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Keywords:

  • cytotoxicity;
  • γδ T cells;
  • multiple myeloma;
  • NKp44;
  • natural cytotoxicity receptors

Summary

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

γδ T cells account for up to 10% of T lymphocytes in the peripheral blood of healthy donors. They can be activated by cytokines such as interleukin (IL)-2, IL-12 and IL-15, express natural killer (NK) cell markers such as NKG2D and show cytotoxic activity against several tumour cells, including multiple myeloma. Here, we present activated polyclonal γδ T cells from healthy donors with an NK T cell-like phenotype expressing the natural cytotoxicity receptor NKp44. Natural cytotoxicity receptors NKp30, NKp44 and NKp46 have been regarded as specific NK receptors; only two γδ T cell clones described so far expressed NKp44. Isolated polyclonal γδ T cells cultured for 7 days according to the cytokine-induced killer cell (CIK) protocol with additional IL-15 revealed a surface expression of NKp44 of 8 ± 7% (n = 22). This could be confirmed by detection of NKp44 mRNA by reverse transcription–polymerase chain reaction (RT–PCR). γδ T cells exhibited a marked cytotoxic activity against myeloma cells, which could be reduced by inhibition of NKp44. To our knowledge, this is the first description of the expression of NKp44 on polyclonal γδ T cells.


Introduction

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

T cells bearing the γδ T cell receptor (TCR) constitute 2–10% of peripheral blood T lymphocytes. Recently, there has been much interest in γδ T cells as immunological effector cells against several tumours, including multiple myeloma, as they exhibit a non-major histocompatibility complex (MHC)-restricted anti-tumour activity in vivo[1]. Although the exact mechanism of γδ T cell-mediated lysis of tumour cells is not entirely clear there is strong evidence that, apart from the T cell receptor, activating natural killer (NK) cell receptors play a fundamental role in cytotoxicity [2].

A thoroughly investigated activating NK receptor is NKG2D, a homodimer forming an activating receptor complex with DNA X-activating protein (DAP)10 [3]. The interactions of NKG2D with its ligands, MHC class I-related proteins A and B (MICA/B) and unique long (UL) 16 binding proteins (ULBP), have been described recently [4]. NKG2D ligands are expressed on most tumours of epithelial origin [5], but may also occur on haematopoietic tumours [6]. Some tumours express MIC RNA without surface expression of MICA/B, which is vital for recognition by NKG2D [7]. Although NKG2D is regarded as one of the key activators in natural cytotoxicity, it is not specific for NK cells but may also occur on CD8+ T cells and γδ T cells [4]. In contrast, natural cytotoxicity receptors (NCR) have been described as possibly the only specific NK cell receptors. They are activating receptors consisting of NKp30 and NKp46, which are expressed on resting and activated NK cells, as well as NKp44, which is expressed on activated NK cells only [8]. Two γδ T cell clones express NKp44, but polyclonal T cells with either αβ TCR or γδ TCR do not express any of the NCR [9–11].

Earlier protocols for the proliferation of γδ T cells have included co-culture with lymphoma cells [12], as well as stimulation with synthetic aminobisphosphonates [13] and various cytokines, including interleukin (IL)-2 and IL-15 [14].

We have previously investigated a protocol used to generate cytokine-induced killer (CIK) cells with additional IL-15 for the proliferation of purified γδ T cells [15]. This appeared to be a more effective way of propagating γδ T cells than the stimulation with aminobisphosphonates. In the present study, we expanded γδ T cells from healthy donors after enrichment to a purity > 90%, as reported previously, in the presence of IL-15 and high-dose IL-2 for 2 weeks. γδ T cells cultured in this way exhibited an NK T cell-like phenotype, including expression of NKp44 and cytotoxic activity against myeloma cells.

