HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carayannopoulos, L. N. Articles by Yokoyama, W. M. Search for Related Content PubMed PubMed Citation Articles by Carayannopoulos, L. N. Articles by Yokoyama, W. M.

Cutting Edge: Murine UL16-Binding Protein-Like Transcript 1: A Newly Described Transcript Encoding a High-Affinity Ligand for Murine NKG2D¹

Leonidas N. Carayannopoulos^*, Olga V. Naidenko, Daved H. Fremont and Wayne M. Yokoyama^2,

^* Division of Pulmonary and Critical Care Medicine, Department of Pathology and Immunology, and Division of Rheumatology and Howard Hughes Medical Institute, Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Murine NKG2D is known to recognize H60 and five RAE1 variants. The human homologue recognizes both inducible MHC class I chain-related gene and constitutive (UL16-binding protein (ULBP)) ligands. Widely expressed, the latter are thought to mark transformed or infected cells for destruction by NK cells in the context of down-regulated cell surface class I (i.e., the "missing self"-response). Unlike MIC and ULBP however, mRNA for the murine ligands appears only in very limited contexts in the mature animal. In this study, we describe a NKG2D ligand termed "murine ULBP-like transcript 1 (MULT1) whose mRNA appears to be widely expressed in adult parenchyma. This molecule possesses MHC class I-like 1 and 2 domains as well as a large cytoplasmic domain. Recombinant MULT1 binds NKG2D with relatively high affinity (K_D 6 nM) and low k_off (0.006s^-1). Expression of MULT1 by normally resistant RMA cells results in their susceptibility to lysis by C57BL/6 splenocytes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Atypical cells, whether transformed (1, 2, 3) or infected (4, 5), frequently demonstrate two phenomena: down-regulation of cell surface class I and up-regulation of NKG2D ligands. In innate responses, down-regulation of target cell class I releases NK cells from tyrosine phosphatase-mediated inhibition (6) and forms the basis of the "missing self"-phenomenon (7). Ligand recognition by the lectin-like immunoreceptor NKG2D provides at least a subset of the activation signals disinhibited by missing self (8). High levels of NKG2D ligand expression may even override normal levels of class I-mediated NK inhibition (2, 9, 10). In adaptive responses, down-regulation of class I on atypical cells would be expected to blunt CTL action; however, simultaneous engagement of NKG2D ligands provides compensatory costimulation (4) and enables activated CTL-mediated killing.

Two MIC and three or more ULBP molecules constitute the known human NKG2D ligand repertoire. mRNA for subsets of these proteins are widely expressed in resting nonlymphoid tissues (2, 11, 12). The known murine NKG2D-binding repertoire encompasses H60 and five RAE1 variants. Though expressed by numerous tumor cell lines (1, 13), mRNA for these molecules in normal tissues appear to be tightly restricted to the early embryo and selected adult cell types (1, 13, 14, 15).

Despite intense interest in this system, the murine ligands for NKG2D have not yet been shown to be expressed in nonlymphoid organs except following transformation (3), unlike their constitutively expressed and/or easily inducible human orthologs. The analogy between human and murine systems being accordingly unsatisfying, we suspected the existence of NKG2D-binding proteins that had eluded detection by the expression cloning systems previously used, both of which were based on transformed cell cDNA libraries (1, 13). In this study, we report the identification of a murine transcript (murine UL16-binding protein-like transcript 1 (MULT1))³ encoding a high-affinity ligand for NKG2D with greater protein sequence similarity to human UL16-binding protein 3 (ULBP3) than to murine RAE1 or H60. Like ULBP, this transcript appears to be widely expressed in nonlymphoid tissues.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Production of RNA

C57BL/6NCR (B6) tissues were perfused free of blood, then organs were harvested and flash frozen in liquid nitrogen. RNA was isolated using TRIzol (Life Technologies, Rockville, MD) as per the instructions, treated with RQ1 DNase (Promega, Madison, WI), and repurified using RNEasy silica columns (Qiagen, Chatsworth, CA). RNA quality was verified by standard formaldehyde gel electrophoresis and RT-PCR of hypoxanthine-guanine phosphoribosyltransferase.

Molecular cloning of MULT1 and generation of transduced cell lines

TBLASTn search (National Center for Biotechnology Information) of the murine expressed sequence tag (EST) database (16) was performed using the extracellular sequences of ULBP1–3 with the expected value set at 100. This yielded a match with a The Institute of Physical and Chemical Research neonatal thymus sequence (accession no. AK020784) herein termed "MULT1." Pfu polymerase (Stratagene, La Jolla, CA) and oligonucleotides 5'-TTTGCAGTGATCACCGCCATGGA(G/A)CT(G/C)ACT(G/C)CCAGT-3'and 5'-ACGGGTCGACTTAATGGTGATGGTGATGGTGATGTGGGATCCCATCAATATCGTC-3' were used to amplify the correspondingsequence from B6 day 1 neonatal thymus RNA following reverse transcription with Superscript reverse transcriptase (Life Technologies). Primers were selected to amplify only MULT1 based on all available similar sequence fragments from the EST database and the Celera murine genome resource (Celera Genomics, Rockville, MD). The resultant amplicon was ligated into the BamHI and XhoI sites of pMXIRES-enhanced green fluorescent protein (EGFP; courtesy of Dr. T. Kitamura, University of Tokyo, Tokyo, Japan) (17). This plasmid was then used to generate RMA (American Type Culture Collection, Manassas, VA) and Ba/F3 cells retrovirally transduced with MULT1 as described elsewhere (18). Sequencing of constructs was performed using BigDye 2.0 (PerkinElmer, Norwalk, CT) exactly according to the manufacturer’s instructions. Sequence data were manipulated using VectorNTI 3.0 (Informax, Bethesda, MD). Secondary structure prediction was performed using 3D-PSSM (19). Alignments were performed using CLUSTALW. Signal and GPI predictions were performed using SignalP (20), and DGPI (21) and big-PI (22), respectively.

