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Keywords:

  • Head and neck cancer;
  • Induced regulatory T cells;
  • NK cell;
  • Perforin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

NK cells play a crucial role in the eradication of tumor cells. Naturally occurring (n) Treg cells and induced (i) Treg cells are two distinct Treg subsets. While the interaction of nTreg cells with NK cells has been investigated in the past, the role of tumor iTreg cells in the modulation of NK-cell function remains unclear. Tumor iTreg cells were generated from CD4+CD25 T cells in the presence of autologous immature DCs, head and neck cancer cells and IL-2, IL-10, and IL-15. The effect of iTreg cells and nTreg cells on the expression of NKG2D, NKp44, CD107a, and IFN-γ by NK cells, as well as NK tumor-cytolytic activity, were investigated. iTreg cells — similar to recombinant TGF-β and nTreg cells — inhibited IL-2-induced activation of NK cells in the absence of target cell contact. Surprisingly, and in contrast to nTreg cells, iTreg cells enhanced NK-cell activity elicited by target cell contact. The cytolytic activity of NK cells activated by iTreg cells was mediated via perforin and FasL. We conclude that tumor iTreg cells inhibited IL-2-mediated NK-cell activity in the absence of target cells, whereas the tumoricidal activity of NK cells was enhanced by iTreg cells. Our data suggest a complex, previously not recognized, differential regulation of human NK activity by iTreg cells in the tumor microenvironment.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Natural and (tumor)-induced regulatory T cells (nTreg and iTreg cells, respectively) represent subpopulations of T regulatory cells involved in the maintenance of self-tolerance and prevention of autoimmunity 1. iTreg cells (also called Tr1 cells) are induced by (tumor-) antigen-stimulation via an IL-10-dependent process in vitro and in vivo. Through secretion of the immunosuppressive cytokines IL-10 and TGF-β, iTreg cells suppress T-cell proliferation and downregulate co-stimulatory receptors and cytokine production of APCs (e.g. DCs) 2. We and others have reported that both Treg subsets accumulate in the blood and tumors of patients with cancer of different entities and contribute to the progression of disease and reduced survival 3–5.

NK cells represent innate effectors and protect the host against foreign invaders such as viruses, parasites, bacteria, or transformed cells 6. Following stimulation, NK cells release large amounts of immunostimulatory cytokines including IFN-γ and TNF-α, and trigger target cell death through the perforin/granzyme pathway or extrinsic pathways of apoptosis (Fas/FasLigand or TRAIL) 7. Expression of activating or inhibitory receptors on NK cells enables self and non-self recognition 8. The NK group family receptor (e.g. NKG2D), the killer cell immunoglobulin-like receptors (KIR, e.g. CD158a and CD158b) and the natural cytotoxicity receptors (e.g. NKp44) coordinate recognition and killing of target cells while avoiding the destruction of autologous healthy tissues 9. Depending on the balance between inhibitory and activating signals engaged by ligands expressed on tumor cells, NK cells are triggered to kill or to ignore target cells. For example, NKG2D interacts with its ligands major histocompatibility complex (MHC) class I-related chains (MICs) A and B (MICA and MICB), contributing to the control of epithelial tumors. In cancer patients, NK cell activation can be hampered by tumor-mediated shedding of MICs 10.

Recently, it has been reported that nTreg cells suppress NK cell effector functions in vitro and in vivo 11, 12. Ghiringhelli et al. have shown that Treg cell-derived TGF-β inhibits NK cell cytolytic activity and downregulates NKG2D but does not inhibit the production of IFN-γ by NK cells stimulated by IL-2Rγ-chain-dependent cytokines 11. Surprisingly, the studies focusing on the interaction of iTreg cells and NK cells are not available, so far.

