The immunoreceptor NKG2D stimulates activation of cytotoxic lymphocytes upon engagement with MHC class I-related NKG2D ligands of which at least some are expressed inducibly upon exposure to carcinogens, cell stress, or viruses. In this study, we investigated consequences of a persistent NKG2D ligand expression in vivo by using transgenic mice expressing MHC class I chain-related protein A (MICA) under control of the H2-Kb promoter. Although MICA functions as a potent activating ligand of mouse NKG2D, H2-Kb-MICA mice appear healthy without aberrations in lymphocyte subsets. However, NKG2D-mediated cytotoxicity of H2-Kb-MICA NK cells is severely impaired in vitro and in vivo. This deficiency concurs with a pronounced down-regulation of surface NKG2D that is also seen on activated CD8 T cells. As a consequence, H2-Kb-MICA mice fail to reject MICA-expressing tumors and to mount normal CD8 T cell responses upon Listeria infection emphasizing the importance of NKG2D in immunity against tumors and intracellular infectious agents.

The C-type lectin-like activating receptor NKG2D is broadly expressed on cytotoxic lymphocytes. In humans, NKG2D is present on most NK cells, CD8 T cells, and γδ T cells in association with the adaptor protein DAP10 (1, 2). In mice, CD8 T cells express NKG2D only upon activation, whereas NK cells, NKT cells, and some γδ T cells constitutively express NKG2D (3, 4). Different from humans, activated mouse NK cells generate a second NKG2D isoform with a shortened cytoplasmic domain (NKG2D-S), which is capable of pairing with both DAP10 and DAP12, whereas the constitutively expressed NKG2D-L isoform exclusively associates with DAP10 (5, 6). DAP10 mediates costimulation of CD8 T cells and triggers cytotoxicity by NK cells, whereas signal transduction via DAP12 augments cytotoxicity and is strictly required for activation of cytokine release (7, 8, 9).

A peculiarity of NKG2D resides in its interaction with a multitude of MHC class I-related ligands of which at least the MIC molecules are expressed inducibly in association with cell stress, infection, or malignant transformation (10, 11, 12). Whereas the ectodomain of NKG2D is fairly conserved in mouse and man, the various MHC class I-related binding partners of NKG2D are highly diverged. In humans, the MHC-encoded MIC molecules MHC class I chain-related proteins A and B (MICA and MICB)3 and five members of the UL16-binding protein (ULBP) family (ULBP1–4; RAET1G) ligate NKG2D and consequently trigger NK cells (13, 14, 15). In vitro, cell stress-inducible MIC molecules are expressed by many tumor cell lines and up-regulated upon infection with human CMV, Mycobacterium tuberculosis, and Escherichia coli (12, 16). In vivo, MIC molecules are not detectable on most healthy tissues, but are expressed on gastrointestinal epithelium, on tumors, and on human CMV-infected cells (8, 16). Recently, MICA expression was reported for tissues affected by autoimmune reactions in patients with rheumatoid arthritis and celiac disease together with evidence for an involvement of NKG2D in the autoimmune pathogenesis of these diseases (17, 18, 19).

In mice, members of the retinoic acid early transcript 1 (RAE-1) protein family, the minor histocompatibility Ag H60, and murine ULBP-like transcript 1 act as ligands of NKG2D (20, 21, 22). Similarly to ULBP molecules they all lack an α3 domain. Recently, up-regulation of RAE-1 molecules on macrophages by various ligands of TLR has been demonstrated (23). RAE-1 expression is also induced by carcinogens and stimulates antitumor activity of γδ T cells (24). RAE-1-transduced tumor cell lines were rejected in vivo due to NK and CD8 T cell responses and induced tumor immunity against the parental cell line supporting a role for NKG2D in tumor immunity (25, 26), though no direct evidence for an involvement of NKG2D was provided.

Recent findings that tumor cells release soluble MIC molecules may account for the failure of tumor surveillance by the NKG2D system in human cancer patients. MICA molecules are shed from tumor cells by metalloproteases resulting in a reduced NKG2D ligand (NKG2DL) surface density (27). Further, soluble MICA (sMICA) was shown to down-regulate NKG2D surface expression and, thereby, to impair the antitumor reactivity of cytotoxic lymphocytes in vitro (28). Substantial levels of sMICA were detected in sera of patients with various malignancies and correlated with a systemic NKG2D down-regulation on peripheral NK and CD8 T cells (27, 29, 30). However, direct evidence for an in vivo impairment of NKG2D-mediated tumor immunosurveillance by persistent MICA expression was lacking. Another study reported NKG2D down-regulation upon coculture with NKG2DL-expressing cells that was at least in part due to signaling of the DAP10 adaptor. In addition, down-regulation of NKG2D was observed for NK cells from NOD mice and attributed to coexpression of RAE-1 (31), but consequences for NK cell activation in vivo were not investigated.

In this study, we explored implications of persistent NKG2DL expression in vivo. We investigated consequences of persistent MICA expression for NKG2D-mediated immunosurveillance as it may occur in cancer patients. In addition, we took advantage of the strongly impaired NKG2D function in H2-Kb-MICA mice to address the role of NKG2D for NK and CD8 T cell responses in vivo.

C57BL/6 mice were obtained from Charles River Wiga. For the generation of mice constitutively expressing MICA, one-cell embryos of (C57BL/6 × SJL)F1/J hybrid mice were microinjected with a 4.75-kb XhoI/KpnI DNA fragment excised from the pHSE plasmid (32) containing the H2-Kb promoter followed by the 1.15-kb MICA*07 open reading frame (accession number AY750850) and the 3′ untranslated region of the β-globin gene. Eggs were transferred into the oviducts of B6 CBA mice (transgenic mouse facility, Fred Hutchinson Cancer Research Center). Offspring were tested for the presence of the MICA transgene by PCR from genomic tail DNA using the oligonucleotides MICAEX2F (5′-GAC TTG ACA GGG AAC GGA AAG G-3′) and MICAEX4R (5′-CCC CCC ACT GCT GGG TGT TG-3′). Several H2-Kb-MICA transgenic lines were obtained, and one of them (tg24) exhibiting high surface MICA expression was backcrossed with C57BL/6 mice at least 12 times for further studies. Litters were tested for MICA transgenes by PCR and/or for MICA surface expression on PBL by flow cytometry using biotinylated anti-MICA/B mAb BAMO1. RAG2-deficient and β2-microglobulin-deficient C57BL/6 mice were kindly provided by H. Schild (University of Mainz, Mainz, Germany). Animals were maintained under specific pathogen-free conditions in the animal facilities of the Department of Immunology at the University of Tübingen. All animal experiments were conducted according to the German animal protection law.

All cell culture media were supplemented with 10% FCS (PAA Laboratories), 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Cambrex), 1 mM sodium pyruvate (c.c. pro), and 50 μM 2-ME. The cell lines RMA (T cell lymphoma), RMA-S (TAP2-deficient RMA variant), and CHO (Chinese hamster ovary carcinoma) were cultured in RPMI 1640 (Cambrex). The RMA cell line was transfected by electroporation (250 V, 950 μF) using 15 μg of the vector RSV.5neo or RSV.5neo containing the MICA*07 open reading frame (15). Stable transfectants (RMA-neo and RMA-MICA*07) were selected in RPMI 1640 supplemented with 1 mg/ml G418 (PAA Laboratories).