Production of recombinant proteins

Biotinylated recombinant soluble (rs) NKG2D ectodomains were produced in insect cells exactly as described previously (23). The rsMULT1 ectodomain sequence from (MG)IEETAS... to ... GSFST was ligated into the NcoI and XhoI sites of pET-15b (Novagen, Madison, WI); the additional N-terminal methionine and glycine were a consequence of the NcoI cloning site. Expression, purification, and refolding of rsMULT1 and rsH60 was as described elsewhere (23). Mass spectrometric analysis (Keck Mass Spectrometry Resource, Yale University, New Haven, CT) of refolded rsMULT1 showed the N-terminal methionine to be removed. NKG2D tetramers were produced by dropwise addition of streptavidin-PE (SAPE; BD PharMingen, San Diego, CA) to biotin-rsNKG2D at a 1:4 molar ratio with gentle mixing. Standard amino acid analysis showed the A₂₈₀:mass relationship for rsMULT1 to be: A_{280(1 mg/ml)} = 1.34 in HEPES-buffered saline.

Cell staining

RMA and Ba/F3 cells infected with pMXIRES-EGFP, pMXIRES-RAE1-EGFP, or pMXIRES-MULT1-EGFP retrovirus were processed on a MoFlo sorter (Cytomation, Fort Collins, CO) to generate pure populations of green fluorescent protein-positive cells with similar geometric mean fluorescence intensity and scatter profiles. These were then stained with appropriate reagents as detailed below. Cytotoxic effectors were stained with 4 µg/ml PK136 (24) conjugated to Alexa488 (Molecular Probes, Eugene, OR) and 4 µg/ml anti-CD3-PerCP (BD PharMingen) and then subjected to flow cytometry to verify phenotype.

Surface plasmon resonance (SPR) experimentation

SPR work, data interpretation, and quality control were performed exactly as described previously (23), except that kinetic and steady-state analyses were performed with R_max set at 40 and 90 response units, respectively. Kinetics experiments were performed four times at four concentrations on two separate surfaces. Steady-state experiments were performed twice, in duplicate, over two separate surfaces. Raw data were analyzed and graphed using BIAeval 3.1 (BIAcore, Piscataway, NJ) and Kaleidagraph 3.5 (Synergy Software, Reading, PA).

Cytotoxicity assay

B6 splenocytes were positively selected for DX5 expression using the MACS (Miltenyi Biotec, Auburn, CA) system, washed twice with RPMI 1640 supplemented with 10% FCS (R10), and incubated in R10 for 10 min with or without 1 µM rsH60 or 1 µM irrelevant recombinant protein purified the same way. These effector cells were then used in standard 4-h ⁵¹Cr release assays with 10,000 targets/well at 37C° and in 5% CO₂. Percent specific lysis was defined as (observed dpm - spontaneous dpm)/(maximal detergent released dpm - spontaneous dpm) x 100. Data points were obtained in triplicate. Experiments were performed independently twice.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Molecular cloning of MULT1

We observed staining of C1498 murine lymphoma cells with tetrameric NKG2D, yet could obtain neither RAE1 nor H60 sequences from C1498 RNA by RT-PCR (data not shown). We therefore searched the mouse EST database using ULBP1–3 query sequences; genomic correlations were obtained using the Celera Discovery Tool (Celera Genomics). Among several related sequences, an EST (accession no. AK020784) encoding a deduced 334-aa protein appeared most promising and was studied in greater detail. Based on its level of identity with ULBP3 (>20%), its placement on mouse chromosome 10 in a region syntenic to human chromosome 6q25, its appearance in several EST libraries, and its predicted MHC class I-like fold (3D-PSSM (19)), the sequence was termed MULT. Distinct, highly related exons (within Loc 237247 on supercontig NW_000022) also exist nearby on B6 mouse chromosome 10. Examination of the Celera genome resource suggested three loci (mCG54954, mCG12640, and mCG51610) may encode similar genes in the DBA/2J mouse. Thus, sequence AK020784 was renamed MULT1 to reflect possible existence of a family of related sequences.

Full-length MULT1 cDNA was obtained from B6 neonatal thymus RNA by RT-PCR. Fig. 1A shows the protein sequence and secondary structural alignment with ULBP3 and RAE1 (25, 26). It encompasses 1- and 2-like domains but no 3-like domain or predicted GPI transamidation site. Uniquely among known NKG2D ligands, MULT1 possesses an extensive intracellular domain. MULT1 has four possible N-linked glycosylation sites, two disulfide bonds, and two unpaired cysteines (verified by mass spectrometric analysis of the refolded, active protein: mass_obs = 21011, mass_{pred(reduced)} = 21015; data not shown).

View larger version (41K): [in this window] [in a new window]   FIGURE 1. MULT1 possesses a similarity to other class I-like proteins and is widely expressed. A, MULT1 sequence was aligned to the ULBP3 and RAE1 sequences for which structural data exists. Gray shading denotes MULT1 cysteines; disulfide bonds are predicted at C44–C60 and at C99–C163. *, Potential N-linked glycosylation sites. Thin black bars mark the leader and transmembrane sequences. Open boxes enclose residues identically or strongly conserved between MULT1 and one of the other sequences; black shading marks residues common to all three sequences. Known secondary structure elements are schematized below the alignment while the 3D-PSSM-predicted secondary structural elements of MULT1 appear above. B, Total RNA (100 ng) from several tissues was subjected to RT-PCR using primers specific for MULT1. The 1.1-kb band corresponds to the full-length MULT1 coding sequence verified by sequence analysis. No bands were obtained in the absence of reverse transcriptase.