In this study, we determined how tumor iTreg cells modulate NK cell function. We provide evidence that in a human in vitro system iTreg cells promote perforin and FasL-dependent cytotoxicity of non-activated NK cells, while IL-2-mediated NK cell activation was inhibited in the presence of iTreg cells. Our data provide new insights into the complex regulation of human NK cells in the tumor microenvironment.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Tumor-induced regulatory T cells share properties with iTreg cells and Tr1 cells found in vivo

iTreg cells used here have been generated according to a protocol described earlier 13 and showed a purity of >99%. They are known to express the inhibitory cytokines IL-10 and TGF-β at high levels, but — in contrast to nTreg cells — they do not express CD25 (IL-2Rα). This phenotype is found in iTreg cells/Tr1 cells of patients with cancer or autoimmune diseases 4, 14–16 (Fig. 1A). Thus, the iTreg cells generated here — in an in vitro model mimicking the tumor microenvironment — displayed typical iTreg cell-/Tr1 cell properties. As shown in Fig. 1B, iTreg cells inhibited the proliferation of activated CD4+ T cells (from 100 to 8%) significantly.

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Figure 1. Phenotypic and functional characteristics of tumor iTreg cells. CD4+CD25 T cells were generated by 10 days in co-culture with autologous immature DCs and mitomycin C-treated PCI-13 cells and in the presence of IL-2, IL-10, and IL-15. (A) Shown are results from flow cytometric analysis of TGF-β and IL-10 expression on CD4+ T cells. Isotype control is depicted as gray histograms; specific antibody as white histograms. (B) Autologous CD4+ responder T cells were labeled with fluorescent CFSE and stimulated with plate-bound anti-CD3 and anti-CD28 in the presence of IL-2 for 5 days. Histograms show proliferation of T cells alone and those co-cultured with iTreg cells at the indicated ratios. Results shown are representative of five individual experiments with different donors.

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Tumor iTreg cells impair IL-2-mediated activation of NK cells

NK cells express a large number of different cell surface receptors that deliver activating signals including the transmembrane receptor NKG2D and the natural cytotoxicity receptor NKp44 17, 18. IL-2-activated NK cells showed 3.8- and 10.7-fold increased expression of NKG2D (Fig. 2A) and NKp44 (Fig. 2B) compared with basal expression of non-stimulated NK cells, respectively. IL-2-induced activation of NK cells was significantly inhibited by tumor iTreg cells, but not by control CD4 T cells, in terms of reduced expression of NKG2D and NKp44 from 3.8- to 1.8-fold and from 10.7- to 3.9-fold, respectively. Also, incubation of IL-2-activated NK cells in the presence of nTreg cells resulted in a significant inhibition of upregulation of NKG2D (2.6–2.0; p=0.01). Similarly, the expression of NKp44 on NK cells was inhibited by nTreg cells in all experiments but without reaching statistical significance (Fig. 2A and B). In agreement with previously published work, which showed a TGF-β-mediated modulation of NK cells by nTreg cells 11, 19, IL-2-activated NK cells cultured in the presence of 1 ng/mL TGF-β, showed no induction of NKG2D.

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Figure 2. IL-2-mediated activation of NK cells is impaired by iTreg cells and nTreg cells. (A and B) NK cells were activated with IL-2 for 36 h in the presence or absence of autologous iTreg cells, nTreg cells, CD4+ T cells (1:2 each), or rhTGF-β (1 ng/mL). Expression levels of (A) NKG2D and (B) NKp44 on CD4CD16+ NK cells were determined by flow cytometry. Graphs show fold-increase of antigen expression compared with basal level (first bar of each histogram). Arithmetic mean values of fold increase+SD from six (nTreg cells and iTreg cells in A and nTreg cells in B) and seven (iTreg cells) independent experiments are shown. (C) After 36 h co-incubation, the supernatants were collected and IFN-γ concentration was measured by ELISA. Results are presented in fold-decrease compared with IL-2-activated NK cells alone. Arithmetic mean values of fold increase+SD from three independent experiments are shown. (D) After 24 h co-incubation, the degranulation marker CD107a on NK cells was measured by flow cytometry. Data are presented as fold increased expression of CD107a compared with that of non-stimulated NK cells. Arithmetic mean values of fold increase+SD from seven independent experiments are shown. Statistical significance was determined by the paired Student's t-test.