Thymocytes and splenocytes were prepared by passing the respective organs through a 40-μm cell strainer (BD Biosciences Europe). For analysis of PBL, blood was collected from the orbital sinus. RBC in PBL and splenocyte suspensions were lysed by ammonium chloride treatment. For isolation of NK cells, nylon wool nonadherent cells were positively selected from splenocytes by magnetic cell sorting using DX5 MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions. Resulting cells were 65–85% NK1.1+. For in vitro analyses DX5-sorted cells were cultured in RPMI 1640 (Cambrex) supplemented with 1500 U/ml human IL-2 (R&D Systems). For analysis of in vivo-activated NK cells, mice were injected i.p. with 300 μg of polyinosinic-polycytidylic acid potassium salt (poly(I:C)) (Sigma-Aldrich). Twelve to 18 h later, DX5-positive cells were isolated from splenocytes as described above. Con A blasts were generated by culturing splenocytes for 48 h in Alpha-MEM (Cambrex) containing 2.5 μg/ml Con A. Hepatocytes were isolated by a two-step perfusion protocol (33). In brief, 60 ml of PBS containing 0.5 mM EGTA and 0.05 M HEPES was used as a first perfusate. Liver was perfused with 60 ml of collagenase (40 U/ml in RPMI 1640; Sigma-Aldrich) and subsequently excised from the body cavity, the gall bladder was removed, and the liver was pushed through a tea strainer and incubated for 10 min at 37°C in collagenase. To get a single-cell suspension, the digested liver was additionally rinsed through a 70-μm cell strainer (BD Biosciences Europe).

For isolation of CD8 T cells, nylon wool nonadherent splenocytes were first depleted for NK cells using DX5 MicroBeads (Miltenyi Biotec). DX5-depleted cells were then positively selected with anti-CD8 (clone Ly-2) MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions. Purified CD8 T cells were either directly analyzed for NKG2D expression or after 3 days culture in anti-CD3 (clone 17A2) coated microwells.

Anti-CD3ε FITC (145-2C11), anti-H2-Kb FITC (AF6-88.5), anti-NK1.1 PE (PK136), anti-CD8 PE (Ly-2), anti-CD19 PE (1D3), anti-γδTCR PE (GL3), anti-TNP-KLH PE (A110-1; rat Ig control), anti-TNP hamster IgG PE (A19-3; hamster Ig control), and anti-CD4 PerCP (GK1.5) were purchased from BD Pharmingen. Anti-NKG2D PE (CX5), anti-NKG2D allophycocyanin (CX5), and anti-NK1.1 FITC (PK136) were obtained from eBioscience. Anti-NKG2D mAb C7 was described elsewhere (3). Rat IgG Abs, anti-CD16/CD32 mAb (clone 2.4G2), anti-IFN-γ mAb (XMG1.2), anti-CD8α mAb (YTS169), and anti-CD62L mAb (Mel-14) were purified from rat serum or hybridoma supernatants with protein G-Sepharose. Abs were Cy5- or FITC-conjugated according to standard protocols. The mAbs AMO1 and BAMO1 recognizing MICA and MICA/B, respectively, were generated and purified as described (27). BAMO1 was biotinylated using EZ-Link Sulfo-NHS-Biotin (Pierce) according to the manufacturer’s protocol. Streptavidin-PE (Molecular Probes) was used as a secondary reagent. Soluble mouse NKG2D was produced in insect cells using the baculovirus system as described elsewhere (15). Binding of FLAG-tagged mouse NKG2D was detected with the biotinylated anti-FLAG mAb M2 (Sigma-Aldrich) in combination with streptavidin-PE. Stained cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest software for evaluation.

Concentrations of sMICA in sera of H2-Kb-MICA and nontransgenic littermates (nontgLM) were determined by sandwich-ELISA using a modified protocol of the previously published MICA-ELISA (27). In brief, plates were coated with 2 μg/ml of the MICA-specific capture mAb AMO1 overnight at 4°C. After blocking with 7.5% BSA-PBS, plates were washed with 0.05% Tween 20. Samples or recombinant sMICA*04 serving as a standard were added in 2% BSA-PBS. After incubation and washing, biotinylated BAMO1 was added at a concentration of 1 μg/ml. After incubation and washing, HRP-conjugated streptavidin (BD Pharmingen) was applied as a 1/1000 dilution. Plates were washed extensively before adding the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) according to the manufacturer’s instructions. HRP activity was stopped by addition of 1 M phosphoric acid, and absorbance was measured at 450 nm.

Cytotoxicity of NK cells in vitro was assessed in a 51Cr release assay. NK effector cells were isolated from splenocytes with DX5-coated beads and either used immediately or after 5 days of cultivation with human IL-2 at 1500 U/ml (R&D Systems). Target cells were labeled with 50 μCi of 51Cr (Amersham) for 1 h at 37°C and washed three times. Effector cells were titrated on target cells and incubated for 4 h at 37°C unless noted otherwise. Spontaneous release of target cells alone was <15% of the maximum release taken from target cells lysed in 1% Triton X-100. Percentage of lysis was calculated as follows: 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release). Data are means of duplicates. For NKG2D blocking studies, NK cells were preincubated for 20 min with the mAb C7 (30 μg/ml) before addition to the target cells. Hamster Ig (ICN Pharmaceuticals) served as a control.

Purified NK cells were cocultured for 17 h with RMA transfectants at a ratio of 2:1 in medium containing 1000 U IL-2/ml. During the final 11 h of culture, GolgiStop (BD Biosciences) was added according to manufacturer’s instructions. Subsequently, cells were stained with FITC-conjugated anti-NK1.1, permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with PE-conjugated anti-IFN-γ mAb (BD Biosciences). After a further 20-min incubation, cells were washed and analyzed by flow cytometry.

RNA was isolated from purified NK cells using TRIzol (Invitrogen Life Technologies) followed by reverse transcription using SuperScript RTII (Invitrogen Life Technologies) according to the manufacturer’s protocol. The resulting cDNA was amplified with primer pairs specific for NKG2D, DAP10, DAP12, and 18S rRNA, respectively, in duplicates (40 cycles: 95°C × 15 s, 60°C × 1 min) using SYBRGreen chemistry on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). PCR products were analyzed on 3% agarose gels for purity und validated by direct sequencing. Data were analyzed by the ΔCT method for relative quantification and calculated as the relative increase in relative copy numbers. Oligonucleotide sequences (forward, reverse) are: 18S rRNA: 5′-CGGCTACCACATCCAAGGAA-3′, 5′-GCTGGAATTACCGCGGCT3′; NKG2D: 5′-ACG TTT CAG CCA GTA TTG TGC-3′, 5′-GGA AGC TTG GCT CTG GTT C-3′; DAP10: 5′-CCC AGG CTA CCT CCT GTT C-3′, 5′-CTA CAA TTA GGA GTG ACA TGA CCG-3′; DAP12: 5′-CTG GGA TTG TTC TGG GTG AC-3′, 5′-CTG AAG CTC CTG ATA AGG CG-3′. The NKG2D primer pair amplifies both the NKG2D-S and NKG2D-L variants.