NKG2D binds to cells expressing full-length MULT1

The cell lines Ba/F3 and RMA were transduced with bicistronic vectors containing cDNA for EGFP alone, RAE1 and EGFP, or MULT1 and EGFP. Transductants were stained with tetramerized NKG2D; results for RMA (Fig. 2) and Ba/F3 (data not shown) were identical. EGFP-alone transductants displayed no reactivity (Fig. 2D), while RAE1/EGFP and MULT1/EGFP transductants bound NKG2D tetramers in a manner linearly related to EGFP expression. Specificity of the interaction was confirmed both by the lack of staining with free SAPE and by the ability of 1 µM (100 x K_D) rsH60 to block binding (Fig. 2, H and I). Irrelevant recombinant protein purified identically from bacteria failed to block staining at the same concentration (data not shown). Thus, full-length MULT1, when expressed in cells, confers specific cell surface binding to NKG2D.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Tetrameric rsNKG2D binds specifically to RMA cells that express MULT1. RMA cells transduced with bicistronic expression cassettes expressing either noncoding, DNA and EGFP (A, D, and G), RAE-1 and EGFP (B, E, and H), or MULT1 and EGFP (C, F, and I) were incubated with the following reagents: A–C, SAPE; D–F, biotinylated rsNKG2D tetramerized around SAPE; and G–I, tetramerized rsNKG2D preincubated for 10 min with 1 µM rsH60. The x-axes represent log(fluorescent intensity) of EGFP while the y-axes represent log(fluorescent intensity) of R-PE. These results are representative of three trials.

Ligands for murine NKG2D demonstrate two binding regimes—a lower affinity interaction demonstrated by RAE1- (K_D 300–800 nM) and a higher affinity interaction demonstrated by RAE1 and H60 (K_D 10–30 nM) (23, 27). To assess the properties of the MULT1-NKG2D interaction, the ectodomain of MULT1 was refolded from bacterial inclusion bodies. Integrity of the refolded protein was verified both by demonstration of a symmetric peak eluting at the volume expected for a 21-kDa monomer on gel filtration and by the ability of excess rsNKG2D to completely shift the migration of rsMULT1 in native polyacrylamide gels (data not shown).

The refolded rsMULT1 protein was applied to SPR experiments over a flowcell decorated with biotin-NKG2D. These studies revealed a K_D of 6 nM (K_Dcalc, 2 nM) and a K_off of 0.006 s^-1, several times lower than that of H60 (Fig. 3). Thus, MULT1 binds NKG2D with the highest affinity of all known ligands and displays a t_1/2 of 2 min, longer than either H60 (20 s) or RAE-1- (5 s).

View larger version (37K): [in this window] [in a new window]   FIGURE 3. rsMULT1 binds NKG2D with high affinity. A and B, Measurements of the affinity of rsMULT1 for immobilized rsNKG2D were performed using SPR. rsMULT1 (0, 0.8, 1.6, 4.0, 7.9, 16, 40, 79, 160 nM) was sequentially analyzed for binding to SPR flowcells derivatized with biotin-antiCD19 (negative control) and biotin-rsNKG2D. Shown are representative net responses derived by subtraction of the control signal from the NKG2D signal (A) and fitting of a 1:1 (monomeric ligand:dimeric NKG2D) Langmuir model, incorporating the displayed KD, to duplicate data points from a representative experiment (B). Symbols in B represent observed data; curves represent best fit of the Langmuir isotherm. Stated affinity refers to mean and SD from four experiments. C, Kinetics of rsMULT1 recognition of rsNKG2D was assayed by flow of rsMULT1 at 4.0, 7.9, 15.8, and 39.5 nM over rsNKG2D. Representative experiments are shown. Symbols in C represent best fits of kinetic models to the data (curves). Stated kinetic parameters represent mean and SEM from four sets of four experiments. Horizontal bars in A and C mark the injection interval for rsMULT1. Analogous experiments performed with 1 µM BSA as analyte showed responses of essentially zero. Absolute response levels of all injections over the control surface were <4 response units.

Neither fresh B6 splenocytes nor IL-2-activated killer cells can lyse RMA lymphoma cells due to syngeneic class I expression by the latter (7). High-level expression of NKG2D ligands, however, appears to overcome class I-mediated inhibition of lymphokine-activated NK cells and NK cell lines, resulting in target lysis (2, 9, 10). We examined the ability of MULT1 overexpression to similarly render cells susceptible to lysis by fresh, unstimulated B6 splenocytes. RMA cells transduced with EGFP alone, RAE1 and EGFP, or MULT1 and EGFP were used as targets for syngeneic DX5⁺ fresh splenocytes in 4-h ⁵¹Cr release assays. As expected, EGFP-only transductants were not significantly lysed. MULT1 transductants were lysed similarly to RAE1 transductants (Fig. 4). Killing of both transductants was greatly inhibited by excess rsH60 (open symbols) but not irrelevant recombinant protein (data not shown), verifying the role of NKG2D in mediating this effect. Similar results were obtained with IL-2-activated killer cells against Ba/F3 transductants (data not shown). These data demonstrate the ability of MULT1 to recruit NK-mediated killing via NKG2D.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Overexpression of MULT1 sensitizes RMA lymphoma cells to lysis by fresh B6 splenocytes. Fresh splenocytes enriched for NK cells (70% NK1.1+CD3-, 5% NK1.1-CD3+) were applied as effectors to a 4-h 51Cr release assay at E:T ratios of 0:1, 1:1, 8:1, and 20:1 against RMA cells transduced with the following: EGFP alone (•), RAE-1 and EGFP (), and MULT1 and EGFP (). Open symbols represent, respectively, lysis of the same transductants by effectors pretreated for 15 min with 1 µM rsH60. This figure shows averaged triplicate data points from a representative independent experiment. Coefficients of variation of triplicate data points were 10% or less.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Evolutionary factors selecting for such a complicated receptor-ligand system are likely 2-fold. First, the functional consequences of NKG2D engagement are pleiotropic, involving T cell costimulation, NK cell activation, macrophage stimulation, and possibly regulation of fetal development (1, 4, 13, 14). Precise recruitment of these diverse functions to particular contexts may require multiple genes with distinct promoter/enhancer sequences, posttranslational controls, and even kinetics of binding. Second, microbes exert enormous selective pressure to diversify immune-related functions, albeit at differing rates (28, 29). Recent evidence suggests that human CMV interferes with the NKG2D system using the UL16 gene product to bind ULBP1 and ULBP2 (2); also, mouse CMV gp40 may down-regulate H60 (30). Pathogen-encoded factors such as these might have selected for NKG2D-binding partners which retain receptor specificity but lack susceptibility to interference or subversion (e.g., ULBP3, which does not bind to UL16), resulting in the current repertoire of dissimilar NKG2D ligands. However, a true understanding of this system awaits description of all of the NKG2D ligands, the mechanisms controlling their cell surface expression, and the functional consequences of the recognition event.

