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The Journal of Immunology, 2005, 175: 2144-2151.
Copyright © 2005 by The American Association of Immunologists

Activation of V{gamma}9V{delta}2 T Cells by NKG2D1

Bladimiro Rincon-Orozco*, Volker Kunzmann{dagger}, Philine Wrobel{ddagger}, Dieter Kabelitz{ddagger}, Alexander Steinle§ and Thomas Herrmann2,*

* Institut für Virologie und Immunobiologie, {dagger} Medizinische Poliklinik, Julius-Maximilians Universität, Würzburg, Germany; {ddagger} Institut für Immunologie, Universitätsklinikum Schleswig-Holstein Campus Kiel, Kiel, Germany; and § Institut für Zellbiologie, Eberhard-Karls-Universität Tübingen, Tübingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human V{gamma}9V{delta}2 T cells recognize phosphorylated nonpeptide Ags (so called phosphoantigens), certain tumor cells, and cells treated with aminobisphosphonates. NKG2D, an activating receptor for NK cells, has been described as a potent costimulatory receptor in the Ag-specific activation of {gamma}{delta} and CD8 T cells. This study provides evidence that V{gamma}9V{delta}2 T cells may also be directly activated by NKG2D. Culture of PBMC with immobilized NKG2D-specific mAb or NKG2D ligand MHC class I related protein A (MICA) induces the up-regulation of CD69 and CD25 in NK and V{gamma}9V{delta}2 but not in CD8 T cells. Furthermore, NKG2D triggers the production of TNF-{alpha} but not of IFN-{gamma}, as well as the release of cytolytic granules by V{gamma}9V{delta}2 T cells. Purified V{gamma}9V{delta}2 T cells kill MICA-transfected RMA mouse cells but not control cells. Finally, DAP10, which mediates NKG2D signaling in human NK cells, was detected in resting and activated V{gamma}9V{delta}2 T cells. These remarkable similarities in NKG2D function in NK and V{gamma}9V{delta}2 T cells may open new perspectives for V{gamma}9V{delta}2 T cell-based immunotherapy, e.g., by Ag-independent killing of NKG2D ligand-expressing tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The majority of circulating human {gamma}{delta} T cells express a TCR comprising the variable segments {gamma}9 and {delta}2 (V{gamma}9V{delta}2 TCR). Functional characteristics of these cells are MHC-unrestricted cytolysis, production of Th 1 type cytokines such as IFN-{gamma} and TNF-{alpha}, and expansion in a variety of bacterial and parasite infections (1, 2, 3). In addition, there is increasing evidence for an important role in host defense and tumor surveillance (4, 5, 6). A hallmark of V{gamma}9V{delta}2 T cells is their unique reactivity toward phosphoantigens (7, 8). The most potent natural phosphoantigen is (E)-4-hydroxy-3-methyl-but-enyl pyrophosphate, a metabolite of the nonmevalonate pathway of isoprenoid synthesis, which is found in plants, bacteria, and protozoa (9). Less potent phosphoantigens are ubiquitous metabolites of isoprenoid synthesis such as isopentenyl pyrophosphate (IPP) (10). Some tumors and lymphomas, such as the B cell lymphoma cell line Daudi, express endogenous ligands, which activate {gamma}{delta} T cells in a V{gamma}9V{delta}2 TCR-dependent fashion (11). Recently, V{gamma}9V{delta}2 T cells were shown to recognize on Daudi cells a complex of an ectopically expressed mitochondrial F1-ATPase and apolipoprotein A1 (12). Finally, V{gamma}9V{delta}2 T cells are stimulated by cognate recognition of human cells pulsed with aminobisphosphonates, e.g., pamidronate or zoledronate, probably as a consequence of their pharmacological action leading to accumulation of mevalonate metabolites (13, 14). All these ligands need to be presented to the V{gamma}9V{delta}2 T cells (15), and there is accumulating evidence that this presentation requires species-specific compounds (16, 17).

Recently, the response of V{gamma}9V{delta}2 T cells to aminobisphosphonates has attracted special attention. Aminobisphosphonates have been found to induce V{gamma}9V{delta}2 T cell proliferation in vitro (13) and in vivo (6), and to trigger V{gamma}9V{delta}2 TCR-mediated lysis of tumor lines as well as reduced survival of autologous myeloma cells in pamidronate-treated bone marrow biopsy specimens (13). Furthermore, in vivo activation and expansion of V{gamma}9V{delta}2 T cells after the combined application of pamidronate and low dose IL-2 coincides with an objective tumor response in patients with lymphoid malignancies (6).

In addition to TCR ligation, activation of V{gamma}9V{delta}2 T cells is modulated by a range of inhibitory and activating NK-cell receptors. Killing by V{gamma}9V{delta}2 T cell clones is inhibited by ligation of killer inhibitory receptors (KIR) leading to modulation of lysis depending on the KIR expressed by effector clones and MHC class I iso- and allotypes expressed by target cells (18, 19). Although there are clonal differences in KIR expression, expression of the activating NK cell receptor NKG2D is universal. NKG2D is a homodimeric C-type lectin-like receptor encoded by orthologous genes in mouse, man, and rat that show a high degree of sequence similarity (20). Nevertheless, species vary considerably in the cell type specificity of expression, types of ligands, usage of signaling pathways, and the cellular functions triggered through NKG2D (20).

In humans, seven ligands of NKG2D have been identified to date: the MHC class I-related proteins A and B (MICA/B) and members of the UL16-binding protein family (ULBP1–4, RAET1G). The NKG2D ligands MICA and MICB are often induced by stress (heat shock, genotoxic stress) (20) and, in the mouse, also by TLR-mediated signals (21), but some epithelial tissues and many tumors (22, 23) have also been found to constitutively express NKG2D ligands. NKG2D ligation provides (co)stimulatory signals in recognition of infected cells and contributes to control of infection (24). Direct evidence for a role of NKG2D in tumor control has been obtained for NK cells and CD8 T cells in mouse lymphoma models (25) and for {gamma}{delta} T cells in a mouse model of skin tumor development (26). In humans, evidence for a contribution of NKG2D ligand interaction to tumor surveillance is less direct, but NKG2D-triggered cytotoxicity of tumor cells has been demonstrated (27, 28, 29). The fact that some tumors and leukemias release NKG2D ligands (22, 30), which may inhibit direct interaction of cell surface expressed ligands with NKG2D of effector cells, provides circumstantial support for the idea of NKG2D as player in tumor surveillance.

In human NK cells, the transmembrane adaptor molecule DAP10 links NKG2D to phosphoinositol 3-kinase-dependent pathways, which are also used by costimulatory molecules such as CD28 (28, 31). NKG2D acts as a costimulatory receptor for the TCR-dependent activation of human CD8 T cells, V{gamma}9V{delta}2, and gut intraepithelial V{gamma}1{delta}1 T cells (24, 27, 32); although the signaling pathways leading to activation of these cell types are poorly understood. The recently reported Ag-independent activation of certain types of human CD8 cells will be addressed in the discussion.

