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The Journal of Immunology, 2001, 166: 1601-1610.
Copyright © 2001 by The American Association of Immunologists

Functional Analysis of the Molecular Factors Controlling Qa1-Mediated Protection of Target Cells from NK Lysis1

Frances Gays*, Karen P. Fraser*, Jennifer A. Toomey*, Austin G. Diamond*, Margaret M. Millrain{dagger}, P. Julian Dyson{dagger} and Colin G. Brooks2,*

* Department of Microbiology and Immunology, The Medical School, Newcastle, United Kingdom; and {dagger} The Transplantation Biology Unit, Medical Research Council Clinical Sciences Centre, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD94/NKG2 receptors on mouse NK cells recognize the nonclassical class I molecule Qa1 and can deliver inhibitory signals that prevent NK cells from lysing Qa1-expressing cells. However, the exact circumstances under which Qa1 protects cells from NK lysis and, in particular, the role of the dominant Qa1-associated peptide, Qdm, are unclear. In this study, we examined in detail the lysis of Qa1-expressing cells by fetal NK cells that express CD94/NKG2 receptors for Qa1 but that lack receptors for classical class I molecules. Whereas mouse L cells and human C1R cells transfected with Qa1 were resistant to lysis by these effectors, Qa1-transfected TAP-deficient human T2 cells showed no resistance despite expressing high levels of surface Qa1. However, these cells could be efficiently protected by exposure to low concentrations of Qdm peptide or certain Qdm-related peptides. By contrast, even prolonged exposure of TAP-deficient RMA/S cells to high doses of Qdm peptide failed to induce levels of surface Qa1 detectable with a Qa1-specific mAb or to protect them from NK lysis, although such treatment induced sensitivity to lysis by Qa1-specific CTL. Collectively, these findings indicate that high surface expression of Qa1 is necessary but not sufficient for protection, and that effective protection requires the expression of sufficient levels of suitable Qa1-peptide complexes to overcome activatory signals. Results obtained with a series of substituted Qdm peptides suggest that residues at positions 3, 4, 5, and 8 of the Qdm sequence, AMAPRTLLL, are important for recognition of Qa1-Qdm complexes by inhibitory CD94/NKG2 receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class I molecules have at least two important functions. The first is to present peptides derived from intracellular parasites to CD8+ T cells. The second, more recently discovered, is to regulate the activity of NK cells (1). This is achieved via the expression on NK cells of receptors for class I molecules, the largest and most divergent families of which are the KIR family in humans and the Ly49 family in mice (2). In addition, both species express the CD94 family of heterodimeric class I receptors that are composed of an invariant CD94 chain linked by a disulfide bond to one of a small set of NKG2 molecules (3).

CD94/NKG2 receptors recognize the nonclassical class I molecule HLA-E in humans (4, 5, 6), and its homolog in mice, Qa1 (7, 8, 9). Remarkably, under normal circumstances a large proportion of the surface expression of each of these molecules is controlled by the availability of a single peptide derived from the leader sequence of certain classical class I molecules. This was first revealed by the pioneering work of Forman and colleagues (10) that demonstrated the existence of different classes of Qa1b-specific alloreactive clones. Type 1 and type 2 clones were both TAP dependent, implying that they recognized a form of Qa1 whose expression was controlled by cytosol-derived peptides. However, whereas type 2 clones would recognize Qa1b in all strains of mice, type 1 clones failed to recognize Qa1b in H-2k strains. Elegant biochemical and genetic studies revealed that the strain specificity of type 1 clones was due to their inability to recognize Qa1 molecules containing peptides derived from the leader sequences of certain classical class I molecules (11). In strains other than H-2k, the class I leader sequence peptide that binds to Qa1 has the sequence AMAPRTLLL and is known as the Qdm peptide, whereas in H-2k strains it has the sequence AMVPRTLLL and is designated Qdm-k. Peptide elution experiments demonstrated directly that Qdm and Qdm-k peptides not only associate with Qa1 molecules, but also are extraordinarily abundant, perhaps accounting for the majority of all Qa1-associated peptide (12, 13).

Subsequent studies demonstrated that the stable assembly and surface expression of HLA-E is similarly dependent on the presence within the cell of peptides, homologous to Qdm, derived from the leader sequences of certain HLA-A, B, and C molecules (14). By using a soluble tetrameric form of HLA-E associated with such peptides, Braud et al. (4) were able to demonstrate that HLA-E bound to CD94/NKG2 receptors on NK cells. Together with the finding that NK cells bearing inhibitory CD94/NKG2 receptors failed to lyse target cells that expressed both an appropriate "donor" class 1 molecule and a functional TAP complex (4, 5, 6), this led to the view that an important function of CD94+ NK cells is to detect and eliminate cells that lack proper expression of either class I molecules or TAP due to mutation or interference by parasites seeking to avoid recognition by cytotoxic T cells.

In adult mice, ~50% of NK cells express CD94/NKG2 receptors that bind soluble Qa1-Qdm complexes (7, 8, 9). Studying the interaction of these cells with target cells bearing Qa1 molecules is complicated by the fact that Qa1 receptor-expressing (Qa1R+) adult NK cells also express a variable selection of Ly49 receptors that can deliver inhibitory (or activatory) signals after interaction with classical class I molecules (7, 8, 15). To avoid this problem we have taken advantage of the fact that fetal NK cells are deficient in the expression of Ly49 receptors (16), lack detectable receptors for classical class I molecules (17), yet express CD94/NKG2 receptors for Qa1 (17, 18). These latter receptors are acquired in a stochastic manner during the development of fetal NK cells in vitro (17), allowing the selection of clones or lines that are composed predominantly of Qa1R- or Qa1R+ cells. In this study, we have used such selected fetal NK lines to examine in detail the circumstances under which target cells can be protected from lysis by Qa1R+ NK cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture media and reagents

Except where stated otherwise, cells were cultured in DMEM (52100-039; Life Technologies, Paisley, U.K.) made up in highly purified water and supplemented with 2x nonessential amino acids, 5 x 10-5 M 2-ME, and 10% FBS (F-7524; Sigma, Poole, U.K.) in a 10% CO2 atmosphere at 37°C. Serum-free AIMV medium (12030-029) was purchased from Life Technologies, and human rIL2 was obtained from Perkin-Elmer/Cetus (Emeryville, CA). Peptides were purchased from Genosys (The Woodlands, TX) or from the University of Newcastle (Newcastle, U.K.) Molecular Biology Unit. All the peptides used were purified by HPLC, were >95% pure, were confirmed to have the correct composition by mass spectrometry, and were soluble in aqueous medium at 30 times the highest concentration used in the assays.

