Expression cloning and function of the rat NK activating and inhibitory receptors NKR-P1A and -P1B

Jennifer Li1,2, Brian A. Rabinovich1,2, Rose Hurren1,2, John Shannon1,2 and Richard G. Miller1,2

Departments of 1 Medical Biophysics and 2 Immunology, University of Toronto and the Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada

Correspondence to: R. G. Miller, Department of Medical Biophysics, Ontario Cancer Institute, Room 9-305, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada. E-mail: miller{at}oci.utoronto.ca
Transmitting editor: W. M. Yokoyama


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have characterized the rat NK receptors NKR-P1A and -P1B. A cDNA library was constructed from the rat NK cell line, RNK-16. Using the pMX retroviral cloning system, the library was expressed in the human NK cell line, YTSeco, and cells staining with the anti-rat mAb 10/78 identified, FACS sorted and cloned. Two genes, corresponding to rat NK receptors NKR-P1A and -P1B, were identified. YTSeco clones expressing either NKR-P1A or -P1B were functionally tested using 51Cr-release redirected lysis assays and calcium flux experiments. This demonstrated that NKR-P1A functions as an activation receptor, as previously shown, and that NKR-P1B functions as an inhibitory receptor, as predicted by the presence of an immunoreceptor tyrosine-based inhibition motif. Although annotated as NKR-P1A specific, we found that mAb 10/78 stained YTSeco clones expressing NKR-P1A or -P1B equally well, as did the mAb 3.2.3 used for the original cloning of rat NKR-P1A.

Keywords: calcium flux, cytotoxicity, lymphokine activated killer cell


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It is now generally accepted that NK cells express two opposing sets of cell-surface receptors. Upon target ligation, the summation of signals received through the activating and inhibitory forms determines whether NK activation is triggered (1,2). Characterization of the inhibitory receptors, including KIR (humans) and Ly49 (mice), led to the fundamental principle that NK rarely kill self (3,4). Among the myriad of activation receptors reported, rat NKR-P1 was one of the first molecules which could be used to reliably identify NK cells (5).

Rat NKR-P1 was identified through characterization of the mAb, 3.2.3, first shown to recognize a structure expressed at high density on rat NK cells. When cross-linked, the 3.2.3 ligand induced a calcium flux (6) and produced redirected lysis of FcR+ targets (7). COS expression cloning was used to identify rat NKR-P1 (now rat NKR-P1A) as the molecule recognized by 3.2.3. Following transfection of COS cells with cDNA from IL-2-activated rat NK cells, those cells expressing cDNA encoding the receptor recognized by 3.2.3 were enriched by panning. When the cDNA was isolated and sequenced, it was found to code for a 30-kDa type II transmembrane protein that was a member of the C-type lectin superfamily (8). Following the cloning of NKR-P1, Ryan et al. definitively showed its importance to the natural cytotoxicity of certain tumor cell lines by restoring the activity of an NKR-P1 mutant of the rat NK leukemia line, RNK-16, via transfection with NKR-P1 (9).

Recently, much of the more detailed characterization of NK cell receptors has been performed with mouse or human NK cells. Individual activating or inhibitory receptors can be assigned to either the C-type lectin or Ig superfamily (2), with specificity determined by the extracellular domains. However, it is thought that a receptor can be readily identified as activating or inhibitory by examination of its transmembrane portion and cytoplasmic tail. All inhibitory receptors contain, in their cytoplasmic tail, one or two characteristic immunoreceptor tyrosine-based inhibition motifs (ITIM) that can bind the phosphatase SHP-1 or -2 and thus initiate an inhibitory signaling cascade (2,10,11). The transmembrane portion of these receptors is uncharged and cell-surface expression is accomplished without the aid of adaptor or accessory molecules. In contrast, activating receptors, including rat NKR-P1, carry a charge in their transmembrane portion such that they can only be expressed on the cell surface with an accessory or adaptor molecule. This molecule usually contains a characteristic motif, the immunoreceptor tyrosine-based activation motif (ITAM), which couples the receptor to the activating signal cascade (1214).

