CD137L expression were cocultured with freshly isolated NK cells in the presence of cytokines and one of two independent clones of anti-CD137 costimulatory receptor (either 4B4-1 or BBK-2 clones) to mimic the activity of membrane-bound CD137L expressed on aAPCs (Fig. 4). Although the anti-CD137 Abs did not affect the expansion of NK cells in the presence of K562 cells forced to express CD86 and CD137L molecules (p = 1.0 at all concentrations) (Fig. 4A, 4B, red squares versus red circles), the Abs increased NK cell proliferation in the presence of parental K562 cells in a dose-dependent manner (p < 0.0001 from 64 to 1000 ng/ml; Fig. 4A, blue squares versus blue circles) (p < 0.0001 from 16 to 1000 ng/ml; Fig. 4B, blue squares versus blue circles). Once plate bound, the anti-CD137 Ab (clone 4B4-1) lost its ability to trigger NK cell proliferation (p = 1.0 at all concentrations) (Fig. 4C, blue squares versus blue circles). In the absence of aAPCs, NK cell proliferation was undetectable regardless of which anti-CD137 clone was used and regardless of the presence of cytokines (Fig. 4A, 4B, green squares versus green circles). Because CD137L was detected at low levels by flow cytometry with the anti-CD137L Ab clone 5F4 (Supplemental Fig. 2), we investigated whether the ability of parental K562 cells to sustain the proliferation of NK cells in the presence of cytokines could be mediated by activation of the CD137 axis. For this purpose, a CD137 IgG1 Fc chimeric protein was added to the coculture of the parental K562 aAPCs and freshly isolated NK cells in the presence of cytokines. In this setting, dose-dependent inhibition of the NK cell proliferation was observed (p < 0.0001 from 16 to 1000 ng/ml) (Fig. 4D, blue squares versus blue circles). Altogether, these data suggest that CD137 costimulates the cytokine-driven proliferation of NK cells, and cell–cell interaction is mandatory for the process. Indeed, cytokine-driven proliferation is fine tuned by the level of CD137 engagement, either by CD137L or agonistic anti-CD137 Abs, only in the presence of aAPCs.

FIGURE 4.
FIGURE 4.

Cell-to-cell interaction, activation of the CD137 axis, and presence of cytokines are required for optimal proliferation of human NK cells. (A) Freshly purified NK cells were cultured in 96-well flat-bottom plates in duplicate for 7 d with the parental K562 (blue symbols), CD86-, and CD137L-expressing K562 (red symbols) or without aAPC (green symbols) in the presence of cytokines and indicated concentrations of reagents. (A) Soluble anti-CD137 Ab clone 4B4-1 (circles) or IgG1 isotype controls (squares). (B) Soluble anti-CD137 Ab clone BBK-2 (circles) or IgG1 isotype controls (squares). (C) Plate-bound anti-CD137 Ab clone 4B4-1 (circles) or IgG1 isotype controls (squares). (D) Soluble CD137 IgG1 Fc (circles) or IgG1 Fc control (squares). Proliferation was determined with the cell proliferation reagent WST-1. Shown are the means ± SEM from 14 (A), 6 (B), 7 (C), and 11 (D) donors. Statistical analysis was performed as described in the Materials and Methods section.

A collaboration/synergy between CD137L, IL-15, and IL-21 determines the extent of NK cell proliferation

To better understand the respective roles of cell–cell interaction, costimulatory receptor ligands CD86 and CD137L, IL-15 (soluble or membrane-bound), IL-2, and IL-21, the following three new aAPCs were engineered: 1) K562 expressing IL-15RA and IL-15, 2) K562 expressing CD137L, IL-15RA, and IL-15, and 3) K562 expressing CD86, CD137L, IL-15RA, and IL-15. The phenotypic analysis of these aAPCs by flow cytometry is depicted in Supplemental Fig. 2.