Effector cells

Fetal NK cell lines and clones, generated from immature cells present in the thymuses of day 14 fetal C57BL/6 mice as described in detail elsewhere (19, 20), and adult NK cells purified as described in detail elsewhere (21), were cultured in medium containing 104 U/ml IL-2. The stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro results in lines and clones of NK cells that contain different proportions of Qa1R+ cells, ranging from 0 to 100% (17). In this study we used as Qa1R- lines two clones, I7 and 1608b, which contained no detectable Qa1R+ cells and, as Qa1R+ lines, the 1198 line and the J1.1 clone that contained >95% Qa1R+ cells. The human NK line NK92 (22) was provided by Dr. H.-G. Klingemann (St. Luke’s Medical Center, Chicago, IL). Qa1-specific CTL clones were generated by stimulating B6-Tlaa (H-2b, Qa-1a) spleen cells with irradiated (15 Gray) B10 (H-2b, Qa-1b) spleen cells, followed by cloning by limiting dilution. Clones were maintained by restimulating at biweekly intervals with C57BL/6 irradiated spleen cells in medium containing 100 U/ml of IL-2. Viable cells were purified by centrifugation on Lymphoprep 3 days after stimulation, cultured for another 4 days in medium containing 100 U/ml IL-2, then refed twice per week with medium containing 5% FBS and 5 U/ml of IL-2. They were used in cytotoxicity assays at between 3 and 7 days after stimulation.

Target cells

Two independent lines of untransfected TAP-deficient T2 cells (23) were provided by Dr. M. Soloski (Johns Hopkins University, Baltimore, MD) and Dr. V. Engelhard (University of Virginia, Charlottesville, VA). Qa1-transfected T2 cells (T2Q), Qa1-transfected C1R cells (C1RQ), and C1R cells transfected with neo alone were also provided by Dr. M. Soloski. Ld-transfected T2 cells, Dd- and Ld-transfected C1R cells, and untransfected C1R cells (24) were provided by Dr. V. Engelhard, all with the permission of Dr. P. Cresswell (Yale University, New Haven, CT). T2 cell lines were grown in DMEM supplemented with HEPES buffer (D6171; Sigma, St. Louis, MO) and 10% FBS. The derivation of control-transfected (F12-) and Qa1-expressing (F12+) transfected L cells (referred to here as L and LQ) has been described previously (13, 17). The derivation and characterization of the RMA and RMA/S cells have been described elsewhere (25, 26).

Cytotoxicity assays

These were performed in a standard manner by incubating serial dilutions of effector cells with 5000 51Cr-labeled target cells for 4 h in V-bottom microtest plates in medium containing 5% FBS. Where appropriate, target cells were preincubated or mixed with peptides before assay as described in detail in Results and the figure legends. Lytic activity was calculated as lytic units per 103 effector cells from the slopes of regression lines fitted to the linear portions of the dose-response curves as described elsewhere (27). All lines and clones used in these studies displayed high levels of cytotoxicity. A lytic activity value of 10 corresponds to 50% lysis with 5000 effectors, i.e., at an E:T ratio of 1:1.

Immunofluorescence and flow cytometry

Aliquots of 1–3 x 105 cells were stained with Abs using a standard indirect immunofluorescence protocol or with soluble tetrameric forms of appropriate MHC molecules. The 6F10 and 4C2 anti-Qa1 mAbs (28) were provided by Dr. M. Soloski. Both of these mAbs gave identical patterns of staining, and the results described are those obtained with the 4C2 mAb. Soluble tetramers of Qa1b (refolded with the Qdm peptide, AMAPRTLLL) and of HLA-E (refolded with the HLA-G-derived peptide VMAPRTLFL) were provided by Dr. M. Salcedo (Pasteur Institute, Paris, France) and Dr. V. Braud (University of Oxford, Oxford, U.K.), respectively. Staining was analyzed on a FACScan (BD Becton Dickinson, San Jose, CA), using forward and side scatter to gate on single viable cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Qa1-transfected TAP-deficient T2 cells express high levels of Qa1 at the surface but are not protected from lysis by Qa1R+ NK cells

As reported previously (17), Qa1R+ fetal NK cells, such as clone J1.1, lyse targets such as YAC-1 and L cells efficiently, but display much lower levels of lysis against L cells transfected with a Qa1b/Db construct (LQ cells) (Fig. 1GoA). They also lyse {beta}2-microglobulin ({beta}2m)3-deficient blast cells, although much less efficiently than YAC or L cells. The lytic activity of Qa1R+ fetal NK cells and their pattern of target cell killing is similar to that of purified IL-2-activated polyclonal adult NK cells with the exception that the latter effectors discriminate less well between L cells and LQ cells due to their lower content of Qa1R+ cells (~50%).



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  		FIGURE 1. Lysis of blast cells and Qa1-transfected tumor cells by NK and CTL effectors. A, The Qa1R+ NK cell clone J1.1 and highly purified adult splenic NK cells that had been cultured for 10 days in IL-2 were titrated on YAC tumor cells, control transfected L cells, L cells transfected with a Qa1b/Db construct (LQ), and blast cells prepared by culturing CD8-depleted spleen cells from B6 and {beta}2m-/- mice with Con A as described elsewhere (17 ). B, Untransfected T2 cells from two independent sources (T2-VE and T2-MS), T2 cells transfected with Ld (T2-Ld), T2 cells transfected with Qa1b (T2Q), control-transfected L cells, and L cells transfected with Qa1b/Db (LQ) were tested for susceptibility to lysis by the Qa1R+ NK clone J1.1, the Qa1R- NK clone I7, the type 1 Qa1b-specific CTL clone a2d, and the type 2 Qa1b-specific CTL clone d12i. The data show percent cytotoxicity as a function of the number of effector cells per well and are representative of three experiments.

 
Whereas L cells transfected with Qa1 are highly resistant to lysis by Qa1R+ NK cells, human TAP-deficient T2 cells transfected with Qa1 (T2Q cells) are not. Fig. 1GoB shows a typical experiment where Qa1R+ fetal NK cells, Qa1R- fetal NK cells, and Qa1-specific CTL clones were tested in paralleled on L cells and T2 transfectants. To illustrate the lytic unit method of quantifying target cell killing that is used elsewhere in this article, the results of this experiment are also shown as lytic activity values in Fig. 2GoA. Similar results were obtained with three other Qa1R+ NK clones (data not shown). That T2Q cells express Qa1 was shown by staining with the Qa1-specific mAb 4C2 (Fig. 3Go). Indeed, the level of staining of T2Q cells was higher than that found on a variety of normal (including blast) and malignant (e.g., RMA) mouse cells and was also higher than on LQ cells. Expression of Qa1 by T2Q cells was confirmed by their efficient lysis by type 1 Qa1-specific CTL clones, such as a2d (Figs. 1GoB and 2). However, they were not lysed by the type 2 Qa1-specific clone d12i. Thus, despite expressing high levels of Qa1 molecules, at least some of which are functional as demonstrated by their recognition by type 1 CTL clones, T2Q cells are not protected from lysis by Qa1R+ mouse NK cells.



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  		FIGURE 2. Lysis of TAP-deficient and TAP-sufficient human and mouse target cells by mouse NK and CTL clones. A, The data shown in Fig. 1GoB converted to lytic activity values as described in Materials and Methods. B, Data from one (of four) experiments in which untransfected C1R cells or C1R cells transfected with Dd, Ld, neo vector alone, or Qa1b (C1RQ and the recloned line C1RQ6) were used as targets. The effector cells were the same as in Fig. 1Go and 2GoA except the type 1 CTL clone used was Bh3d. C, T2Q, C1RQ, and RMA/S cells were incubated overnight at 26°C in AIMV medium with or without 100 µM Qdm peptide. They were then harvested, washed, and tested for sensitivity to lysis by the same effector cells as in Figs. 1Go and 2GoA. The data are representative of five experiments.

