Nature Immunology
3, 1150 - 1155 (2002)
Published online: 11 November 2002; | doi:10.1038/ni857
NKG2D recruits two distinct adapters to trigger NK cell activation and costimulationSusan Gilfillan1, Emily L. Ho2, Marina Cella1, Wayne M. Yokoyama2
& Marco Colonna11 Department of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110, USA. 2 Howard Hughes Medical Institute, Rheumatology Division, Department of Medicine, Barnes-Jewish Hospital and Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110, USA.
Correspondence should be addressed to Marco Colonna mcolonna@pathology.wustl.eduNKG2D is a receptor on natural killer (NK) cells and cytotoxic T lymphocytes that binds major histocompatibility complex (MHC) class I−like ligands expressed primarily on virally infected and neoplastic cells. In vitro studies indicate that NKG2D provides costimulation through an associated adapter, DAP10, which recruits phosphatidylinositol-3 kinase. Here we show that in DAP10-deficient mice, CD8+ T cells lack NKG2D expression and are incapable of mounting tumor-specific responses. However, DAP10-deficient NK cells express a functional NKG2D receptor due to the association of NKG2D with another adapter molecule, DAP12 (also known as KARAP), which recruits protein tyrosine kinases. Thus, NKG2D is a versatile receptor that, depending on the availability of adapter partners, mediates costimulation in T cells and/or activation in NK cells.NKG2D is a C-type lectin-like receptor encoded within the natural killer (NK) gene complex that is constitutively expressed on all NK cells and up-regulated on activated CD8+ T cells,   T cells and macrophages. Among the numerous activating receptors expressed primarily on NK cells1, NKG2D is distinct in that it recognizes a number of disparate major histocompatibility complex (MHC) class I−related molecules; these include MICA and MICB in humans2, the murine minor histocompatibility antigen H-603,
4 and retinoic acid−inducible gene products encoded by the murine genes Raet1a, Raet1b, Raet1c and Raet1d3,
4 and their putative human counterparts, the ULBP genes5,
6. NKG2D ligands do, however, share some characteristics: many are not constitutively expressed in adult tissues, but are inducible and expressed in tumor cells. MICA and MICB are frequently expressed in epithelial tumors and can be induced by stress7,
8. Raet1a, Raet1b, Raet1c and Raet1d (which encode Rae-1 , Rae-1 , Rae-1 and Rae-1, respectively) are expressed early during ontogeny but not in adult tissues3,
4. ULBP expression is low in various tissues but higher in some tumors5. Expression of MIC and ULBP on human tumor cells is sufficient to overcome the inhibitory effects of MHC class I expression on NK cell killing2,
5. Similarly, overexpression of Rae-1 and H-60 on murine tumor cells leads to NKG2D-mediated rejection of the tumors by NK, CD8+ T and T cells9,
10,
11. In addition, NKG2D augments the TCR responses of CD28-CD8+ T cells to targets that have up-regulated MIC due to cytomegalovirus (CMV) infection12. Together, these observations indicate that NKG2D provides first-line surveillance against stressed or "abnormal" cells that have been induced to express one of its ligands.
Lacking intrinsic cytoplasmic signaling motifs, NKG2D is thought to depend solely on the transmembrane adapter DAP10 for cell-surface expression and function13. The majority of transmembrane adapters—including CD3, CD3, CD3 , CD3 , FcR and DAP12 (also known as KARAP)—trigger intracellular signaling pathways that are dependent on the protein tyrosine kinases Syk and/or ZAP-70 via immunoreceptor tyrosine-based activation motifs (ITAMs), defined by the consensus sequence YxxL6−8xYxxL/I, in their cytoplasmic tails14,
15,
16,
17. In contrast, DAP10 contains an YxxM motif that, when phosphorylated, binds the p85 subunit of phosphatidylinositol-3 kinase (PI3K)13,
18,
19 and Grb218. Engagement of NKG2D-DAP10 is expected to induce costimulatory signals analogous to those transmitted by CD28, which shares the DAP10 YxxM motif20,
21. Given the distinct nature of the DAP10 adapter and its only known ligand NKG2D, we decided to further elucidate their functions by generating DAP10-deficient mice.