Freshly isolated splenocytes were washed in PBS and resuspended at a final concentration of 2 × 107 cells/ml in PBS. Cells were labeled either with 8 μM CFSE (Molecular Probes) or with 5–7 μM PKH26 (Sigma-Aldrich) for 4 min at room temperature. The reaction was stopped by adding FCS, and cells were washed once in RPMI 1640 and twice in PBS. Labeled cells were mixed at defined ratios, and a total of 1.5 × 107 cells was adoptively transferred into H2-Kb-MICA mice or nontgLM, respectively. Immediately after adoptive transfer, labeled cells were analyzed in the peripheral blood. Six or 14 h after transfer, mice were sacrificed and PBL, splenocytes, and lymph node cells were analyzed for labeled cells by flow cytometry.

Growth of RMA-neo and RMA-MICA*07 cells was analyzed in H2-Kb-MICA mice, nontgLM, and RAG2-deficient mice. Mice were injected s.c. with 105 RMA-neo in the right flank and 105 RMA-MICA*07 cells in the left flank, respectively. Tumor growth was monitored by measuring tumor surface with a metric caliper at the indicated time points. Animals were sacrificed at day 17 when some tumors reached a size of ∼200 mm2. Data are representative of two independent experiments.

Mice were infected with L. monocytogenes strain EGD or with a L. monocytogenes strain recombinant for a secreted form of OVA (34). For i.v. infection, listeriae were injected into a lateral tail vein. Inocula were controlled by plating serial dilutions on tryptic soy broth agar.

For determination of cytokine expression, at day 9 postinfection, 4 × 106 splenocytes were stimulated for 5 h with 10−6 M of peptides derived from listeriolysin O (LLO190–201, NEKYAQAYPNVS) or OVA (OVA257–264, SIINFEKL). During the final 4 h of culture, 5 μg/ml brefeldin A (Sigma-Aldrich) was added. Cultured cells were incubated for 10 min with rat IgG and anti-CD16/CD32 mAb. Subsequently, cells were stained with PE-conjugated anti-CD4 mAb or anti-CD8α mAb, and after 30 min on ice, fixed for 20 min at room temperature with PBS 4% paraformaldehyde. Cells were washed with PBS 0.1% BSA, permeabilized with PBS 0.1% BSA/0.5% saponin (Sigma-Aldrich), and incubated with rat IgG and anti-CD16/CD32 mAb. After 5 min, FITC-conjugated anti-IFN-γ mAb was added. After a further 20-min incubation, cells were washed with PBS, fixed with PBS 1% paraformaldehyde, and analyzed by flow cytometry.

Modified full-length cDNA of H2-Kb and human β2-microglobulin were kindly provided by D. Busch (Technical University of Munich, Munich, Germany). H2-Kb/OVA257–264-tetramers were generated as described (35). For flow cytometric analysis, at day 9 postinfection, 2 × 106 splenocytes were incubated for 15 min at 4°C with rat IgG, anti-CD16/CD32 mAb, and streptavidin (Molecular Probes) in PBS with 0.5% BSA and 0.01% sodium azide. After incubation, cells were stained for 60 min at 4°C either with Cy5-conjugated anti-CD8α mAb, FITC-conjugated anti-CD62L mAb, and PE-conjugated MHC class I-OVA257–264-tetramers, or with allophycocyanin-conjugated anti-NKG2D mAb, FITC-conjugated anti-CD8α mAb, and PE-conjugated MHC class I- OVA257–264-tetramers. Subsequently, cells were washed with PBS 0.5% BSA/0.01% sodium azide and diluted in PBS. Propidium iodide was added before four-color flow cytometric analysis.

To gain insight into consequences of a persistent NKG2DL expression in vivo, we established transgenic mice constitutively and ubiquitously expressing the human NKG2DL MICA. We corroborated previous data of MIC molecules acting as a ligands of mouse NKG2D by demonstrating that MICA*07-expressing cells bind soluble mouse NKG2D (Fig. 1) (22, 36). To achieve constitutive and ubiquitous MICA expression, a transgene containing the coding sequence of MICA*07 under control of the MHC class I H2-Kb promoter was introduced into the germline of (C57BL/6 × SJL)F1/J mice (Fig. 2 A). Offspring expressing MICA on PBL were selected, and a transgenic line (H2-Kb-MICA) was established that was repeatedly backcrossed with C57BL/6 mice (B6 mice).

FIGURE 1.

MICA*07 ligates mouse NKG2D. Flow cytometric analysis of RMA cells ectopically expressing MICA*07 (RMA-MICA*07) with soluble mouse NKG2D (filled histogram) and anti-MICA/B mAb BAMO1 (gray histogram). Mock-transfected RMA cells (RMA-neo) did not bind mouse NKG2D (open histogram).

FIGURE 1.

MICA*07 ligates mouse NKG2D. Flow cytometric analysis of RMA cells ectopically expressing MICA*07 (RMA-MICA*07) with soluble mouse NKG2D (filled histogram) and anti-MICA/B mAb BAMO1 (gray histogram). Mock-transfected RMA cells (RMA-neo) did not bind mouse NKG2D (open histogram).

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FIGURE 2.

Constitutive MICA expression by H2-Kb-MICA mice. A, H2-Kb-MICA transgene encompassing the H2-Kb promoter, the 1.15-kb MICA*07 coding sequence, and the 3′ untranslated region (UT) of the β-globin gene. B, MICA cell surface expression by PBL, thymocytes, splenocytes, and hepatocytes of H2-Kb-MICA mice as detected by BAMO1. Cells were costained with anti-H2-Kb. C, MICA expression by gated subpopulations of splenocytes and thymocytes from nontgLM (gray histograms) and H2-Kb-MICA mice (filled histograms) as analyzed with BAMO1. D, sMICA levels in sera of H2-Kb-MICA mice detected with a MICA-sandwich-ELISA. The mean of sMICA levels from four representative mice is shown. NontgLM tested negative (N.D., not detectable).

FIGURE 2.

Constitutive MICA expression by H2-Kb-MICA mice. A, H2-Kb-MICA transgene encompassing the H2-Kb promoter, the 1.15-kb MICA*07 coding sequence, and the 3′ untranslated region (UT) of the β-globin gene. B, MICA cell surface expression by PBL, thymocytes, splenocytes, and hepatocytes of H2-Kb-MICA mice as detected by BAMO1. Cells were costained with anti-H2-Kb. C, MICA expression by gated subpopulations of splenocytes and thymocytes from nontgLM (gray histograms) and H2-Kb-MICA mice (filled histograms) as analyzed with BAMO1. D, sMICA levels in sera of H2-Kb-MICA mice detected with a MICA-sandwich-ELISA. The mean of sMICA levels from four representative mice is shown. NontgLM tested negative (N.D., not detectable).