Freshly isolated NK cells were cocultured in the presence or absence of one of each of the five aAPCs and cytokines (alone or in combination). As mentioned previously, in the absence of aAPCs, NK cell proliferation was barely detectable regardless of the combination of cytokines used (Fig. 5A). In all settings with cytokines, the parental K562 cell line supported low but significant NK cell proliferation (p < 0.0001). The rate of NK cell proliferation was higher in the presence of K562 expressing CD86 and CD137L than in the presence of the parental K562 cells (Fig. 5B). Strikingly, IL-21 in combination with at least one of the other cytokines was always more potent in triggering NK cell expansion when K562 cells expressed CD86 and CD137L (p < 0.0001) (Fig. 5B). In the absence of IL-2 and IL-15, IL-21 was less robust in sustaining NK cell proliferation, although the proliferation remained highly significant (p < 0.0001). IL-21 alone was more potent in triggering NK cell expansion when CD137L, IL-15RA, and IL-15 were coexpressed by K562-derived aAPCs (p < 0.0001) (Fig. 5C). No statistically significant differences were observed between the aAPCs expressing IL-15RA and IL-15 and either CD137L and CD86 or CD137L alone (p = 1.0) (Fig. 5D), suggesting that CD86 does not costimulate NK cell proliferation. Altogether, the data show a close collaboration/synergy between IL-15 (either membrane-bound or soluble) and IL-21 that relies on the presence of aAPCs expressing at least CD137L.

FIGURE 5.
FIGURE 5.

Collaboration/synergy between CD137L, IL-15, and IL-21 determines the extent of NK cell proliferation. Freshly purified NK cells were cultured in 96-well flat-bottom plates in duplicate for 7 d with different cytokine combinations in either the presence or absence of the parental K562 tumor cell line (A), in the presence of either the parental or CD86/CD137L–expressing K562 cells (B), in the presence of either IL-15RA/IL-15–expressing or CD137L/IL-15RA/IL-15–expressing K562 cells (C), in the presence of either CD137L/IL-15RA/IL-15–expressing or CD86/CD137L/IL-15RA/IL-15–expressing K562 cells (D). Proliferation was determined by flow cytometry. Shown are the means ± SEM from six donors, and statistical analysis was performed as described in the Materials and Methods section. ***p < 0.001.

IL-2, soluble IL-15, and membrane-bound IL-15 support NK cell proliferation in a similar manner

Freshly isolated NK cells were cocultured in the presence or absence of one of each of the five aAPCs and cytokines (alone or in combination). IL-2 could sustain NK cell expansion in the presence of either the parental K562 cell line or its aAPC derivatives expressing both CD86 and CD137L (p < 0.001) (Fig. 6A). In the presence of aAPCs coexpressing IL-15RA and IL-15, the mitogenic activity of IL-2 was compensated by membrane-bound IL-15 (p = 1.0). Similar observations were made with soluble IL-15, which could support NK cell proliferation in the presence of either the parental K562 cell line or its aAPC derivatives expressing both CD86 and CD137L (p < 0.001) (Fig. 6B). The ability of soluble IL-15 to support NK cell proliferation was compensated by membrane-bound IL-15 (p = 1.0). IL-21 triggered NK cell expansion only when CD137L expressing K562-derived aAPCs were present and synergized with membrane-bound IL-15 (p < 0.0001) (Fig. 6C). In all settings, when combined with IL-21, the mitogenic activities of IL-2 and soluble IL-15 were indistinguishable (p = 1.0) (Fig. 6D). NK cells cocultured in the presence of the parental K562 cell line or one of its four derived aAPCs in the absence of soluble cytokines were stained for flow cytometry analysis (Fig. 7). Each aAPC expressing IL-15RA and IL-15 could sustain low-level proliferation of NK cells in the absence of exogenously added cytokines (Fig. 6). This observation was also supported by the high proportion of CD3−CD56+ cells in the flow cytometry analysis (Fig. 7). Most cells also expressed CD16 and displayed an activated CD69+NKG2D+ phenotype (Fig. 7). Therefore, we can conclude that the mitogenic activities of IL-2, soluble IL-15, and membrane-bound IL-15 are indistinguishable.

FIGURE 6.
FIGURE 6.

IL-2, soluble IL-15, and membrane-bound IL-15 support NK cell proliferation in a similar manner. Freshly purified NK cells were cocultured in 96-well flat-bottom plates in duplicate with aAPCs expressing either or both CD86, CD137L, IL-15RA and/or IL-15 in the presence or absence of IL-2 (A), IL-15 (B), IL-21 (C), or IL-21 in combination with either IL-2 or IL-15 (D). Cells were counted after 1 wk of culture by flow cytometry, and the data are expressed as fold expansion. Shown are the means ± SEM from six donors, and statistical analysis was performed as described in the Materials and Methods section. **p < 0.01, ***p < 0.001.

FIGURE 7.
FIGURE 7.

