Cutting Edge: Tumor-Targeting Antibodies Enhance NKG2D-Mediated NK Cell Cytotoxicity by Stabilizing NK Cell–Tumor Cell Interactions

Jacques Deguine, Béatrice Breart, Fabrice Lemaître and Philippe Bousso
J Immunol December 15, 2012, 189 (12) 5493-5497; DOI: https://doi.org/10.4049/jimmunol.1202065

Abstract

Monoclonal antibodies represent a promising approach to fight a variety of tumors, but their mode of action remains to be fully understood. NK cells can recognize Ab-coated targets, as well as stress ligands, on tumor cells. In this study, we investigated how NK cells integrate both kinds of activating signals. NK cell–mediated killing was maximal with the combined recognition of NKG2D ligands and Ab; surprisingly, only NKG2D engagement substantially enhanced degranulation. Conversely, Ab recognition by NK cells uniquely increased contact stability with tumor cells. Furthermore, using intravital imaging of solid tumors, we showed that Ab recognition favored prolonged interactions between NK cells and targets. Altogether, our results demonstrate that NK cell–mediated killing can be differentially regulated at the level of degranulation and contact stability by distinct activating receptors. Thus, complementary signals mediated by recognition of stress ligands and tumor-specific Abs may contribute to the efficacy of NK cells during mAb therapy.

Introduction

In recent years, mAb therapies have been increasingly used in the clinic with promising success rates and good safety profiles (1). Tumor-targeting Ab can act in a variety of ways, including direct inhibition of signaling pathways, complement activation, and cell-mediated Ab-dependent cytotoxicity (ADCC). Polymorphisms in the receptors for the common portion of Igs FcγR and, in particular, FcγRIII appear to be important factors in therapy efficiency, suggesting a critical role for ADCC (2, 3). FcγRIII is expressed by several immune cells, including macrophages, neutrophils, and NK cells (4). In leukemia models, therapies using a combination of rituximab and IL-2 for NK cell activation suggested a substantial contribution of these cells to tumor elimination (5, 6).

In addition to FcγRIII, NK cells express a variety of activating and inhibitory receptors involved in target recognition (7). Among NK cell–activating receptors, NKG2D was shown to play an important role in tumor cell rejection and tumor immunosurveillance (8–10). Indeed, ligands for this receptor are often overexpressed on transformed cells because their expression can be induced by oncogenes (11) or through the DNA damage response (12).

Activating receptors can act in synergy to drive NK cell responses (13). However, the precise mechanisms underlying this synergy remain poorly understood and could range from a simple addition of activating signals to the cooperation of receptor-specific pathways. To address this question, we took advantage of a solid tumor model that can be targeted through depleting Abs and/or recognition of NKG2D ligands. Although either signal was sufficient to drive NK cell accumulation into the tumor and their local activation, both signals were necessary for rapid tumor cell elimination. In vitro experiments revealed that, although NKG2D signaling enhanced degranulation, it had little influence on adhesion to target cells, whereas FcR engagment improved adhesion but not degranulation. Using two-photon microscopy, we showed that recognition through FcR affected intratumoral NK cell behavior and promoted long-lasting interactions with targets in vivo, whereas NKG2D signals alone failed to promote NK cell arrest, as we described previously (14). Altogether, our results uncover a novel aspect of cooperation between activating receptors in NK cells and suggest that cell dynamics represent a critical regulator of cell-mediated cytotoxicity.

Materials and Methods

Cell lines

EL4 and EG7 tumor cell lines were obtained from American Type Culture Collection and transfected with membrane-bound CFP and YFP, as described previously (15). Because CD4 levels were heterogeneous on EL4 cells, we selected a clone that did not express CD4, which was used as a negative control. Expression of Rae-1β was induced by retroviral infection of EG7 YFP cells using PlatE packaging cells and a plasmid encoding Rae-1β provided by D. Raulet (University of California, Berkeley, Berkeley, CA). Following transduction, cells were cloned, and one clone expressing Rae-1β and displaying YFP and CD4 levels identical to the parental EG7 YFP cell line was selected.

Mice

C57BL/6 mice were obtained from Charles River France. Ncr1GFP/+ knock-in mice (16) were bred in our animal facility; perforin-deficient mice (Pfp−/−) were a kind gift from G. De Saint-Basile and F. Sepulveda (Hôpital Necker, Paris, France). All procedures were performed in agreement with the Pasteur Institute's institutional guidelines for animal care.