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H2-Kb-MICA mice presented phenotypically normal and healthy, and did not display any overt signs of autoimmunity. No significant alterations in frequencies of lymphocyte subsets were observed when splenocytes of H2-Kb-MICA mice and nontgLM were compared (data not shown). Most splenocytes from H2-Kb-MICA mice strongly expressed MICA (Fig. 2,B). In particular, B and NK cells expressed high levels of MICA, whereas MICA expression of CD4+ and CD8+ T cells reached intermediate levels (Fig. 2,C). Accordingly, MICA expression by PBL varied over a broad range (Fig. 2,B). In contrast, most thymocytes representing the CD4+/CD8+ subpopulation had only low amounts of MICA, while other thymocyte subsets displayed a more pronounced MICA expression (Fig. 2,, B and C). As for thymocytes, MICA expression of hepatocytes paralleled H2-Kb expression levels (Fig. 2,B). No expansions of intraepithelial CD4+/CD8αα+ T cells were observed as previously described for transgenic mice with a gut-specific MICA expression (37) (data not shown). Sera from H2-Kb-MICA mice, but not from nontgLM, contained high levels of sMICA (∼50 ng/ml) exceeding sMICA levels previously detected in tumor patients (27, 28, 29), suggesting substantial MICA shedding by nonmalignant cells (Fig. 2 D).

To address functional consequences of constitutive MICA expression, we analyzed expression and functionality of NKG2D on NK cells. NKG2D surface levels of NK cells from H2-Kb-MICA were reduced when compared with NK cells of nontgLM (Fig. 3,A). Differences in surface expression were even more pronounced when poly(I:C)-activated NK cells of H2-Kb-MICA mice and nontgLM were analyzed ex vivo. NKG2D down-regulation coincided with a strong impairment of NKG2D function. In contrast to NK cells from nontgLM, poly(I:C)-activated NK cells from H2-Kb-MICA mice failed to lyse RMA-MICA*07 cells ex vivo (Fig. 3,B). When poly(I:C)-activated NK cells from nontgLM were stimulated ex vivo with RMA-MICA*07 cells, we observed a markedly higher frequency of NK1.1+ IFN-γ producing cells (∼16%) as opposed to stimulation with RMA-neo cells (∼8%). In contrast, frequencies of NK1.1+ cells from H2-Kb-MICA mice producing IFN-γ were only slightly increased when stimulated with RMA-MICA*07 cells as compared with RMA-neo stimulation (Fig. 3,C). To assess a general defect in cytotoxicity, we tested the lytic capacity of NK cells from H2-Kb-MICA mice toward CHO and RMA-S cells. CHO cells are recognized by mouse NK cells via the activating receptor Ly49D in complex with DAP12 (38). Activated NK cells from H2-Kb-MICA mice and nontgLM displayed similar lytic potential for CHO cells arguing against a general impairment of cytotoxicity (Fig. 3,D). Similarly, RMA-S cells were lysed by NK cells from H2-Kb-MICA mice and nontgLM at comparable rates (Fig. 3,E). When NK cells from H2-Kb-MICA mice were cultivated in vitro with high dose of IL-2 (1500 U/ml), NKG2D surface levels increased, but did not reach wild-type levels presumably due to MICA expression by NK cells (Fig. 3,F). Accordingly, RMA-MICA*07 cells were lysed by IL-2 cultivated H2-Kb-MICA NK cells, but less efficiently when compared with lysis by nontransgenic NK cells (Fig. 3,G). CD8 T cells freshly isolated from H2-Kb-MICA mice and nontgLM contained a small fraction of NKG2D-positive cells, which presumably are activated CD8 T cells. Again, NKG2D expression was greatly reduced on cells from H2-Kb-MICA mice as compared with cells from nontgLM (Fig. 3 H). However, after 3 days of stimulation with anti-CD3, NKG2D expression of CD8 T cells from both mice was almost indistinguishable.

FIGURE 3.

NKG2D on H2-Kb-MICA NK cells is dysfunctional. A, NKG2D cell surface expression by resting and poly(I:C)-activated NK cells from H2-Kb-MICA mice is strongly reduced. Purified NK cells from H2-Kb-MICA mice (filled histograms) and nontgLM (gray histograms) were stained ex vivo with the anti-NKG2D mAb CX5 or with an isotype control (open histograms). Mean fluorescence intensities of gated NK1.1 cells are shown. B, In contrast to poly(I:C)-activated nontransgenic NK cells, poly(I:C)-activated H2-Kb-MICA NK cells fail to lyse RMA-MICA*07 cells ex vivo in the presence (anti-NKG2D; control Ig) or absence (w/o mAb) of Abs. Both types of NK cells fail to lyse RMA-neo cells (neg. control). Cytotoxicity was measured by a 12-h chromium-release assay. C, IFN-γ production by H2-Kb-MICA NK cells. Freshly isolated, poly(I:C)-activated NK cells from H2-Kb-MICA mice and nontgLM were cocultured with RMA-neo or RMA-MICA cells. After 17 h, frequencies of IFN-γ producing NK1.1+ cells were evaluated by intracellular staining. Data represent means of triplicates. D and E, Lysis of CHO cells (D) and RMA-S cells (E) by poly(I:C)-activated NK cells ex vivo left out from H2-Kb-MICA mice and nontgLM. F, NKG2D expression of purified H2-Kb-MICA NK cells increases upon in vitro culture. NKG2D surface expression of NK1.1+ cells from H2-Kb-MICA mice (filled histogram) and nontgLM (gray histogram) cultured for 5 days with IL-2 was analyzed with CX5. Isotype controls are shown as open histograms. G, In vitro cultivated H2-Kb-MICA NK cells lyse RMA-MICA*07 cells. Purified NK cells from H2-Kb-MICA mice and nontgLM were cultivated 5 days with IL-2 and subsequently assayed for lysis of RMA-MICA*07 cells. Lysis was blocked by mAb C7 (anti-NKG2D), but not by control Ig (control Ig). RMA-neo cells were not lysed (neg. control). H, NKG2D up-regulation on purified CD8 T cells from H2-Kb-MICA mice and nontgLM upon in vitro stimulation with anti-CD3 mAb is comparable. NKG2D expression by freshly isolated CD8 T cells or by purified CD8 T cells after 3 days of culture in the presence of anti-CD3 mAb was analyzed with PE-conjugated CX5. NKG2D surface expression of CD8+ gated cells from H2-Kb-MICA mice (filled histogram) and nontgLM (gray histogram) are shown. Isotype controls are open histograms.

FIGURE 3.