Three molecules homologous to NKR-P1, generally known as NKR-P1A, -P1B and -P1C, have long been known in C57BL/6 mice (15). More recently, Plougastel et al. have reported the cloning of three new NKR-P1 family members: NKR-P1D, -P1E and -P1F (16). The B product carries an ITIM, can be expressed without an adaptor molecule and can function as an inhibitory receptor (17,18). The A and C products do not contain an ITIM, and can probably only be expressed in association with an ITAM-bearing adaptor molecule, such as FcR{gamma} in the case of NKR-P1C (19). The C product has been directly demonstrated to be an activation receptor (20). At present, there are no functional data available for either NKR-P1D or -P1F, although sequence analyses indicate they are probably inhibitory and activating respectively. NKR-P1E is a pseudogene (16). The original rat NKR-P1 gene, now designated A, appears the most similar to mouse NKR-P1C. Two additional transcripts belonging to this family have been identified in rat and both have been designated as B in GenBank. One, GenBank U56936 (Dissen et al., unpublished), has an ITIM, while the second, GenBank X97477 (21), does not. This second sequence is also labeled as NKR-P1D. We observe that it appears most similar to the newly described mouse NKR-P1F (16). Further, it is quite different from mouse NKR-P1B and -P1D which are nearly identical, and which both have an ITIM. We will refer to the two rat sequences as B (U56936) and B* (X97477) respectively as in a recent review by Lanier (2).

In this study we have used expression cloning to isolate rat NKR-P1A and -P1B cDNAs, and have directly demonstrated that they can function as activating and inhibitory receptors respectively.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies and flow cytometry
Anti-human 2B4 (C1.7) was purchased from Coulter (Fullerton, CA); biotinylated or phycoerythrin (PE)-labeled anti-NKR-P1 (10/78) and anti-TNP (mIgG1 isotype control) were purchased from PharMingen (San Diego, CA). Streptavidin–PE was also purchased from PharMingen. Anti-NKR-P1 (3.2.3) was purchased from Cedarlane (Hornby, Ontario, Canada) and biotinylated using EZ-Link Sulfo-NHS-Biotin from Pierce (Rockford, IL). 2.4G2 (rat anti-mouse Fc{gamma}RIII-{alpha}) was obtained from ATCC (Manassas, VA). Goat anti-mouse Ig was purchased from Jackson (West Grove, PA). Flow cytometric acquisition was completed using a Becton Dickinson FACSCalibur or FACScan and CellQuest software (Becton Dickinson, Mountain View, CA). Analyses were performed using FSC Express (De Novo Software, Thornhill, Ontario, Canada).

Construction of the cDNA library
Poly(A)+ RNA was isolated from the rat NK cell line RNK-16 (generous gift of Dr J.C. Ryan, UCSF, CA) (22) using the FastTrack 2.0 mRNA purification kit (Invitrogen, Carlsbad, CA). The mRNA was further purified by DNase I treatment followed by another selection on oligo(dT) beads. This material was used to make cDNA with the SuperScript plasmid system (Invitrogen). The cDNA library was then ligated into pMXs (generous gift of Dr H. Arase and Dr L. Lanier, UCSF, CA) (23), a variant of pMX (24). DH5{alpha}-FT competent cells (Invitrogen) were then transformed with the library.

Library screening
The cDNA library was transfected into the packaging cell line Plat-E (kind gift of Dr T. Kitamura, University of Tokyo, Japan) (25) using Lipofectamine Plus (Life Technologies, Rockville, MD). Supernatants were harvested 48 h later and used to infect the human NK cell line YTSeco (generous gift of Dr J. C. Zuniga-Pflucker), a variant of YTS into which mouse ecotropic receptor has been transduced (26). The infection was carried out in the presence of polybrene at 10 µg/ml (Sigma, St Louis, MO). Infected cells were stained with PE-labeled anti-NKR-P1 mAb 10/78 and the positive fraction sorted on a FACS Vantage (BD Biosciences, San Jose, CA). Following expansion, positive cells were single-cell cloned using a MoFlo (Dako Cytomation, Fort Collins, CO). The sequence of the integrated cDNA was amplified using PCR primers specific for pMXs as described (23).