 


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  		FIGURE 3. Staining of various cell lines with the Qa1-specific mAb 4C2 with or without incubation with peptide. C1RQ, T2Q, RMA/S, and RMA cells were incubated overnight at 26°C in AIMV medium alone, or AIMV medium containing 100 µM of the Db-binding peptide WMHHNMDLL or Qdm peptide. Cells were then washed and stained with medium, 4C2 anti-Qa1, or mAb 28-11-5S anti-Db. Freshly harvested L, LQ, and Con A blasts cultured for 5 days in IL-2 were stained in parallel. Similar results were obtained in three additional experiments.

 
There are at least five possible explanations for these findings: 1) mouse NK cells do not interact properly with human target cells; 2) the activatory signals delivered by T2 cells to mouse NK cells are so strong that they cannot be overcome by inhibitory signals; 3) the Qa1 molecules on T2 cells, perhaps because of their association with human {beta}2m, cannot be recognized by Qa1 receptors on mouse NK cells; 4) the defect is caused by the TAP deficiency of T2 cells; and 5) human cells lack peptides that can functionally associate with Qa1. The first three possibilities are excluded by the previous finding (7, 17), documented in more detail below, that T2Q cells can be protected from NK lysis by exposure to Qa1-binding peptides. A prediction of the fourth hypothesis would be that transfection of TAP-sufficient human cells with Qa1 would protect them from lysis by mouse NK cells. Therefore, we examined whether human C1R cells that are highly susceptible to lysis by mouse NK cells are protected by transfection with Qa1. Fig. 2GoB shows that, compared with untransfected C1R cells or C1R cells transfected with other class I molecules, both the parent C1RQ line and a recloned subline derived from it, C1RQ6, are partially resistant to lysis by the Qa1R+ clone J1.1, but not by the Qa1R- clone I7. C1RQ cells are also killed well by type 1 CTL clones and, in contrast to T2Q cells, to a moderate extent by the type 2 CTL clone d12i (Fig. 2GoB). However, C1RQ cells do not show higher staining with the Qa1-specific mAb than T2Q cells (Fig. 3Go). The implication of these findings is that C1R expresses forms of Qa1 on its surface that are lacking on T2Q cells. The most likely candidates would be forms that contain particular peptides.

Qa1-expressing human cells, but not RMA/S cells, can be protected from mouse NK cells by the Qdm peptide

When T2Q cells were incubated overnight at 26° with 100 µM Qdm peptide in serum-free medium, then washed free of exogenous peptide, they acquired a high level of resistance to lysis by Qa1R+ but not by Qa1R- NK cells (Fig. 2GoC), confirming and extending previous findings. When C1RQ cells that are already partially resistant to Qa1R+ NK cells were incubated overnight with Qdm peptide, they became almost completely resistant to Qa1R+ NK cells. However, when RMA/S cells were pretreated with Qdm peptide in the same manner, they failed to acquire any detectable resistance. This was not due to a failure of RMA/S cells to express Qa1 or an inability of the Qa1 molecules on RMA/S cells to bind Qdm peptide because when RMA/S cells were incubated with Qdm peptide, they acquired a level of sensitivity to lysis by Qa1-specific CTL clones that was similar to that of wild-type RMA cells (Fig. 2GoC). T2Q and C1RQ cells also showed increased sensitivity to lysis by the type 2 CTL clone d12i after incubation with Qdm peptide, but no change in their innate sensitivity to lysis by type 1 CTL clones. Despite the significant changes in the sensitivity of T2Q, C1RQ, and RMA/S cells to lysis by NK cells and/or Qa1-specific CTL, incubation with Qdm peptide led to no detectable increase in the staining of these cells by the Qa1-specific mAb 4C2 (Fig. 3Go). Indeed, neither RMA nor RMA/S cells, with or without incubation with Qdm peptide, showed detectable staining for Qa1. The same was true of all mouse tumor cell lines examined (data not shown), and normal Con A blasts also were stained only weakly (Fig. 3Go). Curiously, incubation of T2Q and C1RQ cells with Qdm peptide led to a significant and highly reproducible decrease in staining.

Conditions required for peptide-mediated protection of T2Q cells

To facilitate analysis of the involvement of different residues of the Qdm peptide in the protection of Qa1-expressing target cells from Qa1R+ NK cells and to address some controversial issues concerning the loading of class I molecules on TAP-deficient human cells using exogenous peptides, we examined in some detail the conditions required for protection of T2Q cells with Qdm. Overnight incubation of T2Q cells with peptide at 26°C was not necessary for protection from lysis by Qa1R+ NK cells because it could be achieved equally well by incubation at 37°C, provided the incubation with peptide was performed in serum-free medium (Fig. 4GoA). In serum-containing medium, Qdm peptide was rapidly inactivated (data not shown), perhaps due to the action of peptidase enzymes present in serum, although sufficient Qdm peptide survived to sensitize T2Q cells to lysis by the CTL clone d12i (Fig. 4GoA). Protection could also be achieved by adding Qdm peptide to target cells just before dispensing them into cytotoxicity assays (Fig. 4GoB). Under these conditions, equally good protection could be achieved with T2Q cells previously cultured in either serum-free or serum-containing medium and at either 26°C or 37°C. Surprisingly, lower doses of peptide were required for protection in the "instant loading" system than in the overnight loading system (Fig. 4GoC). In both systems, sensitization of cells to lysis by Qa1-specific CTL could be achieved with lower doses of peptide than were required for protection. Experiments in which T2Q cells were pulsed with Qdm peptide at room temperature (~22°C) for various lengths of time, followed by washing to remove unbound peptide, revealed that significant protection against NK cell lysis and maximal sensitization to lysis by Qa1-specific CTL was achieved within 15 min of exposure of T2Q cells to Qdm peptide (Fig. 4GoD).



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  		FIGURE 4. Parameters of protection of T2Q by Qdm peptide. A, Protection by preloading. T2Q cells were incubated overnight at 26°C or 37°C in AIMV or serum-containing medium (D10F) containing 51Cr with or without 100 µM Qdm peptide. They were then washed and added to wells containing the Qa1R+ clone J1.1, the Qa1R- clone 1608b, and the type 2 Qa1-specific CTL clone d12i. B, Protection by adding peptide into the assay. T2Q cells were labeled with 51Cr, washed, and resuspended in D5F with or without 100 µM Qdm peptide. After 10 min of incubation at room temperature (~22°C), they were added to wells containing the same effectors as in A. C, Titration of Qdm peptide in preloading and inclusion assays. T2Q cells were incubated overnight at 26°C in AIMV with 51Cr and different concentrations of Qdm peptide or were prelabeled overnight with 51Cr and mixed with different concentrations of Qdm peptide just before adding to the assay. D, Kinetics of acquisition of the protected and sensitized states. T2Q cells were labeled with 51Cr for 1 h at 37°C, washed, resuspended in AIMV, divided into aliquots, and incubated at room temperature. At various times during incubation, Qdm peptide was added to give 100 µM. At the end of the incubation period, cells were washed twice to remove unbound peptide and added to wells containing the Qa1R+ NK line 1198 and the Qa1-specific CTL clone d12i. All experiments shown are representative of at least three similar experiments. Note that the sensitivity of cells to lysis is reported as "lytic activity" values derived from linearized titration data as described in Materials and Methods.

