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NK Cells Can Recognize Different Forms of Class I MHC¹

Ruey-Chyi Su^*, Sam K. P. Kung^*, Jean Gariépy^*, Brian H. Barber and Richard G. Miller^*,2

^* Department of Medical Biophysics, Ontario Cancer Institute, and Department of Immunology, University of Toronto, Toronto, Ontario, Canada

	Abstract

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NK recognition and lysis of targets are mediated by activation receptor(s) whose effects may be over-ridden by inhibitory receptors recognizing class I MHC on the target. Incubation of normal lymphoblasts with a peptide that can bind to their class I MHC renders them sensitive to lysis by syngeneic NK cells. By binding to class I MHC, the peptide alters or masks the target structure recognized by an inhibitory NK receptor(s). This target structure is most likely an "empty" dimer of class I heavy chain and ß₂m as opposed to a "full" class I trimer formed by binding of specific peptide that is recognized by CTL.

	Introduction

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It is now widely accepted that NK cells recognize and lyse target cells through the interplay of two families of receptors (1, 2, 3). Activating receptors, when occupied, trigger lysis of the target cell being recognized. The activating signal, however, can be overridden by a dominant negative signal from an inhibitory receptor when the latter interacts with its ligand (if present) on the target cell. The ligand(s) for activating receptors remains unknown, but there is a general agreement that the ligand for some (perhaps all) inhibitory receptors is associated with MHC class I alleles, with a particular receptor being specific for a limited number of class I alleles. C1R is an HLA-A, HLA-B null human tumor cell line sensitive to lysis by polyclonal human NK cells. Storkus et al. found that transfection of some (but not all) HLA-A or HLA-B molecules into C1R protected it from NK lysis (4). By performing exon shuffling and point mutation experiments, Storkus et al. showed that the 1-2 region of MHC class I appears to be critical in determining the specificity of MHC class I as an inhibitory ligand (5), and that the amino acids in the peptide binding site of MHC class I molecules appear to be important in the protection (6). In addressing whether occupation of the peptide binding site was important, Storkus et al., using C1R cells transfected with protective human HLA-A or HLA-B MHC class I molecules, found that addition of peptide that could bind to a protective MHC class I reversed protection, i.e., sensitivity to lysis was restored upon peptide binding (7). Similar observations have been obtained in a more physiologic setting in which normal, untransformed lymphoblasts and syngeneic (polyclonal) mouse NK cells were used, respectively, as target and effector cells (8, 9). They found that the lymphoblasts, which are resistant to lysis by syngeneic mouse NK cells, could be rendered sensitive to lysis if peptides that could bind to the MHC class I of the normal cells were included in the assay. Eight peptides, capable of binding K^b, D^b, K^d, or L^d class I molecules, were tested. All eight peptides tested (seven of which included CTL epitopes and one of which did not) could sensitize normal targets for lysis if they could bind to the class I of the target, but otherwise had no effect (9). One possible explanation of these results, consistent with those of Storkus et al. (7), is that binding of peptide to MHC class I is altering or masking an inhibitory ligand recognized by an inhibitory receptor and thus sensitizing the cells to lysis. Identification of human KIRs³ (p58.1, p58.2, or p70) and murine Ly49A molecules as NK inhibitory receptors specific for particular MHC class I alleles facilitates a detailed study of specific receptor-ligand interactions. Ly49A is known to recognize D^d (10, 11), and recent evidence indicates that recognition requires that D^d is loaded with peptide (12, 13). Both groups used mutant cell lines lacking functional peptide transporter molecules so that only empty (and unstable) MHC class I molecules appear on the cell surface. These can bind and be stabilized by high affinity class I-binding exogenous peptide (14, 15). Both groups used Ly-49A⁺ NK cells as effector cells and the mouse mutant cell lines RMA-S (12) and LKD8 (13), transfected with D^d as target cells. Addition of peptide that could bind to D^d was shown to protect the cell lines from lysis by Ly49A⁺ mouse NK cells. The extent of protection correlated with the extent to which the added peptide stabilized D^d expression (12). Both groups suggested that the role of peptide was to promote the assembly and cell surface expression of MHC class I and that there was no peptide specificity in Ly49A recognition of the D^d molecule. In a similar study, Malnati et al. used RMA-S cells transfected with HLA-B27 as targets, human NK clones expressing KIR receptors specific for HLA-B27 as effectors, and exogenous synthetic peptide ligands of HLA-B27 to stabilize surface expression of the HLA molecules on RMA-S cells (16). One of the four peptide ligands specific for HLA-B27 tested provided protection from lysis by the specific NK clones (16). The protection was independent of the peptide binding affinity to HLA-B27. By performing further analysis of HLA-B27-specific peptides using amino acid substitutions, Peruzzi et al. found that the side chains of the seventh and eighth amino acids of "protective" peptides were conserved and may be involved in NK recognition (17). This involvement may be either indirect, by affecting the conformation of the KIR binding site, or direct, through interference with KIR binding to the class I heavy chain (18).