This work demonstrates Ag-independent NKG2D-mediated activation of V{gamma}9V{delta}2 T cells, a process that shares aspects of NK cell activation, such as the expression of CD25 and triggering of cytotoxicity. This NKG2D-mediated triggering of V{gamma}9V{delta}2 T cell functions may be also of clinical interest, because it opens the possibility of Ag-independent targeting of V{gamma}9V{delta}2 T cells to NKG2D ligand-expressing tumors or (infected) tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

Daudi cells are derived from a {beta}2-microglobulin-deficient B cell lymphoma and do not express surface MHC class I molecules. RMA are T cell lymphoma cells, which have been transfected with MICA*07 cDNA in RSV.5-Neo or with the vector alone. C1R cells are EBV-transformed B cells that have lost most HLA class I alleles and express only Cw0401 and trace amounts of B3503. C1R-MICA, C1R-ULBP2, and C1R-Neo are C1R cells that have been transfected with cDNA for MICA*01 and ULBP2 in RSV.5-Neo, respectively, or with the vector alone as previously described (22). Raji is an EBV-transformed human B cell lymphoma and THP1 is a human monocytic line. The human melanoma Mewo was obtained from the American Type Culture Collection.

Generation of V{gamma}9V{delta}2 T cell lines and isolation of V{gamma}9V{delta}2T effector cells

PBMC were isolated from freshly drawn heparinized blood by centrifugation over Ficoll-Hypaque gradient as described by the manufacturer (Amersham). PBMC were finally resuspended at a concentration of 1 x 106 per milliliter in culture medium, which was RPMI 1640 (Life Technologies) supplemented with 10% FCS, 100 mM sodium pyruvate, 0.05% (w/v) of glutamine, 10 mM nonessential amino acids, and 100 µM 2-ME (Life Technologies). Culture was performed for up to 15 days in presence of 1 µg/ml IPP (Sigma-Aldrich) and 100 IU/ml recombinant human IL-2 (Strathmann) in a 24-well tissue culture plate (Greiner) at 37°C in 5% CO2. Half of the medium was replaced by fresh medium with IPP and IL-2 on days 3, 7, and 10. Cells were analyzed by flow cytometry at day 10. At this time point, ~20–70% of CD3+ cells were {gamma}{delta} T cells. From these cells, {gamma}{delta} T cells were isolated using MACS. Cells tested for cytotoxicity and granule release were negatively selected. Briefly, in vitro-stimulated PBMC were preincubated with primary Abs specific for CD4, CD8, and CD16 (BD Biosciences), followed by goat anti-mouse magnetic microparticles (MACS system; Miltenyi Biotec). After washing, cells were passed through a strong magnetic field and the purity of effluent cells was tested by staining with PE-labeled anti-V{gamma}2 TCR—V{gamma}9 TCR according to the nomenclature used in this paper—from BD Biosciences. In some experiments, negative selection was performed exactly as previously described elsewhere (13, 33). In either case, 99% of isolated cells were CD3 positive and consisted of 75–95% {gamma}{delta} T cells with <5% CD8 cells. Approximately 1% of the cells were CD16 positive. The purified cells will be referred to as effector {gamma}{delta} T cells throughout this paper. Viability of the cells was confirmed by trypan blue exclusion test and forward/side-scatter gating. Cells used for RNA preparation and immunoblot were positively sorted. Briefly, in vitro-stimulated PBMC were preincubated with primary Abs specific for V{gamma}9 TCR (BD Biosciences), followed by goat anti-mouse magnetic microparticles (MACS system; Miltenyi Biotec). After washing, cells were passed through a strong magnetic field, and attached {gamma}{delta} T cells were collected by washing the column outside the magnetic field. Purity was determined by staining with PE-labeled anti-V{gamma}9 TCR mAb. Generation, phenotypic and functional characterization of V{gamma}9V{delta}2 T cell lines EP3 and HO has been described in detail elsewhere (34).

In vitro stimulation with immobilized Abs or NKG2D ligand

Ninety-six U-bottom plates (Greiner) were coated overnight at 4°C with 12 µg/ml mAb to NKG2D (clone 149810) (R&D Systems) or 0.5 µg/ml for CD3 (clone HIT3a; BD Biosciences) or 10 µg/ml mouse IgG1 as isotype control in 0.05 M carbonate buffer, pH 9.6, 100 µl per well. Coating with MICA-Fc recombinant protein (R&D Systems) was done for 2 days at 10 µg/ml. Subsequently, plates were thoroughly washed with PBS three times, and 5 x 105 cells per well of PBMC were isolated by Ficoll-Hypaque were cultured in medium plus 50 UI/ml IL-2 and kept in culture for 24 or 96 h at 37°C and 5% CO2. Cells cultured for 24 h were stained for CD69, and the cells cultured for 96 h were stained for CD25. Expression of activation markers was analyzed by two-color flow cytometry against {gamma}{delta} TCR, CD16, or CD8. Stimulation for measurement of cytokine production by intracellular staining or granule release was performed on the same types of plates as described above.

Staining of cell surface Ags and intracellular staining

Cells were suspended at a concentration of 1 x 105 per 100 µl of staining buffer (PBS, 0.1% BSA) and stained by adding 1 µg of fluorochrome-conjugated mAb specific for a cell surface Ag. If not stated otherwise, Abs were from BD Biosciences. CD3 (clone HIT3a), CD4 (clone RPA-T4), CD8 (clone RPA-T8), CD19 (clone HIB19), CD25 (clone M-A251), CD28 (clone CD28.2), CD69 (clone FN50), V{delta}2 TCR (clone B6), V{gamma}9 TCR (clone B3.1), NKG2D (clone 149810) (R&D Systems). After 30 min at 4°C, cells were washed and the cell pellet was resuspended in staining buffer and analyzed by flow cytometry on a FACScan or FACSCalibur (BD Biosciences) using CellQuest software.

For intracellular cytokine staining, IPP-activated PBMC (see above) were washed and living cells were isolated by Ficoll-Hypaque, resuspended in medium plus 50 U/ml IL-2, and transferred at 5 x 105 cells/200 µl of culture plates coated with Ab or ligand. After 1 h, 1 µg/ml protein transport inhibitor brefeldin A (BD Biosciences) was added, and after 5 h, cells were resuspended in staining buffer and 1 µg of FITC or PE-conjugated mAb V{delta}2 TCR was added. Subsequently, cells were washed once with staining buffer, fixed and permeabilized for 20 min at 4°C using cytofix/cytoperm (BD Biosciences). Fixed cells were washed twice with Perm/Wash buffer (BD Biosciences) and resuspended in 50 µl of Perm/Wash buffer. Later, 1 µg of anti-IFN-{gamma} FITC or anti-TNF-{alpha} PE (BD Biosciences) or isotype-matched control mAb were added, incubated for 30 min at 4°C, and washed twice with Perm/Wash buffer. Finally, samples were resuspended in staining buffer and analyzed by flow cytometry. The detection of perforin was performed essentially the same way as for intracellular cytokines but without addition of brefeldin A or IL-2. Abs used were 1 µg of V{gamma}9 TCR FITC and 1 µg of anti-perforin PE (BD Biosciences).