 
To test the stability of the protected and sensitized states, T2Q cells were loaded overnight at 26°C with various peptides, washed to remove exogenous peptide, then incubated for different lengths of time at 37°C before adding to cytotoxicity assays. The protected and sensitized states induced by Qdm peptide were stable for at least 4 h at 37°C (Fig. 5GoA). The protected and sensitized states induced by a Qdm-like peptide derived from the leader sequence of HLA-Cw4 were also stable (Fig. 5GoB). By contrast, both the protected and sensitized states induced by the analog peptide P2T, bearing a M to T substitution at position 2 of the Qdm sequence, were lost within 1 h of incubation at 37°C (Fig. 5GoB). However, P2T gave strong protection and sensitization when included in cytotoxicity assays (Fig. 5GoC).



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  		FIGURE 5. Stability of the peptide-induced protected state. A, T2Q cells were incubated overnight at 26°C in AIMV containing 51Cr with or without 100 µM Qdm peptide. They were then washed to remove unbound peptide and incubated at 37°C for various times before adding to wells containing the Qa1R+ NK line 1198 and the Qa1-specific CTL clone d12i. B, Similar experiment but using the P2T and HLA-Cw4 peptides. C, T2Q cells were incubated overnight at 26°C in AIMV medium alone or AIMV containing 100 µM Qdm, P2T, or HLA-Cw4 peptides. They were then washed, labeled with 51Cr for 1 h at 37°C, washed again, and added to assays in the presence or absence of 100 µM of the corresponding peptide. Each experiment was performed on three occasions with similar results. Note that the sensitivity of cells to lysis is reported as "lytic activity" values derived from linearized titration data as described in Materials and Methods.

 
Quantitative analysis of the protective capacity of Qdm-related peptides

In the light of these findings, we investigated the ability of a series of Qdm-related peptides to protect and sensitize T2Q cells in both the peptide inclusion assay and a preloading assay in which T2Q cells were incubated overnight with peptide at 26°C, washed, then incubated for 1 h at 37°C before adding to assays. The simplicity of the peptide inclusion assay permitted detailed quantitative analysis of the protective and sensitization capacity of different peptides. Two typical experiments are shown in Fig. 6Go, A and B. Because the preloading system was not so amenable to quantitation, a single dose of peptide (100 µM) was tested in these assays, as illustrated in Fig. 5GoC. A summary of all the results obtained in both systems is shown in Table IGo.



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  		FIGURE 6. Quantitative analysis of the ability of peptides to protect and sensitize target cells in the peptide inclusion assay. Typical experiments in which various peptides were titrated for their ability to protect and sensitize T2Q and RMA/S cells to lysis by the Qa1R+ NK cell line 1198 and Qa1-specific CTL clones. A, The ability of the peptide P1S to protect T2Q cells from lysis by NKL 1198 and to sensitize T2Q and RMA/S cells to lysis by the Qa1-specific CTL clones a2d and d12i. B, Comparison of the ability of Qdm and Qdm-k peptides to protect cells from lysis by NKL 1198 and sensitize them to lysis by d12i. In both experiments, killing by NKL 1198 is plotted as a percentage of that found in the absence of peptide, and killing by CTL as a percentage of that found with Qdm peptide at 10 µM.

 

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  		Table I. Ability of Qdm-related peptides to sensitize target cells to lysis by CTL and protect them from lysis by NKC

 
The quantitative data obtained in the peptide inclusion system show that none of the natural or synthetic Qdm-related peptides tested possessed a better sensitization or protective capacity than Qdm. Most of them possessed a significantly reduced sensitization and/or protective capacity but none were devoid of both activities, demonstrating that they must all bind to Qa1. By contrast, two Qdm-unrelated heat shock protein-derived peptides recognized by Qa1-restricted Salmonella-induced T cells (29) failed to give detectable sensitization to Qa1-specific CTL or protection from NK cells. These latter peptides also failed to inhibit sensitization when admixed in a 100,000-fold excess with 1 nM Qdm peptide (data not shown), indicating that they bind extremely poorly to Qa1 compared with Qdm.

In line with the much lower expression of Qa1 on RMA/S cells than on T2Q, ~1000-fold higher concentrations of peptide were required to sensitize RMA/S cells to lysis by d12i than to sensitize T2Q cells to lysis by the same clone (Fig. 6GoA; Table IGo). This result, combined with the finding that ~30-fold higher concentrations of Qdm peptide were required to protect T2Q cells from NK cells than were required to sensitize them to lysis by d12i (Fig. 6GoB; Table IGo), strongly suggests that the failure of Qdm peptide to protect RMA/S cells from NK lysis is due to a failure to create a sufficient number of Qa1-peptide complexes on RMA/S cells.

Of the naturally occurring homologs of the Qdm peptide, Qdm-k was as potent as Qdm in sensitizing T2Q cells to lysis by d12i, but displayed a ~30-fold lower capacity than Qdm to protect T2Q cells from NK lysis (Fig. 6GoB; Table IGo). As expected (11), Qdm-k was completely incapable of sensitizing cells to lysis by type 1 Qa1-specific CTL clones, such as a2d (Table IGo). The relative ability of five HLA-derived peptides to protect human T2Q cells from lysis by mouse NK cells (Table IGo) showed no correlation with their ability to protect against lysis by human NK cells (4, 5, 6, 14). However, the finding that the Qdm-like peptide derived from HLA-Cw4, which is expressed in C1R cells (30), is able to protect efficiently against mouse NK cells may explain the partial resistance of C1RQ cells to lysis by mouse NK cells. The corresponding peptide derived from HLA-A2 that differs from the HLA-Cw4 peptide only at position 8 had extremely weak protective activity against mouse NK cells, yet bound well to Qa1 as judged by its potent ability to sensitize RMA/S cells to lysis by the Qa1-specific CTL clone a2d. This suggests that the P8L residue of the Qdm peptide is important for recognition by inhibitory CD94/NKG2 receptors. This conclusion is supported by the finding that the P8V peptide also binds well to Qa1 as shown by its ability to induce thermostable up-regulation of Qa1 on LQ cells (13) and its efficient promotion of CTL lysis (Ref. 13 and Table IGo) but has very poor protective activity. The P4P and P5R residues of the Qdm peptide may also be important for recognition by inhibitory CD94/NKG2 receptors because the substituted Qdm analog peptides P4A and P5A promote thermostable up-regulation of Qa1 on LQ cells (13) and efficiently promote lysis by some CTL (Ref. 13 and Table IGo) but had weak or no protective activity.