In summary, binding of peptide to MHC class I has been shown to sensitize targets to NK lysis (7, 8, 9), as well as to protect targets from NK lysis (12, 13, 16, 17). We here try to reconcile these apparently contradictory findings by assessing the possibility that NK cells can recognize different forms of MHC class I molecules. There are four possible forms of MHC class I molecules expressed on the normal cell surface. The majority exist as trimolecular complexes, each composed of a properly folded heavy chain (H) containing the peptide binding groove, a noncovalently associated ß₂m molecule, and a peptide (p) that can bind to MHC class I with high affinity (therefore, p_H) in the peptide-binding groove (thus p_H-H-ß₂m) (19). Three other unstable forms of MHC class I, ß₂m-H, p_H-H, and H (perhaps in decreasing order of stability) can be found (19, 20). In addition, p_L-H-ß₂m molecules in which the peptide is either too long or lacks the proper binding motif and thus binds with low affinity (therefore, p_L) are probably also present. For the cell line RMA-S (H-2^b), H-ß₂m and H have been shown directly to have half-lives of <30 min, and at least one particular p_L-H-ß₂m has been inferred indirectly to have a comparably short lifetime and is likely to give rise to H-ß₂m, whereas p_H-H-ß₂m appears to have a lifetime much greater than 4 h (19). Only two of these four forms of MHC class I molecule (the trimolecular complex of H chain, ß₂m and peptide bound with high affinity, and the bimolecular complex in which the peptide is not present) are likely to be expressed in appreciable numbers on the surface of a normal cell (14, 15, 19, 20, 21). Approximately 10% of D^b molecules expressed on the cell surface are likely to be bimolecular MHC class I (H-ß₂m) molecules because 1) about 10% of D^b molecules on EL4 cells can be bound very rapidly by exogenous peptide (half-time of 9.3 ± 1.1 min at 37°C) (14); and 2) the binding of exogenous peptide to purified D^b H chain was determined to have a half-time of 13 h, presumably because most of it was denatured, while binding of peptide to purified H-ß₂m bimolecules had a half-time of <10 min at 22°C (20). Although both H chain and H-ß₂m bimolecule can potentially bind exogenous peptide, added peptide is most likely to bind to H-ß₂m bimolecular MHC class I because H chain is very unstable at 37°C. We therefore refer to H-ß₂m bimolecular MHC class I as empty MHC class I. All four forms of MHC class I molecule, but particularly p_H-H-ß₂m and empty H-ß₂m molecules (because of their appreciable abundance on the cell surface), might be recognized by NK inhibitory receptors involved in self-recognition.