Results
Generation of DAP10-deficient mice
The DAP10 targeting construct was designed to replace exon 1 and intron 1 of the gene encoding DAP10 with the MC1-neor gene flanked by loxP sites (Fig. 1a). MC1-neor was then deleted by breeding mice heterozygous for the MC1-neor insertion (DAP10n/+ mice) to mice expressing a Cre transgene22. DAP10-/+ mice were crossed to produce mice homozygous for the deletion (Fig. 1b); these mice expressed virtually no full-length mRNA that was detectable by northern blot analysis of spleen RNA (Fig. 1c). Immunoblot analysis confirmed that no DAP10 protein was present in either activated NK or CD8+ T cells from the DAP10-/- animals (Fig 1d). Because the gene encoding DAP12 (Hcst) is located only 307 bp from the 3' end of the gene encoding DAP10 (Tyrobp), we ensured that our targeting had not caused an unanticipated effect on DAP12 expression (Fig. 1c,d).
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DAP10-/- mice were born at the expected frequency, bred well and were phenotypically normal. Histological analysis of heart, lung, liver, kidney, stomach, intestine, brain, thymus and skin from mice aged 6 weeks revealed no major abnormalities. Phenotyping of the immune system by flow cytometry demonstrated that the major lymphoid populations (, CD4+ and CD8+ T cells, T cells and B cells) were present at the expected frequency and numbers in the thymus, lymph nodes (LNs), spleen and bone marrow from DAP10-/- mice. NK cells were also present in normal numbers and frequency in the bone marrow, spleen, liver and peripheral blood—as were NK1.1+ T cells—in the DAP10-/- mice (data not shown). Thus, DAP10 is not critical for normal development nor is required for the development of a phenotypically normal immune system, including the NK cell population.
Expression of NKG2D in WT and DAP10-/- mice
Because DAP10 is only known to associate with NKG2D and is required for NKG2D cell-surface expression in transfectants13, we assessed the expression of this receptor in mutant mice using monoclonal antibodies (mAbs) specific for murine NKG2D23. NKG2D was expressed on all NK1.1+CD3- NK cells and on  50% of NK1.1+CD3lo NKT cells in littermate controls (Fig. 2a). In contrast, no NKG2D could be detected on NKT cells from the DAP10-/- mice (Fig. 2). However, low NKG2D expression was consistently observed on NK1.1+CD3- NK cells during analyses of spleen (Fig. 2a), bone marrow (Fig. 2b), peripheral blood, liver and thymus (data not shown) from the DAP10-/- mice. Expression of receptors recognized by an antibody specific for NKG2A, NKG2E and NKG2C (NKG2A/E/C) as well as the DAP12-associated receptors Ly49D and Ly49H was normal (Fig. 2b and data not shown). Thus, NKG2D expression is low but can still be detected on NK cells, but not T cells, freshly isolated from DAP10-/- mice.
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Expression of NKG2D on activated NK and T cells
This discrepancy in NKG2D expression was magnified on NK and T cells activated in vitro. DAP10-/- and DAP10+/+ spleen and LN cells were incubated in interleukin 2 (IL-2)−containing medium on plates coated with either or TCR mAbs. The majority of and CD8+ T cells from DAP10+/+ mice expanded in these cultures expressed high amounts of NKG2D, whereas similar populations expanded from DAP10-/- mice expressed no NKG2D on the cell surface (Fig. 3). In contrast, wild-type expression of NKG2D was observed on DAP10-/- NK cells 3 days after culture in IL-2. NKG2D on NK cells was also up-regulated after intraperitoneal injection of poly(I C) (Fig. 3). Thus, NKG2D was not expressed on resting T cells in the DAP10-/- mice and was not induced upon T cell activation. NKG2D was, however, expressed on the surface of resting NK cells and was further up-regulated by in vitro and in vivo activation.