	Footnotes

 
¹ This work supported by a Howard Hughes Medical Institute Transitional Award (to L.N.C.), a Cancer Research Institute Fellowship (to O.V.N.), National Institutes of Health GM62414 and a Burroughs-Wellcome Fund Award (to D.H.F.), and by National Institutes of Health grants to W.M.Y., who is an investigator for the Howard Hughes Medical Institute.

² Address correspondence and reprint requests to Dr. Wayne M. Yokoyama, Division of Rheumatology and Howard Hughes Medical Institute, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8045, St. Louis, MO 63110. E-mail address: yokoyama{at}imgate.wustl.edu

³ Abbreviations used in this paper: MULT, murine UL16-binding protein-like transcript; ULBP, UL16-binding protein; EGFP, enhanced green fluorescent protein; EST, expressed sequence tag; SAPE, streptavidin-PE; rs, recombinant soluble; SPR, surface plasmon resonance.

Received for publication July 16, 2002. Accepted for publication August 22, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Diefenbach, A., A. M. Jamieson, S. D. Liu, N. Shastri, D. H. Raulet. 2000. Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat. Immunol. 1:119.[Medline]
2. Cosman, D., J. Mullberg, C. L. Sutherland, W. Chin, R. Armitage, W. Fanslow, M. Kubin, N. J. Chalupny. 2001. ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity 14:123.[Medline]
3. 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 - T cells. Science 294:605.[Abstract/Free Full Text]
4. Groh, V., R. Rhinehart, J. Randolph-Habecker, M. S. Topp, S. R. Riddell, T. Spies. 2001. Costimulation of CD8 T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat. Immunol. 2:255.[Medline]
5. Tieng, V., C. Bouguenec, L. du Merle, P. Bertheau, P. Desreumaux, A. Janin, D. Charron, A. Toubert. 2002. Binding of Escherichia coli adhesin AfaE to CD55 triggers cell surface expression of the MHC class I-related molecule MICA. Proc. Natl. Acad. Sci. USA 99:2977.[Abstract/Free Full Text]
6. Binstadt, B. A., K. M. Brumbaugh, P. J. Leibson. 1997. Signal transduction by human NK cell MHC-recognizing receptors. Immunol. Rev. 155:197.[Medline]
7. Karre, K., H. G. Ljunggren, G. Piontek, R. Kiessling. 1986. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319:675.[Medline]
8. Pende, D., C. Cantoni, P. Rivera, M. Vitale, R. Castriconi, S. Marcenaro, M. Nanni, R. Biassoni, C. Bottino, A. Moretta, L. Moretta. 2001. Role of NKG2D in tumor cell lysis mediated by human NK cells: cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin. Eur. J. Immunol. 31:1076.[Medline]
9. 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.[Abstract/Free Full Text]
10. Cerwenka, A., J. L. Baron, L. L. Lanier. 2001. Ectopic expression of retinoic acid early inducible-1 gene (RAE-1) permits natural killer cell-mediated rejection of a MHC class I-bearing tumor in vivo. Proc. Natl. Acad. Sci. USA 98:11521.[Abstract/Free Full Text]
11. Radosavljevic, M., B. Cuillerier, M. J. Wilson, O. Clement, S. Wicker, S. Gilfillan, S. Beck, J. Trowsdale, S. Bahram. 2002. A cluster of ten novel MHC class I related genes on human chromosome 6q24.2-q25.3. Genomics 79:114.[Medline]
12. Hankey, K. G., C. B. Drachenberg, J. C. Papadimitriou, D. K. Klassen, B. Philosophe, S. T. Bartlett, V. Groh, T. Spies, D. L. Mann. 2001. MIC expression in renal and pancreatic allografts. Transplantation 73:304.
13. Cerwenka, A., A. B. H. Bakker, T. McClanahan, J. Wagner, J. Wu, J. H. Phillips, L. L. Lanier. 2000. Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice. Immunity 12:721.[Medline]
14. Zou, Z., M. Nomura, Y. Takihara, T. Yasunaga, K. Shimada. 1996. Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells: a novel cDNA family encodes cell surface proteins sharing partial homology with MHC class I molecules. J. Biochem. 119:319.[Abstract/Free Full Text]
15. Malarkannan, S., P. P. Shih, P. A. Eden, T. Horng, A. R. Zuberi, G. Christianson, D. Roopenian, N. Shastri. 1998. The molecular and functional characterization of a dominant minor H antigen, H60. J. Immunol. 161:3501.[Abstract/Free Full Text]
16. Lennon, G., C. Auffray, M. Polymeropoulos, M. B. Soares. 1996. The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics 33:151.[Medline]
17. Onishi, M., S. Kinoshita, Y. Morikawa, A. Shibuya, J. Phillips, L. L. Lanier, D. M. Gorman, G. P. Nolan, A. Miyajima, T. Kitamura. 1996. Applications of retrovirus-mediated expression cloning. Exp. Hematol. 24:324.[Medline]
18. Morita, S., T. Kojima, T. Kitamura. 2000. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7:1063.[Medline]
19. Kelley, L. A., R. M. MacCallum, M. J. Sternberg. 2000. Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol. 299:499.[Medline]
20. Nielsen, H., J. Engelbrecht, S. Brunak, G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1.[Abstract/Free Full Text]
21. Buloz, D., and J. Kronegg. 1999. DGPI, Geneva, Switzerland.
22. Eisenhaber, B., P. Bork, F. Eisenhaber. 1999. Prediction of potential GPI-modification sites in proprotein sequences. J. Mol. Biol 292:741.[Medline]
23. Carayannopoulos, L. N., O. V. Naidenko, J. Kinder, E. L. Ho, D. H. Fremont, W. M. Yokoyama. 2002. Ligands for NKG2D display heterogeneous binding behavior. Eur. J. Immunol. 32:597.[Medline]
24. Koo, G. C., J. R. Peppard. 1984. Establishment of monoclonal anti-NK-1.1 antibody. Hybridoma 3:301.[Medline]
25. Radaev, S., B. Rostro, A. G. Brooks, M. Colonna, P. D. Sun. 2001. Conformational plasticity revealed by the cocrystal structure of NKG2D and its class I MHC-like ligand ULBP3. Immunity 15:1039.[Medline]
26. Li, P., G. McDermott, R. K. Strong. 2002. Crystal structures of RAE-1 and its complex with the activating immunoreceptor NKG2D. Immunity 16:77.[Medline]
27. O’Callaghan, C. A., A. Cerwenka, B. E. Willcox, L. L. Lanier, P. J. Bjorkman. 2001. Molecular competition for NKG2D: H60 and RAE1 compete unequally for NKG2D with dominance of H60. Immunity 15:201.[Medline]
28. Klein, J., Y. Satta, C. O’Huigin, N. Takahata. 1993. The molecular descent of the major histocompatibility complex. Annu. Rev. Immunol. 11:269.[Medline]
29. Khakoo, S. I., R. Rajalingam, B. P. Shum, K. Weidenbach, L. Flodin, D. G. Muir, F. Canavez, S. L. Cooper, N. M. Valiante, L. L. Lanier, P. Parham. 2000. Rapid evolution of NK cell receptor systems demonstrated by comparison of chimpanzees and humans. Immunity 12:687.[Medline]
30. Krmpotic, A., D. H. Busch, I. Bubic, F. Gebhhardt, H. Hengel, M. Hasan, A. A. Scalzo, U. H. Koszinowski, S. Jonjic. 2002. MCMV glycoprotein gp40 confers virus resistance to CD8 T cells and NK cells in vivo. Nat. Immunol. 3:529.[Medline]