In this report, we first reinvestigate and further characterize the experimental system developed by Chadwick et al. in which incubation of normal lymphoblasts with class I binding peptide sensitizes them to lysis by syngeneic NK cells (8, 9). We conclude that the peptide is most likely altering or masking the ligand recognized by an inhibitory receptor. This ligand appears to be empty MHC class I, as defined above. Second, to reconcile this conclusion with the fact that the inhibitory receptor Ly49A recognizes the trimolecular complex of class I D^d plus peptide, we have investigated the lysis of normal lymphoblasts by syngeneic Ly49A⁺ and Ly49A^- NK cells in the presence and the absence of class I binding peptide. The results are consistent with the conclusion that Ly49A recognizes D^d plus peptide but, at the same time, suggest that there are additional inhibitory receptors that recognize empty MHC class I molecules. We propose a model in which a small change in the total inhibitory signal delivered by several inhibitory receptors can switch a cell from resistance to sensitivity to lysis by NK cells.

	Materials and Methods

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NK generation

The method used for producing activated NK cells (LAK cells) was identical with that used previously (8, 9, 22). Briefly, 2 x 10⁶ nylon wool nonadherent spleen cells from B6 athymic nude mice (The Jackson Laboratory, Bar Harbor, ME) were cultured at 37°C for 3 to 4 days in 5 ml of -MEM supplemented with 10% FCS, 50 µM 2-ME, and 10 mM HEPES buffer (hereafter referred to as CM), containing 500 U/ml mouse rIL-2. In some experiments, as specified, B6 CD8 knockout mice (23), BALB/c athymic nude mice (The Jackson Laboratory), or normal BALB/c mice (The Jackson Laboratory) depleted of T cells using anti-CD4/CD8 Abs and Dynabeads (Dynal, Oslo, Norway), all depleted of nylon wool adherent cells, were used. Mouse rIL-2 was obtained as a supernatant from a cell line transfected with the IL-2 gene (24). These cells were cultured in 25-cm² flasks at 37°C in a 10% CO₂ in air incubator. Yields typically exceeded 5000 U/ml of rIL-2.

Target cell generation

Target cells were B6 Con A (ICN Pharmaceuticals Canada, Montreal, Canada) blast cells produced by incubating 10⁷ B6 splenocytes for 3 days in 10 ml of CM supplemented with Con A (2 µg/ml). On day 3, Con A blast cells were harvested on Lympholyte M (Cedarlane, Hornby, Canada) and ⁵¹Cr labeled by incubating about 6 x 10⁶ cells for 90 min at 37°C with 360 µCi of Na⁵¹CrO₄ (New England Nuclear, Boston, MA) in 150 µl of PBS containing 67% FCS. They were then washed three times with CM containing 1% FCS to remove nonincorporated Na⁵¹CrO₄.

MHC class I binding peptides

The effect of MHC class I binding peptides on normal lymphoblast sensitivity to NK lysis was assessed by pulsing lymphoblasts with the experimental peptide (at a concentration of 1 ng/ml in CM unless stated otherwise) for 45 min at 4°C before the assay. The peptides used were a D^b-restricted epitope of influenza nucleoprotein, ASNENMETM, (Flu-NP_366–374) (25), a K^b-restricted epitope of chicken OVA, SIINFEKL, (OVAp_258–265) (26); a D^d-restricted epitope of HIV gp160, RGPGRAFVTI, (HIVp_318–327) (27); a K^d-restricted epitope of influenza nucleoprotein, TYQRTRALV, (Flu-NP_147–155) (25, 28); and an L^d-restricted epitope referred to as Tum^-, ISTQNHRALDLVAAK, (Tum-_12–26) (29). Both Flu-NP peptides (>90% purity) were synthesized and purified by the Alberta Peptide Institute (Edmonton, Canada). Chicken OVA, SIINFEKL (OVAp_258–265), and its derivatives, biotinylated OVA peptide, (bio)-XSIINFEKL, where X is aminocaproic acid (a linker between biotin and the peptide), and SIINFEK(bio)L were prepared by the Ontario Cancer Institute Biotechnology Laboratory, using an Applied Biosystems Peptide Synthesizer (Applied Biosystems, Foster City, CA). HIVp (>90% purity) was a gift from Dr. D. Williams (University of Toronto). Flu-NP(D^b), OVAp, HIVp, and Flu-NP(K^d) peptides are natural ligands for D^b, K^b, D^d, and K^d, respectively, and bind to D^b, K^b, D^d and K^d with high affinities (25, 26, 27, 28). Tum^- peptide binds specifically to L^d molecules after being processed to its optimal length by proteases in serum. During the pulsing condition used in the current study, unprocessed Tum^- peptide cannot bind to L^d (9).