Materials and methods

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

Cell separation and culture

Peripheral blood mononuclear cells were derived from buffy coats of healthy donors and isolated by density gradient centrifugation on a Ficoll (Lymphoprep, Nycomed, Oslo, Norway) cushion. Isolated cells were allowed to adhere at a density of 5 × 106 cells/ml for 1 h. Non-adherent cells were taken for positive selection of γδ T cells, CD4 and CD8 positive T cells, which was performed using magnetic activated cell sorting from Miltenyi Biotec, Bergisch Gladbach, Germany, according to the manufacturer’s instructions. The achieved purity was > 90%. All T cells were incubated at 37°C in a humidified atmosphere of 5% CO2 at 3 × 106 cells/ml in RPMI-1640 (gibco BRL, Karlsruhe, Germany) supplemented with 10% fetal calf serum (PAA, Cölbe, Germany), 25 mm HEPES (gibco BRL), 100 U/ml penicillin and 100 µg/ml streptomycin (Seromed, Berlin, Germany). Cells were cultured according to the CIK protocol [16] as follows: 1000 U/ml human recombinant interferon (IFN)-γ (Roche, Mannheim, Germany) was added on day 0. After 24 h of incubation, 50 ng/ml of anti-CD3 (Orthoclone OKT 3, Cilag GmbH, Sulbach, Germany), 100 U/ml IL-1β, 300 U/ml IL-2 (both Roche, Mannheim, Germany) and 20 ng/ml IL-15 (R&D Systems, Wiesbaden, Germany) were added. For most experiments fresh medium, including IL-2 and IL-15, was added every 3 days. To evaluate the different influence of IL-2, IL-15 and anti-CD3 the γδ T cells were split into four groups for the experiments:

  • (i) 
    CIK protocol: these cells received all cytokines of day 0 and day 1, as above, and 300 U/ml IL-2 every 3 days;
  • (ii) 
    CIK + IL-15: these cells received all cytokines of day 0 and day 1, as above, and 300 U/ml IL-2 and 20 ng/ml IL-15 every 3 days;
  • (iii) 
    CIK + IL-15–IL-2: these cells received all cytokines of day 0 and day 1 except IL-2, as above, and 20 ng/ml IL-15 every 3 days; and
  •  (iv) 
    CIK − anti-CD3: these cells received all cytokines of day 0 and day 1 except anti-CD3, as above, and 300 U/ml IL-2 every 3 days.

Human tumour cell lines RPMI-8226, U266, OPM-2, NK92 and NKL were purchased from DSMZ (Braunschweig, Germany) and cultured as described previously [17–21].

Flow cytometry

A total of 105 cells were labelled according to the manufacturer’s instructions and resuspended in 500 µl phosphate-buffered saline (PBS)/1% bovine serum albumin (BSA) for flow cytometric analysis on a Coulter Epics XL Cytometer (Coulter Immunotech, Krefeld, Germany). Antibodies were purchased from Coulter Immunotech [IgG1 fluorescein isothiocyanate (FITC) and phycoerythrin (PE) clone 679·1Mc7, anti-γδ TCR FITC, clone immu 510, anti-NKp44 PE clone Z231, anti-CD8 FITC clone B9·11, anti-CD25 PE clone B1·49·9, anti-CD28 FITC clone CD28·2], from BD Pharmingen (anti-Vδ2 FITC clone B6, anti-CD4 FITC clone RPA-T4, anti-CD11a FITC clone HI111, anti-CD40L PE clone TRAP1, anti-CD54 PE clone HA58, anti-CD56 FITC clone B159, anti-CD94 PE clone HP-3D9, anti-HLA-ABC FITC clone G46-2·6, anti-HLA-DR PE clone G46-6) and R&D Systems (anti-NKG2D PE clone 149810). Antibodies against MICA (AMO1), MICB (BMO1), ULBP1 (AUMO1), ULBP2 (BUMO2) and ULBP3 (CUMO2) were kindly provided by Dr Steinle, Tuebingen, Germany, and were used as described [6].

Reverse transcription–polymerase chain reaction (RT–PCR)

Total RNA was extracted from γδ T cells, CD4+ αβ T cells and NK92 after 14 days of culture cells using the QIAamp RNA Blood Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Ten µl (i.e. about 200 ng) of total RNA were reverse-transcribed for 60 min at 37°C using oligo-dT primers (♯C1101, Promega, Mannheim, Germany) at a concentration of 10 µmol/l and Omniscript reverse transciptase (Qiagen), according to the standard protocol provided by the manufacturer. Primers used for cDNA amplification were as follows: NKp44 sense 5′-TCT CTA AGT CCG TCA GAT TC-3′, NKp44 anti-sense 5′-GAT GGT AGA TGG AGA CTC AG-3′, DAP 12 sense 5′-TCA TGG GGG GAC TTG AAC C-3′, DAP 12 anti-sense 5′-GAT TCG GGC TCA TTT GTA ATA C-3′, β-actin sense 5′-CTG GCA TCG TGA TGG ACT C-3′, β-actin anti-sense 5′-TCT CTT GCT CGA AGT CCA GG-3′, MIC sense 5′-ACA CCC AGC AGT GGG GGG AT-3′, MICA anti-sense 5′-GCA GGG AAT TGA ATC CCA GCT-3′, MICB anti-sense 5′-AGC AGT CGT GAG TTT GCC CAC-3′. Amplification of 2 µl of the resulting cDNA was performed for 35 cycles (denaturation: 1 s at 95°C, annealing: 5 s at 57°C for NKp44 (60°C for DAP 12 or β-actin, 56°C for MICA/B), extension: 15 s (NKp44), 40 s (DAP 12), 20 s (β-actin) or 35 s (MICA/B) at 72°C, respectively) on a LightCycler in a total volume of 10 µl using the FastStart DNA SYBR Green I kit (Roche Diagnostics, Mannheim, Germany). The reaction mix contained 3·5 mmol/l Mg2+ and 0·5 µmol/l of each of the respective sense-specific primers.