The results obtained in the preloading system, in which the stability of the protected state for 1 h at 37°C in the absence of peptide was tested, generally correlated well with those obtained in the peptide inclusion system. Thus, peptides that gave strong protection at low concentrations (<300 nM) in the inclusion system generally protected well in the preloading system, whereas peptides that gave strong protection only at high concentrations in the inclusion system gave weak or no protection in the preloading system.

The binding of Qa1 peptide complexes to CD94/NKG2 receptors is species and temperature dependent

The remarkable functional conservation of the Qa1/HLA-E-mediated NK protection mechanism led us to examine whether the molecular interaction between the receptors and ligands had been conserved between the two species. The data in Fig. 7GoA demonstrate that it has not, because soluble HLA-E molecules fail to bind to mouse NK cells and soluble Qa1 molecules fail to bind to human NK cells. In view of the temperature sensitivity of the protected state induced by certain peptides, we examined whether the binding of Qa1-Qdm complexes to CD94/NKG2 receptors on NK cells might also be temperature dependent. To our surprise, we found that the binding of Qa1 tetramers to NK cells increased dramatically between 0°C and 37°C.



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  		FIGURE 7. A, The mouse NK clone J1.1 and the human NK clone NK92 were stained with medium (light line) or with Qa1 or HLA-E tetramers (bold lines). B, Adult mouse NK cells that contain both Qa1R- and Qa1R+ populations were stained with Qa1 tetramer at 0, 21, or 37°C.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The interaction between class I receptors on NK cells and the class I molecules on the cells they contact is potentially of immense biological importance because it most likely determines the life and death fate of the contacted cells. Therefore, understanding the molecular nature of this interaction and the conditions under which it delivers effective inhibitory signals to NK cells is of considerable interest. To investigate the circumstances under which cells expressing Qa1 molecules are protected from NK cell lysis we have used as effectors fetal NK cells that express CD94/NKG2 receptors for Qa1, and perhaps also receptors for an as yet uncharacterized nonclassical class I molecule expressed on EL4/RMA cells, but that lack receptors for classical class I molecules (17, 18). Our results indicate that protection is dependent on the association of Qa1 with a peptide, on the exact nature of this peptide, and on the expression of the resulting Qa1-peptide complexes reaching a sufficient level to overcome the activating signals delivered by the target cells.

The dependence on peptide was demonstrated by the finding that in the absence of exogenously added peptides, T2Q cells that express high levels of surface Qa1 are completely unprotected from lysis by Qa1R+ NK cells. The level of Qa1 expression on unprotected T2Q cells was, if anything, higher than that found on highly protected C1RQ and LQ cells and did not increase during acquisition of the protected state in the presence of exogenous peptide. It can be concluded from these observations that the association of Qa1 with an appropriate peptide is essential for recognition and delivery of an inhibitory signal by CD94/NKG2 receptors on NK cells and that the majority of the Qa1 molecules present on T2Q cells are presumably expressed in a form that cannot be recognized and/or provoke the delivery of inhibitory signals by CD94/NKG2 receptors. The exact form in which Qa1 molecules are expressed on T2Q cells is unclear, but the weight of evidence would suggest that they are present in a properly conformed but peptide-empty state. Thus, although TAP-deficient T2 cells do express some peptide-loaded endogenous HLA molecules, this is only at very low levels, and apparently arises from the occasional inefficient loading of some HLA molecules with peptides generated in the endoplasmic reticulum (31). Given the highly restricted peptide-binding repertoire of Qa1, it is unlikely that a peptide able to form stable complexes with Qa1 would be present in the endoplasmic reticulum of TAP-deficient T2 cells. More importantly, it has been directly demonstrated that mouse, unlike human, class I molecules transfected into T2 cells are expressed at the surface at high levels in a peptide-empty state (31). This might be explained by: 1) the high affinity of human {beta}2m for mouse class I {alpha}-chains renders peptide-empty mouse class I {alpha}-chain/human {beta}2m dimers stable both in the endoplasmic reticulum and at the cell surface (32); and 2) mouse class I {alpha}-chain/human {beta}2m dimers do not associate efficiently with the human peptide loading machinery and, hence, are not retained in the endoplasmic reticulum.

The importance of achieving an adequate level of expression of Qa1 peptide complexes for protection from NK lysis was demonstrated by the finding that, whereas T2Q cells could be protected by incubation with as little as 10 nM Qdm peptide, RMA/S cells could not be protected even with doses of Qdm peptide as high as 100 µM. This was not due to a failure of RMA/S cells to express Qa1 or to an inability of these molecules to be loaded with exogenous Qdm peptide, because RMA/S cells exposed to high levels of Qdm peptide became highly sensitive to lysis by Qa1-specific CTL. In addition, several previous studies, most importantly those using HLA-E transfected RMA/S cells, have demonstrated that both transfected and endogenous class I molecules expressed in RMA/S cells can be loaded to sufficient levels to protect the cells from NK lysis (5, 6, 33, 34, 35, 36). The implication is that the level of expression of Qa1-peptide complexes that can be achieved on RMA/S cells by exposing them to Qdm peptide is insufficient to overcome the activatory signals that RMA/S deliver to NK cells. This conclusion is in line with the findings that: 1) ~1000-fold higher concentrations of Qdm peptide are required to sensitize RMA/S cells to lysis by Qa1-specific CTL than are required to sensitize T2Q cells to lysis by the same CTL; and 2) even after incubation with high concentrations of Qdm peptide at 26°C, RMA/S cells show no detectable staining with the Qa1-specific mAb 4C2, indicating that the level of expression of Qa1 on peptide-loaded RMA/S cells is no more than ~1% of the level of Qa1 found on T2Q, C1RQ, and LQ transfectants. Indeed, untransfected mouse cells in general, including a wide range of tumor cells, fresh spleen cells, and T cell blasts, show low or undetectable staining with mAb 4C2, suggesting that, like HLA-E in humans, Qa1 is generally expressed at much lower levels than classical class I molecules. Although normal blast cells are resistant to lysis by Qa1R+ NK cells (17) (Fig. 1Go), none of the many mouse tumor cell lines we have tested showed any detectable resistance to lysis by Qa1R+ NK cells, although, as judged from their efficient lysis by Qa1-specific CTL, they do express Qa1 (data not shown).

Taken together, these data suggest that whereas normal blast cells that deliver low levels of activating signals to NK cells (as judged by the relatively high E:T ratios required for the lysis of class I deficient blasts by NK cells from various sources (Fig. 1Go and Refs. 37, 38, 39, 40)) can be protected by the low levels of Qa1-Qdm complexes normally expressed on these cells, in vitro grown tumor cells that deliver much stronger activating signals are not protected unless the levels of Qa1 are boosted by transfection, and perhaps also by the provision of additional exogenous or endogenous sources of Qdm peptide (the Qa1b/Db construct used to transfect L cells included the Db leader sequence (13)). This suggests that the physiological level of expression of Qa1 is set just sufficient to counteract the very low levels of NK cell activating signals delivered by normal cells, thereby allowing the system to be sensitive to small changes in the supply of Qdm peptide, and at the same time to be readily overridden by signals arising from augmented expression of activating structures. In this context it is interesting to note that large proportions of {gamma}{delta} T cells express not only inhibitory CD94/NKG2 receptors (41, 42), but also receptors, including TCRs and activating NKG2D receptors, that may recognize heat shock proteins and other stress-induced molecules (43, 44, 45).