Peptide pulsing and cytotoxicity assay

Methods for measuring lytic activity were identical with those used previously (9, 22). After three washes, ⁵¹Cr-labeled Con A lymphoblasts were incubated with peptide in 3 ml of CM for 45 min at 4°C and washed again before being used in a 4.5-h ⁵¹Cr release assay performed in 96-well V-bottom microtiter plates using 2000 targets/well, dispensed in 100-µl aliquots and effectors at an E:T cell ratio as indicated or at 30:1, also added in 100-µl aliquots. For experiments where preincubation of NK cells with F(ab')₂ anti-Ly49A mAb (JR9-318) was required, the preincubation was performed at 37°C for 30 to 45 min while preparing target cells for the assay. Specific lysis was calculated as % specific lysis = (E - S)/(T - S) x 100, where each value represents the mean ± SE of five replicates. E is the experimental mean of ⁵¹Cr released, S is the amount of ⁵¹Cr released when the target cells were cultured in medium alone, and T is the total amount of ⁵¹Cr released in the presence of 2% acetic acid. Dialyzed FCS (12 kDa cutoff) was regularly used in place of regular FCS during the ⁵¹Cr labeling, pulsing, and assay stages (14, 30).

CTL generation and maintenance

Generation of peptide-specific CTL was performed as described previously (31). Briefly, lymphocytes from normal C57BL/6 (B6) mice were depleted of B cells by passage through nylon wool and cultured at 5 to 6 x 10⁶ cells/ml in 10 ml of CM in the presence of 1 ng/ml of peptide (Flu-NP or OVAp) and 5 U/ml of mouse rIL-2 (24). On day 7, CTL were harvested on Lympholyte M (Cedarlane) and used in the cytotoxicity assay. To maintain a CTL line, 10⁶ cells were harvested after 7 to 10 days of culture and cultured with 2 x 10⁶ irradiated (15 gray) B6 spleen cells in the presence of 1 ng/ml of peptide and 5 U/ml of mouse rIL-2 as described above.

Cold target competition assay

B6 radiolabeled Con A lymphoblasts, either pulsed or unpulsed with peptide (1 ng/ml), were tested as targets using either B6 NK cells or peptide-specific B6 CTL lines as effectors, as described in the cytotoxicity assay except that unlabeled B6 Con A lymphoblasts, either pulsed (1 ng/ml) or unpulsed, were included in the wells at zero-, one-, three-, or fivefold multiplicities of the labeled targets as indicated. Cold and hot targets were premixed before the addition of effector cells (i.e., NK cells or CTL lines). A 4.5-h ⁵¹Cr release assay was performed in 96-well V-bottom microtiter plates, and specific ⁵¹Cr release was measured. Specific lysis was calculated as described in the cytotoxicity assay section.