The amplification product was run on a 2% agarose gel and stained with ethidium bromide. In addition the PCR product of NKp44 amplification was sequenced by GATC (Konstanz, Germany).

Cytotoxic activity

For analysis of cytotoxic activity a standard 51chromium release assay was performed. In brief, tumour cells were labelled with 10 µCi 51chromium for 2 h, washed twice and co-cultured with effector cells in 150 µl at various effector : target ratios for 4 h. Fifty µl supernatant were taken for determination of radioactivity on a Top Count NXT (Canberra Packard, Rüsselsheim, Germany). For blocking experiments, 10 µl antibody was added to 100 µl of cell suspension and incubated for 30 min at 37°C. Before use as effector cells, the blocked cells were washed and counted. Blocking antibodies were purchased from R&D Systems (anti-NKG2D clone 149810), Coulter Immunotech (IgG1 isotypic control clone 679·1Mc7, anti-NKp44 clone Z231) and Miltenyi (anti-γδ TCR clone 11F2).

Cytotoxicity calculation was performed using the following formula:

  • image

Statistical analysis

Statistical analysis was carried out using the paired Student’s t-test. A P-value < 0·05 was considered statistically significant.

Results

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

Immunophenotype γδ T cells were enriched to a purity of > 90% and cultured according to the protocol mentioned above. After 2 weeks of culture with IL-2 and IL-15 γδ T cells showed an NK-T cell-like phenotype in flow cytometric analysis (Fig. 1). Most of the cells (62 ± 17%, n = 6) expressed the Vδ2 chain.

image

Figure 1. Immunophenotyping of γδ T cells on day 14 of culture of three independent experiments with cells from three different donors is presented (mean ± standard deviation).

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Because IL-15 has been shown to promote NK cell differentiation [22], expression of NCR was also evaluated. There was no expression of NKp30 and NKp46 (data not shown). However, after 7 days of culture a slight but significant expression of NKp44 up to 26% could be shown (mean 8 ± 7%, n = 22). To detect the baseline level of NKp44 and the development over time, NKp44 was then measured on day 1, day 7 and day 14 in cell cultures from four different donors. There was very little expression of NKp44 on day 1. After 1 week of culture the expression of NKp44 was 15 ± 9% (< 0·05) in this group. Although one cell culture from one donor had an even higher expression of NKp44 (31·5%, Fig. 2a) after 14 days of culture, the average expression was not increased further on day 14 (13 ± 7%, Fig. 2b). The purity of γδ T cells on day 14 was confirmed to be > 97% (mean 97·6 ± 0·6%).

image

Figure 2. (a) Flow cytometric analysis of natural killer (NK)p44 expression on day 14 of culture. One representative example of 20 independent experiments with cells from 20 different donors is shown. (b) Flow cytometric analysis of NKp44 on γδ T cells on day 1, day 7 and day 14. Mean ± standard deviation are presented of four independent experiments with cell cultures from four different donors. (c) Flow cytometric analysis of NKp44 on γδ T cells on day 14. Mean ± standard deviation are presented of three independent experiments with cell cultures from three different donors. (a) Cytokine-induced killer cell (CIK) protocol as described in Materials and nethods; (b) CIK protocol plus interleukin (IL)-15; (c) CIK protocol plus IL-15 without IL-2; (d) CIK protocol without anti-CD3.

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In contrast, isolated CD4+ and CD8+ αβ T cells were consistently negative for surface expression of NKp44 after 1 or 2 weeks of culture with the CIK protocol and additional IL-15 (data not shown). Immunophenotyping of the NK cell lines NK92 and NKL revealed strong expression of NKG2D on both cell lines (93·8% and 99·8%, respectively). There was no expression of NKp44 on NKL cells, whereas NK92 cells expressed NKp44 in 62·4%.