Measurement of granule release

Plates were coated as described in In vitro stimulation with immobilized antibodies or NKG2D ligand, and 1 x 105 cells of effector {gamma}{delta} T cells were added in 200 µl of medium. After incubation for 4 h at 37°C in 5% CO2, supernatants were taken and tested with a standard N-benzyloxycarbonyl lysine thiobenzyl ester (BLT3) esterase assay (35). Positive control were cells cultured with 50 ng/ml PMA (Sigma-Aldrich) plus 1 µg/ml ionomycin (Sigma-Aldrich). Total content was estimated by analyzing supernatant of cells treated with 0.1% Triton X-100 (Sigma-Aldrich) in parallel.

Analysis of DAP10 transcripts

RNA was extracted from distinct cellular subsets using an RNeasy minikit (Qiagen). The concentration of RNA was determined by measuring the absorbance at 260 nm. One microgram of total RNA was used to produce cDNA using oligo(dT)18 primers as described in the First Strand cDNA Synthesis Kit (Fermentas). Possible contamination with genomic DNA was reduced by addition of RNA-free DNase (Qiagen). Serial dilution (0.01, 0.1, and 1 µg/ml) of cDNA were tested in the PCR. The following primers were used: human DAP10 reverse 5'-GATGTAGTTGTACGGTCCGT-3', forward 5'-TCTGGGTCACATCCTCTTCC-3', actin; forward 5'-CCACGTCATCACTATTGGCAACGA-3', reverse 5'-GAGCAGTAATCTCCTTCTGCATCC-3'. Reaction was performed at 92°C for 2 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 1 min, and finally 72°C for 7 min. Reaction products were analyzed by electrophoresis on 2% agarose gel and visualized by ethidium bromide staining running appropriate m.w. standards (Fermentas) in parallel.

Immunoblot of DAP10

A total of 5 x 106 {gamma}{delta} T cells and cells serving as positive and negative controls were lysed in cell extraction buffer (20% glycerol, 350 mM KCl, 20 mM HEPES (pH 7.6), 1 mM MgCl2, 0.5 mM EDTA (pH 8.0), 0.1 mM EGTA (pH 8.0), 1% Nonidet P-40, 5 mM DTT) (Sigma-Aldrich) and 1x complete protease inhibitor mixture (Roche Applied Science). Samples were quantified by micro-BCA protein assay kit (Pierce), and 10 µg of protein of each sample was analyzed in discontinuous 15% SDS-PAGE. Proteins were transferred onto a Roti-PVDF membrane (Carl Roth) by semidry blotting. DAP10 protein was detected with goat anti-DAP10 antisera (Santa Cruz Biotechnology) followed by anti-goat polyclonal HRP (Santa Cruz Biotechnology). Enzymatic activity was visualized by the chemiluminescence system ECL (Amersham Pharmacia).

Fluorometric assessment of T lymphocyte Ag-specific lysis (FATAL)

A total of 1 x 106 target cells were stained with PKH-26 (final concentration of 2.5 µM) according to the manufacturer’s instructions (Sigma-Aldrich). PKH-26-labeled target cells were subsequently labeled with a final concentration of 2.5 µm CFSE (Molecular Probes). Finally, the target cells were resuspended in medium and dispensed in duplicates at 5 x 104 cells per well into 96-well U-bottom plates. Effector cells were added at various E:T ratios, and mixed with the target cells. The FATAL assay was performed for up to 5 h at 37°C, 5% CO2. The cell pellet was resuspended in 150 µl of 1% paraformaldehyde, and cells were analyzed by flow cytometry (36). For cytotoxicity assays that were performed with blocking mAbs, either isotype control or blocking Abs against NKG2D were added to effector cells for 30 min before the addition of tumor cell targets (final concentration, 20 µg/ml). Unconjugated Ab against NKG2D (M585) was a kind gift of Dr. D. Cosman (Amgen, Seattle, WA) (37).

Determination of cellular cytotoxicity by 51Cr release assay

Target cells were labeled with 100 µCi of sodium 51Cr and used in standard 4-h 51Cr release assays with titrated numbers of V{gamma}9V{delta}2 T cells as effector cells. Specific lysis was calculated as follows: percent specific lysis = cpm-test – cpm-spontaneous/cpm-max – cpm-spontaneous, where spontaneous release was determined in medium only, and maximal release was determined in Triton X-100-lysed target cells. Inhibition of cellular cytotoxicity by mAb was performed with a final concentration of 10 µg/ml NKG2D-specific mAb M585 or V{gamma}9-specific mAb 7A5 (38) and isotype-specific controls. Abs were added to effectors 30 min before addition of target cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NKG2D ligation induces expression of activation markers on V{gamma}9V{delta}2 T cells

Total PBMC were cultured in the presence of 50 U/ml IL-2 in tissue culture plates coated with mAb specific for CD3 or NKG2D, with a MICA-Fc construct or isotype-specific control mAb. After 24 h, V{gamma}9V{delta}2 T cells, NK cells, and CD8 cells were analyzed by two-color staining for expression of the activation marker CD69, and after 96 h for CD25 expression. Different cell populations were identified by electronic gating on expression of {gamma}{delta} TCR, CD16, or CD8. Fig. 1 depicts a representative example (Fig. 1A, dot plot) and frequencies of CD69-positive cells and CD25-positive cells determined in three independent experiments with blood from three different donors (Fig. 1B, histogram). Little or no changes were found for CD8 cells, except after stimulation with anti-CD3. The proportion of CD69-positive cells increased strongly following NKG2D ligation, which results from an activation of most if not all V{gamma}9V{delta}2 T cells as it is demonstrated by a shift in the mean fluorescence. For NK cells, background expression of CD69 was considerably higher and effects of NKG2D ligation were less pronounced. Determination of CD25 expression after 4 days of culture confirmed NKG2D-mediated activation of V{gamma}9V{delta}2 T cells and NK cells.



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  		FIGURE 1. Differential induction of CD69 and CD25 expression in CD8 cells, V{gamma}9V{delta}2 T cells, and NK cell after ligation of NKG2D. A, The graph provides a representative example analysis of CD69 expression. V{gamma}9V{delta}2 T cells from PBMC were stained with anti-CD69-FITC vs anti-V{gamma}9TCR-PE after 24 h of stimulation in medium plus 50 U/ml IL-2 with indicated stimuli: NS (nonstimulated; immobilized isotype control), MICA (immobilized recombinant MICA-Fc), NKG2D (immobilized anti-NKG2D), IPP, CD3 (immobilized anti-CD3). B, PBMC were cultured for 24 h (upper panel) or 96 h (lower panel) in medium plus 50 U/ml IL-2 with indicated stimuli. Proportion of CD69-positive cells (upper panel), mean fluorescence intensity (middle panel), and proportion of CD25-positive cells (lower panel) were determined by two-color immuno flow cytometry. Error bars indicate SEM of duplicates stained for each sample.