Conjugate formation assay

FITC (green dye, Sigma, St. Louis, MO)-labeled LAK cells were prepared as described by Kung et al. (32). Briefly, day 3 or day 4 B6 LAK cells (10–12 x 10⁶) were incubated with a FITC solution (10 µg/ml PBS, final concentration) at 37°C for 18 min. Excess FITC was removed by centrifuging the cells through 5 ml of 6% BSA/PBS. The cells were then washed twice with 1% BSA/PBS. PKH26 (red dye, Sigma)-labeled target cells were prepared according to the manufacturer’s protocol. Briefly, YAC-1 and B6 Con A lymphoblasts were washed twice with serum-free medium and then incubated with PKH26 dye (4 x 10^-6 M) in labeling buffer (diluent C; 10⁷ cells/ml) at 25°C for 3 to 5 min. The staining reaction was stopped by adding an equal volume of 1% BSA/PBS. The cells were washed three times with 10% CM to remove excess PKH26 dye. The conjugation formation assay used was described by Cavarec et al. (33). FITC-labeled LAK cells were pelleted and incubated with the PKH26-labeled target cells (B6 Con A lymphoblasts, B6 Con A lymphoblasts pulsed with OVAp peptide, or YAC-1) at an E:T cell ratio of 3:1 for 10 min at 37°C. At the end of incubation, the effector-target mixture was resuspended in 1 ml of 1% BSA/PBS and kept at 4°C before being analyzed for its fluorescence. For negative controls, LAK cells and target cells, at an E:T cell ratio of 3:1, were mixed and vortexed without any cocentrifugation before the analysis with FACScan.

Time delay experiment with or without brefeldin A (BFA)

B6 Con A lymphoblasts were pulsed with peptide and washed free of unbound peptide as described in the cytotoxicity assay section. The cells were then incubated in CM at 37°C with or without BFA (5 µg/ml; Sigma-Aldrich Canada, Oakville, Canada) for varying lengths of time before being tested as targets in a 4.5-h ⁵¹Cr release assay using either syngeneic B6 NK cells or peptide specific B6 CTL lines.

Flow cytometry/FACS analysis

To measure newly emerged empty MHC class I molecules, day 3 Con A-activated lymphoblasts were prepulsed with nonlabeled OVAp peptide (10 ng/ml) for 45 min to fill empty K^b molecules, washed free of unbound peptide, and then incubated at 37°C for 0 or 90 min in the presence or the absence of BFA before being pulsed with biotinylated OVAp peptide (100 ng/ml). To measure the effect of BFA on the existing empty MHC class I molecules, day 3 Con A-activated lymphoblasts were incubated at 37°C for 0 or 4 h in the presence or the absence of BFA before being pulsed with biotinylated OVAp peptide (100 ng/ml). FITC-conjugated mAb 5F1, purified from the hybridoma 5F1-2-14 (34), was used to detect the expression of peptide-K^b complexes on the cell surface immediately after the pulsing with biotinylated OVAp peptide, as it has been shown (34) that this mAb does not recognize empty K^b molecules. OVAp_X-bio (does not bind to MHC class I) and OVAp_K-bio were used in the staining assay. The binding of biotinylated OVAp was visualized with R-phycoerythrin-conjugated streptavidin (Sigma), which binds to biotin, and analyzed using the LYSIS II program (Becton Dickinson, Mountain View, CA).

Cell sorting for Ly49A⁺ and Ly49A^- NK subsets

Day 3 BALB/c LAK cultures were harvested and resuspended in 1% BSA/PBS (10⁷ cells/ml). The cells were then incubated with 4 µg of JR9-318 mAb (35) (obtained from Dr. D. Raulet with the permission of Dr. J. Roland, Pasteur Institute, Paris, France) per 10⁶ cells at 4°C on a rotator for 45 min. JR9-318 mAb recognizes the NK inhibitory receptor, Ly49A (35). Stained cells were washed with cold 1% BSA/PBS and then incubated with sheep anti-mouse IgG conjugated to Dynabeads (Dynal; one bead per cell) for 45 min at 4°C on a rotator. Ly49A⁺ cells, bound to the magnetic beads, were separated from Ly49A^- cells, and both were cultured in 5 ml of CM containing 500 U/ml mouse rIL-2 (24) for an additional 3 to 4 days as described above. Ly49A⁺ cells that were bound to the beads dissociated from the beads during the overnight incubation, and the beads were then removed.