Role of DAP12 in NKG2D expression
One explanation for the unexpected expression of NKG2D on DAP10-/- NK cells is the association with another adapter. DAP12 is a plausible candidate because it is expressed in NK cells but not activated CD8+ T cells (Fig. 1). To address this possibility, we performed immunoblot analysis on NKG2D immunoprecipitates. NKG2D associated with DAP12 in NK cells from both wild-type and DAP10-/- mice (Fig. 4). As expected, NKG2D coimmunoprecipitated with DAP10 in NK cells and CD8+ T cells from wild-type but not DAP10-/- mice. Therefore, the association of NKG2D with DAP12 could explain the unexpected expression of NKG2D on NK cells in the DAP10-/- mice. However, because DAP12 is not expressed in the majority of CD8+ T cells, NKG2D apparently has no suitable adapter partner and hence is not expressed on the cell surface of DAP10-/- T cells.
Functional association of NKG2D with DAP12
To functionally evaluate the intracellular promiscuity of NKG2D, we examined DAP10-/- NK cell−mediated cytoxicity. Target cells used included RMAS, a TAP2-deficient cell line that expresses low amounts of MHC class I but no NKG2D ligands4,
24; an RMAS cell line stably transfected with Rae-1 (RMAS.Rae-1); YAC, a prototypic NK cell target expressing NKG2D ligands4; and Chinese hamster ovary (CHO) cells which are killed in a Ly49D-DAP12−dependent manner25. Purified NK cells were expanded in IL-2 for 5 days and challenged with all target cells. Both DAP10-/- and DAP10+/+ NK cells killed RMAS−Rae-1 transfectants more proficiently than they killed the parental RMAS cells (Fig. 5a). However, DAP10-/- killing of RMAS.Rae-1 cells was consistently less efficient than that of DAP10+/+ NK cells. An NKG2D mAb blocked both DAP10+/+ and DAP10-/- NK cell killing of RMAS.Rae-1 target cells (Fig. 5a), demonstrating that the Rae-1−dependent lysis was mediated by NKG2D in both wild-type and DAP10-/- NK cells.
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This was confirmed by a series of experiments that used YAC cells as targets. Freshly isolated NK cells from both DAP10+/+ and DAP10-/- mice lysed YAC cells, although the DAP10-/- NK cells were slightly less efficient (Fig. 5b). However, the NKG2D mAb blocked killing by both DAP10+/+ and DAP10-/- NK cells. Thus, DAP10-/- primary and IL-2−activated NK cells retained some NKG2D-mediated activity, which was most likely due to association with the DAP12 adapter. NK cell function was not globally affected by the abrogation of DAP10: DAP10+/+ and DAP10-/- NK cells killed CHO cells equally well and, as expected, the NKG2D mAb had no effect (Fig. 5c). Thus, DAP10-/- NK cells killed NKG2D ligand−negative cells in a Ly49-DAP12−mediated manner as effectively as their wild-type counterparts.
Rejection of tumors in DAP10-/- mice
Tumors induced by small inoculi of RMAS cells are primarily rejected by NK cells, as RMAS expresses low amounts of MHC class I24. However, large inoculi overcome NK surveillance unless they have high cell surface expression of stimulatory ligands9,
10,
26,
27,
28. To further test DAP10-/- NK cell function in vivo, we challenged DAP10-/- and control littermates with either RMAS or RMAS.Rae-1 cells and monitored tumor growth. After subcutaneous (s.c.) injection of 5  106 or more RMAS cells, almost all DAP10-/- and control mice developed tumors (Table 1). Similarly, 5/6 DAP10-/- and 6/6 control littermates developed tumors after being challenged with a large inoculum (3 106 cells) of RMAS.Rae-1 cells expressing intermediate amounts of Rae-1 (data not shown). In contrast, only 3/32 DAP10-/- and 0/31 littermate controls developed exponentially growing tumors after s.c. injection of RMAS transfectants expressing high amounts of Rae-1 (Table 1). Thus, expression of Rae-1 on RMAS promoted tumor rejection in both wild-type and DAP10-/- mice, indicating that NK cells can mount an NKG2D-mediated antitumor response in the absence of DAP10. This was consistent with the cytotoxicity data and provided additional evidence that DAP12 can substitute for DAP10 in vivo.