NKG2D on H2-Kb-MICA NK cells is dysfunctional. A, NKG2D cell surface expression by resting and poly(I:C)-activated NK cells from H2-Kb-MICA mice is strongly reduced. Purified NK cells from H2-Kb-MICA mice (filled histograms) and nontgLM (gray histograms) were stained ex vivo with the anti-NKG2D mAb CX5 or with an isotype control (open histograms). Mean fluorescence intensities of gated NK1.1 cells are shown. B, In contrast to poly(I:C)-activated nontransgenic NK cells, poly(I:C)-activated H2-Kb-MICA NK cells fail to lyse RMA-MICA*07 cells ex vivo in the presence (anti-NKG2D; control Ig) or absence (w/o mAb) of Abs. Both types of NK cells fail to lyse RMA-neo cells (neg. control). Cytotoxicity was measured by a 12-h chromium-release assay. C, IFN-γ production by H2-Kb-MICA NK cells. Freshly isolated, poly(I:C)-activated NK cells from H2-Kb-MICA mice and nontgLM were cocultured with RMA-neo or RMA-MICA cells. After 17 h, frequencies of IFN-γ producing NK1.1+ cells were evaluated by intracellular staining. Data represent means of triplicates. D and E, Lysis of CHO cells (D) and RMA-S cells (E) by poly(I:C)-activated NK cells ex vivo left out from H2-Kb-MICA mice and nontgLM. F, NKG2D expression of purified H2-Kb-MICA NK cells increases upon in vitro culture. NKG2D surface expression of NK1.1+ cells from H2-Kb-MICA mice (filled histogram) and nontgLM (gray histogram) cultured for 5 days with IL-2 was analyzed with CX5. Isotype controls are shown as open histograms. G, In vitro cultivated H2-Kb-MICA NK cells lyse RMA-MICA*07 cells. Purified NK cells from H2-Kb-MICA mice and nontgLM were cultivated 5 days with IL-2 and subsequently assayed for lysis of RMA-MICA*07 cells. Lysis was blocked by mAb C7 (anti-NKG2D), but not by control Ig (control Ig). RMA-neo cells were not lysed (neg. control). H, NKG2D up-regulation on purified CD8 T cells from H2-Kb-MICA mice and nontgLM upon in vitro stimulation with anti-CD3 mAb is comparable. NKG2D expression by freshly isolated CD8 T cells or by purified CD8 T cells after 3 days of culture in the presence of anti-CD3 mAb was analyzed with PE-conjugated CX5. NKG2D surface expression of CD8+ gated cells from H2-Kb-MICA mice (filled histogram) and nontgLM (gray histogram) are shown. Isotype controls are open histograms.

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Reduced NKG2D staining was not due to epitope masking by sMICA, because complexes formed between recombinant MICA and NKG2D bound anti-NKG2D and NKG2D staining of H2-Kb-MICA NK cells was not increased following mild acid treatment (data not shown). An impact of different transcriptional regulation on NKG2D expression was excluded by real-time PCR that did not reveal differences in NKG2D, DAP10, or DAP12 transcripts between NK cells from H2-Kb-MICA mice and nontgLM (Fig. 4 A).

FIGURE 4.

NKG2D-down-regulation by MICA. A, Transcript levels of DAP10, DAP12, and NKG2D are unaltered in NK cells from H2-Kb-MICA mice as compared with nontgLM. Bars show the relative copy numbers in purified unstimulated NK cells from three individual H2-Kb-MICA mice (▪) and three nontgLM (▦). B and C, NKG2D down-regulation by cell-bound and sMICA. B, Splenocytes from nontgLM (5 × 105) were coincubated with CFSE-labeled splenocytes (1 × 106) either from H2-Kb-MICA mice or from nontgLM for 12 h in vitro and, subsequently, NKG2D surface expression of CFSE-negative cells analyzed by flow cytometry. C, Splenocytes from nontgLM were incubated for 12 h in sera (1/2 dilution with medium) from H2-Kb-MICA mice and nontgLM, respectively, and, subsequently, analyzed for NKG2D surface expression. The fluorescence intensities of unlabeled NKG2D-expressing cells after incubation were 55.0 ± 0.5 (nontransgenic splenocytes coculture), 37.9 ± 1.2 (H2-Kb-MICA splenocytes coculture), 49.3 ± 0.4 (nontransgenic serum), and 46.6 ± 1.3 (H2-Kb-MICA serum) representing means of triplicates of y mean fluorescence intensities. D, NKG2D down-regulation in vivo. A total of 8 × 106 CFSE-labeled nylon wool nonadherent splenocytes from B6 mice were injected i.v. into H2-Kb-MICA mice or nontgLM. After 10 h, splenocytes were isolated and analyzed for NKG2D surface expression on CFSE-labeled cells with allophycocyanin-conjugated CX5.

FIGURE 4.

NKG2D-down-regulation by MICA. A, Transcript levels of DAP10, DAP12, and NKG2D are unaltered in NK cells from H2-Kb-MICA mice as compared with nontgLM. Bars show the relative copy numbers in purified unstimulated NK cells from three individual H2-Kb-MICA mice (▪) and three nontgLM (▦). B and C, NKG2D down-regulation by cell-bound and sMICA. B, Splenocytes from nontgLM (5 × 105) were coincubated with CFSE-labeled splenocytes (1 × 106) either from H2-Kb-MICA mice or from nontgLM for 12 h in vitro and, subsequently, NKG2D surface expression of CFSE-negative cells analyzed by flow cytometry. C, Splenocytes from nontgLM were incubated for 12 h in sera (1/2 dilution with medium) from H2-Kb-MICA mice and nontgLM, respectively, and, subsequently, analyzed for NKG2D surface expression. The fluorescence intensities of unlabeled NKG2D-expressing cells after incubation were 55.0 ± 0.5 (nontransgenic splenocytes coculture), 37.9 ± 1.2 (H2-Kb-MICA splenocytes coculture), 49.3 ± 0.4 (nontransgenic serum), and 46.6 ± 1.3 (H2-Kb-MICA serum) representing means of triplicates of y mean fluorescence intensities. D, NKG2D down-regulation in vivo. A total of 8 × 106 CFSE-labeled nylon wool nonadherent splenocytes from B6 mice were injected i.v. into H2-Kb-MICA mice or nontgLM. After 10 h, splenocytes were isolated and analyzed for NKG2D surface expression on CFSE-labeled cells with allophycocyanin-conjugated CX5.

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Previous reports described NKG2D down-regulation by NK cells upon exposure to NKG2DL-expressing cells in vitro and in vivo (23, 28, 31). We observed that down-regulation of surface NKG2D on nontransgenic splenocytes was most pronounced after cocultivation with splenocytes from MICA transgenic mice in vitro, and only marginally following treatment with sera from H2-Kb-MICA mice, whereas incubation with control cells and sera from nontgLM, respectively, had no effect (Fig. 4, B and C). In vivo, NKG2D surface expression on splenocytes from B6 mice was down-regulated after adoptive transfer into H2-Kb-MICA mice, but not after transfer into nontgLM (Fig. 4 D). Altogether, these data suggest that reduced surface NKG2D on H2-Kb-MICA NK cells results in NKG2D dysfunction and that NKG2D down-regulation is primarily caused by a persistent exposure to cell-bound MICA in vivo.