F(ab')₂ fragment generation

For F(ab')₂ fragment generation, 2 mg of affinity-purified anti-Ly49A mAb (JR9-318) was resuspended in 1 ml of 0.1 M sodium citrate buffer (pH 3.5) and then digested with 10 µg of pepsin (Boehringer Mannheim, Mannheim, Germany) at 37°C for 4 to 5 h. The reaction was stopped by adding 0.5 vol of 1 M Tris to the mixture. After a centrifugation at 10,000 rpm for 30 min, the supernatant was collected and mixed with protein A-Sepharose beads (Sigma, St. Louis, MO) to remove undigested Ab and Fc fragments. The purity and the binding activity of F(ab')₂ fragments were checked by 10% SDS-PAGE and flow cytometry, respectively.

	Results

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Normal Con A lymphoblasts become sensitive to lysis mediated by syngeneic NK cells after being pulsed with MHC class I binding peptide

The effect of pulsing normal Con A lymphoblasts with MHC class I binding peptide was studied. Effector cells were splenocytes from B6 (H-2 K^b, D^b) athymic nude (T cell-deficient) or CD8 knockout mice (23) depleted of B cells by passage through nylon wool and cultured for 3 to 4 days in a high concentration of mouse rIL-2 (8, 22). This procedure produces a population of highly enriched and activated NK cells (often referred to as LAK or lymphokine-activated killer cells). Target cells were B6 Con A lymphoblasts pulsed for 45 min at 4°C with the D^b-binding peptide Flu-NP_366–374 (Flu-NP) (25), the K^b-binding peptide OVA_258–265 (OVAp) (26), or the D^b-binding peptide GAD-Flu-NP (Flu-NP with three additional amino acids added to the N-terminus of the Flu-NP_366–374 peptide). Both Flu-NP and OVAp are natural ligands of an optimum length that can bind with high affinity to D^b or K^b MHC class I, respectively, in <30 min (36). In contrast, GAD-Flu-NP peptide might bind to D^b MHC class I with a relatively low affinity (14, 36). With varying E:T cell ratios, significant lysis of Flu-NP- or OVAp-pulsed target cells was always observed for E:T cell ratios of 3:1 to 10:1, and usually reached a maximum value at ratios of 10:1 to 30:1 (Fig. 1A). Normal Con A lymphoblasts pulsed with medium alone were, as expected, resistant to NK-mediated lysis. When normal Con A lymphoblasts pulsed with varying concentrations of Flu-NP or OVAp peptide were used in the assay, significant lysis over background of Flu-NP- or OVAp-pulsed target cells was seen for peptide concentrations as low as 1 pg/ml with the lysis values plateauing in the 10 to 100 pg/ml range (Fig. 1B). When normal Con A lymphoblasts were pulsed with a too-long peptide, GAD-Flu-NP, no increase in lysis was observed over the whole dose-response range (Fig. 1B). Pulsing normal Con A lymphoblasts with peptides that could not bind to either D^b or K^b MHC class I did not sensitize these target cells to lysis mediated by syngeneic NK cells (data not shown). Furthermore, no significant lysis of normal Con A lymphoblasts was observed when NK cells were pulsed with Flu-NP for 45 min at 4°C and then used as effector cells (data not shown). Thus, sensitization to lysis required that the target cells be exposed to the added peptide and that the added peptide have both the correct length and the correct motif to bind to a MHC class I molecule expressed.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Normal cells become more sensitive to NK lysis after being incubated with peptide that can bind to their MHC class I molecules. A, Percent lysis vs E:T ratio for target cells pulsed with Flu-NP (closed square), OVAp (closed diamond), or no peptide (open diamond). B6 Con A blasts were pulsed with the peptide indicated (10 ng/ml) and then tested as targets at varying E:T cell ratios, as indicated on the abscissa, in a 4.5-h 51Cr release cytotoxicity assay using syngeneic B6 NK cells. This experiment is representative of >40 such experiments. B, Peptide dose-response curve for Flu-NP (closed square), OVAp (closed diamond), or Flu-NP with GAD added on the N-terminus (open circle). B6 Con A blasts were pulsed with varying concentrations of peptide as indicated on the abscissa, washed free of unbound peptide, and then tested as targets in a 4.5-h 51Cr release cytotoxicity assay using syngeneic B6 NK cells. The middle parts of both dose-response curves have been reproduced at least three times for both peptides using B6 NK cells derived from B6 normal mice, B6 CD8 knockout mice (23), and B6 athymic nude mice. This experiment is representative of two independent experiments.