Phenotype of NK cells upon activation by K562-derived aAPCs forced to express IL-15RA and IL-15 in the absence of exogenously added cytokines. NK cells were cultured as described in the legend of Fig. 5 with either one of the five aAPCs. PBMCs, freshly purified NK cells, and NK cells after 1 wk of culture at 37°C were stained for the expression of CD3, CD56, CD16, CD69, and NKG2D. The data are representative of eight experiments.

In contrast to either soluble or membrane-bound IL-15, CD86 and CD137L do not affect the K562 target cell sensitivity to NK cell natural cytotoxicity

To evaluate whether CD86, CD137L, and IL-15 influence NK cell effector function, freshly purified and 1-wk–activated NK cells were compared for their ability to induce cell lysis of the parental K562 cell line and its aAPC derivatives. The three aAPCs displaying IL-15 on their cell surfaces were more sensitive to freshly isolated NK cell lysis regardless of CD137L and/or CD86 expression compared with the parental K562 cell line or its aAPC derivatives forced to express CD137L and CD86 (p < 0.0001 at ratios 1.25:1 to 10:1) (Fig. 8A). However, in the presence of soluble IL-15, all five K562 cell lines and derivatives were similarly sensitive to resting NK cell–mediated cytolysis (p = 1.0 at all ratio) (Fig. 8C). By contrast, the presence (Fig. 8D) or absence (Fig. 8B) of soluble IL-15 did not modify activated NK cell effector functions regardless of the target cells used (p = 1.0 at all ratios). As previously shown (Fig. 2B), after 1 wk of expansion in the presence of aAPCs and cytokines, NK cells were much more lytic than their resting counterparts. Therefore, we can conclude that IL-15, in either soluble or membrane-bound form, activates NK cell cytolytic activity, whereas CD86 and CD137L have no significant effect.

FIGURE 8.
FIGURE 8.

In contrast to either soluble or membrane-bound IL-15, CD86 and CD137L do not affect the K562 target cell sensitivity to NK cell natural cytotoxicity. The natural cytotoxicity of freshly isolated (A and C) or activated (B and D) NK cells in either the presence (C and D) or absence (A and B) of soluble IL-15 against the parental K562 (blue squares), CD86- and CD137L-expressing K562 (blue circles), IL-15RA– and IL-15–expressing K562 (red squares), CD137L-, IL-15RA–, and IL-15–expressing K562 (red triangles), or CD86-, CD137-, IL-15RA–, and IL-15–expressing K562 (red circles) target cells in a 4-h calcein release assay at different E:T ratios in 96-well flat-bottom plates in duplicate. Shown are the means ± SEM from five donors, and statistical analysis was performed as described in the Materials and Methods section.

Discussion

Cell therapy is currently facing two major difficulties: the production of immune effector cells under good manufacturing practice conditions and in sufficient quantity to treat at least one patient. In this work, we have addressed the numerical expansion of NK cells and tried to gain more insight into the molecular mechanisms underlying NK cell proliferation. We confirmed that aAPCs could be used in large-scale assays to support NK cell expansion, which requires the collaboration among close cell–cell contact between aAPCs and NK cells (signal 1), one costimulatory molecule (signal 2), and two cytokines (signal 3).