To determine the different potential of IL-15 and IL-2 to induce expression of NKp44 on activated γδ T cells we tested four different culture conditions (as described above). After 14 days of culture with the CIK protocol in the absence of IL-15, γδ T cells expressed NKp44 in 7% ± 4% (Fig. 2c). This was not dependent on anti-CD3, as omission of anti-CD3 did not alter expression of NKp44 (Fig. 2c). However, those culture conditions which included IL-15 led to a higher expression of NKp44, regardless of the presence or absence of IL-2 (Fig. 2c). It seems, therefore, that stimulation with either IL-2 and IL-15 leads to expression of NKp44 on γδ T cells. Although the difference between these cytokines was not significant in our hands, IL-15 appeared to be more effective than IL-2. The addition of both cytokines did not lead to a higher expression of NKp44 than IL-15 alone.

RT–PCR

RNA was isolated from γδ T cells, CD4+ αβ T cells and NK92 cells, as described above, and RT–PCR was performed for mRNA of β-actin and NKp44. NKp44 mRNA could be detected in NK92 cells and in γδ T cells, but not in CD4+ αβ T cells (Fig. 3a). In addition, DAP12, the signal transduction molecule for NKp44 [23], could be detected in γδ T cells as well as CD4+ αβ T cells and NK92 cells by RT–PCR (Fig. 3b). The sequence of the NKp44 cDNA was confirmed by sequencing the PCR product.

image

Figure 3. Polymerase chain reaction (PCR) products from reverse transcription–polymerase chain reaction (RT–PCR) of (a) natural killer (NK)p44 (160 bp); (b) DNA X-activating protein (DAP)12 [353 base pairs) bp]; and (c) β-actin (234 bp). (a) Lanes: 0 = 25 bp ladder, 1 = NKp44 cDNA of γδ T cells of donor 1, 2 = NKp44 cDNA of CD4+ αβ T cells of donor 1, 3 = NKp44 cDNA of γδ T cells of donor 2, 4 = NKp44 cDNA of CD4+ αβ T cells of donor 2, 5 = NKp44 cDNA of γδ T cells of donor 3, 6 = NKp44 cDNA of CD4+ αβ T cells of donor 3, 7 = NKp44 cDNA of NK92, 8 = H2O control with NKp44 primer. (b) Lanes: 0 = 25 bp ladder, 1 = DAP12 cDNA of γδ T cells of donor 1, 2 = DAP 12 cDNA of CD4+ αβ T cells of donor 1, 3 = DAP12 cDNA of γδ T cells of donor 2, 4 = DAP12 cDNA of CD4+ αβ T cells of donor 2, 5 = DAP12 cDNA of γδ T cells of donor 3, 6 = DAP12 cDNA of CD4+ αβ T cells of donor 3, 7 = DAP12 cDNA of NK92, 8 = H2O control with DAP12 primer. (c) Lanes: 0 = 25 bp ladder, 1 = β-actin cDNA of γδ T cells of donor 1, 2 = β-actin cDNA of CD4+ αβ T cells of donor 1, 3 = β-actin cDNA of γδ T cells of donor 2, 4 = β-actin cDNA of CD4+ αβ T cells of donor 2, 5 = β-actin cDNA of γδ T cells of donor 3, 6 = β-actin cDNA of CD4+ αβ T cells of donor 3, 7 = β-actin cDNA of NK92, 8 = H2O control with β-actin primer. Three representative examples of five independent experiments are shown.

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Cytotoxic activity of activated γδ T cells

Cytotoxic activity of γδ T cells against myeloma cell lines was evaluated in a standard chromium release assay on day 14 of culture. Prior to their use in the chromium release assay, OPM-2, U266 and RPMI-8226 cells were analysed flow cytometrically for expression of MHC class I molecules. There was a high expression of classical MHC class I molecules (U266 62%, RPMI-8226 99·7%, OPM-2 97·6%), suggesting that natural cytotoxicity against these cell lines is unlikely. Regarding NKG2D ligands, mRNA of MICB could be detected by RT–PCR in all three myeloma cell lines, but there was no expression of MICA mRNA. In addition, immunophenotyping for NKG2D ligands revealed no surface expression for MICA/B, ULBP1, ULBP2 and ULBP3 on all tested cell lines. Not surprisingly, natural killer cells (NKL and NK92 cell lines) showed only very little cytotoxic activity against either cell line (data not shown). However, γδ T cells cultured as described exhibited cytotoxic activity against all three cell lines, with the highest lysis of RPMI-8226 cells (Fig. 4a). γδ T cell cytotoxicity was exerted partially via the TCR, as blocking of the γδ TCR resulted in a significant reduction of cytotoxic activity. The NCR NKp44 seems to have an additional effect on the activity of γδ T cells, because blockage resulted in a significant (< 0·05 for all three cell lines) reduction of cytotoxic activity (Fig. 4a). In contrast, the addition of irrelevant antibody (isotypic control) or anti-NKG2D did not have any effect on the lysis of myeloma cells (Fig. 4b and data not shown).