To address functional consequences of NKG2D down-regulation for NK cell activation in vivo, we took advantage of H2-Kb-MICA splenocytes. In contrast to Con A-activated splenocytes (Con A blasts) from B6 mice, H2-Kb-MICA Con A blasts were subject of substantial lysis by freshly isolated poly(I:C)-activated B6 NK cells, and lysis was blocked by addition of anti-NKG2D Ab (Fig. 5,A). To test the efficacy of NKG2D-mediated NK cytotoxicity in vivo, we adoptively cotransferred CFSE-labeled MICA-transgenic and PKH26-labeled nontransgenic splenocytes into nontgLM. Within 6 h, the relative number of MICA-expressing splenocytes in the peripheral blood was reduced to one-third (Fig. 5,B), indicating an efficient and preferential elimination of MICA-expressing cells in vivo. A similar preferential elimination of H2-Kb-MICA splenocytes was observed when lymph nodes and spleens were analyzed (Fig. 5,D). However, when CFSE-labeled MICA-transgenic splenocytes were adoptively cotransferred together with PKH26-labeled nontransgenic splenocytes into H2-Kb-MICA mice, relative numbers of MICA-transgenic splenocytes were only slightly decreased indicating that NKG2D-mediated activation of NK cytotoxicity is deficient in H2-Kb-MICA mice (Fig. 5, C and D). To address functionality of NKG2D-independent NK cytotoxicity, we adoptively transferred CFSE-labeled splenocytes from β2-microglobulin-deficient mice. Both H2-Kb-MICA mice and nontgLM exhibited similar clearance rates of β2-microglobulin-deficient cells excluding a general impairment of NK cytotoxicity in H2-Kb-MICA mice (Fig. 5 E).

FIGURE 5.

Deficient NKG2D-mediated natural cytotoxicity in H2-Kb-MICA mice. A, Poly(I:C)-activated B6 NK cells ex vivo lyse Con A blasts from H2-Kb-MICA mice (w/o mAb). Lysis was inhibited by addition of the mAb C7 (anti-NKG2D), but not by control Ig (control Ig). Con A blasts from B6 mice were not lysed (neg. control). BD, H2-Kb-MICA splenocytes are readily eliminated in nontransgenic mice, but not in H2-Kb-MICA mice. Splenocytes of H2-Kb-MICA mice (CFSE-labeled) and nontgLM (PKH26-labeled) were adoptively cotransferred into nontgLM (B) or in H2-Kb-MICA mice (C), and their presence among PBL was analyzed by flow cytometry 1 min and 6 h postinjection (p.i.). In addition to PBL, spleen and lymph nodes were analyzed 6 h p.i. and all results are summarized in D. The number of MICA-negative splenocytes was set as 1 and the relative number of MICA-positive splenocytes recovered from nontgLM (▦) or H2-Kb-MICA (▪) was calculated accordingly. Data represent the means of the relative number of transferred MICA-positive splenocytes from three mice per indicated organ. Results are representative of two independent experiments. E, Splenocytes of CFSE-labeled β2-microglobulin-deficient (β2M−/−) and PKH26-labeled wild-type controls were adoptively cotransferred and analyzed by flow cytometry 1 min and 14 h p.i. in PBL, spleens, and lymph nodes of nontgLM (▦) or H2-Kb-MICA mice (▪).

FIGURE 5.

Deficient NKG2D-mediated natural cytotoxicity in H2-Kb-MICA mice. A, Poly(I:C)-activated B6 NK cells ex vivo lyse Con A blasts from H2-Kb-MICA mice (w/o mAb). Lysis was inhibited by addition of the mAb C7 (anti-NKG2D), but not by control Ig (control Ig). Con A blasts from B6 mice were not lysed (neg. control). BD, H2-Kb-MICA splenocytes are readily eliminated in nontransgenic mice, but not in H2-Kb-MICA mice. Splenocytes of H2-Kb-MICA mice (CFSE-labeled) and nontgLM (PKH26-labeled) were adoptively cotransferred into nontgLM (B) or in H2-Kb-MICA mice (C), and their presence among PBL was analyzed by flow cytometry 1 min and 6 h postinjection (p.i.). In addition to PBL, spleen and lymph nodes were analyzed 6 h p.i. and all results are summarized in D. The number of MICA-negative splenocytes was set as 1 and the relative number of MICA-positive splenocytes recovered from nontgLM (▦) or H2-Kb-MICA (▪) was calculated accordingly. Data represent the means of the relative number of transferred MICA-positive splenocytes from three mice per indicated organ. Results are representative of two independent experiments. E, Splenocytes of CFSE-labeled β2-microglobulin-deficient (β2M−/−) and PKH26-labeled wild-type controls were adoptively cotransferred and analyzed by flow cytometry 1 min and 14 h p.i. in PBL, spleens, and lymph nodes of nontgLM (▦) or H2-Kb-MICA mice (▪).

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Tumor cells ectopically expressing NKG2DL stimulate tumor immunity. For example, RAE-1-expressing RMA cells have been shown to be rejected by NK cells and/or CD8 T cells (25, 26). We adopted this experimental setting to evaluate functional consequences of NKG2D impairment in vivo and injected 105 RMA-neo and 105 RMA-MICA*07 cells into the right and left flank, respectively, of nontgLM. As expected, RMA-neo cells gave rise to tumors in all mice, but RMA-MICA*07 cells were rejected analogous to previous findings with RAE-1-expressing RMA cells (25, 26) (Fig. 6). To assess a potential contribution of T cells recognizing putative MICA peptides presented by MHC class I in the rejection of RMA-MICA cells, we tested RAG2-deficient mice. RMA-MICA*07 cells were rejected by RAG2-deficient mice, whereas RMA-neo cells expanded to tumors, demonstrating that rejection of RMA-MICA*07 cells occurred independently of T cell recognition, but rather is due to the NKG2D-mediated activation of NK cells reaffirming the functionality of MICA as surrogate ligand of mouse NKG2D. However, when we challenged H2-Kb-MICA mice with injections of RMA cells, both RMA-neo and RMA-MICA*07 gave rise to tumors demonstrating that the NKG2D-mediated tumor rejection is strongly impaired in these mice (Fig. 6).

FIGURE 6.

NKG2D-mediated tumor rejection is impaired in H2-Kb-MICA mice. H2-Kb-MICA mice, nontgLM, and B6 RAG2-deficient mice, respectively, were inoculated s.c. with 1 × 105 RMA-neo (into the right flank) and 1 × 105 RMA-MICA*07 cells (left flank). Each panel displays the tumor growth of RMA-neo (○) and RMA-MICA (•) in four mice monitored for 17 days postinoculation.

FIGURE 6.

NKG2D-mediated tumor rejection is impaired in H2-Kb-MICA mice. H2-Kb-MICA mice, nontgLM, and B6 RAG2-deficient mice, respectively, were inoculated s.c. with 1 × 105 RMA-neo (into the right flank) and 1 × 105 RMA-MICA*07 cells (left flank). Each panel displays the tumor growth of RMA-neo (○) and RMA-MICA (•) in four mice monitored for 17 days postinoculation.