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IL-2 activation of NK cells resulted in a substantial release of IFN-γ after 36 h. Both Treg subtypes and TGF-β, which served as a positive control in this assay (data not shown) 20, impaired IL-2-induced IFN-γ secretion from NK cells, with the effect of nTreg cells on NK cells being less prominent (Fig. 2C).

Cytotoxicity of NK cells is mediated by granule exocytosis and the release of perforin and granzymes to kill virally infected or neoplastic cells. A sensitive marker for NK cell granule exocytosis is CD107a, also referred to as lysosomal-associated membrane protein-1 (LAMP-1), which is increased following NK cell activation. Treatment of NK cells with IL-2 resulted in strong degranulation (4.5-fold compared with basal expression) in terms of upregulation of CD107a assessed by flow cytometry (Fig. 2D). Co-culture with both iTreg cells and nTreg cells as well as rh-TGF-β significantly downregulated the IL-2-induced CD107a expression almost to basal levels (p<0.01; Fig. 2D and data not shown).

Tumor iTreg cells enhance target cell killing by NK cells in the absence of IL-2 pre-activation

After we have shown the interference of iTreg cells and nTreg cells with IL-2-induced NK activation, we next investigated the activation of NK cells by tumor target cell contact. To specifically focus on NK activation induced by target cell contact only, we performed these experiments in the absence of IL-2 stimulation.

Co-culture with Colo699 adenocarcinoma cells slightly induced degranulation (expression of CD107a) compared with non-stimulated NK cells (Fig. 3A). To our surprise, the addition of iTreg cells significantly enhanced degranulation of NK cells (10.4% versus 39.5%; p<0.001). In contrast, co-culture of NK cells with target cells in the presence of nTreg cells did not result in enhanced degranulation (Fig. 3A). Enhanced NK activity in the presence of iTreg cells was confirmed in a chromium release assay showing stronger lysis of target cells under these conditions (15.8% versus 38.1% at effector target ratio 5:1; p<0.001; Fig. 3B). We observed comparable effects in terms of NK cell degranulation, when additional tumor cell targets including Colo699, positive or negative for MICA, the human erythroleukemia line K562 or the squamous cell carcinoma line PCI-13 was used. Furthermore, the iTreg cell induction protocol was modified and the cell line Colo699 was used instead of PCI-13. In all conditions, iTreg cells significantly enhanced NK cell degranulation (Fig. 3C). We also added the supernatant of anti-CD3-activated iTreg cells to NK cells to evaluate an iTreg cells derived soluble factor responsible for iTreg cell–NK cell interaction but could not detect significant effects on NK cell degranulation. Further, iTreg cells were tested negative for surface expression of potentially NK activating NKG2D ligands, ULBP1, ULBP2, ULBP3, MICA, and MICB (data not shown).

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Figure 3. Tumor induced iTreg cells promote NK-cell cytotoxicity. NK cells were co-cultured overnight with iTreg cells (1:2) or nTreg cells (1:2) without additional IL-2. (A) Colo699 tumor cells were added as NK target cells for additional 6 h on the following day. Degranulation was measured as the percentage of CD107a+ CD56+ NK cells. Arithmetic mean values of fold increase+SD from four (nTreg cells) and seven (iTreg cells) independent experiments are shown. (B) Influence of iTreg cells on NK-cell cytotoxicity against Colo699 tumor cells was assessed in a 6-h chromium release assay. NK cells were co-cultured overnight with iTreg cells (1:2). On the following day, chromium-labeled tumor cells were added to the co-culture at the indicated effector (NK): target cell ratios. Data are presented as mean of seven independent experiments+SEM. (C) NK cells were co-cultured with iTreg cells overnight. Different tumor cells were used as targets and added for an additional 6 h to the co-culture. iTreg cell generation was performed as indicated in the figure with PCI-13 or Colo699 (MICA) tumor cells. Percentages in the upper right quadrants indicate degranulation of NK cells (CD56+CD107a+). Dot plots are representative of 2–7 experiments. Statistical significance was determined by the paired Student's t-test.