Cytotoxicity assay
The ability of YTSeco clones, which had integrated either NKR-P1A or -P1B, to kill the FcR+ murine mastocytoma P815 in the presence of various mAb as indicated was assayed using a 51Cr-release assay as previously described (27). Briefly, YTSeco effectors were incubated with 0.5 or 1 µg of mAb in a 100-µl volume in 96-well V-bottom microtiter plates for 15 min at room temperature. The mAb used were anti-human 2B4 (C1.7), biotinylated anti-NKR-P1 (10/78) and anti-TNP (mIgG1 isotype control). 51Cr-labeled P815 (1000 cells/well) were added to these effectors in 100 µl aliquots in complete medium to give a final volume of 200 µl/well and the cultures incubated for 4 h at 37°C. Supernatants were sampled and counted. Specific lysis was calculated as (ES)/(TS) x 100 in which each value represents the mean ± SE of three replicates. E is the experimental mean of 51Cr released when the target was incubated with effectors, S the value when it was incubated in medium alone and T the total release when cells were incubated with 2% acetic acid.

Calcium flux assay
We used a protocol similar to that used by Lipp et al. (28) and Novak et al. (29). Briefly, in order to measure intracellular calcium mobilization, YTSeco clones expressing NKR-P1A or -P1B were loaded in 4 ìM Fluo-4 AM and 8 µM Fura Red AM (both from Molecular Probes, Eugene, OR) for 50 min at 30°C. Cells were washed to remove excess reagent. Then, 2 µg of anti-human 2B4 (C1.7) or biotinylated anti-NKR-P1 (10/78) mAb was used to induce receptor cross-linking. This staining was carried out for 10 min at room temperature. Cells were washed again and warmed to 37°C after beginning continuous FACS on a FACSCalibur (BD Bioscience). Then cross-linking goat anti-mouse Ig (24 µg/ml) was added. Efficient loading of the cells was verified by inducing a Ca2+ flux using ionomycin (Sigma). Data were analyzed using the FloJo flow cytometry system (Tree Star, San Carlos, CA) and exported into a Microsoft Excel spreadsheet. They are presented as Fluo-4 (FL1) intensity divided by Fura Red (FL3) intensity over time.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The rat RNK-16 cell line was used to make a cDNA library in the retroviral vector pMXs. The human NK line YTS, transduced with the mouse ecotropic receptor, YTSeco, was infected with this library and cells staining with the mAb 10/78-PE, known to recognize rat NKR-P1A, were sorted by FACS. It was verified beforehand that YTSeco did not stain with 10/78 (not shown). Also, both PCR and Western blotting demonstrated that YTSeco expressed the adaptor molecules DAP-10 and -12 (not shown). Figure 1(A) shows the fluorescence versus forward scatter profile after the initial infection. Cells staining positive with 10/78–PE were sorted, expanded in culture and reanalyzed, at which time ~42% of cells appeared to stain with 10/78 (Fig. 1B). These cells were cloned by single-cell sorting so that PCR amplification of the insertion using pMXs-specific primers could be carried out. The sequences obtained fell into two groups corresponding to either rat NKR-P1A (GenBank M62891) or NKR-P1B (GenBank U56936). In all, 22 clones corresponding to rat NKR-P1B and four clones corresponding to rat NKR-P1A were sequenced. No other sequences, in particular nothing corresponding to B* (GenBank X97477), were found. Figure 1(C) shows 10/78 staining of two YTSeco clones, one transduced with NKR-P1A and the other with NKR-P1B. Although there is some difference in 10/78 staining intensity in the two clones shown, these intensities did not vary systematically over the whole data set and we could not use staining intensity to predict whether a clone had integrated NKR-P1A or -P1B.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1. Expression cloning of NKR-P1A and -P1B identified by the mAb 10/78. (A) Flow cytometry profiles of YTSeco stained with mAb 10/78 following transduction with the RNK-16 retroviral library and (B) following regrowth after one round of sorting. The horizontal line indicates the threshold used for defining positive cells. (C) mAb 10/78 staining of typical examples of YTSeco-derived NKR-P1A and -P1B clones grown up after single-cell sorting.