Peptide binding to MHC class I appears to alter or mask an inhibitory signal

NK recognition is thought to be mediated by an activating receptor whose effects may then be overridden by an inhibitory receptor (1, 2, 3). Our results are most easily explained by assuming that binding of the added peptide to the MHC class I on the target cell surface altered or masked an inhibitory signal recognized by NK cells. However, in principle, the peptide binding to MHC class I might either create a target structure that is recognized by an NK-activating receptor (i.e., similar to T cell recognition) or alter or mask an inhibitory structure that is recognized by an NK inhibitory receptor. In an attempt to distinguish between these possibilities, we performed cold target competition experiments. Radiolabeled, peptide-pulsed, normal B6 Con A lymphoblasts were incubated with B6 NK cells and varying numbers of cold B6 targets that had or had not been pulsed with peptide. The results (Fig. 2) show that the cold targets were equally effective competitors regardless of whether they were pulsed with OVAp peptide (Fig. 2A). This implies that both peptide-pulsed and unpulsed cold targets were equally effective in forming conjugates with NK cells. We tested this directly by measuring the ability of B6 NK cells to form conjugates with B6 Con A lymphoblasts pulsed or not pulsed with OVAp and, as a control, with YAC-1 (Fig. 3). The NK cells were stained with a green fluorescent dye (FITC), and the Con A lymphoblasts were stained with a red fluorescent dye (PKH26), mixed, and centrifuged together (experiment, conjugates should form) or kept suspended (control, conjugates much less likely to form). Events detected in the flow cytometer that showed both green and red fluorescence were scored as conjugates. In agreement with the competition results (Fig. 2), comparable numbers of conjugates formed using both pulsed and unpulsed Con A lymphoblasts (Fig. 3). As only peptide-pulsed lymphoblasts are lysed, the observations are consistent with the conclusion that binding of the added peptide to the MHC class I on Con A lymphoblast target cells is altering or masking an inhibitory structure recognized by NK cells, leading to the lysis of target cells (see Discussion).

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Cold targets compete for NK-mediated lysis of hot targets regardless of whether they have been pulsed with peptide, whereas only peptide-pulsed cold targets can compete for CTL-mediated lysis. A, B6 radiolabeled Con A lymphoblasts, either pulsed (filled symbols) or unpulsed (open symbols) with OVAp (1 ngm/ml), were tested as targets using B6 NK cells as in Figure 1B, except that unlabeled B6 Con A lymphoblasts, either pulsed (1 ng/ml; diamonds) or unpulsed (circles), were included at zero-, one-, three-, or fivefold multiplicities of the labeled targets, as indicated on the abscissa. Cold and hot targets were premixed before the addition of effector cells (i.e., NK cells). The figure is representative of six independent experiments. B, Same as in A, except that an OVAp-specific CTL line was used at an effector to hot target ratio of 50:1. An additional group was included in which Flu-NP-pulsed (1 ng/ml) targets were tested as cold competitors (filled square). The figure is representative of four independent experiments.