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Induced Treg cells promote perforin- and FasL-dependent natural NK cytotoxicity

Next, we sought to identify the cytolytic effector mechanism responsible for the increased cytotoxicity of NK cells when co-cultured with iTreg cells. To exclude that iTreg cells themselves exhibit cytotoxicity on tumor cells, we tested iTreg cells for the expression of perforin and FasL as well as their capacity to lyse tumor cells. iTreg cells neither expressed perforin nor FasL nor induced tumor cell lysis when co-cultured with tumor cells (data not shown). To investigate if the observed enhanced cytotoxicity of NK cells is mediated by soluble factors, we added the supernatant of iTreg cells/NK cells co-cultures to the tumor cells but could not detect any tumor cell lysis (data not shown). These observations suggested that tumor cell lysis is mediated by a direct cell–cell interaction between NK cells and target cells; thus, we used concanamycin A (CMA) and inhibitory antibodies to block perforin-, FasL-, and TRAIL-mediated cytotoxicity, respectively.

As depicted in Fig. 4, tumoricidal activity of non-stimulated NK cells in the absence of iTreg cells was predominantly mediated by perforin. This is illustrated by the reduction of tumor cell lysis by CMA (Fig. 4A), while inhibitory antibodies, which blocked FasL and TRAIL had no effect (Fig. 4B and C). Consistent with our data in Fig. 3, NK cells showed a significantly higher cytotoxicity towards tumor cells when they were co-cultured with iTreg cells overnight prior to the addition of 51-Cr-labeled target cells (triangles in Fig. 4A–C). This effect was significantly reduced if NK cells were pretreated with CMA or inhibitory antibodies to FasL, while anti-TRAIL antibodies had a minor or no effect (Fig. 4A–C). In summary, natural cytolytic activity of NK cells is mainly mediated by perforin, while death receptor pathways like FasL and TRAIL play a minor role. In contrast, iTreg cell-induced cytotoxicity of NK cells is mediated by perforin and FasL-associated pathways.

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Figure 4. Perforin and FasL – but not TRAIL – contribute to increased cytotoxicity of NK cells. NK cells were co-cultured with autologous iTreg cells overnight at a ratio of 1:2 prior to the addition of chromium-labeled tumor cells (COLO699) at the indicated effector/target cell ratios (NK cells/tumor cells). (A) To inhibit perforin-mediated cytotoxicity, NK cells were pre-incubated for 2 h with concanamycin A (CMA) previous to the addition of Cr-labeled tumor cells. (B and C) Chromium release assay was conducted in the presence of neutralizing antibodies to (B) FasL and (C) TRAIL or corresponding isotype controls. Data are presented as mean±SEM of at least three independent experiments. Statistical significance was determined by the paired Student's t-test.

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In the next series of experiments, we wanted to further characterize the phenotype of NK cells after co-culture with iTreg cells to potentially explain increased NK cell activity. For that purpose, we stained iTreg cells and NK cells for the expression levels of NKG2D, NKp44, CD158a, CD158b, and perforin after 18 h co-culture. CD4CD16+ NK cells showed upregulation of the activation marker NKG2D (Fig. 5A and D) after co-incubation with iTreg cells. While the inhibitory KIRs CD158a and CD158b were not modulated on NK cells (Fig. 5B), the expression of perforin was clearly enhanced after co-culture with iTreg cells (Fig. 5C and D). These data indicate that iTreg cells are able to activate NK cells resulting in the upregulation of NKG2D and perforin in the absence of IL-2 pre-stimulation. In summary, our findings thus far demonstrate that iTreg cells impair IL-2-mediated NK activation, provided that NK cells have no target cell contact. In contrast, target cell-induced NK activation is enhanced by iTreg cells.

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Figure 5. Co-culture with iTreg cells modulates activation status of NK cells. (A–C) NK cells were co-cultured without iTreg cells (black bars) or with iTreg cells (white bars) at a ratio of 1:2 overnight and subjected to FACS analysis. The change in the median expression of (A) the activating receptors NKG2D, NKp44, and (B) inhibitory receptors, CD158a and CD158b, as well as (C) intracellular expression of perforin was analyzed on CD4CD16+ NK cells. Data are presented as mean+SD of three or four independent experiments. (D) FACS analysis of gated CD4CD16+ NK cells showing the expression of NKG2D, NKp44, and perforin by NK cells alone (gray histograms) and co-cultured with autologous iTreg cells (open curves). Shown are data from one representative experiment out of 3–4 individual donors. Statistical significance was determined by the paired Student's t-test.