This article has been cited by other articles:

				R. W. McGilvray, R. A. Eagle, N. F.S. Watson, A. Al-Attar, G. Ball, I. Jafferji, J. Trowsdale, and L. G. Durrant NKG2D Ligand Expression in Human Colorectal Cancer Reveals Associations with Prognosis and Evidence for Immunoediting Clin. Cancer Res., November 15, 2009; 15(22): 6993 - 7002. [Abstract] [Full Text] [PDF]

				W. B. Tabayoyong, J. G. Salas, S. Bonde, and N. Zavazava HOXB4-Transduced Embryonic Stem Cell-Derived Lin-c-kit+ and Lin-Sca-1+ Hematopoietic Progenitors Express H60 and Are Targeted by NK Cells J. Immunol., November 1, 2009; 183(9): 5449 - 5457. [Abstract] [Full Text] [PDF]

				J. Arapovic, T. Lenac, R. Antulov, B. Polic, Z. Ruzsics, L. N. Carayannopoulos, U. H. Koszinowski, A. Krmpotic, and S. Jonjic Differential Susceptibility of RAE-1 Isoforms to Mouse Cytomegalovirus J. Virol., August 15, 2009; 83(16): 8198 - 8207. [Abstract] [Full Text] [PDF]

				W. Cao, L. Bover, M. Cho, X. Wen, S. Hanabuchi, M. Bao, D. B. Rosen, Y.-H. Wang, J. L. Shaw, Q. Du, et al. Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction J. Exp. Med., July 6, 2009; 206(7): 1603 - 1614. [Abstract] [Full Text] [PDF]

				B. M. Carreno, J. R. Garbow, G. R. Kolar, E. N. Jackson, J. A. Engelbach, M. Becker-Hapak, L. N. Carayannopoulos, D. Piwnica-Worms, and G. P. Linette Immunodeficient Mouse Strains Display Marked Variability in Growth of Human Melanoma Lung Metastases Clin. Cancer Res., May 15, 2009; 15(10): 3277 - 3286. [Abstract] [Full Text] [PDF]

				T. J. Nice, L. Coscoy, and D. H. Raulet Posttranslational regulation of the NKG2D ligand Mult1 in response to cell stress J. Exp. Med., February 16, 2009; 206(2): 287 - 298. [Abstract] [Full Text] [PDF]

				A. Cerwenka New twist on the regulation of NKG2D ligand expression J. Exp. Med., February 16, 2009; 206(2): 265 - 268. [Abstract] [Full Text] [PDF]

				E. M. Mace, S. J. Monkley, D. R. Critchley, and F. Takei A Dual Role for Talin in NK Cell Cytotoxicity: Activation of LFA-1-Mediated Cell Adhesion and Polarization of NK Cells J. Immunol., January 15, 2009; 182(2): 948 - 956. [Abstract] [Full Text] [PDF]