The results obtained in this study provide some potential insights into the nature of the interaction between CD94/NKG2 receptors and Qa1 molecules. The finding that human C1R and T2 cells transfected with the Qa1{alpha}-chain alone are efficiently protected from lysis by NK cells (in the case of T2 cells in the presence of exogenous Qdm peptide) demonstrates that mouse {beta}2m is not essential for Qa1-mediated protection. Likewise, the finding that L cells transfected with a chimeric Qa1 molecule that contains the {alpha}1 and {alpha}2 domains of Qa1 associated with the {alpha}3 domain of Db demonstrates that the {alpha}3 domain of Qa1 is also not needed for protection. Together these results suggest that inhibitory CD94/NKG2 receptors interact primarily or exclusively with the {alpha}1/{alpha}2 domains of Qa1.

The fact that T2Q cells expressing abundant levels of "empty" Qa1 molecules are completely unprotected, but become strongly and rapidly protected after addition of exogenous peptide, argues compellingly against the possibility, suggested for some Ly49 class I receptors (46), that inhibitory CD94/NKG2 receptors recognize empty peptide-receptive Qa1 molecules. This raises the critical question of whether CD94/NKG2 receptors are peptide selective, as has been suggested for some mouse Ly49 receptors (36) and human KIRs (47), perhaps even interacting directly with peptide, or whether peptide is merely required to promote expression of correctly conformed class I molecules, as has been suggested for Ly49A (34, 35). Our studies of natural and synthetic analogs of the Qdm peptide fail to answer these questions but do lead to a number of clear conclusions.

First, the method used for measuring protection is critical. Whereas most Qdm-related peptides give good protection when added directly into cytotoxicity assays at high concentration, many of them fail to protect when used in a pretreatment assay at the same concentration. Similar observations have recently been made by others studying HLA-E-mediated protection from human NK cells (33). The finding that there is a close correlation between the amount of peptide required to give good protection in the inclusion assay and whether a single high concentration of peptide protects in the preloading assay suggests that the difference between the two assays is primarily one of sensitivity, the peptide inclusion assay being capable of detecting much weaker interactions between peptides and surface Qa1 molecules than the preloading assay. This is supported by the finding that higher concentrations of peptides are needed to induce protection in the preloading assay. The protected state induced by low-affinity peptides such as P2T was found to be lost rapidly when cells were incubated at 37oC in the absence of peptide, in agreement with similar findings concerning HLA-E mediate protection in humans (33). Whether this is due to an innate instability and faster dissociation of the Qa1-peptide complexes formed by these peptides compared with those formed by peptides such as Qdm or is simply due to their being present initially at much lower concentrations and, therefore, decaying to subthreshold levels more rapidly, is uncertain. Regardless of explanations, it is clear from the data presented here and elsewhere that results obtained by adding a single high dose of peptide into the cytotoxicity assay would be an unreliable indicator of the protective activity of peptides in vivo. The finding that the binding of soluble Qa1-Qdm complexes to CD94/NKG2 receptors on NK cells is optimal at 37°C, as is the binding of soluble class I molecules to TCRs (48), also suggests that caution is necessary in the interpretation of data concerning the binding of HLA-E and Qa1 peptide complexes to CD94/NKG2 receptors at other temperatures.

Second, although the Qa1- and HLA-E-based protection systems display a remarkable degree of functional homology, they are actually highly species specific. Thus, just as Qdm and Qdm-k peptides bind poorly to HLA-E and only weakly protect human cells from lysis by human NK cells (14, 33), peptides derived from the leader sequences of HLA-A2, B8, and G, which are strongly protective when associated with HLA-E (4, 5, 6), have little or no protective activity in mice. There seems to have been a close coevolution between class I leader sequences and the other molecules involved in protection from NK lysis in the two species. In addition, the finding that HLA-E and Qa1 tetramers containing protective peptides fail to bind to NK cells of the opposite species demonstrates that the receptors and ligands of the two species are no longer compatible. This, together with the finding that HLA-A2 and HLA-G leader peptides that are highly protective in humans have negligible ability to protect T2Q cells from mouse NK cells, excludes the possibility that peptide-mediated protection of T2Q cells from mouse NK cells could be caused by peptides being loaded onto HLA-E molecules. It is interesting that the HLA-derived peptide that protected cells best against lysis by mouse NK cells is identical with a nine amino acid peptide found within the cytomegalovirus gpUL40 protein that can protect cells from lysis by human NK cells and is thought to form part of a complex immune evasion strategy used by the virus (49).

Third, based on the assumption that peptides that increase the thermostable expression of a Qa1b/Db construct on LQ cells and/or sensitize target cells to lysis by CTL at low concentrations must bind efficiently to Qa1, the P4A, P5A, and P8V peptides, and the HLA-A2-derived peptide bound well to Qa1 but gave little or no protection from NK lysis. These results suggest that the P at P4, the R at P5, and the L at P8 of the AMAPRTLLL sequence of the Qdm peptide play an important role in the recognition of Qa1-Qdm complexes by inhibitory CD94/NKG2 receptors. Based on the crystal structure of HLA-E associated with the HLA-B8-derived peptide (50), the P4 and P5 residues would be expected to be solvent exposed; therefore, it is possible that they interact directly with receptors on NK cells. Alternatively, substitutions at these positions may cause conformational changes elsewhere in the peptide or in Qa1 itself. The latter possibility may explain the influence of the P8 residue, which in the HLA-E crystal structure has a close interaction with the walls of the groove. It was also observed that a natural allelic form of the Qdm peptide, Qdm-k, bearing an A to V substitution at P3 of Qdm, had an identical capacity to sensitize T2Q cells to lysis by CTL but a 30-fold weaker capacity to protect them from NK cells. This same substitution is responsible for the well-documented failure of type 1 Qa1-specific CTL clones to recognize Qa1b in H-2K strains (11). In the HLA-E crystal structure, the P3A residue of the peptide projects downward into the groove. If the same were true of the Qa1-Qdm structure, an A to V substitution at this position would most likely affect recognition by CD94/NKG2 receptors and the TCRs of type 1 clones by altering the disposition of the solvent exposed P4 and P5 residues or of main chain contact residues. Based on the finding that the HLA-Cw7-derived peptide binds well to HLA-E but that the resulting complex does not bind to soluble CD94/NKG2 complexes or protect cells from NK lysis (33, 51), the T residue at P6 of the bound peptide has been implicated in recognition by human CD94/NKG2 complexes. As judged by the fact that a conservative T to S substitution at this position of Qdm effectively abrogated functional recognition by both CTL and NK cells in this study, it is likely that in the mouse this residue is important for the binding of the Qdm peptide to Qa1. The finding in this study that certain substitutions at P2 of the Qdm peptide sequence were extremely well tolerated is surprising, given the critical importance of this residue for the binding of peptides to HLA-E (14, 50), and may suggest that the structure of the B pocket in Qa1 is significantly different to that in HLA-E.