IL-2, IL-15, and IL-21 belong to the family of cytokines that share the γ-chain (CD132) among their receptor subunits, including IL-4, IL-7, and IL-9. In addition to the common γ-chain, IL-2 and IL-15 share another subunit referred to as β-chain (CD122) to form a dimeric IL-2/IL-15R that is constitutively expressed at the surface of lymphocytes. The binding of IL-2, IL-15, and IL-21 induces receptor oligomerization that leads to the juxtaposition of the intracellular domains and, consequently, activation of the constitutively bound JAK1 and JAK3 kinases. This signaling complex induces the activation of the PI3K, MAPK, and STAT downstream signaling pathways (19, 20). The activation of JAK kinases induces preferentially the recruitment and activation of either STAT5 or STAT3 to the IL-2/IL-15R or IL-21R, respectively. The synergy observed between IL-15 and IL-21 in the proliferation of NK cells might therefore rely on the coactivation of STAT3 and STAT5. We cannot exclude that it may also be due to the engagement of a higher number of common γ-chains in the presence of both cytokines because each cytokine alone may not engage the common γ-chains associated with the other receptor. Expression of CD137 on NK cells is induced by the presence of cytokines (21, 22), and its engagement by CD137 ligand–positive aAPCs reinforce the activation of the MAPK signaling pathway, whereas CD137 activates the NF-κB pathway and inhibits apoptosis. One possibility is that activation of the NF-κB pathway and inhibition of apoptosis must occur first for the cytokines to drive NK cell proliferation through the activation of the PI3K, MAPK, and STAT signaling pathways. In a two-compartment model of NK cell proliferation, Zhao and French (23) showed that IL-15 influenced NK cell expansion by modulating division rates to a greater extent than death rates. In our hands, although the presence of IL-15 and IL-21 is mandatory, the balance between proliferation and apoptosis is finely regulated by CD137 engagement. Either K562 aAPCs expressing CD137L or cytokine alone (or in combination) induced only low or no proliferation of freshly purified NK cells (Fig. 5). Remarkably, aAPCs expressing CD137L and either membrane-bound IL-15 (6) or IL-21 (18) are usually used to produce many NK cells but are never coexpressed together or combined with either soluble IL-15 or IL-21. In many studies, aAPCs expressing CD86 were used. However, in our hands, the presence of CD86 in the K562 tumor cell line was not required for the proliferation of NK cells.

CD137-deficient mice have reduced NK/NKT cell numbers, but the cytolytic activity of the remaining NK cells is not different from that of wild-type mice (24). In agreement with this finding, our data (Fig. 8) and observations made by other groups have shown that CD137 signaling did not affect the cytotoxic potential of NK cells (21, 25). Moreover, and in contrast to its minimal role in effector functions, our data suggest that the cytokine-driven NK cell proliferation is finely tuned by the level of CD137L on aAPCs: low or high expression of CD137L induces a small or large numerical expansion of NK cells, respectively. A similar conclusion can be drawn regarding the viability of NK cells, but it required 3–4 wk to observe a negative correlation between the level of CD137L expression and the percentage of dying cells. The low activation of CD137 on NK cells by the parental K562 tumor cell line CD137Ldull could be advantageously compensated by the addition of agonistic anti-CD137 Abs in the culture medium. In this setting, the cytokine-driven NK cell proliferation was dependent on and correlated with the concentration of agonistic anti-CD137 and, consequently, on the level of CD137 engagement. Therefore, CD137 is a good candidate to provide the second signal leading to the proliferation of NK cells in the presence of cytokines. In its absence, cytokines support a low level of NK cell proliferation. Surprisingly, although CD137 knockout mice have been developed (24) and characterized by a reduced number of NK and NKT cells, the role of CD137L in the development of NK cells has not been described in the literature (26).

Among the genetic alterations affecting NK cell number and function (27–29), mutations in the IL-2/IL-15R β-chain (30) and γ-chain (31) and mutations in JAK3 (32, 33) have been described. In mouse studies, reduced numbers or a lack of NK cells have also been reported in mice with deficiencies in IL-15 (34, 35), IL-15RA (35, 36), IL-2RB (37), IL-2RG (38), JAK3 (39), and STAT5 (40), supporting an important and unavoidable role of IL-15 and its signaling pathway in the development of NK cells. In addition to its role in NK cell development, IL-15 supports mild NK cell proliferation (Fig. 6) either bound to its high-affinity receptor or soluble in the presence of the parental K562 tumor cell line. aAPCs forced to express IL-15RA/IL-15 complexes are more sensitive to NK cell killing than the parental K562 tumor cell line, and soluble IL-15 similarly triggered NK cell effector function (Fig. 8).

In contrast to CD137 and IL-15, IL-21 is probably not involved in the development of NK cells. In mouse and human IL-21 or IL-21R deficiency, the number of NK cells and their functions appeared normal (41, 42). IL-21 alone did not support the expansion of NK cells in vitro (Figs. 5, 6, reviewed in Ref. 42, 43) but collaborated with IL-15 and Flt3-L for both the proliferation and maturation of NK cells from bone marrow CD34+ hematopoietic progenitors (43, 44). By contrast, IL-21 was shown to block IL-15–induced expansion of resting NK cells (42, 45). Therefore, we cannot exclude that depending on the biological environment (i.e., cytokines and/or signaling molecules) IL-21 can either stimulate or inhibit the proliferation of NK cells. Our data suggest also that IL-21 supports NK cell expansion only when CD137 or IL-2/IL-15R is triggered, and its effect is exacerbated in the presence of aAPCs forced to express both CD137L and IL-15RA/IL-15 (Figs. 5, 6). Although CD8 and various subsets of CD4 T cells have been described as sources of IL-21, IL-21 seems to be mostly produced by NKT cells (46, 47). Therefore, by producing IL-21, CD4 and CD8 T cells as well as NKT cells may support the proliferation of NK cells under pathological conditions.