				J. D. Wu, C. L. Atteridge, X. Wang, T. Seya, and S. R. Plymate Obstructing Shedding of the Immunostimulatory MHC Class I Chain-Related Gene B Prevents Tumor Formation Clin. Cancer Res., January 15, 2009; 15(2): 632 - 640. [Abstract] [Full Text] [PDF]

				N. Nausch, I. E. Galani, E. Schlecker, and A. Cerwenka Mononuclear myeloid-derived "suppressor" cells express RAE-1 and activate natural killer cells Blood, November 15, 2008; 112(10): 4080 - 4089. [Abstract] [Full Text] [PDF]

				C. Germain, E. Campigna, I. Salhi, S. Morisseau, I. Navarro-Teulon, J.-P. Mach, A. Pelegrin, and B. Robert Redirecting NK cells mediated tumor cell lysis by a new recombinant bifunctional protein Protein Eng. Des. Sel., November 1, 2008; 21(11): 665 - 672. [Abstract] [Full Text] [PDF]

				H. Guo, A. Samarakoon, B. Vanhaesebroeck, and S. Malarkannan The p110{delta} of PI3K plays a critical role in NK cell terminal maturation and cytokine/chemokine generation J. Exp. Med., September 29, 2008; 205(10): 2419 - 2435. [Abstract] [Full Text] [PDF]

				W. Cao, X. Xi, Z. Wang, L. Dong, Z. Hao, L. Cui, C. Ma, and W. He Four novel ULBP splice variants are ligands for human NKG2D Int. Immunol., August 1, 2008; 20(8): 981 - 991. [Abstract] [Full Text] [PDF]

				T. Baba, S. Iwasaki, T. Maruoka, A. Suzuki, U. Tomaru, H. Ikeda, T. Yoshiki, M. Kasahara, and A. Ishizu Rat CD4+CD8+ Macrophages Kill Tumor Cells through an NKG2D- and Granzyme/Perforin-Dependent Mechanism J. Immunol., March 1, 2008; 180(5): 2999 - 3006. [Abstract] [Full Text] [PDF]

				C. Li, B. Ge, M. Nicotra, J. N. H. Stern, H. D. Kopcow, X. Chen, and J. L. Strominger JNK MAP kinase activation is required for MTOC and granule polarization in NKG2D-mediated NK cell cytotoxicity PNAS, February 26, 2008; 105(8): 3017 - 3022. [Abstract] [Full Text] [PDF]

				A. Takada, S. Yoshida, M. Kajikawa, Y. Miyatake, U. Tomaru, M. Sakai, H. Chiba, K. Maenaka, D. Kohda, K. Fugo, et al. Two Novel NKG2D Ligands of the Mouse H60 Family with Differential Expression Patterns and Binding Affinities to NKG2D J. Immunol., February 1, 2008; 180(3): 1678 - 1685. [Abstract] [Full Text] [PDF]

				Y. Ito, T. Kanai, T. Totsuka, R. Okamoto, K. Tsuchiya, Y. Nemoto, A. Yoshioka, T. Tomita, T. Nagaishi, N. Sakamoto, et al. Blockade of NKG2D signaling prevents the development of murine CD4+ T cell-mediated colitis Am J Physiol Gastrointest Liver Physiol, January 1, 2008; 294(1): G199 - G207. [Abstract] [Full Text] [PDF]

				S. Vilarinho, K. Ogasawara, S. Nishimura, L. L. Lanier, and J. L. Baron Blockade of NKG2D on NKT cells prevents hepatitis and the acute immune response to hepatitis B virus PNAS, November 13, 2007; 104(46): 18187 - 18192. [Abstract] [Full Text] [PDF]

				G. Galazka, A. Jurewicz, W. Orlowski, M. Stasiolek, C. F. Brosnan, C. S. Raine, and K. Selmaj EAE Tolerance Induction with Hsp70-Peptide Complexes Depends on H60 and NKG2D Activity J. Immunol., October 1, 2007; 179(7): 4503 - 4512. [Abstract] [Full Text] [PDF]

				B. K. Choi, Y. H. Kim, W. J. Kang, S. K. Lee, K. H. Kim, S. M. Shin, W. M. Yokoyama, T. Y. Kim, and B. S. Kwon Mechanisms Involved in Synergistic Anticancer Immunity of Anti-4-1BB and Anti-CD4 Therapy Cancer Res., September 15, 2007; 67(18): 8891 - 8899. [Abstract] [Full Text] [PDF]

				S. Malarkannan, J. Regunathan, H. Chu, S. Kutlesa, Y. Chen, H. Zeng, R. Wen, and D. Wang Bcl10 Plays a Divergent Role in NK Cell-Mediated Cytotoxicity and Cytokine Generation J. Immunol., September 15, 2007; 179(6): 3752 - 3762. [Abstract] [Full Text] [PDF]

				J. A. Campbell, D. S. Trossman, W. M. Yokoyama, and L. N. Carayannopoulos Zoonotic orthopoxviruses encode a high-affinity antagonist of NKG2D J. Exp. Med., June 11, 2007; 204(6): 1311 - 1317. [Abstract] [Full Text] [PDF]

				A. Averdam, H. Kuhl, M. Sontag, T. Becker, A. L. Hughes, R. Reinhardt, and L. Walter Genomics and Diversity of the Common Marmoset Monkey NK Complex J. Immunol., June 1, 2007; 178(11): 7151 - 7161. [Abstract] [Full Text] [PDF]

				J. Regunathan, Y. Chen, S. Kutlesa, X. Dai, L. Bai, R. Wen, D. Wang, and S. Malarkannan Differential and Nonredundant Roles of Phospholipase C{gamma}2 and Phospholipase C{gamma}1 in the Terminal Maturation of NK Cells J. Immunol., October 15, 2006; 177(8): 5365 - 5376. [Abstract] [Full Text] [PDF]