Flow cytometry

For tumor injections, mice were anesthetized, and a mixture of 2 × 106 EG7 YFP or EG7 Rae-1β YFP tumor cells and 2 × 105 EL4 CFP tumor cells was injected s.c. in the left leg. When indicated, 100 μg functional grade anti-CD4 Ab (clone GK1.5; BioLegend) was injected i.v. 4 d posttumor injection. Tumor cells and spleen were harvested 24 h later and dissociated in RPMI 1640 containing 1 mg/ml collagenase and 0.05 mg/ml DNAse (Sigma). After blocking of FcRs with anti-CD16/32, cells were stained with e450–anti-CD3 (17A2), PE–Cy7–NK1.1 (PK136), and PercP–Cy5.5–anti-CD11b (M1/70; eBioscience). For intracellular staining, cells were fixed using the Cytofix/Cytoperm kit (BD Biosciences) and stained with allophycocyanin–anti-granzyme B (GB11; Caltag). Acquisitions were performed on a FACS Canto II (BD) and analyzed with FlowJo software (TreeStar).

Cell conjugation, degranulation, and killing assays

Splenic NK cells were purified from C57BL/6 mice that were injected i.p. with 50 μg polyinosinic-polycytidylic acid 18 h earlier. Purification was performed by negative selection using an NK cell isolation kit (Miltenyi Biotec), yielding purities > 80%. When indicated, tumor cells were coated with GK1.5 at 20 μg/ml for 20 min at 4°C, washed, and resuspended in complete RPMI 1640. Effector cells and target cells were mixed at a 2:1 ratio and centrifuged for 1 s at 300 × g. After 6 h, absolute cell numbers were determined using counting beads (Invitrogen), and specific lysis was determined as 100 × (1 − [cell number in presence of effectors/cell number in absence of effectors]). To assess degranulation, cells were incubated for 4 h in the presence of Alexa Fluor 647–conjugated anti-CD107a. Cells were then washed, resuspended in PBS 0.5% FCS containing e450–anti-NK1.1, and incubated at 4°C before washing and FACS acquisition. For clustering experiments, NK cells and target cells were mixed at a 1:4 ratio in complete RPMI 1640, centrifuged for 1 s at 300 × g, and incubated for 1 h at 37°C before immediate acquisition.

Intravital imaging and immunofluorescence

Imaging of tumors was performed as described before (14, 15) using an upright microscope (DM6000B; Leica) with a water-dipping 25×/1.05 NA objective (Olympus). Excitation was provided with a Ti:sapphire laser (Coherent) tuned at 950 nm. Signals were collected using four nondescanned detectors (Leica) and dichroic filters and mirrors (513/20 for GFP, 542/25 for YFP, mirror 520; Semrock). For immunofluorescence, sections were stained with anti-cleaved caspase 3 Ab (Cell Signaling), followed by DyLight649–anti-rabbit IgG (Jackson ImmunoResearch). Videos were analyzed using Imaris software (Bitplane).

Statistical analysis

All data are presented as mean ± SEM. Statistical analyses were performed using Prism software (GraphPad) and the unpaired Student t test (comparison of two samples) or ANOVA followed by the Tukey posttest (comparison of four samples). The p values < 0.05 were considered significant.

Results and Discussion

Tumor-targeting Ab recognition enhances NKG2D-mediated killing

To study the effect of the combined engagement of NKG2D and FcR, we first induced expression of the NKG2D ligand Rae-1β in EG7 YFP tumor cells by retroviral infection (Fig. 1A). Control and transduced tumor cells expressed similar levels of the CD4 molecule; thus, both could be targeted using a depleting anti-CD4 Ab, such as GK1.5 (17). First, we assessed the NK cell–mediated lysis of control and Rae-1β–expressing cells coated with anti-CD4 Ab. Each signal was able to induce NK cell-mediated cytotoxicity, but the combination of both signals significantly enhanced tumor cell lysis (Fig. 1B). Interestingly, FcR- and NKG2D-mediated recognition appeared to have an additive effect on cytotoxicity, an observation that was confirmed by performing killing assays on target cells expressing various levels of NKG2D ligands (Supplemental Fig. 1A–C). NK cells can kill targets through several mechanisms, such as the exocytosis of lytic granules and the expression of death-inducing molecules like FasL. We found that NK cells from perforin-deficient animals were largely impaired in their ability to lyse tumor cells expressing Rae-1β and/or coated with Abs (Fig. 1C), suggesting that lytic granule secretion was the main killing pathway involved in both cases.

FIGURE 1.
FIGURE 1.