 
Cytokine production after NKG2D ligation

PBMC cultured for 7–15 days with IPP and IL-2 were Ficoll purified and activated with immobilized NKG2D Ab or MICA-Fc or as positive controls anti-CD3, respectively, 1 µg of IPP per milliliter plus 5 x 105 Daudi cells. Fig. 2 shows representative staining for TNF-{alpha} or IFN-{gamma} after 5 h of restimulation in the presence of 50 U/ml IL-2 (left), and summarizes data of three donors (right). It shows increased frequencies of TNF-{alpha}-producing cells after restimulation with NKG2D-specific mAb (3.4–6.2%), respectively, recombinant MICA-Fc (2–3.6%), which was similar to what was found after coculture with Daudi plus IPP (2.2–5.3%), but much weaker than after restimulation with CD3 (47–67% positive cells). Only in one of the donors did NKG2D induce some IFN-{gamma} production (2.6% producing cells vs 1.0% in the nonstimulated culture). Induction of IFN-{gamma} after coculture with Daudi plus IPP was consistent, although much weaker than after CD3 stimulation.



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  		FIGURE 2. Analysis of IFN-{gamma} and TNF-{alpha} production in V{gamma}9V{delta}2 T cells by intracellular staining after 5 h of stimulation. V{gamma}9V{delta}2 T cells were stimulated with the indicated stimuli: NS (nonstimulated; immobilized isotype control), MICA (immobilized recombinant MICA-Fc), NKG2D (immobilized anti-NKG2D), IPP (Daudi + IPP), CD3 (immobilized anti-CD3). The left graph provides a representative example for intracellular staining with anti-IFN-{gamma} FITC or anti-TNF-{alpha} PE vs anti-V{gamma}9TCR-PE or anti-V{gamma}9TCR-FITC after 5 h of stimulation. The right graph summarizes data from three donors. Error bars indicate SEM of duplicates stained for each sample.

 
Release of cytolytic granules after NKG2D ligation

Release of cytolytic granules was measured by two methods; reduction in intensity of intracellular perforin staining and the determination of esterase activity in the BLT assay. For the first experiment IPP-induced V{delta}9V{delta}2 T cell lines were ligated as described above for resting V{gamma}9V{delta}2 T cells and perforin content was determined. Fig. 3, left, shows a uniform decrease in intracellular staining for perforin in V{gamma}9V{delta}2 T cells after stimulation with immobilized NKG2D mAb. Results for different stimulation conditions were as follows (arbitrary units of mean fluorescence intensity): nonstimulated (NS), 19.2 and 18.0; MICA-Fc, 14.3 and 14.4; anti-NKG2D, 10.8 and 12.5; anti-CD3, 12.2 and 12.0; for PMA plus ionomycin, 5.4 and 6.4. Thus, NKG2D and CD3 ligation leads to the same degree of reduction of intracellular perforin, which most likely can be attributed to the release of cytolytic granules.



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  		FIGURE 3. NKG2D-dependent granule release by effector V{gamma}9V{delta}2 T cells. Left, Intracellular staining for perforin after incubation of V{gamma}9V{delta}2 T cell line with immobilized anti-NKG2D (NKG2D) or isotype-specific Ab (NS). The histogram shows cells electronically gated for expression of V{gamma}9V{delta}2 TCR. The dotted line shows staining with a isotype-matched Ab. Right, Determination of secreted granule-associated enzymatic activity by BLT esterase after incubation of purified V{gamma}9V{delta}2 T cells with immobilized anti-NKG2D ({cjs2106}) anti-CD3 ({cjs2117}) or PMA and ionomycin ({blacksquare}), isotype-specific control ({square}). One hundred percent content of granule was estimated by analysis of supernatants of cells treated with 0.2% Triton X-100.

 
NKG2D-mediated triggering of cytolytic capacity was also tested by measuring the release of active esterase activity (BLT assay) known to be associated with cytotoxic granules. Negatively selected effector V{gamma}9V{delta}2 T cells were restimulated for 4 h with immobilized Ab. Fig. 3, right, shows the results of two independent experiments performed with V{gamma}9V{delta}2 effector cells (90 and 93% purity) and with CD3 or NKG2D-specific mAbs. Cross-linking of NKG2D was at least as efficient as cross-linking of CD3. The extent of granule release after CD3 ligation reached ~80% of what was found after stimulation with PMA plus ionomycin, respectively, >50% of total granule content, indicating the T cells are the main, if not exclusive, source of esterase activity.

MICA- and NKG2D-dependent lysis by in vitro-activated V{gamma}9V{delta}2 T cells

NKG2D-triggered cytotoxicity was tested with V{gamma}9V{delta}2 T cells purified from IPP-induced polyclonal cells, which will be referred to as effector {gamma}{delta} T cells in this paper. To exclude TCR-mediated effects by residual Ag, effector {gamma}{delta} T cells were purified on a Ficoll gradient and then by negative selection, resulting in a purity of 70–95% of {gamma}{delta} T cells as assessed by a {gamma}{delta} TCR-specific mAb. More than 97% of the cells were CD3 positive; <1–3% of them expressed CD8 or CD16. An example of purified cells is given in Fig. 4, upper part. The effector cells were tested for lysis of different target cells. Targets were mouse RMA cells transfected either with MICA expression vector (RMA-MICA) or control vector (RMA-Neo). These cells were chosen because mouse cells neither express nor present ligands for the V{gamma}9V{delta}2 TCR (16, 17). In addition, the species difference between effector and target cells makes ligation of KIRs of effector {gamma}{delta} T cells by target MHC class I molecules rather unlikely. Daudi cells express endogenous ligands for the V{gamma}9V{delta}2 TCR and are negative for surface MHC class I and MICA. Other targets were the B cell lymphoblastoma C1R transfected with MICA (C1R-MICA) or ULBP2 (C1R-ULBP2), or with vector alone (C1R-Neo) and the melanoma line Mewo for which V{gamma}9V{delta}2 TCR-mediated lysis as well as MICA expression has been previously demonstrated (34). Fig. 4, lower part, depicts MICA expression of the target cells used in experiments of Fig. 5. RMA-MICA cells showed uniform expression of MICA. Daudi cells expressed no MICA. C1R cells (C1R-Neo) expressed very low levels of endogenous MICA and ULBP2. C1R-ULBP2 cells expressed ULBP2 at high levels (data not shown, and Ref. 22).



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  		FIGURE 4. Phenotype of effector and target cells. Upper panel, Generation and isolation of V{gamma}9V{delta}2 T- effector cells. PBMC (left) were stimulated in vitro in the presence of 100 U/ml IL-2 and 1 µg/ml IPP for 10 days (middle) and isolated by negative selection. Phenotype was determined by two-flow cytometry using the indicated Abs. Proportion of V{gamma}9V{delta}2 T cells is indicated in the upper right quadrant. Lower panel, MICA expression of target cells was determined by one-color flow cytometry. Only RMA-MICA cells express MICA.