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The role of NKG2D in generating a CD8+ T cell response
NKG2D has been implicated in generating an antitumor T cell11 and CD8+ T cell9,
12,
29 response. Although RMAS expresses low amounts of MHC class I, CD8+ T cells specific for RMAS are elicited by "vaccination" with an RMAS transfectant expressing high amounts of CD8026,
27. Additionally, vaccination with an RMAS line expressing CD70, a ligand for the costimulatory molecule CD27, elicits memory CD8+ T cells that specifically recognize the parental TAP-sufficient MHC class Ihi RMA tumor cell line28. We assessed the antitumor CD8+ T cell responses generated by RMAS.Rae-1 cells in DAP10+/+ and DAP10-/- mice that remained tumor-free 6−8 weeks after being challenged with RMAS.Rae-1 cells. Mice were boosted with an intravenous injection of RMAS.Rae-1 cells 3−5 days before analysis. CD8+ T cells were purified and incubated with RMAS.Rae-1 cells in vitro, and interferon- (IFN-) and IL-2 secretion were measured by intracellular staining. A population of IFN--secreting CD8+ T cells was detected in wild-type but not in DAP10-/- mice (Fig. 6a).
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Further analysis of NKG2D expression revealed that most of the IFN-−producing T cells expressed NKG2D. Production of IFN- was also evident after incubation of DAP10+/+CD8+ T cells with the parental RMAS line, showing that the CD8+ T cell response was tumor-specific and not solely dependent on Rae-1 expression. No IFN- was observed with an unrelated cell line or with CD8+ T cells from naïve mice, demonstrating the specificity of the CD8+ T cell response (data not shown). This indicated that CD8+ T cells specific for TAP-2−deficient RMAS cells were generated in wild-type but not DAP10-/- mice that had been "vaccinated" with RMAS.Rae-1 cells.
In addition, NKG2D was down-regulated on CD8+ T cells after incubation with RMAS.Rae-1 but not RMAS tumor cells (Fig. 6b). This, again, reflected NKG2D engagement of a ligand, which was probably important for the priming or generation of tumor-specific CD8+ T cells. We also analyzed the lymphocytes infiltrating the tumors that arose in DAP10+/+ and DAP10-/- mice after injection of RMAS cells expressing intermediate amounts of Rae-1. Consistent with the induction of an antitumor CD8+ T cell response in DAP10+/+ mice, we found that the proportion of NKG2D+CD8+ T cells within the tumor was much higher than that observed in the spleens of the same animals. This suggested that NKG2D+CD8+ T cells were preferentially recruited to or, more likely, expanded within the tumors (Fig. 7). Together, these results indicated that DAP10-/- mice failed to generate a CD8+ T cell memory response to tumors expressing NKG2D ligands.
Discussion
Our analysis of DAP10-/- mice shows that NKG2D not only uses conformational plasticity to recognize a large number of ligands but also has the capacity to associate with two adapter proteins, each linked to a distinct intracellular signaling pathway. However, the association with different signaling chains is cell type− and activation state−dependent. In T cells, NKG2D associates only with DAP10 and therefore is limited to the YxxM "costimulatory" pathway. As a result, DAP10-/- mice show impaired development of tumor-specific CD8+ T cell responses that could be due to the absence of NKG2D-DAP10 signaling during either antigen-specific T cell priming or effector activation. In contrast, in NK cells—which express both DAP10 and DAP12—NKG2D can activate ZAP-70 or Syk protein tyrosine kinases through the ITAM of DAP12 and the p85 subunit of PI3K and Grb2 through the YxxM motif of DAP10. Consequently, NK cells can reject tumors expressing NKG2D ligands even in the absence of DAP10.