				K. Abdool, E. Cretney, A. D. Brooks, J. M. Kelly, J. Swann, A. Shanker, E. W. Bere Jr., W. M. Yokoyama, J. R. Ortaldo, M. J. Smyth, et al. NK Cells Use NKG2D to Recognize a Mouse Renal Cancer (Renca), yet Require Intercellular Adhesion Molecule-1 Expression on the Tumor Cells for Optimal Perforin-Dependent Effector Function J. Immunol., August 15, 2006; 177(4): 2575 - 2583. [Abstract] [Full Text] [PDF]

				T. Lenac, M. Budt, J. Arapovic, M. Hasan, A. Zimmermann, H. Simic, A. Krmpotic, M. Messerle, Z. Ruzsics, U. H. Koszinowski, et al. The herpesviral Fc receptor fcr-1 down-regulates the NKG2D ligands MULT-1 and H60 J. Exp. Med., August 7, 2006; 203(8): 1843 - 1850. [Abstract] [Full Text] [PDF]

				B. Meresse, S. A. Curran, C. Ciszewski, G. Orbelyan, M. Setty, G. Bhagat, L. Lee, M. Tretiakova, C. Semrad, E. Kistner, et al. Reprogramming of CTLs into natural killer-like cells in celiac disease J. Exp. Med., May 15, 2006; 203(5): 1343 - 1355. [Abstract] [Full Text] [PDF]

				S. Gasser and D. H. Raulet The DNA damage response arouses the immune system. Cancer Res., April 15, 2006; 66(8): 3959 - 3962. [Abstract] [Full Text] [PDF]

				H. Zhou, Y. Luo, C. D. Kaplan, J. A. Kruger, S.-H. Lee, R. Xiang, and R. A. Reisfeld A DNA-based cancer vaccine enhances lymphocyte cross talk by engaging the NKG2D receptor Blood, April 15, 2006; 107(8): 3251 - 3257. [Abstract] [Full Text] [PDF]

				J. D. Bui, L. N. Carayannopoulos, L. L. Lanier, W. M. Yokoyama, and R. D. Schreiber IFN-Dependent Down-Regulation of the NKG2D Ligand H60 on Tumors J. Immunol., January 15, 2006; 176(2): 905 - 913. [Abstract] [Full Text] [PDF]

				S. Skov, M. T. Pedersen, L. Andresen, P. Thor Straten, A. Woetmann, and N. Odum Cancer Cells Become Susceptible to Natural Killer Cell Killing after Exposure to Histone Deacetylase Inhibitors Due to Glycogen Synthase Kinase-3-Dependent Expression of MHC Class I-Related Chain A and B Cancer Res., December 1, 2005; 65(23): 11136 - 11145. [Abstract] [Full Text] [PDF]

				M. J. Smyth, J. Swann, E. Cretney, N. Zerafa, W. M. Yokoyama, and Y. Hayakawa NKG2D function protects the host from tumor initiation J. Exp. Med., September 6, 2005; 202(5): 583 - 588. [Abstract] [Full Text] [PDF]

				M. A. Markiewicz, L. N. Carayannopoulos, O. V. Naidenko, K. Matsui, W. R. Burack, E. L. Wise, D. H. Fremont, P. M. Allen, W. M. Yokoyama, M. Colonna, et al. Costimulation through NKG2D Enhances Murine CD8+ CTL Function: Similarities and Differences between NKG2D and CD28 Costimulation J. Immunol., September 1, 2005; 175(5): 2825 - 2833. [Abstract] [Full Text] [PDF]

				A. K. Kriegeskorte, F. E. Gebhardt, S. Porcellini, M. Schiemann, C. Stemberger, T. J. Franz, K. M. Huster, L. N. Carayannopoulos, W. M. Yokoyama, M. Colonna, et al. NKG2D-independent suppression of T cell proliferation by H60 and MICA PNAS, August 16, 2005; 102(33): 11805 - 11810. [Abstract] [Full Text] [PDF]

				R. Takaki, Y. Hayakawa, A. Nelson, P. V. Sivakumar, S. Hughes, M. J. Smyth, and L. L. Lanier IL-21 Enhances Tumor Rejection through a NKG2D-Dependent Mechanism J. Immunol., August 15, 2005; 175(4): 2167 - 2173. [Abstract] [Full Text] [PDF]

				H. Zhou, Y. Luo, J.-f. Lo, C. D. Kaplan, M. Mizutani, N. Mizutani, J.-D. Lee, F. J. Primus, J. C. Becker, R. Xiang, et al. DNA-based vaccines activate innate and adaptive antitumor immunity by engaging the NKG2D receptor PNAS, August 2, 2005; 102(31): 10846 - 10851. [Abstract] [Full Text] [PDF]

				A. A. Dandekar, K. O'Malley, and S. Perlman Important Roles for Gamma Interferon and NKG2D in {gamma}{delta} T-Cell-Induced Demyelination in T-Cell Receptor {beta}-Deficient Mice Infected with a Coronavirus J. Virol., August 1, 2005; 79(15): 9388 - 9396. [Abstract] [Full Text] [PDF]

				K. Wiemann, H.-W. Mittrucker, U. Feger, S. A. Welte, W. M. Yokoyama, T. Spies, H.-G. Rammensee, and A. Steinle Systemic NKG2D Down-Regulation Impairs NK and CD8 T Cell Responses In Vivo J. Immunol., July 15, 2005; 175(2): 720 - 729. [Abstract] [Full Text] [PDF]