Ab treatment enhances NKG2D-mediated lysis in vitro and in vivo. (A) Expression of CD4 (left panel) and Rae-1 (right panel) in EG7 YFP and EG7 Rae-1β YFP tumor cells. Unstained controls are shown in gray. Tumor cells precoated or not with anti-CD4 Ab were cocultured for 6 h with NK cells from wild-type (B) or perforin-deficient (C) mice at a 2:1 E:T ratio. (D and E) A mixture of EL4 CFP and either EG7 YFP or EG7 Rae-1β YFP was injected s.c. into wild-type recipients. When indicated, 100 μg of anti-CD4 Ab was injected i.v. at day 4, and tumors were harvested at day 5 to measure the ratio of remaining tumor cells (D) and verify Ab coating ex vivo with anti-rat IgG (E). (F) Ratios of EG7/EL4 (left) and EG7 Rae-1β/EL4 (right) in Ab-treated animals were normalized to untreated controls. Among infiltrating cells (CFP− YFP−), NK cell (NK1.1+ CD3−) numbers (G) and intracellular granzyme B content (H) were quantified. Data are representative of two or three independent experiments that were pooled for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. ns, Not significant.

We then assessed the effect of NKG2D- and FcR-mediated lysis in solid tumors in vivo. Because NKG2D ligands alone can induce tumor cell rejection (9), we measured EG7 elimination at an early time point using a second tumor cell line that does not express NKG2D ligands or CD4 as an internal control. Although we could demonstrate efficient and specific Ab coating of EG7 YFP cells in vivo (Fig. 1D, 1E), Ab treatment alone was not sufficient to induce tumor cell rejection at this early time point. In contrast, it significantly enhanced the lysis of Rae-1β–expressing cells (Fig. 1D, 1F) (33.6 ± 14% specific lysis versus −8.4 ± 11% in the absence of Rae-1β, p = 0.039). This effect correlated with an increase in cleaved caspase 3 in tumor cells, indicating that the tumor cells likely underwent apoptosis (Supplemental Fig. 1D, 1E). Interestingly, either Ab treatment or Rae-1β expression was sufficient to increase intratumoral NK cell accumulation (Fig. 1G) and to induce the upregulation of the effector molecule granzyme B in the tumor (Fig. 1H). Altogether, we demonstrated that the combination of FcR and NKG2D engagement increased the efficiency of NK cell killing. Furthermore, our results suggest that this effect is not due to an increase in NK cell numbers, but rather to an enhanced lytic activity on a per cell basis.

NKG2D-mediated, but not FcR-mediated, recognition increases NK cell degranulation

To investigate how Ab coating modifies NKG2D-mediated killing, we assessed degranulation of NK cells in the presence of either activating signal using LAMP-1 (CD107a) surface expression as a marker. Although coculture of NK cells with EG7 Rae-1β tumor cells induced degranulation, we were surprised to find that Ab coating did not increase NK cell degranulation upon coculture with Rae-1β–expressing or control tumor cells (Fig. 2A, 2B). These results are reminiscent of a study demonstrating that distinct NK cell receptors can recruit distinct adaptors for degranulation (18). However, in contrast with this study, we found that FcR was a poor inducer of degranulation, a difference that could be linked to the method of ADCC used (direct versus redirected lysis with anti-CD16).

FIGURE 2.
FIGURE 2.

NKG2D and FcR engagement enhance degranulation and contact stability, respectively. (A) NK cells were cocultured with control (No Ab) or Ab-coated (Anti-CD4) EG7 YFP or EG7 Rae-1β YFP in the presence of anti–LAMP-1. Acquisition of YFP signal by NK cells (NK1.1+) and staining with anti–LAMP-1 were analyzed by flow cytometry. Numbers indicate the percentage of LAMP-1+ cells among NK cells. (B) Percentage of LAMP-1+ NK cells normalized to the percentage in the presence of EG7 cells without Ab. (C) Mean fluorescence intensity of YFP in LAMP-1+ NK cells divided by the mean fluorescence intensity in the presence of EG7 cells without Ab. Data are pooled from four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns, Not significant.

Using this assay, we also noticed that NK cells cocultured with YFP-expressing tumor cells acquired YFP fluorescence, a phenomenon likely reflecting trogocytosis or related processes (19). Interestingly, Ab coating of tumor cells, but not Rae-1β expression, increased YFP intensity within degranulating NK cells (Fig. 2C). Because trogocytosis occurs during cell–cell interactions, these results raise the possibility that Ab coating primarily enhances the stability of NK cell–tumor cell contacts but has little effect on the intensity of degranulation.

Ab recognition increases the stability of NK cell–tumor cell interactions

To assess this possibility and determine the influence of NKG2D ligands and Ab coating on NK cell–tumor cell contacts, we measured NK cell conjugation to tumor cells using a flow cytometry–based assay. Confirming our previous study (14), Rae-1β expression only modestly increased the conjugation of NK cells to their targets (Fig. 3A, 3B). In contrast, we observed that Ab coating of tumor cells significantly increased the formation of stable conjugates independently of Rae-1β expression, suggesting that FcR engagement was sufficient for efficient adhesion. Interestingly, we observed similar results using three distinct targets and specific Abs, indicating that this might be a general feature of NK cell responses to Ab-coated targets (Supplemental Fig. 2).