image

Figure 4. Cytotoxic activity against the myeloma cell lines U266, OPM-2 and RPMI-8226. Cytotoxicity was measured in a standard chromium release assay. All experiments were performed in triplicate. (a) Median ± standard deviation of four independent experiments with effector cells from four different donors is shown. The effector : target ratio in all experiments was 20 : 1. For statistical significance the Student’s t-test was performed. (b) Representative examples of five (RPMI-8226), eight (OPM-2) and six (U266) independent experiments with effector cells from different donors are presented. The plot shows mean ± standard deviation of triplicates of one experiment.

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Discussion

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

γδ T cells have been shown to express a variety of activating as well as inhibitory NK cell markers. Among these, the activating marker NKG2D has been widely studied. In the present study we detected surface expression of NKG2D similar to other reports [24]. NKp44 is a novel activating NK cell receptor described first in 1998 on only activated NK cells [9]. T cells cultured in unfractionated peripheral blood mononuclear cells (PBMC) in the presence of IL-2 did not express NKp44, even after 30 days of culture. Cantoni et al. showed very low levels of surface NKp44 on two clones of γδ T cells derived from one donor after stimulation with IL-2 [8]. Because NCR have been detected only on NK cells, they are regarded as specific NK cell receptors. However, we found marked expression of NKp44 after 7 days of culture on purified γδ T cells without co-expression of NKp30 or NKp46. Isolated αβ T cells, on the other hand, did not express any NCR whereas the NK cell line NK92 was also positive for NKp44, as determined by flow cytometry. These data were confirmed by RT–PCR of NKp44 mRNA extracted from γδ T cells, CD4+ αβ T cells and NK92 cells.

Myeloma cells have been discussed as one of the main targets of lysis by γδ T cells [13]. This is of particular interest, as multiple myeloma is still an incurable disease for the vast majority of patients. Despite promising in vitro results, clinical investigations concerning potential immunotherapeutic strategies including idiotype and dendritic cells (DC) vaccination have been unsatisfying [25–28]. A study published recently investigated γδ T cells as effector cells in the treatment of myeloma and lymphoma, confirming the role of γδ T cells as potential effector cells in the immunotherapy of multiple myeloma [29]. It has been demonstrated previously that γδ T cells exert their cytolytic activity mainly via the non-MHC-restricted TCR, possibly recognizing mevalonate metabolites [30] or pyrophosphates presented by tumour cells [31]. On the other hand, it is well known that activating NK cell receptors also play an important role. This has been shown particularly for NKG2D, which is present on most γδ T cells. Ligands for NKG2D include MICA/B, which are clearly recognized by γδ T cells and may also be expressed by tumours of haematopoietic origin [6]. The myeloma cells studied here did not express any NKG2D ligands and consequently there was no effect of blockage of NKG2D on γδ T cells. However, our blocking experiments suggest that NKp44 participates in cytotoxicity of γδ T cells against myeloma cells. Viral haemagglutinins have been postulated as ligands for NKp44 [32], which explains one mechanism by which virally infected cells are recognized by NK cells. The ligands for NKp44 on tumour cells are not yet known. None the less, Carbone et al. found NCR-dependent cytolysis of myeloma cells by NK cells, thus supporting our results for γδ T cells [33].

Our data show, for the first time, that NKp44 is also present on activated polyclonal γδ T cells and plays a role in their cytotoxic activity against myeloma.

Acknowledgements

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

This study was supported by a Bonfor research grant (M. v. L.-T., J. N.), a grant by the Hector foundation (E. S., S. F., I. S. W.) and the Deutsche Krebshilfe (I. S. W.). We thank Annette Brause for excellent technical assistance, Dr A. Steinle, University of Tuebingen, for providing antibodies against NKG2D ligands and Professor T. Sauerbruch, University of Bonn and D. Messmer, University of California San Diego, for critical reading of the manuscript.

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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