The relative contributions of DAP12 and DAP10 pathways to NKG2D function in wild-type NK cells are likely to change with their state of activation. We have observed that NKG2D expression and NKG2D-mediated lysis in culture are optimal early after IL-2 activation of NK cells, whereas they decline at later time points of culture (data not shown). In an accompanying study published in this issue, Raulet and colleagues show that NKG2D includes two isoforms generated by alternative splicing of the same gene30. One isoform is associated only with DAP10, whereas the other is associated with both DAP10 and DAP12, but both apparently retain similar ligand specificity. Consistent with our study, the DAP12-associated isoform of NKG2D increases upon IL-2 and poly(IC)-induced NK cell activation and slowly decreases over time in culture. These observations may also explain the apparent discrepancy between our data and a published study that demonstrated the association of DAP10, but not DAP12, with NKG2D in a B cell transfection system and in coimmunoprecipitation experiments with a human NK cell line19. The reported NKG2D-DAP10 specificity probably describes the human NKG2D equivalent of the mouse DAP10-specific isoform; whether a "promiscuous" isoform of NKG2D is expressed in humans remains to be tested.
These NKG2D signaling options may reflect the disparate nature of NK and T cells. Individual T cells require the clonotypically expressed T cell receptor for specific recognition of a peptide−MHC class I complex and rely on a number of costimulatory molecules, such as NKG2D-DAP10, to modify the strength and duration of the signal. In this situation, an additional receptor that directly activates the cell, such as NKG2D-DAP12, would jeopardize T cell specificity. In contrast, individual NK cells simultaneously express a broad variety of activating receptors that are dominated by inhibitory receptors, with the functional outcome determined by signal integration31. In this context, NKG2D-DAP10−mediated "costimulation" may augment stimulation induced by other NK cell−activating receptors recognizing different ligands23,
32. Alternatively, the same MHC class I−like ligand can trigger both activation and costimulation through the NKG2D-DAP12 and NKG2D-DAP10 complexes, permitting innate host defense early in an immune response. Therefore, NKG2D has adapted to appropriately serve both cell types in surveillance for abnormal and stressed cells.
Methods
Generation of DAP-/- mice.
A targeting construct in which exon 1 and intron 1 of DAP10 were replaced by MCI-neor flanked by loxP sites was made by amplifying a 1.9-kb fragment 5' of DAP10 exon 1 and a 6.5-kb 3' fragment containing exon 2 from phage DNA with the oligo pairs 5'-TAGTAGGCGGCCGCAGCCGCAGAGCTAACTTAGACTCTGA-3', 5'-TAGTAGGCGGCCGCGCTGGGCTGGGACCTGAAGGGATCTGG-3' (NotI sites are underlined) and 5'-TAGTAGACTAGTAGTGGCTGCAAGTCAGACATCGGCA-3', 5'-TAGTAGACTAGTCTGGACATGCTAACACCACCCAAGAGAGTAG-3' (SpeI sites are underlined). The PCR fragments were digested with NotI and SpeI, respectively, and cloned into pmmNeoFlox-8 (a gift of R. Torres, Denver, CO), which contains MCI-neor flanked by loxP sites. The construct was electroporated into E14.1 embryonic stem (ES) cells33 and G418-resistant clones were screened by Southern blot analysis with multiple enzyme digests and external probes located 5' and 3' of the targeting vector as well as a neor internal probe. One correctly targeted clone was injected into C57BL6 blastocysts and two chimeras were obtained. One of these transmitted the targeted allele when bred with C57BL/6 mice. The MCI-neor insertion was deleted by crossing DAP10n/+ mice with B6N14 mice expressing a Cre transgene under the CMV promoter22. Mice were genotyped by either Southern blot analysis or PCR analysis of tail DNA with the oligos DAP105', 5'-GGTCCTCTTGCCACTCCA-3'; Neo 3', 5'-TTGCCGAATATCATGGTGGAAAAT-3'; DAP10 leader, 5'-GAAGCAGGAACAGGAGGTAG-3'; and DAP10 exon 2, 5'-GATGTCTGACTTGCAGCCAC-3'. These probes distinguished between all three genotypes: the endogenous (+) band was 263 bp, the neo insertion (n) was 365 bp and targeted with neo-deleted (-) was 302 bp. Southern blotting of genomic DNA isolated from ES clones or tail tissues and RNA blot analysis of spleen RNA from DAP10-/- and DAP10+/+ mice was done as described33. DAP10, DAP12 and HPRT probes were amplified from either LN or liver cDNA with the following primers: DAP10, 5'-TACCTCCTGTTCCTGCTTCTG-3' and 5'-GCCTCTGCCAGGCATGTTGAT-3'; DAP12, 5'-GAGCCCTCCTGGTGCCTTCTG-3' and 5'-TGGTCTCTGACCCTGAAGCTC-3'; and HPRT, 5'-GCTGGTGAAAAGGACCTCT-3', 5'-CACAGGACTAGAACACCTGC-3'.