 
The mouse mastocytoma tumor line, P815, is not lysed by YTSeco. Further, because P815 is FcR+, it is capable of cross-linking antibodies recognizing receptors on YTSeco in a redirected lysis assay. This allowed us to detect and distinguish between activating and inhibitory forms transduced into the parental cell line. YTSeco clones which had integrated either rat NKR-P1A or -P1B genes were added at various ratios to 51Cr-labeled P815 cells in the presence of anti-rat NKR-P1 (10/78), anti-rat 2B4 or both. Cross-linking of 2B4, an activation receptor expressed on YTSeco, served as the positive control. Addition of 10/78 resulted in lysis above background when the YTSeco-derived clone had integrated the rat NKR-P1A gene. We also observed augmentation of lysis above that seen with anti-2B4 alone, when anti-NKR-P1 was added simultaneously (Fig. 2A). When anti-NKR-P1 was added to YTSeco-derived clones which had integrated rat NKR-P1B, no lysis was observed (Fig. 2B). Importantly, when anti-NKR-P1 and anti-2B4 were added together to NKR-P1B clones, lysis observed from 2B4 cross-linking alone was abolished (Fig. 2B). In both cases, addition of anti-TNP, an isotype-matched control mAb, gave no killing. These data establish for the first time that rat NKR-P1A and -P1B function as activating and inhibitory receptors respectively.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2. NKR-P1A is an activation receptor, whereas NKR-P1B is an inhibitory receptor, as shown by a redirected lysis assay. YTSeco clones transduced with NKR-P1A (A) or -P1B (B) were tested for their ability to kill P815 following addition of mAb anti-2B4 (C1.7), anti-NKR-P1 (10/78) or both together, as well as isotype-matched control anti-TNP mAb. Effectors were incubated with 0.5 or 1 µg of mAb in a 100 µl for 15 min at room temperature and then tested for their ability to kill P815 in a 4-h 51Cr-release assay at the indicated E:T ratios.

 
We also performed intracellular calcium flux experiments using NKR-P1A+ or -P1B+ YTSeco clones to confirm the functional data obtained in the redirected lysis assay. As shown in Fig. 3(A), cross-linking of NKR-P1 or 2B4 on NKR-P1A+ clones, using 10/78 or C1.7 respectively, resulted in a calcium flux as measured by the ratio of Fluo-4 to Fura Red fluorescence over time. Co-engagement of NKR-P1A and 2B4 produced a calcium flux suggestive of a synergistic relationship between these two NK cell receptors. These data confirm that NKR-P1A functions as an activation receptor. By contrast, cross-linking of NKR-P1B+ clones with 10/78 resulted in no discernible calcium mobilization (Fig. 3B). Cross-linking of 2B4 resulted in a calcium flux and this was completely abrogated by co-engagement of NKR-P1B. Therefore, as anticipated, NKR-P1B functions as an inhibitory receptor.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3. Calcium mobilization determined by the ratio of Fluo-4 (FL1) to Fura Red (FL3) fluorescence over time. YTSeco clones transduced with rat NKR-P1A (A) or -P1B (B) were loaded with the fluorescence indicators Fluo-4 and Fura Red, and preincubated for 10 min at room temperature with either anti-NKR-P1 (10/78), anti-2B4 (C1.7) or both. The cells were then washed and analyzed continuously at 37°C by using a FACSCalibur. Goat anti-mouse Ig was added after the analysis began, indicated with an arrow, and was by itself not able to induce a calcium flux (data not shown).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have shown directly that the rat NK receptors NKR-P1A and -P1B function as activating and inhibitory receptors respectively. The genes for these receptors were isolated by expression cloning from a cDNA library derived from the rat NK line RNK-16. The library was expressed in the human NK line YTSeco and the receptors identified using the mAb 10/78. Clearly this mAb recognizes both A and B forms of NKR-P1. It would be useful to have a mAb that could distinguish between them. The mAb initially used to define rat NKR-P1A, 3.2.3 (8) can induce a calcium flux in RNK-16 and can mediate redirected lysis by RNK-16 (6,7). However, we found that this mAb is not NKR-P1A specific as it also produced equivalent staining of YTSeco clones that had integrated either NKR-P1A or -P1B (data not shown). Since we cloned NKR-P1B from an RNK-16 cDNA library, mRNA for NKR-P1B must be present and, because surface expression of NKR-P1B does not require an adaptor molecule, it is likely to be expressed. This implies that the level of surface expression of NKR-P1B relative to NKR-P1A is sufficiently low that the balance of signaling on the activation/inhibition teeter-totter (1) must favor the activation side. Studies using the mAb 10/78 or 3.2.3 should be interpreted in light of the fact that both can recognize activating and inhibitory NKR-P1 receptors.

Although the results described here are in good agreement with the fact that rat NKR-P1B contains an ITIM, the precise definition of an ITIM is something of a moving target. Previously, this was described as I/V/LxYxxL/V (10,30), a motif contained in both rat (VVYADL) and mouse (LVYADL) NKR-P1B receptors, which have been demonstrated to be inhibitory, and by the mouse NKR-P1D (LVYADL), which is believed to be inhibitory. However, it is not contained in any of the other NKR-P1 receptors. Recently, the ITIM has been redefined as I/V/L/SxYxxL/V with there being several well-documented examples of inhibitory receptors with serine two residues upstream of the tyrosine (3133). However, this definition cannot be complete in itself as mouse NKR-P1C, known to function as an activating receptor, carries a satisfactory ITIM (SIYLGL) according to this new broader definition.