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Tumor iTreg cells enhance degranulation by IL-2-activated NK cells in the presence of target cells

In the final series of experiments, we investigated NK activation induced by a combination of IL-2 and target cell contact. Under these conditions, NK degranulation was induced from 8 to 26% (p=0.02) compared with resting NK cells (Fig. 6). Induced Treg cells further promoted this NK cell function compared with IL-2-activated NK cells with tumors alone (from 26 to 54%; p=0.01, Fig. 6). In contrast, nTreg cells did not further modulate degranulation of IL-2-activated NK cells towards target cells.

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Figure 6. iTreg cells promote degranulation of IL-2-activated NK cells upon target cell contact. NK cells were co-cultured overnight with iTreg cells (1:2, left panel) or nTreg cells (1:2, right panel) in the presence of IL-2 (100 U/mL). On the following day, Colo699 tumor cells were added as NK target cells for an additional 6 h. Data are presented as percentage of CD107a (degranulation marker)-positive CD56+ NK cells. Arithmetic mean values of CD107a+ cells+SD from four independent experiments are shown. Statistical significance was determined by the paired Student's t-test.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

In agreement with previous reports, we found that IL-2 induces activation of primary human peripheral blood NK cells resulting in upregulation of activating receptors, NKG2D and NKp44, as well as increased degranulation and IFN-γ secretion 21, 22. These effects were significantly impaired in the presence of tumor iTreg cells, nTreg cells or TGF-β. Our results are in agreement with published reports, where other types of Treg cells were described to suppress NK cell functions under various experimental conditions, in most instances in a TGF-β-dependent manner 11, 12, 19, 23–27.

Unexpectedly, we found that degranulation and the subsequent tumoricidal activity of naive NK cells were enhanced by iTreg cells. iTreg cell-enhanced cytotoxicity of NK cells was perforin- and FasLigand-dependent, while death receptor TRAIL was not involved. Consistent with the upregulation of activating receptors NKG2D and NKp44, the expression of inhibitory KIRs CD158a and CD158b on NK cells remained at basal levels in the co-culture with tumor iTreg cells. In conjunction, these data suggest that tumor iTreg cells negatively interfere with IL-2-mediated NK-cell activation, while the IL-2-independent activation of NK cells by target cell contact is augmented in the presence of iTreg cells. Importantly, the activation of NK cells by a combination of IL-2 and target cell contact is further promoted in the presence of iTreg cells.

It is well established that NK cells activated by IL-2 are highly cytolytic to many tumor targets and thus NK cell-activating cytokines like IL-2 are frequently incorporated into current immunotherapeutic strategies and clinical trials 28. We found that tumor iTreg cells interfere with IL-2-mediated activation of NK cells in the absence of target cells (tumor cells). Therefore, our findings may have potential relevance in therapeutic settings, where IL-2 stimulation is used and considerable numbers of iTreg cells are present in the circulation or the malignant tissue. In these cases, tumor iTreg cells could limit the target cell-independent effects and possibly side-effects of IL-2-activated NK cells. According to our data, this effect of iTreg cells would, for example, affect target-cell-independent cytokine secretion of NK cells. By our experiments we cannot determine whether the inhibitory activity of iTreg cells also requires the activation of iTreg cells by IL-2, which is present in the system. On the other hand, we feel that our system reflects a physiological situation, such as therapeutic IL-2 application, where both NK and iTreg cells will be simultaneously exposed to the cytokine. In this situation, iTreg cells will inhibit NK in the absence of target (Fig. 2), while in the presence of target cells iTreg cells will be non-inhibitory and rather enhance NK degranulation (Fig. 6).