				C. Adam, S. King, T. Allgeier, H. Braumuller, C. Luking, J. Mysliwietz, A. Kriegeskorte, D. H. Busch, M. Rocken, and R. Mocikat DC-NK cell cross talk as a novel CD4+ T-cell-independent pathway for antitumor CTL induction Blood, July 1, 2005; 106(1): 338 - 344. [Abstract] [Full Text] [PDF]

				D. Garrity, M. E. Call, J. Feng, and K. W. Wucherpfennig The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure PNAS, May 24, 2005; 102(21): 7641 - 7646. [Abstract] [Full Text] [PDF]

				M. Hasan, A. Krmpotic, Z. Ruzsics, I. Bubic, T. Lenac, A. Halenius, A. Loewendorf, M. Messerle, H. Hengel, S. Jonjic, et al. Selective Down-Regulation of the NKG2D Ligand H60 by Mouse Cytomegalovirus m155 Glycoprotein J. Virol., March 1, 2005; 79(5): 2920 - 2930. [Abstract] [Full Text] [PDF]

				A. Krmpotic, M. Hasan, A. Loewendorf, T. Saulig, A. Halenius, T. Lenac, B. Polic, I. Bubic, A. Kriegeskorte, E. Pernjak-Pugel, et al. NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145 J. Exp. Med., January 18, 2005; 201(2): 211 - 220. [Abstract] [Full Text] [PDF]

				J. Regunathan, Y. Chen, D. Wang, and S. Malarkannan NKG2D receptor-mediated NK cell function is regulated by inhibitory Ly49 receptors Blood, January 1, 2005; 105(1): 233 - 240. [Abstract] [Full Text] [PDF]

				M. J. Smyth, J. Swann, J. M. Kelly, E. Cretney, W. M. Yokoyama, A. Diefenbach, T. J. Sayers, and Y. Hayakawa NKG2D Recognition and Perforin Effector Function Mediate Effective Cytokine Immunotherapy of Cancer J. Exp. Med., November 15, 2004; 200(10): 1325 - 1335. [Abstract] [Full Text] [PDF]

				M. B. Lodoen, G. Abenes, S. Umamoto, J. P. Houchins, F. Liu, and L. L. Lanier The Cytomegalovirus m155 Gene Product Subverts Natural Killer Cell Antiviral Protection by Disruption of H60-NKG2D Interactions J. Exp. Med., October 18, 2004; 200(8): 1075 - 1081. [Abstract] [Full Text] [PDF]

				D. B. Rosen, M. Araki, J. A. Hamerman, T. Chen, T. Yamamura, and L. L. Lanier A Structural Basis for the Association of DAP12 with Mouse, but Not Human, NKG2D J. Immunol., August 15, 2004; 173(4): 2470 - 2478. [Abstract] [Full Text] [PDF]

				L. Bacon, R. A. Eagle, M. Meyer, N. Easom, N. T. Young, and J. Trowsdale Two Human ULBP/RAET1 Molecules with Transmembrane Regions Are Ligands for NKG2D J. Immunol., July 15, 2004; 173(2): 1078 - 1084. [Abstract] [Full Text] [PDF]

				J. A. Hamerman, K. Ogasawara, and L. L. Lanier Cutting Edge: Toll-Like Receptor Signaling in Macrophages Induces Ligands for the NKG2D Receptor J. Immunol., February 15, 2004; 172(4): 2001 - 2005. [Abstract] [Full Text] [PDF]

				J. Koike, H. Wakao, Y. Ishizuka, T.-a. Sato, M. Hamaoki, K.-i. Seino, H. Koseki, T. Nakayama, and M. Taniguchi Bone Marrow Allograft Rejection Mediated by a Novel Murine NK Receptor, NKG2I J. Exp. Med., January 5, 2004; 199(1): 137 - 144. [Abstract] [Full Text] [PDF]

				V. Voigt, C. A. Forbes, J. N. Tonkin, M. A. Degli-Esposti, H. R. C. Smith, W. M. Yokoyama, and A. A. Scalzo Murine cytomegalovirus m157 mutation and variation leads to immune evasion of natural killer cells PNAS, November 11, 2003; 100(23): 13483 - 13488. [Abstract] [Full Text] [PDF]

				M. J. Miley, S. M. Truscott, Y. Y. L. Yu, S. Gilfillan, D. H. Fremont, T. H. Hansen, and L. Lybarger Biochemical Features of the MHC-Related Protein 1 Consistent with an Immunological Function J. Immunol., June 15, 2003; 170(12): 6090 - 6098. [Abstract] [Full Text] [PDF]

				C. Dunn, N. J. Chalupny, C. L. Sutherland, S. Dosch, P.V. Sivakumar, D. C. Johnson, and D. Cosman Human Cytomegalovirus Glycoprotein UL16 Causes Intracellular Sequestration of NKG2D Ligands, Protecting Against Natural Killer Cell Cytotoxicity J. Exp. Med., June 2, 2003; 197(11): 1427 - 1439. [Abstract] [Full Text] [PDF]

				M. Lodoen, K. Ogasawara, J. A. Hamerman, H. Arase, J. P. Houchins, E. S. Mocarski, and L. L. Lanier NKG2D-mediated Natural Killer Cell Protection Against Cytomegalovirus Is Impaired by Viral gp40 Modulation of Retinoic Acid Early Inducible 1 Gene Molecules J. Exp. Med., May 19, 2003; 197(10): 1245 - 1253. [Abstract] [Full Text] [PDF]

				B. A. Rabinovich, J. Li, J. Shannon, R. Hurren, J. Chalupny, D. Cosman, and R. G. Miller Activated, But Not Resting, T Cells Can Be Recognized and Killed by Syngeneic NK Cells J. Immunol., April 1, 2003; 170(7): 3572 - 3576. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carayannopoulos, L. N. Articles by Yokoyama, W. M. Search for Related Content PubMed PubMed Citation Articles by Carayannopoulos, L. N. Articles by Yokoyama, W. M.