Our present state of knowledge argues strongly for an expansion of the dogma that the presence of an ITIM confers inhibitory activity, while a charged residue in the transmembrane domain identifies an activation receptor. In humans, analysis of the KIR2DL4 receptor provides unique insights as this molecule contains both an ITIM (VTYAQL) and an arginine residue in its transmembrane domain. The receptor functions as an activation receptor as its stimulation results in IFN-{gamma} production from resting NK (34) and it can mediate lysis of P815 (35). However, the isolated cytoplasmic tail is capable of binding SHP-1 and -2 (35). Also, when the charged residue in the transmembrane domain is mutated, the molecule can function as an inhibitory receptor (35,36). Presumably, binding of an adaptor molecule, necessary to neutralize the charge in the transmembrane domain and allow expression, blocks the function of the ITIM by making it physically impossible for SHP-1 and -2 to bind.

Since the first discovery of NKR-P1, this molecule and its mouse homologues have played a critical role in the development of NK biology. To date, however, the ligands and physiological significance of both the activation and inhibitory forms of the NKR-P1 family of receptors remain unknown. This report provides the first formal functional demonstration of the inhibitory function of rat NKR-P1B. Because of the lack of reagents to distinguish between rat NKR-P1A and -P1B, the strain-specific expression pattern of these molecules is not known. Nevertheless, we have previously shown that mouse NKR-P1B is expressed and functional in SJL mice, the only known strain in which the function of this inhibitory receptor can be demonstrated (17,18). Interestingly, SJL was originally selected for its poor NK activity (37), which is widely accepted as the reason for many spontaneous tumors in this strain (37,38). It is tempting to speculate that the SJL phenotype is linked to dominant expression of the inhibitory NKR-P1B form instead of an activating form such as NKR-P1C (NK1.1), expressed by B6 mice that do not display the same susceptibility. When we examined NK cells from F344, the rat strain from which RNK-16 was derived (39), for ability to kill FcR+ cells upon cross-linking with mAb 10/78 (specific for both NKR-P1A and -P1B), lysis was observed. This suggests that if normal F344 NK cells express NKR-P1B, its negative signaling is overbalanced by positive signaling through NKR-P1A. We believe there is merit in investigating a connection between the relative expression of NKR-P1A and -P1B and susceptibility to disease associated with poor NK function including cancer (40) and autoimmunity (41).


    Acknowledgements
 
We thank Dr T. Kitamura for his generous gifts of Plat-E, pMX and pMX-GFP. We are grateful to Drs H. Vaziri, T. Kitamura and H. Arase for technical advice regarding library construction and expression cloning. We also thank C. Cantin for his help with cell sorting. This work was supported by a grant from CIHR (Canada) to R. G. M. We are grateful to Dr D. Burshtyn for her helpful comments on the current status of the ITIM motif.