In contrast, iTreg cells seemed to promote natural cytotoxicity of unstimulated resting NK cells. This situation reflects the steady-state or homeostatic conditions within a given tumor tissue or tumor microenvironment. The clinical correlates for our in vitro findings are those patients and clinical studies of solid as well as non-solid tumors in which investigators found tumor-infiltrating Treg cells to be a good prognostic factor 29–32. Examples include lymphomas as Hodgkin lymphoma where investigators found a positive correlation between high Treg cell infiltration and higher rates of survival 32. Consistent with our in vitro data, other groups have reported that an improved survival was associated with high density of tumor-infiltrating FoxP3+ Treg cells in colorectal cancer 30, 33. Further, Badoual et al. reported that Treg cells are positively correlated with locoregional control in patients with head and neck cancer. They concluded that this effect may be facilitated by Treg cells which downregulate harmful inflammatory reactions, which could favor tumor progression 29. Our data suggest that an additional mechanism to explain these findings may be direct activation of naive NK cells by tumor iTreg cells.

On the other hand, many clinical studies suggest that Treg cells contribute to tumor-induced immune suppression, and elimination of Treg cells may represent a possible new therapeutic option 5, 34. However, at present there is no clear evidence from human clinical trials demonstrating the clinical efficacy of this approach. It is important to note that tumor-induced Treg cells may have different effects in the natural tumor microenvironment and the immunotherapeutic setting. This is reflected by the differential effect of iTreg cells on IL-2-stimulated versus unstimulated NK cells in our study. Thus, depletion of Treg cells alone or in combination with immunotherapy may have different outcomes 35–37.

Multiple cellular communication molecules and pathways, including the NKG2D-MICA system, may be involved in iTreg cells-NK cell cross-talk 38. It was shown that the engagement of NKG2D on activated T cells and NK cells promoted antitumor NK and T-cell responses against epithelial MICA+ tumor cells 39. We also observed stronger killing of MICA+ tumor cells compared with MICA cells and induction of NKG2D on NK exposed to iTreg cells. However, iTreg cells enhanced NK cell cytotoxicity against tumor cell targets independent of MICA expression on target cells (Fig. 3C). Also, we could not detect any NKG2D ligands on iTreg cells, which suggests that a mechanism other than direct NKG2D-ligand interaction is involved in the iTreg cell–NK cell cross-talk. Further, it was reported that NK cells can spontaneously lyse certain transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. It was recently reported that NK cells were capable of lysing pathogen-induced Treg cells, which expressed UL16-binding protein 40. In contrast, it was demonstrated that Treg cells in a tumor microenvironment kill NK cells in a granzyme-B-dependent fashion 41. It was even shown that NK cells are able to induce Treg cells, which resulted in immune suppression 42, and underscores the complex cross-talk between these two immune cell subsets. Although we have not yet identified the molecular mechanism of NK activation by iTreg cells, our data suggest that direct contact between both cell types is required. We have also observed that the parallel execution of the perforin and the FasL cytolytic pathway is utilized by iTreg cell-activated NK cells.

To our knowledge, this is the first report about enhancement of anti-tumoral NK cell-function which is mediated by induced regulatory T cells. Without any doubt, still much has to be learned about the interaction of NK cells and regulatory T cells in the tumor microenvironment. A better understanding of the cellular cross-talk between regulatory T cells and cells of the innate immune system will aid future rationale therapeutic manipulation of this T-cell subset in cancer therapy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Ethics statement

This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board (Ethical Committee) of University of Duisburg-Essen. Blood donors provided written informed consent for the collection of samples.

Reagents and cell culture conditions

Fetal calf serum (Biochrom AG, Berlin, Germany) was heat inactivated for 30 min at 56°C (ΔFCS). RPMI 1640 culture medium, L-glutamine, streptomycin, and penicillin were purchased from Invitrogen (Karlsruhe, Germany). The following mAbs were used for flow cytometry: anti-CD4-PerCP, anti-CD16-Pe-Cy7, anti-CD56-PE, anti-CD107a-FITC, anti-CD25-PE (Dako, Berlin, Germany), anti-CD25-FITC (Immunotools, Friesoythe, Germany), anti-IL-10-PE, anti-NKG2D-PE, anti-TGF-β (R&D Systems, Wiesbaden, Germany), anti-NKp44-PE (Beckman Coulter, Krefeld, Germany), and anti-TRAIL (Acris, Herford, Germany). All corresponding isotypes were purchased from BD Bioscience (Heidelberg, Germany). For intracellular staining, the BD Cytofix/Cytoperm Kit (BD Bioscience) was used. For stimulation, we used anti-CD3 mAb from Beckman Coulter, rh-IL-2 from Stratmann (Hamburg, Germany), rh-GM-CSF and rh-IL-4 from R&D Systems, rh-IL-10, rh-IL-15, and rh-TGF-β from Peprotech-Tebu (Frankfurt, Germany).