FIGURE 3.
FIGURE 3.

Ab therapy enhances NK cell–tumor cell adhesion and modifies NK cell behavior in vivo. (A and B) SNARF-stained NK cells were cocultured with control (No Ab) or Ab-coated (Anti-CD4) EG7 YFP or EG7 Rae-1β YFP tumor cells. Numbers indicate the percentage of conjugated NK cells in each condition. (C) EG7 Rae-1β YFP tumor cells (red) were injected into Ncr+/GFP mice that were left untreated (top panel) or received 100 μg anti-CD4 4 d after tumor injection (bottom panel). NK cell (green) behavior was assessed by intravital microscopy at day 5. Trajectories of individual NK cells during 30 min of imaging are represented in white and are overlaid with the first image of representative videos. Short trajectories observed in the presence of anti-CD4 indicate constrained NK cell movement. Data are representative of three individual experiments. (D) Mean velocity (left panel) and mean arrest coefficient (right panel; defined as percentage of time when velocity < 2 μm/min) of NK cells in individual movies. Each symbol represents one movie. (E) Representative example of the distribution of mean velocities (left panel) and arrest coefficients (right panel) of NK cells in an individual movie. Each symbol represents one NK cell. *p < 0.05, **p < 0.01, ***p < 0.001. ns, Not significant.

To assess the in vivo relevance of our findings, we used two-photon imaging to study NK cell behavior in response to tumor-targeting Ab. Intravital imaging of tumors was performed in Ncr1+/GFP mice, in which endogenous NK cells specifically express GFP. Specifically, we compared the dynamics of NK cells in EG7 Rae-1β tumors in the presence or absence of Ab treatment. As observed previously, NK cells remained largely motile in EG7 Rae-1β tumors. Strikingly, Ab treatment decreased NK cell velocity (6.4 ± 1.2 μm/min without treatment, 3.3 ± 0.4 μm/min after Ab injection, p = 0.009). In addition, many NK cells were arrested in the proximity of tumor cells following Ab treatment, as evidenced by their arrest coefficient (defined as percentage of time when velocity < 2 μm/min: 20 ± 5% without treatment, 53 ± 7% after Ab injection, p = 0.03) (Fig. 3C–E, Supplemental Video 1). Taken together, these results demonstrate that Ab therapy alters the dynamics of NK cells in solid tumors expressing NKG2D ligands and favors the formation of prolonged interactions between effector and target cells.

General conclusions

In the current study, we demonstrated that tumor-targeting Ab can act in concert with NKG2D ligands to promote efficient tumor elimination. Previous reports demonstrated that engagement of multiple NK cell–activating receptors results in additive or even synergistic intracellular signaling (13). Interestingly, our data suggest an additional mode of cooperation in which individual NK cell receptors control either degranulation or stability of contact. It is tempting to speculate that optimal NK cell killing is achieved when degranulation is both efficiently induced and targeted at the immunological synapse. A recent study provided evidence that a tumor-targeting Ab favors tumor conjugation to macrophages and neutrophils. Our in vivo imaging data demonstrate that NK cell interactions with tumors cells are also stabilized in the presence of a tumor-targeting Ab. Based on our in vitro experiments, we propose that Ab-mediated conjugation not only triggers ADCC, it may also improve the cytotoxic activity mediated by NKG2D or other activating receptors. Indeed, tumor cells may require extensive interactions with cytotoxic cells before elimination (20), so that enhanced conjugation improves killing efficiency. Consistent with this idea, it was reported that expression of ULBP enhanced rituximab-mediated tumor cell killing by NK cells (21). Fusion proteins between NKG2D ligands and tumor-targeting Ab were also used to increase ADCC efficiency (22). Such therapeutic strategies aiming at increasing stress ligands on tumor cells in combination with Ab treatment may benefit from the complementary effect of NK cell degranulation and arrest on tumor cells.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank D. Raulet for providing the plasmid encoding Rae-1β, members of the Bousso laboratory for helpful comments on the manuscript, and M. Hasan and the Center for Human Immunology at Institut Pasteur for technical support.

Footnotes

  • J.D. and P.B. designed the study and wrote the manuscript; J.D., F.L., and B.B. performed experiments; and J.D. analyzed data.

  • This work was supported by Institut Pasteur, INSERM, and the Fondation pour le Recherche Médicale, as well as by a European Research Council Starting Grant (“Lymphocyte Contacts”).

  • The online version of this article contains supplemental material.

  • Abbreviation used in this article:

    ADCC
    Ab-dependent cytotoxicity.

  • Received July 27, 2012.
  • Accepted October 15, 2012.

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