Immunoblot analysis.
Cell lysates of day 5 IL-2−cultured NK cells (2 105 cells) and day 4 anti-CD3−stimulated CD8+ T cells (1 106) from DAP10+/+ and DAP10-/- mice were separated by SDS-PAGE and immunoblotted with DAP10- and DAP12-specific rabbit antisera raised against CPAQEDGRVYINMPGRG (DAP10) and CESPYQELQGQRPEVYSD (DAP12) peptides. Negative and positive controls included lysates from 293 cells untransfected or transiently transfected with a cDNA encoding NH2-terminal Flag-DAP10 fusion protein, respectively.
Flow cytometric analysis.
The mAbs phycoerythrin (PE)−anti- (GL3), PE−anti-CD4 (H129.19), fluorescein isothiocyanate (FITC)−anti-CD8 (53-6.7), FITC−anti-CD8 (53-5.8), PE−anti-NK1.1 (PK136), FITC−anti-CD3 (145-2C11), FITC−anti-Ly49D (4E5) and biotin−anti-NKG2A/C/E (20D5) were from PharMingen (San Diego, CA). Allophycocyanin-streptavidin was from Molecular Probes (Eugene, OR). mAbs A10 and C7 to NKG2D were purified and biotinylated23. Thymus, LN, spleen, bone marrow and liver suspensions were prepared and stained essentially as described33. Cells were analyzed on a FACScalibur with Cell Quest software (Becton Dickinson, San Jose, CA).
Immunoprecipitations.
NKG2D was immunoprecipitated from digitonin lysates of 107 wild-type and DAP10-/- NK cells 4−6 days after activation in IL-2 with the C7 and A10 NKG2D mAbs23. In addition, we immunoprecipitated NKG2D from 4 107 activated CD8+ T cells from wild-type and DAP10-/- mice activated on day 4 with plate-bound CD3 mAb. Immunoprecipitates were analyzed by immunoblotting with DAP12 and DAP10 antisera.
Cytotoxicity assays.
NK cells were purified with DX5 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) as described3. Purified DX5+ cells were >90% NK1.1+CD3- NK cells were cultured for different time periods in 1000 U/ml of recombinant human IL-2 (Roche, Basel, Switzerland) and 5% of heat-inactivated human serum. Cytotoxic activity of freshly purified NK cells and NK cells cultured in IL-2 was tested against target cells in standard 4-h 51Cr-release assays. Rae-1 was expressed in RMAS cells as an NH2-terminal Flag-peptide fusion protein by cloning Rae-1 cDNA into pFLAG-CMV1 (Sigma, St. Louis, MO). RMAS cells were cotransfected with pFLAG-CMV1−Rae-1 and a neor gene and G418-resistant clones were sorted for high expression of Flag−Rae-1 by cell sorting with the Flag mAb M2 (Sigma).
Priming of tumor specific T cells and determination of intracellular IFN- .
DAP10+/+ and DAP10-/- mice were challenged with 2 106 RMAS.Rae-1 cells. After 6−8 weeks, mice were challenged intravenously with 3 105 RMAS.Rae-1- cells and, after 5 additional days, CD8+ T cells were purified from spleens with CD8 microbeads (Miltenyi Biotech). Purified cells were incubated at a 1:2 ratio with RMAS or RMAS/Rae-1 cells for 6 h. In the last 4 h, 2  g/ml of monensin (Sigma) was added. Cells were then fixed, permeabilized with the Cytofix-Cytoperm kit (PharMingen) and stained.
Received 17 August 2002; Accepted 7 October 2002; Published online: 11 November 2002.
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