    Abbreviations
 
ITAM—immunoreceptor tyrosine-based activation motif

ITIM—immunoreceptor tyrosine-based inhibition motif

PE—phycoerythrin


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Su, R. C., Kung, S. K., Gariepy, J., Barber, B. H. and Miller, R. G. 1998. NK cells can recognize different forms of class I MHC. J. Immunol. 161:755.[Abstract/Free Full Text]
  2. Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.[CrossRef][ISI][Medline]
  3. Kärre, K. 1991. MHC gene control of the natural killer system at the level of the target and the host. Semin. Cancer Biol. 2:295.[Medline]
  4. Kärre, K., Ljunggren, H. G., Piontek, G. and Kiessling, R. 1986. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319:675.[ISI][Medline]
  5. Chambers, W. H., Vujanovic, N. L., DeLeo, A. B., Olszowy, M. W., Herberman, R. B. and Hiserodt, J. C. 1989. Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells. J. Exp. Med. 169:1373.[Abstract]
  6. Ryan, J. C., Niemi, E. C., Goldfien, R. D., Hiserodt, J. C. and Seaman, W. E. 1991. NKR-P1, an activating molecule on rat natural killer cells, stimulates phosphoinositide turnover and a rise in intracellular calcium. J. Immunol. 147:3244.[Abstract/Free Full Text]
  7. Chambers, W. H., Vujanovic, N. L., DeLeo, A. B., Olszowy, M. W., Herberman, R. B. and Hiserodt, J. C. 1989. Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells. J. Exp. Med. 169:1373.[Abstract]
  8. Giorda, R., Rudert, W. A., Vavassori, C., Chambers, W. H., Hiserodt, J. C. and Trucco, M. 1990. NKR-P1, a signal transduction molecule on natural killer cells. Science 249:1298.[ISI][Medline]
  9. Ryan, J. C., Niemi, E. C., Nakamura, M. C. and Seaman, W. E. 1995. NKR-P1A is a target-specific receptor that activates natural killer cell cytotoxicity. J. Exp. Med. 181:1911.[Abstract]
  10. Burshtyn, D. N., Yang, W., Yi, T. and Long, E. O. 1997. A novel phosphotyrosine motif with a critical amino acid at position –2 for the SH2 domain-mediated activation of the tyrosine phosphatase SHP. J. Biol. Chem. 272:13066.[Abstract/Free Full Text]
  11. Long, E. O. and Wagtmann, N. 1997. Natural killer cell receptors. Curr. Opin. Immunol. 9:344.[CrossRef][ISI][Medline]
  12. Wu, J., Song, Y., Bakker, A. B., Bauer, S., Spies, T., Lanier, L. L. and Phillips, J. H. 1999. An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285:730.[Abstract/Free Full Text]
  13. Moretta, A., Bottino, C., Vitale, M., Pende, D., Cantoni, C., Mingari, M. C., Biassoni, R. and Moretta, L. 2001. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19:197.[CrossRef][ISI][Medline]
  14. Lanier, L. L. 2001. On guard—activating NK cell receptors. Nat. Immunol. 2:23.[CrossRef][ISI][Medline]
  15. Giorda, R. and Trucco, M. 1991. Mouse NKR-P1. A family of genes selectively coexpressed in adherent lymphokine-activated killer cells. J. Immunol. 147:1701.[Abstract/Free Full Text]
  16. Plougastel, B., Matsumoto, K., Dubbelde, C. and Yokoyama, W. M. 2001. Analysis of a 1-Mb BAC contig overlapping the mouse Nkrp1 cluster of genes: cloning of three new Nkrp1 members, Nkrp1d, Nkrp1e, and Nkrp1f. Immunogenetics 53:592.[CrossRef][ISI][Medline]
  17. Kung, S. K., Su, R. C., Shannon, J. and Miller, R. G. 1999. The NKR-P1B gene product is an inhibitory receptor on SJL/J NK cells. J. Immunol. 162:5876.[Abstract/Free Full Text]
  18. Carlyle, J. R., Martin, A., Mehra, A., Attisano, L., Tsui, F. W. and Zuniga-Pflucker, J. C. 1999. Mouse NKR-P1B, a novel NK1.1 antigen with inhibitory function. J. Immunol. 162:5917.[Abstract/Free Full Text]
  19. Arase, N., Arase, H., Park, S. Y., Ohno, H., Ra, C. and Saito, T. 1997. Association with FcRgamma is essential for activation signal through NKR-P1 (CD161) in Natural Killer (NK) Cells and NK1.1+ T cells. J. Exp. Med. 186:1957.[Abstract/Free Full Text]
  20. Karlhofer, F. M. and Yokoyama, W. M. 1991. Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL-2-activated NK cells possess additional specific stimulation pathways. J. Immunol. 146:3662.[Abstract/Free Full Text]