Finally, we found that two heat shock protein-derived peptides, one of endogenous mouse origin and one of bacterial origin, that bind to Qa1 and are recognized by Salmonella-induced CTL, have no protective activity and do not inhibit protection by the Qdm peptide. Thus, although these peptides might be generated in substantial quantities in Salmonella-infected or other stressed cells, they would not be expected to interfere directly with Qa1-Qdm-mediated NK protection. However, the intriguing possibility remains that stress proteins or peptides derived from them might interact with activatory receptors on NK or related cells, counteracting the normal inhibitory signals and promoting the elimination of diseased cells.


   Acknowledgments
 
We thank the many colleagues who generously provided us with reagents used in this work. We are especially grateful to Dr. Mark Soloski for providing us with Qa1-transfected human cells and the anti-Qa1 mAbs, Dr. J. Gray for his skill and advice in the preparation of peptides, Drs. V. Braud and C. O’Callaghan for helpful discussions, and Dr. E. Simpson for constructive comments on the manuscript.


   Footnotes
 
1 This work was supported by grants from the Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Cancer Research Campaign, U.K. Dr. Karen Fraser is the recipient of a William Ross Scholarship from the Cancer Research Campaign, U.K. Back

2 Address correspondence and reprint requests to Dr. Colin Brooks, Department of Microbiology and Immunology, The Medical School, Newcastle, NE2 4HH, U.K. Back