 


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  		FIGURE 5. Analysis of NKG2D-dependent cytotoxicity by V{gamma}9V{delta}2 T cells. Shown is lysis in a 5-h cytotoxicity assay at the indicated E:T ratio (A–D, killing by purified polyclonal V{gamma}9V{delta}2 T cells). A, Comparison of lysis of RMA MICA ({blacktriangleup}), RMA Neo ({blacktriangledown}), Daudi (•). B, Lysis of RMA MICA in the presence ({triangleup}) or absence of 5 µg/ml anti-NKG2D ({blacktriangleup}). C, Lysis of Daudi in the presence ({circ}) or absence of 5 µg/ml anti-NKG2D (•). D, Lysis of CR1 Neo ({blacksquare}) CR1 MICA ({square}) and CR1 ULBP2 ({diamond}). The effector cells were prepared as shown in Fig. 4. MICA expression of target cells is shown in the same figure.

 
Fig. 5 shows results of cytotoxicity assays using purified polyclonal T cells as effectors (see Fig. 4, top) and the FATAL method as readout. Fig. 6 shows experiments with two previously described highly purified (>98%) V{gamma}9V{delta}2 T cell lines as effectors tested in a standard 51Cr release assay (34). Fig. 5A depicts data from an experiment representative of six independent experiments performed with cells from four different donors. In all experiments, lysis of RMA-MICA cells and of Daudi cells was quite similar, whereas no dose-dependent lysis was found for RMA-Neo cells. In Fig. 5, B and C, involvement of NKG2D in killing was tested by addition of 20 µg/ml unconjugated anti-NKG2D mAb (M585) (39) and only killing of the NKG2D ligand-expressing RMA-MICA cells was inhibited. Finally, the lysis of C1R-Neo cells, C1R-MICA cells, and C1R-ULBP2 cells was compared (Fig. 5D). Some killing was found for C1R-Neo cells, which express low levels of endogenous ULBP2 and MICA. Lysis was increased for C1R cells after transfection with either MICA or ULBP2.



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  		FIGURE 6. Analysis of TCR- and NKG2D-dependent cytotoxicity by V{gamma}9V{delta}2 T cell lines. Lysis in a 4-h cytotoxicity assay at the indicated E:T ratios and combinations. V{gamma}9V{delta}2 T cells line EP2 (left column) or HO (right column). Upper panel, Lysis of RMA Neo; middle panel, lysis of RMA MICA; lower panel, lysis of Mewo cells. Abs were added as indicated. No Ab ({diamond}), anti-TCR ({blacksquare}), anti-NKG2D ({blacktriangleup}), anti-TCR plus anti-NKG2D (•), isotype control ({triangleup}).

 
NKG2D-mediated lysis was also observed with highly purified V{gamma}9V{delta}2 T cell lines HO and EP3 as effectors (34). As shown above with the FATAL method, only RMA-MICA but not RMA-Neo cells were lysed also by the >98% pure {gamma}{delta} lines using the 51Cr release assay as an independent readout system. To test the respective contribution of NKG2D and TCR to this lysis, Ab inhibition experiments were performed as previously described (34). The presence of NKG2D-specific mAb M585 blocked lysis of RMA-MICA very efficiently, while only weak if any effects of TCR-specific mAb were found (Fig. 6, middle row). The results contrast with those obtained with Mewo melanoma as target cells, which express ligands of V{gamma}9V{delta}2 TCR and NKG2D. In this case, blockade of lysis by TCR-specific mAb (70–80% inhibition) was very efficient, compared with NKG2D-specific mAb (30–50% inhibition). Combining both mAb only negligibly increased the anti-TCR inhibition (Fig. 6, lower row). These results confirm NKG2D ligation is sufficient to trigger lysis of RMA-MICA and reproduce previous results showing inhibition of V{gamma}9V{delta}2 T cell-mediated killing of Mewo cells by TCR-specific mAb (34).

DAP10 expression of V{gamma}9V{delta}2 T cells

Very recently, it was reported that cytokine-induced CD8+ killer cells cultured with high doses of IL-2 show NKG2D-mediated cytotoxicity. This cytolytic activity coincided with IL-2 induced expression of DAP10. Therefore, we tested DAP10 expression in V{gamma}9V{delta}2 T cells. Semiquantitative RT-PCR analysis, as shown in Fig. 7, reveals DAP10 mRNA in resting and activated V{gamma}9V{delta}2 T cells. cDNA for this analysis was generated from positively selected resting {gamma}{delta} T cells (95% purity, no NK cells) or V{gamma}9V{delta}2 T cells after 7 days of culture with IPP and intermediate (100 U/ml; 96% purity, no NK cells) or high concentration of IL-2 (300 U/ml; 93% purity, no NK cells). Positive controls were cDNA of the monocytic line THP1 and PBMC and the negative control was cDNA of B cell lymphoma Raji.



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  		FIGURE 7. Determination of DAP10 expression in V{gamma}9V{delta}2 T cells by RT-PCR and immunoblot. The left panels show a RT-PCR for DAP10 and {beta}-actin using serial dilutions of cDNA from the indicated cell populations. The right panels show DAP10 protein level as tested by immunoblot analysis of the indicated cell types.

 
Expression of DAP10 at the protein level was also tested by immunoblot analysis of detergent lysates of V{gamma}9V{delta}2 T effector cells, human PBMC, and Daudi cells. DAP10 is strongly expressed in V{gamma}9V{delta}2 T effector cells and in human PBMC


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well established that NKG2D ligation provides a powerful costimulus for Ag-mediated activation of CD8 and {gamma}{delta} T cells. In this study, we show that V{gamma}9V{delta}2 T cells can be activated through NKG2D independently of Ag, similar to the NKG2D-mediated activation of NK cells.

Originally, NKG2D was described as a stimulating receptor for NK cells and as a costimulatory receptor for different types of T cells. In the mouse, this functional dichotomy correlates with occurrence of activation-dependent splice-variants of NKG2D. NKG2D-L binds DAP10, which carries a YXNM motif and links it to phosphoinositol 3-kinase-dependent pathways also associated with costimulatory receptors like CD28. NKG2D-S is expressed only after activation, associates with both DAP10 and DAP12, and carries an ITAM motif typical of activating receptors such as TCR/CD3 (40). Nevertheless, some NK cell functions appear to be DAP12 independent, because NK cell cytotoxicity of DAP12-deficient mice can be triggered by NKG2D ligation (41). In humans, NKG2D does not bind to DAP12, and this lack of physical interaction with DAP12 correlates well with the reported lack of NKG2D induced cytokine production (42). Recently it was also reported that stimulation of NK cells from total PBMC with immobilized NKG2D ligands MICA and ULBP1, but not with anti-NKG2D mAb, induces CD25 expression and proliferation, respectively, and that NK cell lines release IFN-{gamma} after such treatment (43). In our experiments, stimulation of resting V{gamma}9V{delta}2 T cells led to quite similar results with respect to induction of CD25, so it appears that the NKG2D-specific mAb 149810 can mimic the natural ligand