  21. Appasamy, P. M., Kenniston, T. W., Brissette-Storkus, C. S. and Chambers, W. H. 1996. NKR-P1dim/TCR alpha beta+ T cells and natural killer cells share expression of NKR-P1A and NKR-P1D. Nat. Immun. 15:259.[ISI][Medline]
  22. Reynolds, C. W., Bere, E. W., Jr and Ward, J. M. 1984. Natural killer activity in the rat. III. Characterization of transplantable large granular lymphocyte (LGL) leukemias in the F344 rat. J. Immunol. 132:534.[Abstract/Free Full Text]
  23. Arase, H., Saito, T., Phillips, J. H. and Lanier, L. L. 2001. Cutting edge: the mouse NK cell-associated antigen recognized by DX5 monoclonal antibody is CD49b (alpha(2) integrin, very late antigen-2). J. Immunol. 167:1141.[Abstract/Free Full Text]
  24. Onishi, M., Kinoshita, S., Morikawa, Y., Shibuya, A., Phillips, J., Lanier, L. L., Gorman, D. M., Nolan, G. P., Miyajima, A. and Kitamura, T. 1996. Applications of retrovirus-mediated expression cloning. Exp. Hematol. 24:324.[ISI][Medline]
  25. Morita, S., Kojima, T. and Kitamura, T. 2000. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7:1063.[CrossRef][ISI][Medline]
  26. Cohen, G. B., Gandhi, R. T., Davis, D. M., Mandelboim, O., Chen, B. K., Strominger, J. L. and Baltimore, D. 1999. The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10:661.[ISI][Medline]
  27. Rabinovich, B. A., Shannon, J., Su, R. C. and Miller, R. G. 2000. Stress renders T cell blasts sensitive to killing by activated syngeneic NK cells. J. Immunol. 165:2390.[Abstract/Free Full Text]
  28. Lipp, P. and Niggli, E. 1993. Ratiometric confocal Ca2+-measurements with visible wavelength indicators in isolated cardiac myocytes. Cell Calcium 14:359.[ISI][Medline]
  29. Novak, E. J. and Rabinovitch, P. S. 1994. Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence ratios. Cytometry 17:135.[ISI][Medline]
  30. Burshtyn, D. and Long, E. 1997. Regulation through inhibitory receptors: lessons from natural killer cells. Trends Cell Biol. 7:473.[CrossRef][ISI]
  31. Blery, M., Kubagawa, H., Chen, C.-C., Vely, F., Cooper, M. D. and Vivier, E. 1998. The paired Ig-like receptor PIR-B is an inhibitory receptor that recruits the protein-tyrosine phosphatase SHP. Proc. Natl Acad. Sci. USA 95:2446.[Abstract/Free Full Text]
  32. Vely, F. and Vivier, E. 1997. Commentary: conservation of structural features reveals the existence of a large family of inhibitory cell surface receptors and noninhibitory/activatory counterparts. J. Immunol. 159:2075.[Abstract]
  33. Ravetch, J. V. and Lanier, L. L. 2000. Immune inhibitory receptors. Science 290:84.[Abstract/Free Full Text]
  34. Rajagopalan, S., Fu, J. and Long, E. O. 2001. Cutting edge: induction of IFN-gamma production but not cytotoxicity by the Killer Cell Ig-Like Receptor KIR2DL4 (CD158d) in resting NK cells. J. Immunol. 167:1877.[Abstract/Free Full Text]
  35. Faure, M. and Long, E. O. 2002. KIR2DL4 (CD158d), an NK cell-activating receptor with inhibitory potential. J. Immunol. 168:6208.[Abstract/Free Full Text]
  36. Yusa, S.-I., Catina, T. L. and Campbell, K. S. 2002. SHP-1- and phosphotyrosine-independent inhibitory signaling by a killer cell Ig-like receptor cytoplasmic domain in human NK cells. J. Immunol. 168:5047.[Abstract/Free Full Text]
  37. Kaminsky, S. G., Nakamura, I. and Cudkowicz, G. 1983. Selective defect of natural killer and killer cell activity against lymphomas in SJL mice: low responsiveness to interferon inducers. J. Immunol. 130:1980.[Abstract/Free Full Text]
  38. Lin, T. Z. and Ponzio, N. M. 1991. Syngeneic B lymphoma cells provide a unique stimulus to natural killer (NK) cells in genetically low-NK SJL/J mice. J. Leukoc. Biol. 49:48.[Abstract]
  39. Reynolds, C. W., Bere, E. W., Jr and Ward, J. M. 1984. Natural killer activity in the rat. III. Characterization of transplantable large granular lymphocyte (LGL) leukemias in the F344 rat. J. Immunol. 132:534.[Abstract/Free Full Text]
  40. Miller, J. S. 2002. Biology of natural killer cells in cancer and infection. Cancer Invest. 20:405.[CrossRef][ISI][Medline]
  41. Shi, F., Ljunggren, H. G. and Sarvetnick, N. 2001. Innate immunity and autoimmunity: from self-protection to self-destruction. Trends Immunol. 22:97.[CrossRef][ISI][Medline]