3 Abbreviation used in this paper: {beta}2m, {beta}2-microglobulin. Back

Received for publication October 30, 2000. Accepted for publication November 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ljunggren, H. G., K. Karre. 1990. In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol. Today 11:237.[Medline]
  2. 1997. Immunol. Rev. 155:.
  3. Lopez-Botet, M., M. Carretero, T. Bellon, J. J. Perez-Villar, M. Llano, F. Navarro. 1998. The CD94/NKG2 C-type lectin receptor complex. Curr. Top. Microbiol. Immunol. 230:41.[Medline]
  4. Braud, V. M., D. S. Allan, C. A. O’Callaghan, K. Soderstrom, A. D’Andrea, G. S. Ogg, S. Lazetic, N. T. Young, J. I. Bell, J. H. Phillips, et al 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391:795.[Medline]
  5. Borrego, F., M. Ulbrecht, E. H. Weiss, J. E. Coligan, A. G. Brooks. 1998. Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J. Exp. Med. 187:813.[Abstract/Free Full Text]
  6. Lee, N., M. Llano, M. Carretero, A. Ishitani, F. Navarro, M. Lopez-Botet, D. E. Geraghty. 1998. HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc. Natl. Acad. Sci. USA 95:5199.[Abstract/Free Full Text]
  7. Vance, R. E., J. R. Kraft, J. D. Altman, P. E. Jensen, D. H. Raulet. 1998. Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b). J. Exp. Med. 188:1841.[Abstract/Free Full Text]
  8. Salcedo, M., P. Bousso, H. G. Ljunggren, P. Kourilsky, J. P. Abastado. 1998. The Qa-1b molecule binds to a large subpopulation of murine NK cells. Eur. J. Immunol. 28:4356.[Medline]
  9. Vance, R. E., A. M. Jamieson, D. H. Raulet. 1999. Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells. J. Exp. Med. 190:1801.[Abstract/Free Full Text]
  10. Soloski, M. J., A. DeCloux, C. J. Aldrich, J. Forman. 1995. Structural and functional characteristics of the class IB molecule, Qa-1. Immunol. Rev. 147:67.[Medline]
  11. Aldrich, C. J., A. DeCloux, A. S. Woods, R. J. Cotter, M. J. Soloski, J. Forman. 1994. Identification of a Tap-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen. Cell 79:649.[Medline]
  12. DeCloux, A., A. S. Woods, R. J. Cotter, M. J. Soloski, J. Forman. 1997. Dominance of a single peptide bound to the class I(B) molecule, Qa-1b. J. Immunol. 158:2183.[Abstract]
  13. Cotterill, L. A., H. J. Stauss, M. M. Millrain, D. J. Pappin, D. Rahman, B. Canas, P. Chandler, A. Stackpoole, E. Simpson, P. J. Robinson, P. J. Dyson. 1997. Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide. Eur. J. Immunol. 27:2123.[Medline]
  14. Braud, V., E. Y. Jones, A. McMichael. 1997. The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur. J. Immunol. 27:1164.[Medline]
  15. Smith, H. R., H. H. Chuang, L. L. Wang, M. Salcedo, J. W. Heusel, W. M. Yokoyama. 2000. Nonstochastic coexpression of activation receptors on murine natural killer cells. J. Exp. Med. 191:1341.[Abstract/Free Full Text]
  16. Manoussaka, M., A. Georgiou, B. Rossiter, S. Shrestha, J. A. Toomey, P. V. Sivakumar, M. Bennett, V. Kumar, C. G. Brooks. 1997. Phenotypic and functional characterization of long-lived NK cell lines of different maturational status obtained from mouse fetal liver. J. Immunol. 158:112.[Abstract]
  17. Toomey, J. A., M. Salcedo, L. A. Cotterill, M. M. Millrain, Z. Chrzanowska-Lightowlers, J. Lawry, K. Fraser, F. Gays, J. H. Robinson, S. Shrestha, et al 1999. Stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro accounts in part but not in whole for the ability of these cells to distinguish between class I-sufficient and class I-deficient targets. J. Immunol. 163:3176.[Abstract/Free Full Text]
  18. Sivakumar, P. V., A. Gunturi, M. Salcedo, J. D. Schatzle, W. C. Lai, Z. Kurepa, L. Pitcher, M. S. Seaman, F. A. Lemonnier, M. Bennett, et al 1999. Cutting edge: expression of functional CD94/NKG2A inhibitory receptors on fetal NK1.1+Ly-49- cells: a possible mechanism of tolerance during NK cell development. J. Immunol. 162:6976.[Abstract/Free Full Text]
  19. Brooks, C. G., A. Georgiou, R. K. Jordan. 1993. The majority of immature fetal thymocytes can be induced to proliferate to IL-2 and differentiate into cells indistinguishable from mature natural killer cells. J. Immunol. 151:6645.[Abstract]
  20. Brooks, C. G. 1999. Cloning and culturing of mouse NK cells. Methods Mol. Biol. In press.
  21. Brooks, C. G., H. Spits. 2000. NK cells and LAK cells. M. J. Dallman, and J. R. Lamb, eds. Haemopoietic and Lymphoid Cell Culture 147. Cambridge University Press, Cambridge, U.K.
  22. Klingemann, H. G., E. Wong, G. Maki. 1996. A cytotoxic NK-cell line (NK-92) for ex vivo purging of leukemia from blood. Biol. Blood Marrow Transplant. 2:68.[Medline]
  23. Salter, R. D., D. N. Howell, P. Cresswell. 1985. Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids. Immunogenetics 21:235.[Medline]
  24. Storkus, W. J., D. N. Howell, R. D. Salter, J. R. Dawson, P. Cresswell. 1987. NK susceptibility varies inversely with target cell class I HLA antigen expression. J. Immunol. 138:1657.[Medline]
  25. 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]
  26. Gays, F., M. Unnikrishnan, S. Shrestha, K. P. Fraser, A. R. Brown, C. M. Tristram, Z. M. Chrzanowska-Lightowlers, C. G. Brooks. 2000. The mouse tumor cell lines EL4 and RMA display mosaic expression of NK- related and certain other surface molecules and appear to have a common origin. J. Immunol. 164:5094.[Abstract/Free Full Text]
  27. Brooks, C. G., G. R. Flannery. 1980. Quantitative studies of natural immunity to solid tumours in rats: persistence of natural immunity throughout reproductive life, and absence of suppressor cells in infant rats. Immunology 39:187.[Medline]
  28. Lo, W. F., H. Ong, E. S. Metcalf, M. J. Soloski. 1999. T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules. J. Immunol. 162:5398.[Abstract/Free Full Text]
  29. Lo, W. F., A. S. Woods, A. DeCloux, R. J. Cotter, E. S. Metcalf, M. J. Soloski. 2000. Molecular mimicry mediated by MHC class Ib molecules after infection with Gram-negative pathogens. Nat. Med. 6:215.[Medline]
  30. Zemmour, J., A. M. Little, D. J. Schendel, P. Parham. 1992. The HLA-A,B "negative" mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon. J. Immunol. 148:1941.[Abstract]
  31. Wei, M. L., P. Cresswell. 1992. HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides. Nature 356:443.[Medline]
  32. Anderson, K. S., J. Alexander, M. Wei, P. Cresswell. 1993. Intracellular transport of class I MHC molecules in antigen processing mutant cell lines. J. Immunol. 151:3407.[Abstract]
  33. Brooks, A. G., F. Borrego, P. E. Posch, A. Patamawenu, C. J. Scorzelli, M. Ulbrecht, E. H. Weiss, J. E. Coligan. 1999. Specific recognition of HLA-E, but not classical, HLA class I molecules by soluble CD94/NKG2A and NK cells. J. Immunol. 162:305.[Abstract/Free Full Text]
  34. Correa, I., D. H. Raulet. 1995. Binding of diverse peptides to MHC class I molecules inhibits target cell lysis by activated natural killer cells. Immunity 2:61.[Medline]
  35. Orihuela, M., D. H. Margulies, W. M. Yokoyama. 1996. The natural killer cell receptor Ly-49A recognizes a peptide-induced conformational determinant on its major histocompatibility complex class I ligand. Proc. Natl. Acad. Sci. USA 93:11792.[Abstract/Free Full Text]
  36. Franksson, L., J. Sundback, A. Achour, J. Bernlind, R. Glas, K. Karre. 1999. Peptide dependency and selectivity of the NK cell inhibitory receptor Ly-49C. Eur. J. Immunol. 29:2748.[Medline]
  37. Correa, I., L. Corral, D. H. Raulet. 1994. Multiple natural killer cell-activating signals are inhibited by major histocompatibility complex class I expression in target cells. Eur. J. Immunol. 24:1323.[Medline]
  38. Ljunggren, H. G., L. Van Kaer, H. L. Ploegh, S. Tonegawa. 1994. Altered natural killer cell repertoire in Tap-1 mutant mice. Proc. Natl. Acad. Sci. USA 91:6520.[Abstract/Free Full Text]
  39. Grigoriadou, K., C. Mâenard, B. Pâerarnau, F. A. Lemonnier. 1999. MHC class Ia molecules alone control NK-mediated bone marrow graft rejection. Eur. J. Immunol. 29:3683.[Medline]
  40. Nutt, S. L., B. Heavey, A. G. Rolink, M. Busslinger. 1999. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401:556.[Medline]
  41. Carena, I., A. Shamshiev, A. Donda, M. Colonna, G. D. Libero. 1997. Major histocompatibility complex class I molecules modulate activation threshold and early signaling of T cell antigen receptor-{gamma}/{delta} stimulated by nonpeptidic ligands. J. Exp. Med. 186:1769.[Abstract/Free Full Text]
  42. Battistini, L., G. Borsellino, G. Sawicki, F. Poccia, M. Salvetti, G. Ristori, C. F. Brosnan. 1997. Phenotypic and cytokine analysis of human peripheral blood {gamma}{Delta} T cells expressing NK cell receptors. J. Immunol. 159:3723.[Abstract]
  43. O’Brien, R. L., Y. X. Fu, R. Cranfill, A. Dallas, C. Ellis, C. Reardon, J. Lang, S. R. Carding, R. Kubo, W. Born. 1992. Heat shock protein Hsp60-reactive {gamma} {delta} cells: a large, diversified T-lymphocyte subset with highly focused specificity. Proc. Natl. Acad. Sci. USA 89:4348.[Abstract/Free Full Text]
  44. 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]
  45. Cerwenka, A., A. B. 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]
  46. Su, R. C., S. K. Kung, E. T. Silver, S. Lemieux, K. P. Kane, R. G. Miller. 1999. Ly-49CB6 NK inhibitory receptor recognizes peptide-receptive H-2Kb. J. Immunol. 163:5319.[Abstract/Free Full Text]
  47. Malnati, M. S., M. Peruzzi, K. C. Parker, W. E. Biddison, E. Ciccone, A. Moretta, E. O. Long. 1995. Peptide specificity in the recognition of MHC class I by natural killer cell clones. Science 267:1016.[Abstract/Free Full Text]
  48. Whelan, J. A., P. R. Dunbar, D. A. Price, M. A. Purbhoo, F. Lechner, G. S. Ogg, G. Griffiths, R. E. Phillips, V. Cerundolo, A. K. Sewell. 1999. Specificity of CTL interactions with peptide-MHC class I tetrameric complexes is temperature dependent. J. Immunol. 163:4342.[Abstract/Free Full Text]
  49. Tomasec, P., V. M. Braud, C. Rickards, M. B. Powell, B. P. McSharry, S. Gadola, V. Cerundolo, L. K. Borysiewicz, A. J. McMichael, G. W. Wilkinson. 2000. Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science 287:1031.[Abstract/Free Full Text]
  50. O’Callaghan, C. A., J. Tormo, B. E. Willcox, V. M. Braud, B. K. Jakobsen, D. I. Stuart, A. J. McMichael, J. I. Bell, E. Y. Jones. 1998. Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E. Mol. Cell 1:531.[Medline]
  51. Llano, M., N. Lee, F. Navarro, P. Garcia, J. P. Albar, D. E. Geraghty, M. Lopez-Botet. 1998. HLA-E-bound peptides influence recognition by inhibitory and triggering CD94/NKG2 receptors: preferential response to an HLA-G-derived nonamer. Eur. J. Immunol. 28:2854.[Medline]



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