For cell culture assays, complete medium (Rxx10) consisting of RPMI 1640 supplemented with 10% v/v ΔFCS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and L-glutamine (2 mmol/L) was used.

All cells were cultured in this medium and incubated in a humidified atmosphere at 37°C with 5% CO2.

Separation of PBMCs and purification of NK cells, T cells, and generation of immature DCs

With the permission and supervision of the Local Ethical Committee, human peripheral blood mononuclear cells (PBMCs) were purified from heparinized venous whole blood from healthy donors by density gradient separation using Biocoll according to manufacturer's guidelines (Biochrom AG).

NK cells were purified from PBMCs using NK Cell Isolation Kit from Miltenyi Biotec (Bergisch Gladbach, Germany) according to the manufacturer's instructions to deplete non-NK cells. The purity of NK cells was confirmed by flow cytometry, and contamination with T cells and B cells was always below 1%.

CD4+CD25 T cells were isolated from PBMCs using Regulatory T Cell Separation Kit from Miltenyi Biotec according to the manufacturer's instructions. CD4+CD25 T cells were used for generation of autologous responder T cells.

CD4+CD25+ nTreg cells were separated from PBMCs using the CD4+CD25+ regulatory T cell Isolation Kit from Miltenyi Biotec according to manufacturer's instruction. To this end, lymphocytes were depleted of non-CD4+ T cells and positively selected for CD4+CD25+ T cells.

Monocytes within PBMCs were separated from lymphocytes by plastic adherence. Monocytes were differentiated into immature DCs (iDCs) within 7 days in the presence of IL-4 and GM-CSF (500 IU/mL each with medium change on days 3 and 5).

Tumor cell lines

PCI-13 cells, a HLA-A2+ human squamous cell carcinoma of the head and neck (HNSCC), were used to generate tumor iTreg cells. PCI-13 was a kind gift from the Whiteside Laboratory at the University of Pittsburgh Cancer Institute 43. Colo699 (human lung adenocarcinoma cell line) cells were used as target cells in cytotoxicity assays. Transduction of cells with an adenovirus encoding the human NKG2D-ligand MICA (Ad-MICA) was performed earlier in our laboratory 44. The human erythroleukemia line K562 was obtained from DSMZ (Braunschweig). All tumor cell lines were routinely tested and confirmed to be mycoplasma free.

iTreg cell generation

CD4+CD25 T cells were co-cultured with autologous iDCs and mitomycin C treated (0.5  mg/mL, for 30 min) PCI-13 cells at a ratio of 10:1:1 with 106 T cells/mL in Rxx10 medium for 10 days. IL-2 (10 IU/mL), IL-10 (20 IU/mL), and IL-15 (20 IU/mL) were added on days 0, 3 and 6. On day 9, culture medium was replaced by fresh medium containing anti-CD3 antibody (1 μg/mL). On day 10, lymphocytes were harvested and used for phenotypic, functional analyses or co-culture assays.

Proliferation assay

CD4+CD25 T cells were fluorescently labeled using carboxyfluorescein diacetate succinimidyl ester (CFSE) according to manufacturer's instructions (CellTrace from Molecular Probes/Invitrogen). iTreg cells and autologous CFSE-labeled CD4+ T cells were co-cultured at different ratios for 5 days in the presence of plate-bound anti-CD3 (1 μg/mL), anti-CD28 (2 μg/mL), and IL-2 (100 IU/mL). Proliferation of CFSE-labeled CD4+ T cells was assessed by flow cytometry and data analysis was performed using Diva6 software.