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To address the role of NKG2D in the immune defense against infectious pathogens and for the generation of T cell responses, we scrutinized the immune response of H2-Kb-MICA mice upon infection with L. monocytogenes. The control of the intracellular pathogen L. monocytogenes involves both cells of the innate and of the acquired immune system (39). To evaluate consequences of NKG2D impairment for the early control of L. monocytogenes by innate mechanisms, H2-Kb-MICA mice and nontgLM were infected with 5 × 104 listeriae, and the bacteria titers in spleen and liver were determined at days 2 and 3 postinfection. Both groups of mice did not differ significantly in their bacterial load, indicating that NKG2D-mediated effector functions of NK cells are not relevant for the early control of L. monocytogenes (data not shown). For the analysis of Listeria-specific T cell responses, we applied a L. monocytogenes strain recombinant for a secreted form of OVA (34). This strain induces a strong OVA-specific CD8+ T cell response, which can be detected with H2-Kb/OVA257–264-tetramers. At the peak of the primary T cell response against L. monocytogenes (day 9 postinfection), we analyzed tetramer-positive T cells in both H2-Kb-MICA mice and nontgLM. Costaining with anti-NKG2D mAb revealed a significant NKG2D expression on the majority of tetramer-positive cells from nontransgenic mice. This is in accord with earlier studies reporting that CD8 T cells, but not CD4 T cells, show induced NKG2D expression several days after antigenic activation (4). In contrast to nontransgenic mice, NKG2D surface expression of tetramer-positive cells from H2-Kb-MICA mice was strongly reduced (Fig. 7,A). Interestingly, spleens of H2-Kb-MICA mice contained significant lower frequencies and total numbers of tetramer-positive CD8 T cells (Fig. 7,B and data not shown). These results were confirmed by the analysis of frequencies and numbers of CD8+ T cells responding to in vitro peptide stimulation with IFN-γ production (Fig. 7,C and data not shown). When we compared the frequencies of CD62Llow cells among CD8+ T cells of infected mice, H2-Kb-MICA mice had a significantly reduced percentage of CD62Llow cells (20.0 ± 3.7% and 32.8 ± 3.8% in H2-Kb-MICA mice and littermate controls, respectively), revealing a general impairment of the anti-Listeria CD8+ T cell response in H2-Kb-MICA mice. In contrast, Listeria-specific CD4+ T cell responses were only marginally affected in H2-Kb-MICA mice. Compared with littermate controls, H2-Kb-MICA mice showed similar frequencies and only slightly reduced total numbers of CD4+ T cells responding to the immunodominant Listeria epitope LLO190–201 (Fig. 7 D and data not shown).

FIGURE 7.

Impaired anti-Listeria CD8 T cell response in H2-Kb-MICA mice. H2-Kb-MICA mice and nontgLM were i.v. infected with 5 × 103 listeriae encoding a secreted form of OVA. On day 9 postinfection, spleen cells of infected mice were analyzed. A, NKG2D surface expression by H2-Kb/OVA257–264-tetramer-positive cells. Only viable CD8-gated splenocytes stained with anti-NKG2D mAb CX5 are shown. B, Frequencies of H2-Kb/OVA257–264-tetramer-positive CD62LlowCD8 T cells in H2-Kb-MICA mice (▪) vs nontgLM (▦) as assessed by flow cytometry. C, Frequencies of IFN-γ-producing CD8+ T cells after in vitro stimulation with peptide OVA257–264 analyzed by intracellular cytokine staining. D, Frequencies of IFN-γ-producing CD4+ T cells after in vitro stimulation with peptide LLO190–201 analyzed by intracellular cytokine staining. BD, Values represent means of three infected mice per group.

FIGURE 7.

Impaired anti-Listeria CD8 T cell response in H2-Kb-MICA mice. H2-Kb-MICA mice and nontgLM were i.v. infected with 5 × 103 listeriae encoding a secreted form of OVA. On day 9 postinfection, spleen cells of infected mice were analyzed. A, NKG2D surface expression by H2-Kb/OVA257–264-tetramer-positive cells. Only viable CD8-gated splenocytes stained with anti-NKG2D mAb CX5 are shown. B, Frequencies of H2-Kb/OVA257–264-tetramer-positive CD62LlowCD8 T cells in H2-Kb-MICA mice (▪) vs nontgLM (▦) as assessed by flow cytometry. C, Frequencies of IFN-γ-producing CD8+ T cells after in vitro stimulation with peptide OVA257–264 analyzed by intracellular cytokine staining. D, Frequencies of IFN-γ-producing CD4+ T cells after in vitro stimulation with peptide LLO190–201 analyzed by intracellular cytokine staining. BD, Values represent means of three infected mice per group.

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A hallmark of the NKG2D/NKG2DL-system is the inducible surface expression of at least some of the MHC class I-related NKG2DL in response to cell stress, microbial infection, or malignant transformation, thereby marking dysfunctional cells for elimination by cytotoxic lymphocytes via NKG2D-mediated mechanisms (“induced-self” hypothesis) (10, 11, 40). Conversely, a sustained NKG2DL expression as described in patients with malignant or autoimmune diseases may desensitize NKG2D-mediated immune responses. Here, we analyzed transgenic mice constitutively expressing MICA to address functional consequences of persistent NKG2DL expression in vivo. MICA was chosen because it is the best-characterized NKG2DL with regard to expression in normal and diseased tissues, regulation of expression and soluble release, and availability of biochemical and structural data (10, 12). Although the amino acid sequence of the MICA ectodomain is fairly divergent from any mouse NKG2DL, crystal structures of MICA and RAE1 α1/α2 platform domains interacting with NKG2D are highly related as are crystal structures of human and mouse NKG2D ectodomains (41, 42, 43). Structural homology is reflected in functional equivalence of MICA/B and RAE1 with regard to mouse NKG2D ligation and NKG2D-mediated activation of mouse NK cells qualifying MICA as a bona fide surrogate ligand of mouse NKG2D (Fig. 1) (22, 36).

MICA expression in H2-Kb-MICA mice generally parallels H2-Kb expression with a strong expression by B and NK cells and an intermediate to low expression by thymocytes and hepatocytes. A disparate expression of Kb and MICA molecules was only observed for peripheral CD4 and CD8 T cells that remains to be addressed. MICA-expressing splenocytes were used to evaluate the efficiency of NKG2D-mediated activation of natural cytotoxicity in vivo. Hitherto, NKG2D-mediated cytotoxicity has been extensively demonstrated in vitro using tumor cell lines expressing NKG2DL. Previous studies also demonstrated that ectopic expression of NKG2DL by tumors induces strong NK and CTL responses in vivo and that rejection of NKG2DL-expressing tumor cells is perforin-dependent (25, 26, 44).

By adoptive transfer of MICA-expressing splenocytes, we here demonstrate that ectopic expression of NKG2DL renders “normal” syngeneic cells highly susceptible to cytotoxicity in vivo, vividly underscoring that NKG2DL-expression potently overrides inhibitory signals by MHC class I molecules for NK cell activation.

To our surprise, H2-Kb-MICA mice were vital, fertile, and did not exhibit any overt signs of autoimmunity despite a strong MICA surface expression. A previous study reported hyperkeratosis and leukocytosis in transgenic mice with a MICB cDNA under control of a chicken β-actin promoter (45). The difference in phenotype of these mice as compared with the H2-Kb-MICA mice may be due to a different tissue expression of the MIC molecules. It turned out that in H2-Kb-MICA mice “tolerance” toward MICA-expressing cells is established by down-modulation of NKG2D, which is due to permanent exposure to MICA. In fact, down-modulation of surface NKG2D by NK cells after exposure to NKG2DL-expressing cells in vitro and in vivo has been previously reported (23, 31). It remains to be investigated whether a high local NKG2D ligand expression also leads to a systemic NKG2D down-regulation and dysfunction or rather stimulates local immune reactions. Previous studies on rejection of NKG2DL-expressing tumor cell lines and on the role of NKG2D in the pathogenesis of diabetes in NOD mice, respectively, suggest the latter (25, 26, 46).