Up-regulation of the activation markers CD25 and CD69 was quite similar in NK cells and resting V{gamma}9V{delta}2 T cells. This observation and detection of DAP10 in resting and activated V{gamma}9V{delta}2 T cells would be consistent with an overlap of signaling pathways used by NKG2D-mediated activation in NK cells and V{gamma}9V2{delta} T cells. The fact that activation does not cover all cellular functions, e.g., production of IFN-{gamma} or proliferation (B. Rincon-Orozco and T. Herrmann, unpublished data) probably is due to the lack of a second signal, which, in human NK cells, appears to be partially dispensable and which, in mice, is provided by the ITAM-bearing and NKG2D-binding molecule DAP12. It will be of special interest to learn whether, in V{gamma}9V{delta}2 T cells, other signaling pathways could provide such an additional signal that would then allow complete activation of V{gamma}9V{delta}2 T cell functions in an Ag-independent but NKG2D-triggered fashion. The most obvious candidates to deliver such signals would be receptors binding ITAM-bearing adaptor molecule. Interestingly, expression of DAP12 (B. Rincon-Orozco and T. Herrmann, unpublished data) was found in activated V{gamma}9V{delta}2 T cells, which increases the range of potential activating cell surface receptors beyond TCR or FcRIIIA to DAP12 associated receptors. An alternative way to boost NKG2D-mediated V{gamma}9V{delta}2 T cell activation would be coligation of NKG2D with other "coreceptors" like CD28. In the case of CD28, which shows some similarity to NKG2D with respect to signaling, combinations of mAb specific for CD28 and CD27 (44) or CD28 and CD2 (45) activate {alpha}{beta} T cells rather efficiently. It is now tempting to speculate that similar regimes could induce substantial NKG2D-triggered and Ag-independent activation of V{gamma}9V{delta}2 T cells. Finally, numerous soluble mediators such as IL-2, IL-15, or poly:IC-induced IFN-{alpha} initiate either partial activation of V{gamma}9V{delta}2 T cells or are indispensable for growth and survival after Ag induced activation (33, 46, 47). Their role in NKG2D induced activation also needs to be investigated in greater detail.

As already mentioned, NKG2D was originally described as a stimulatory receptor for NK cells, but its capacity to enhance activation by other activating receptors suggests it may also function as a costimulatory molecule. Conversely, with the exception of some V{gamma}1{delta}1 T cells, where discrimination between stimulation and costimulation is hardly possible because NKG2D ligands appear to bind simultaneously to TCR and NKG2D (48), until very recently, NKG2D was viewed exclusively as a costimulatory molecule for T cells. This view has now been modified as some subtypes of in vitro-activated human CD8 cells (49, 50, 51) and certain subpopulations of mouse CD8 cells (52) can be directly activated by NKG2D. Relevant to our work was identification of a human CD8 T cell clone that lysed allogenic tumor cells and whose lysis was inhibited by NKG2D but not by MHC class I or TCR-specific mAbs (49). It has also been reported that in vitro-activated CD8 cells could kill K562-FcR (K30) cells loaded with an ULBP4-Fc construct (ULBP4 is designated as LETAL in the cited paper; Ref. 50). Finally, cytokine-induced killer cells generated by culture in the presence of 300 U/ml IL-2 lysed NKG2D ligand-expressing target cells in a NKG2D-dependent but TCR-independent manner. Interestingly, this lysis was not found in CD8 cells generated with 100 U/ml IL-2 for which DAP10 expression could not be detected (51). In contrast we show in this study that resting and activated {gamma}{delta} T cells express DAP10 and are stimulated by NKG2D ligation, which again would be consistent with a role for DAP10 in their activation.

The fact that direct NKG2D-mediated activation of V{gamma}9V{delta}2 T cells has not been described in earlier studies may be due to several reasons. First, previous work has focused on the function of NKG2D as costimulatory receptor, and many experiments measured IFN-{gamma} production, which is not induced by ligation of NKG2D. Nevertheless, there is still a discrepancy between our results on the lysis of MICA and ULBP2-expressing C1R cells and a report where such lysis was not found (32). One explanation could be the choice of effector cells in both studies, namely monoclonal vs polyclonal {gamma}{delta} T cell populations. It appears quite possible that expression of KIR p58.1 by monoclonal effector cells, which bind to HLA-Cw0401 of C1R cells, could have inhibited lysis, although such effects would not be detected with a polyclonal effector cell population, where only a minority of cells express p58.1 (53).

In summary, our results indicate that the effects of NKG2D ligation are remarkably similar for V{gamma}9V{delta}2 T cells and NK cells, suggesting that V{gamma}9V{delta}2 T cells may take over functions of NK cells in destroying NKG2D ligand-expressing tumor cells or stressed or infected cells. Practically, this implies that V{gamma}9V{delta}2 T cell-directed therapy may be applicable for the treatment of tumors that do not express or present V{gamma}9V{delta}2 TCR ligands. In contrast, such "unspecific" activation may be deleterious for the host. In various infectious diseases, V{gamma}9V{delta}2 T cells are massively expanded with sometimes more than half of the population of peripheral blood T cells being V{gamma}9V{delta}2 T cells. In such pathological conditions, where, as consequence of infection, expression of NKG2D ligands may be induced, bystander activation of V{gamma}9V{delta}2 T cells could occur. Finally, it seems likely that the effects of NKG2D ligation could be strongly enhanced by Ag-independent signals given by cytokines or ligation of cell surface molecules, the level of expression in which may also be increased during infection or inflammatory processes, allowing a sustained Ag-independent V{gamma}9V{delta}2 T cell activation.


   Acknowledgments
 
We thank Katrin Wiemann for generation of the RMA transfectants and Judith Engert for help with the cytotoxicity assays.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Interdiziplinäres Zentrum für klinische Forschung, B26 (to V.K. and T.H.) and the European Graduate College "Gene Regulation in and by Microbial Pathogens" (to B.R.O. and T.H.), and forms part of the Ph.D. thesis of B.R.O. and the M.D. thesis of P.W. Back

2 Address correspondence and reprint requests to Dr. Thomas Herrmann, Institut für Virologie und Immunbiologie, Versbacherstrasse 7, 97078 Würzburg, Germany. E-mail address: herrmann-t{at}vim.uni-wuerzburg.de Back

3 Abbreviations used in this paper: BLT, N-benzyloxycarbonyl lysine thiobenzyl ester; IPP, isopentenyl pyrophosphate; MICA, MHC class I related protein A; KIR, killer inhibitory receptor; FATAL, fluorometric assessment of T lymphocyte Ag-specific lysis. Back