Flow cytometry

For phenotypic analysis, cultured cells were harvested, washed, and incubated in PBS (supplemented with 3% human serum and 0.1% sodium azide) containing optimal dilution of each fluorochrome-conjugated mAb for 25 min at 4°C in the dark. Cells were subsequently washed and fixed with 2% v/v paraformaldehyde (PFA) or proceeded to intracellular staining for TGF-β or IL-10. For intracellular staining, washed cells were incubated with BD Cytofix/Cytoperm solution for 30 min at 4°C in the dark, washed twice with BD Perm/Wash buffer, and incubated with fluorescent-labeled mAbs diluted in the same buffer for 25 min at 4°C in the dark. All mAbs used were pre-titrated on fresh PBMCs to determine their optimal working dilutions. Respective isotype controls were used in all experiments. After staining, cells were immediately subjected to measurement in a FACS Canto II flow cytometer. The collected data were analyzed with Diva6 software (both from Becton Dickinson, Heidelberg, Germany).

Interaction of Treg cells with IL-2-activated NK cells

NK cells were activated with IL-2 (100 IU/mL) and co-cultured with control iTreg cells, nTreg cells, or CD4 cells at a ratio of 1:2 (NK cell/T cell). After 36 h, supernatants and cells were harvested. Surface expression of NKG2D and NKp44 on NK cells was assessed, and supernatants were analyzed for IFN-γ by ELISA according to manufacturer's guidelines (R&D Systems). In some experiments, rh-TGF-β (1 ng/mL) was added to IL-2-activated NK cells.

NK cells were activated or not with IL-2 (100 IU/mL) and co-cultured with autologous nTreg cells, iTreg cells, or with TGF-β for 18 h. CD107a FITC and in some experiments Colo699 tumor cells were added for further 6 h to the co-culture system. During the last 5 h, the BD Golgi-Stop was present in the co-culture. At the end, NK cells were stained with CD56-PE as described in the section Flow cytometry and analyzed by flow cytometry. CD107a (LAMP-1) is a marker for NK degranulation and its level of expression correlates with NK cytotoxicity 45.

Chromium release assay

Cytotoxicity was determined in a standard 6-h chromium release assay. NK cells were seeded in 96-well plates at different concentrations to be used as effector cells and were incubated for 18 h with or without iTreg cells at a ratio of 1:2 (NK/iTreg cells). Target cells were labeled with Na251CrO4 (Hartmann, Analytik, Braunschweig, Germany) for 1.5 h at 37°C, washed, and added at a concentration of 1×105 cells/well resulting in the indicated effector/target ratios.

To study the underlying mechanisms of NK cell induced tumor cell death, neutralizing anti-FasL (BD Pharmingen), anti-TRAIL (BioVender), or isotype control antibody was added to the co-culture system. To inhibit perforin-mediated cytolysis, CMA (Sigma-Aldrich, Taufkirchen, Germany) was added to the NK cells 2 h prior to co-culture with target cells. The radioactive content of the supernatant was measured in a gamma counter (Berthold, Wildbad, Germany). Specific lysis was determined according to the following formula: specific lysis (%)=100×(Exp−Spo)/(Max−Spo), where Exp is the experimental release, Spo is the spontaneous release, and Max is the maximum release. Assays were performed as triplicates/quadruplicates, and data are depicted as means±standard deviation (SD).

The experimental design of the Treg cell-NK co-culture experiments is illustrated in the Supporting Information Fig. S1.

Statistical analysis

Student's t-test for means (two-tailed, paired samples) from at least three individual experiments was used to calculate significance, and p-values equal or below 0.05 were considered as significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Kirsten Bruderek for her excellent technical assistance. We also thank Johannes Schulte for his help with the chromium release assays. Antibodies directed against ULBP1, ULBP2, ULBP3, MICA, and MICB were a kind gift from Annette Paschen (UK Essen). Research described in this article was supported in part by the IFORES program of the Medical Faculty, University Duisburg-Essen (to S. B.) and the Deutsche Forschungsgemeinschaft (DFG 4190/1-1 to C. B.).

Conflict of interest: The authors have declared no conflict of interest.

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  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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