Although many acellular protocols were developed, the numerical expansion of NK cells is more effective in the presence of accessory cells (48). Starting from a PBMC population compared with a purified NK cell cultivation yielded a larger number of NK cells and required cell–cell contact with CD14+ cells as well as soluble factors (49). Our data emphasize the importance of cell–cell interaction between aAPCs and NK cells and are supported by CD137L being a membrane-associated costimulator receptor ligand, and its expression level, either low in parental K562 cells or high in aAPCs forced to express it, is correlated with low or high NK cell proliferation, respectively. Furthermore, the low cytokine-driven NK cell proliferation supported by the parental K562 tumor cell line could be entirely abolished by the presence of a CD137 IgG1 Fc fusion protein. The high cytokine-driven numerical expansion induced by the aAPC forced to express CD137L could be mimicked by an agonist anti-CD137 Ab in the presence of the parental K562 tumor cell line. However, in its absence, the agonistic Ab has no effect on the proliferation of NK cells. Furthermore, at steady-state, IL-15 is membrane associated and bound to its high-affinity IL-15RA. The expression of IL-15 is very low and cannot be detected by any standard procedure (50). However, using IL-15–CFP knock-in mice, niches where IL-15 transcription can occur were identified, such as medullary thymic epithelial cells with high MHC class II expression, stromal cell subsets (bone marrow, lymph node, and spleen), and blood endothelial cells. Under pathological conditions, IL-15 is expressed by a wide range of cell types, leading to the hypothesis that it could be a danger signal (51) as supported by our data (Fig. 8). Pathological conditions may be more relevant to our settings than steady-state conditions, and membrane-bound IL-15 can be replaced by either soluble IL-2 or IL-15 but requires the presence of the parental K562 tumor cell line. Although we found no difference between soluble and membrane-bound IL-15, the latter must be more stable and efficient at sustaining NK cell proliferation because it may reach a higher concentration in the immunological synapse. Coexpression of IL-15 and IL-15RA by dendritic cells is required for NK cell activation (35, 52). Similarly, in our hands, IL-15 sustained NK cell proliferation and survival only in the presence of either the parental K562 tumor cell line or aAPCs. Finally, IL-2, IL-15, IL-21, and an agonistic anti-CD137 Ab in the absence of the K562 cell line or aAPCs, could not sustain the proliferation and survival of NK cells (Figs. 4, 5). Therefore, it seems that the coengagement of the cytokine receptors and CD137, although necessary, is not sufficient to sustain NK cell proliferation. Other membrane proteins expressed on the surface of the K562 tumor cell line are required to engage an unidentified receptor on the NK cells. It is tempting to speculate that, in addition to integrins and adhesion molecules, a signal allowing the discrimination between normal or stressed cells may control the proliferation of NK cells. The identification of such a ligand, if it exists, which could be compared with the complex MHC/peptide required to activate T cells, would have a wide therapeutic application in antitumor immunity.

Disclosures

All authors are full-time employees of Sanofi.

Acknowledgments

We thank Bruno Dumas for help in gene synthesis and scientific discussion as well as support and Christophe Marcireau for providing the PiggyBac transposon vector and help in designing the cloning strategy. We also would like to acknowledge Marc Trombe’s help with K562 CD86/CD137L aAPC cell sorting. We would like to express our gratitude to Nizar El-Murr, Georges Azar, Julie-Ann Gavigan, and Dmitri Wiederschain for editing the manuscript and providing supportive comments. We also wish to deeply thank Marie Mangin, Mathieu Boucher, Fanny Windenberger, and Laurent Andrieu for carrying out the statistical analysis. The authors acknowledge the healthy blood donors who made this work possible.

Footnotes

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    aAPC
    artificial APC
    ADCC
    Ab-dependent cellular cytotoxicity
    ALL
    acute lymphoblastic leukemia
    CAR
    chimeric Ag receptor
    FDA
    Food and Drug Administration
    RT
    room temperature.

  • Received August 16, 2018.
  • Accepted May 27, 2019.

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