Received for publication August 18, 2004. Accepted for publication May 30, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. De Libero, G.. 1997. Sentinel function of broadly reactive human {gamma}{delta} T cells. Immunol. Today 18: 22-26. [Medline]
  2. Chen, Z. W., N. L. Letvin. 2003. V{gamma}2V{delta}2+ T cells and anti-microbial immune responses. Microbes Infect. 5: 491-498. [Medline]
  3. Eberl, M., H. Jomaa, A. C. Hayday. 2004. Integrated immune responses to infection—cross-talk between human {gamma}{delta} T cells and dendritic cells. Immunology 112: 364-368. [Medline]
  4. Dieli, F., M. Troye-Blomberg, J. Ivanyi, J. J. Fournie, M. Bonneville, M. A. Peyrat, G. Sireci, A. Salerno. 2000. V{gamma}9/V{delta}2 T lymphocytes reduce the viability of intracellular Mycobacterium tuberculosis. Eur. J. Immunol. 30: 1512-1519. [Medline]
  5. Wang, L., A. Kamath, H. Das, L. Li, J. F. Bukowski. 2001. Antibacterial effect of human V{gamma}2V{delta}2 T cells in vivo. J. Clin. Invest. 108: 1349-1357. [Medline]
  6. Wilhelm, M., V. Kunzmann, S. Eckstein, P. Reimer, F. Weissinger, T. Ruediger, H. P. Tony. 2003. {gamma}{delta} T cells for immune therapy of patients with lymphoid malignancies. Blood 102: 200-206. [Abstract/Free Full Text]
  7. Morita, C. T., H. K. Lee, D. S. Leslie, Y. Tanaka, J. F. Bukowski, E. Marker-Hermann. 1999. Recognition of nonpeptide prenyl pyrophosphate antigens by human {gamma}{delta} T cells. Microbes Infect. 1: 175-186.
  8. Belmant, C., E. Espinosa, F. Halary, Y. Tang, M. A. Peyrat, H. Sicard, A. Kozikowski, R. Buelow, R. Poupot, M. Bonneville, J. J. Fournie. 2000. A chemical basis for selective recognition of nonpeptide antigens by human {delta} T cells. FASEB J. 14: 1669-1670. [Abstract/Free Full Text]
  9. Hintz, M., A. Reichenberg, B. Altincicek, U. Bahr, R. M. Gschwind, A. K. Kollas, E. Beck, J. Wiesner, M. Eberl, H. Jomaa. 2001. Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human {gamma}{delta} T cells in Escherichia coli. FEBS Lett. 509: 317-322. [Medline]
  10. Tanaka, Y., C. T. Morita, E. Nieves, M. B. Brenner, B. R. Bloom. 1995. Natural and synthetic non-peptide antigens recognized by human {gamma}{delta} T cells. Nature 375: 155-158. [Medline]
  11. Bukowski, J. F., C. T. Morita, Y. Tanaka, B. R. Bloom, M. B. Brenner, H. Band. 1995. V{gamma}2V{delta}2 TCR-dependent recognition of non-peptide antigens and Daudi cells analyzed by TCR gene transfer. J. Immunol. 154: 998-1006. [Abstract]
  12. Scotet, E., L. O. Martinez, E. Grant, R. Barbaras, P. Jeno, M. Guiraud, B. Monsarrat, X. Saulquin, S. Maillet, J. P. Esteve, et al 2005. Tumor recognition following V{gamma}9V{delta}2 T cell receptor interactions with a surface F1-ATPase-related structure and apolipoprotein A-I. Immunity 22: 71-80. [Medline]
  13. Kunzmann, V., E. Bauer, J. Feurle, F. Weissinger, H. P. Tony, M. Wilhelm. 2000. Stimulation of {gamma}{delta} T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 96: 384-392. [Abstract/Free Full Text]
  14. Gober, H. J., M. Kistowska, L. Angman, P. Jeno, L. Mori, G. De Libero. 2003. Human T cell receptor {gamma}{delta} cells recognize endogenous mevalonate metabolites in tumor cells. J. Exp. Med. 197: 163-168. [Abstract/Free Full Text]
  15. Morita, C. T., E. M. Beckman, J. F. Bukowski, Y. Tanaka, H. Band, B. R. Bloom, D. E. Golan, M. B. Brenner. 1995. Direct presentation of nonpeptide prenyl pyrophosphate antigens to human {gamma}{delta} T cells. Immunity 3: 495-507. [Medline]
  16. Kato, Y., Y. Tanaka, H. Tanaka, S. Yamashita, N. Minato. 2003. Requirement of species-specific interactions for the activation of human {gamma}{delta} T cells by pamidronate. J. Immunol. 170: 3608-3613. [Abstract/Free Full Text]
  17. Krei{beta}, M., M. Krejci, B. Rinco-Orosco, S. Wiehr, M. Wilhelm, V. Kunzmann, T. Herrmann. 2003. Tissue and species specificity of presentation of ligands of the human V{gamma}9{delta}2 TCR. Immunobiology 208: 56
  18. Fisch, P., E. Meuer, D. Pende, S. Rothenfusser, O. Viale, S. Kock, S. Ferrone, D. Fradelizi, G. Klein, L. Moretta, et al 1997. Control of B cell lymphoma recognition via natural killer inhibitory receptors implies a role for human V{gamma}9/V{delta}2 T cells in tumor immunity. Eur. J. Immunol. 27: 3368-3379. [Medline]
  19. Halary, F., M. A. Peyrat, E. Champagne, M. Lopez-Botet, A. Moretta, L. Moretta, H. Vie, J. J. Fournie, M. Bonneville. 1997. Control of self-reactive cytotoxic T lymphocytes expressing {gamma}{delta} T cell receptors by natural killer inhibitory receptors. Eur. J. Immunol. 27: 2812-2821. [Medline]
  20. Raulet, D. H.. 2003. Roles of the NKG2D immunoreceptor and its ligands. Nat. Rev. Immunol. 3: 781-790. [Medline]
  21. Hamerman, J. A., K. Ogasawara, L. L. Lanier. 2004. Cutting edge: Toll-like receptor signaling in macrophages induces ligands for the NKG2D receptor. J. Immunol. 172: 2001-2005. [Abstract/Free Full Text]
  22. Salih, H. R., H. Antropius, F. Gieseke, S. Z. Lutz, L. Kanz, H. G. Rammensee, A. Steinle. 2003. Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood 102: 1389-1396. [Abstract/Free Full Text]
  23. Groh, V., S. Bahram, S. Bauer, A. Herman, M. Beauchamp, T. Spies. 1996. Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium. Proc. Natl. Acad. Sci. USA 93: 12445-12450. [Abstract/Free Full Text]
  24. Groh, V., R. Rhinehart, J. Randolph-Habecker, M. S. Topp, S. R. Riddell, T. Spies. 2001. Costimulation of CD8{alpha}{beta} T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat. Immunol. 2: 255-260. [Medline]
  25. Diefenbach, A., E. R. Jensen, A. M. Jamieson, D. H. Raulet. 2001. Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity. Nature 413: 165-171. [Medline]
  26. Girardi, M., D. E. Oppenheim, C. R. Steele, J. M. Lewis, E. Glusac, R. Filler, P. Hobby, B. Sutton, R. E. Tigelaar, A. C. Hayday. 2001. Regulation of cutaneous malignancy by {gamma}{delta} T cells. Science 294: 605-609. [Abstract/Free Full Text]
  27. Bauer, S., V. Groh, J. Wu, A. Steinle, J. H. Phillips, L. L. Lanier, T. Spies. 1999. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285: 727-729. [Abstract/Free Full Text]