A down-modulation of NKG2D has also been reported in human cancer patients, where tumors express and release substantial amounts of sMICA (28). Interestingly, H2-Kb-MICA mice also contain high serum levels of sMICA that exceed levels observed in cancer patients at least 10-fold (29) and thus is not a peculiarity of malignant cells. This is in line with the detection of sMICA in patients with autoimmune diseases and raises the question whether MICA shedding is of physiological relevance, e.g., in regulating MIC cell surface levels. Characterization of the MIC shedding activity may resolve this issue. Conflicting results exist regarding the down-regulation of NKG2D by sMICA. Whereas MICA-containing sera of patients with malignancies reportedly cause systemic NKG2D down-regulation correlating with reduced NKG2D expression on CD8 T cells and NK cells in cancer patients (28, 30, 47), NKG2D surface expression is not altered in patients with rheumatoid arthritis and celiac disease despite similar sera levels of sMICA (17, 18). Our in vitro experiments indicate that NKG2D down-regulation in H2-Kb-MICA mice is mainly due to engagement of cell-bound MICA as sMICA had little effect on NKG2D surface expression of nontransgenic NK cells. Possibly, MICA released from tumor cells and benign cells, respectively, may be subjected to different posttranslational modifications differentially affecting NKG2D down-regulation.

Irrespective of the exact molecular mechanism, our results now provide direct in vivo evidence that persistent MICA expression results in down-modulation of NKG2D and impacts tumor immunity as H2-Kb-MICA mice failed to reject MICA-expressing RMA cells in contrast to nontgLM or RAG2-deficient mice. Though our mouse model does not mirror the localized MICA expression by tumors, it does strongly support the notion that ligand-induced NKG2D down-modulation is detrimental for tumor immunosurveillance.

In H2-Kb-MICA mice, down-modulation of surface NKG2D on NK cells was not complete raising the possibility that the observed dysfunction may in part also be due to an impaired signal transduction. However, lysis of CHO cells by H2-Kb-MICA NK cells was similar to lysis by nontransgenic NK cells suggesting that the DAP12 signaling pathway is not affected. Upon in vitro cultivation, H2-Kb-MICA NK cells acquired higher NKG2D expression levels and regained functional activity demonstrating that NKG2DL-induced silencing of NKG2D is reversible. Failure to completely restore NKG2D surface expression levels may also be due to a MICA-NKG2D cis-interaction within same cells as postulated for activated NK cells from NOD mice (31).

In humans, MICA expression has also been reported for thymic epithelial cells raising the possibility that NKG2D is involved in thymic selection of T cells (48). Because MICA costimulates T cells via NKG2D and represents a ligand of some human γδ TCR, constitutive MICA expression might also affect the generation and/or expansion of lymphocyte subpopulations bearing NKG2D or γδ TCR (1, 8, 15, 49, 50). However, we did not detect any major alterations in the total number or composition of lymphocyte subpopulations in naive H2-Kb-MICA mice. In particular, numbers of splenic NK cells, CD8 T cells, and γδ T cells were not significantly altered as compared with nontgLM. Recently, a transgenic mouse with a gut-specific MICA expression directed by the T3b-promoter was described with expansions of CD4+CD8αα+ intraepithelial T cells (37). But a functional involvement of NKG2D was not examined and also no other mechanisms presented that may account for this expansion. In H2-Kb-MICA mice, frequencies of CD4+CD8αα+ intraepithelial T cells did not significantly differ from nontgLM.

Because H2-Kb-MICA mice exhibit a profound NKG2D dysfunction, we implemented these mice to address the relevance of NKG2D in staging immune responses toward infectious pathogens. Infection with L. monocytogenes was chosen, because listeriae activate both innate and adaptive immune responses. Further, induction of RAE1-expression by macrophages incubated with L. monocytogenes has recently been reported (23). When we compared the pathogen load of H2-Kb-MICA mice and nontgLM at days 2 and 3 postinfection, we found no significant differences, indicating that NKG2D dysfunction on NK cells had no major impact at an early stage of infection. However, NKG2DL expression induced by L. monocytogenes may trigger cytokine release by NK cells that in turn may contribute to the generation of an anti-Listeria-specific T cell response. NK cells of H2-Kb-MICA mice showed an impaired IFN-γ production upon NKG2D triggering ex vivo that may also generate a suboptimal generation of an anti-Listeria Th1 response in vivo. However, H2-Kb-MICA NK cells should still be able to respond to other IFN-γ inducing stimuli (i.e., cytokines such as IL-12 and IL-18) present during listeriosis. T cells are of major importance for the immune control and clearance of Listeria (39). We analyzed the anti-Listeria T cell response at day 9 postinfection and found the number and frequency of Listeria-specific CD8 T cells strongly reduced in H2-Kb-MICA, while Listeria-specific CD4 T cells were not affected. At present, it is unclear why the CD8 T cell response is impaired in Listeria-infected H2-Kb-MICA mice. Down-regulation of NKG2D on activated CD8 T cells may reduce costimulatory signals for proliferation and cell survival. In fact, it has been reported that NKG2D-mediated signal transduction via DAP10 also involves activation of the serine/threonine kinase Akt that promotes cellular proliferation (51). Alternately, an impaired activation of NK cells by NKG2D may result in reduced cytokine secretion and/or cell lysis generating a suboptimal Th1 response.

In summary, we demonstrate that the persistent expression of MICA results in a pronounced down-modulation of NKG2D on NK cells and activated CD8 T cells in vivo. The resulting NKG2D dysfunction strongly impacts immunity against NKG2DL-expressing tumor cells and impairs the generation and/or expansion of Listeria-specific CD8 T cells emphasizing an important function of NKG2D for immunity against tumors and intracellular pathogens.

We gratefully acknowledge the excellent technical assistance by Beate Pömmerl and Jessica Bigott, and the experimental assistance by Anouk Feitsma. We thank Keesook Li of the transgenic mouse facility of the Fred Hutchinson Cancer Research Center. We are grateful to Adrian Hayday and colleagues for sharing unpublished data and thank Stefan Bauer for critical reading of the manuscript. The pHSE vector is a kind gift of Hanspeter Pircher.

The authors have no financial conflict of interest.

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 grants from Interdisziplinäres Zentrum für Klinische Forschung Tübingen (Project IIA1; to A.S.), Deutsche Krebshilfe (10-1921-Sa I; to A.S.), Deutsche Forschungsgemeinschaft (SFB633; to H.-W.M.) and the National Institutes of Health (IA-30581; to T.S.).

3

Abbreviations used in this paper: MICA, MHC class I chain-related protein A; MICB, MHC class I chain-related protein B; ULBP, UL16-binding protein; RAE-1, retinoic acid early transcript 1; NKG2DL, NKG2D ligand; poly(I:C), polyinosinic-polycytidylic acid potassium salt; sMICA, soluble MICA; nontgLM, nontransgenic littermate; LLO190–201, listeriolysin O.

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