  28. Wu, J., Y. Song, A. B. Bakker, S. Bauer, T. Spies, L. L. Lanier, J. H. Phillips. 1999. An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285: 730-732. [Abstract/Free Full Text]
  29. Groh, V., R. Rhinehart, H. Secrist, S. Bauer, K. H. Grabstein, T. Spies. 1999. Broad tumor-associated expression and recognition by tumor-derived {gamma}{delta} T cells of MICA and MICB. Proc. Natl. Acad. Sci. USA 96: 6879-6884. [Abstract/Free Full Text]
  30. Groh, V., J. Wu, C. Yee, T. Spies. 2002. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature 419: 734-738. [Medline]
  31. Billadeau, D. D., J. L. Upshaw, R. A. Schoon, C. J. Dick, P. J. Leibson. 2003. NKG2D-DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathway. Nat. Immunol. 4: 557-564. [Medline]
  32. Das, H., V. Groh, C. Kuijl, M. Sugita, C. T. Morita, T. Spies, J. F. Bukowski. 2001. MICA engagement by human V{gamma}2V{delta}2 T cells enhances their antigen-dependent effector function. Immunity 15: 83-93. [Medline]
  33. Kunzmann, V., E. Kretzschmar, T. Herrmann, M. Wilhelm. 2004. Polyinosinic-polycytidylic acid-mediated stimulation of human {gamma}{delta} T cells via CD11c dendritic cell-derived type I interferons. Immunology 112: 369-377. [Medline]
  34. Kabelitz, D., D. Wesch, E. Pitters, M. Zoller. 2004. Characterization of tumor reactivity of human V{gamma}9V{delta}2 {gamma}{delta} T cells in vitro and in SCID mice in vivo. J. Immunol. 173: 6767-6776. [Abstract/Free Full Text]
  35. Takayama, H., G. Trenn, M. V. Sitkovsky. 1987. A novel cytotoxic T lymphocyte activation assay: optimized conditions for antigen receptor triggered granule enzyme secretion. J. Immunol. Methods 104: 183-190. [Medline]
  36. Sheehy, M. E., A. B. McDermott, S. N. Furlan, P. Klenerman, D. F. Nixon. 2001. A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis. J. Immunol. Methods 249: 99-110. [Medline]
  37. Sutherland, C. L., N. J. Chalupny, K. Schooley, T. VandenBos, M. Kubin, D. Cosman. 2002. UL16-binding proteins, novel MHC class I-related proteins, bind to NKG2D and activate multiple signaling pathways in primary NK cells. J. Immunol. 168: 671-679. [Abstract/Free Full Text]
  38. Janssen, O., S. Wesselborg, B. Heckl-Ostreicher, K. Pechhold, A. Bender, S. Schondelmaier, G. Moldenhauer, D. Kabelitz. 1991. T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor {gamma}{delta}+ T cells. J. Immunol. 146: 35-39. [Abstract]
  39. Campbell, K. S., M. Colonna. 1999. DAP12: a key accessory protein for relaying signals by natural killer cell receptors. Int. J. Biochem. Cell Biol. 31: 631-636. [Medline]
  40. Diefenbach, A., E. Tomasello, M. Lucas, A. M. Jamieson, J. K. Hsia, E. Vivier, D. H. Raulet. 2002. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D. Nat. Immunol. 3: 1142-1149. [Medline]
  41. Zompi, S., J. A. Hamerman, K. Ogasawara, E. Schweighoffer, V. L. Tybulewicz, J. P. Di Santo, L. L. Lanier, F. Colucci. 2003. NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases. Nat. Immunol. 4: 565-572. [Medline]
  42. Wu, J., H. Cherwinski, T. Spies, J. H. Phillips, L. L. Lanier. 2000. DAP10 and DAP12 form distinct, but functionally cooperative, receptor complexes in natural killer cells. J. Exp. Med. 192: 1059-1068. [Abstract/Free Full Text]
  43. Andre, P., R. Castriconi, M. Espeli, N. Anfossi, T. Juarez, S. Hue, H. Conway, F. Romagne, A. Dondero, M. Nanni, et al 2004. Comparative analysis of human NK cell activation induced by NKG2D and natural cytotoxicity receptors. Eur. J. Immunol. 34: 961-971. [Medline]
  44. Sunder-Plassmann, R., W. F. Pickl, O. Majdic, W. Knapp, W. Holter. 1995. Crosslinking of CD27 in the presence of CD28 costimulation results in T cell proliferation and cytokine production. Cell Immunol. 164: 20-27. [Medline]
  45. Van Lier, R. A., M. Brouwer, L. A. Aarden. 1988. Signals involved in T cell activation: T cell proliferation induced through the synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies. Eur. J. Immunol. 18: 167-172. [Medline]
  46. Boullier, S., Y. Poquet, T. Debord, J. J. Fournie, M. L. Gougeon. 1999. Regulation by cytokines (IL-12, IL-15, IL-4 and IL-10) of the V{gamma}9V{delta}2 T cell response to mycobacterial phosphoantigens in responder and anergic HIV-infected persons. Eur. J. Immunol. 29: 90-99. [Medline]
  47. Garcia, V. E., D. Jullien, M. Song, K. Uyemura, K. Shuai, C. T. Morita, R. L. Modlin. 1998. IL-15 enhances the response of human {gamma}{delta} T cells to nonpeptide microbial antigens. J. Immunol. 160: 4322-4329. [Abstract/Free Full Text]
  48. Wu, J., V. Groh, T. Spies. 2002. T cell antigen receptor engagement and specificity in the recognition of stress-inducible MHC class I-related chains by human epithelial {gamma}{delta} T cells. J. Immunol. 169: 1236-1240. [Abstract/Free Full Text]
  49. Maccalli, C., D. Pende, C. Castelli, M. C. Mingari, P. F. Robbins, G. Parmiani. 2003. NKG2D engagement of colorectal cancer-specific T cells strengthens TCR-mediated antigen stimulation and elicits TCR independent anti-tumor activity. Eur. J. Immunol. 33: 2033-2043. [Medline]
  50. Conejo-Garcia, J. R., F. Benencia, M. C. Courreges, P. A. Gimotty, E. Khang, R. J. Buckanovich, K. A. Frauwirth, L. Zhang, D. Katsaros, C. B. Thompson, et al 2004. Ovarian carcinoma expresses the NKG2D ligand Letal and promotes the survival and expansion of CD28- antitumor T cells. Cancer Res. 64: 2175-2182. [Abstract/Free Full Text]
  51. Verneris, M. R., M. Karami, J. Baker, A. Jayaswal, R. S. Negrin. 2004. Role of NKG2D signaling in the cytotoxicity of activated and expanded CD8+ T cells. Blood 103: 3065-3072. [Abstract/Free Full Text]
  52. Dhanji, S., S. J. Teh, D. Oble, J. J. Priatel, H. S. Teh. 2004. Self-reactive memory-phenotype CD8 T cells exhibit both MHC-restricted and non-MHC-restricted cytotoxicity: a role for the T-cell receptor and natural killer cell receptors. Blood 104: 2116-2123. [Abstract/Free Full Text]
  53. Rothenfusser, S., A. Buchwald, S. Kock, S. Ferrone, P. Fisch. 2002. Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human {gamma}{delta} T lymphocytes. Cell Immunol. 215: 32-44. [Medline]



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