View larger version (40K): [in this window] [in a new window]   FIGURE 3. B6 Con A lymphoblasts, whether pulsed or not pulsed with peptide, are equally effective in forming conjugates with B6 LAK cells. Targets (A, B6 Con A lymphoblasts; B, B6 Con A lymphoblasts pulsed with OVAp; or C, YAC-1 cells (labeled with PKH26, detected by FL2; fluorescence intensity is shown on the y-axis)) were pelleted and incubated with effectors (B6 LAK cells (labeled with FITC, detected by FL1; fluorescence intensity is shown on the x-abscissa) at an E:T cell ratio of 3. Effector-target conjugates were detected as PKH26+FITC+ events, shown in the boxed area. As negative controls, FITC-labeled B6 LAK cells and PKH26-labeled targets (D, B6 Con A lymphoblasts; E, B6 Con A lymphoblasts pulsed with OVAp; F, Yac-1 cells; at an E:T cell ratio of 3) were mixed and vortexed without any cocentrifugation before the analysis with FACScan. Fewer effector-target conjugates (PKH26+FITC+ events) were formed when the effectors and targets were not brought together by centrifugation. Note that 10,000 events/sample were analyzed, but only the first 2,000 events were presented in the plot for the clarity and neatness of the presentation. The figure is representative of three independent experiments.

Peptide-pulsed target cells remain sensitive to lysis mediated by CTL for a much longer period of time than by NK cells

To gain insight into the nature of the effect produced by peptide pulsing, the lifetime of the lysis-sensitive state was measured. Flu-NP-pulsed B6 Con A lymphoblasts were washed free of unbound peptide and incubated for increasing lengths of time at 37°C before NK cells were added. The results show that the sensitivity of these Flu-NP-pulsed target cells to NK-mediated lysis returned to that of unpulsed targets following an incubation of 60 to 90 min (Fig. 4A). As a control, a CTL line specific for the same peptide was generated (31) and tested against the same target cells pulsed with the same peptide in the same assay. The peptide-pulsed targets retained full sensitivity to CTL-mediated lysis following up to 4 h of incubation (Fig. 4B).

View larger version (13K): [in this window] [in a new window]   FIGURE 4. The NK target structure formed by pulsing with peptide is short lived compared with the CTL target structure formed by pulsing the same targets with the same peptide. A, Time delay experiment, NK cells. B6 Con A lymphoblasts were pulsed with 1 ng/ml Flu-NP (filled square), a nonbinding peptide, Tum- (filled circle), or no peptide (open diamond) and washed free of unbound peptide as described in Figure 1. The cells were then incubated in CM at 37°C for varying lengths of time, as indicated on the abscissa, before being tested as targets in a 4.5-h 51Cr release assay using syngeneic B6 NK cells as described in Figure 1. The figure is representative of eight independent experiments. B, Time delay experiment, CTL. B6 Con A lymphoblast targets were pulsed with Flu-NP (filled square) or no peptide (open diamond), washed free of unbound peptide, and incubated for varying lengths of time in CM exactly as described in A. They were then tested for their ability to be lysed by a CTL line specific for the peptide using conditions identical with those used for the NK cytotoxicity assay, except that the E:T cell ratio was 10:1. The figure is representative of three independent experiments.

It is possible that the expression of newly synthesized MHC class I molecules is involved in the loss of sensitivity to NK lysis of peptide-pulsed target cells after the 90 min of preincubation. BFA is a fungal metabolite that reversibly disrupts the Golgi apparatus, resulting in the blocking of transport to the cell surface of newly synthesized protein (39). In particular, BFA has been shown to block the transport of MHC class I molecules to the cell surface (40). We tested the effect of including BFA in the preincubation step of the experiments shown in Figure 4; Flu-NP-pulsed B6 Con A lymphoblasts were incubated in the absence of free exogenous peptide with or without BFA for varying lengths of time before NK cells or CTL were added. In the presence of BFA, the sensitivity of the Flu-NP-pulsed target cells to NK-mediated lysis remained high for at least 2 h instead of rapidly falling (Fig. 5A). CTL-mediated lysis of Flu-NP pulsed target cells was not affected in the presence of BFA (Fig. 5B). Furthermore, background lysis of normal Con A lymphoblasts was not affected by BFA; BFA did not sensitize normal cells to NK lysis in the absence of peptide. The presence of BFA also prevented the loss of sensitivity to NK lysis for OVAp-pulsed Con A lymphoblasts (Fig. 5C). Thus, we conclude that preventing the appearance of newly synthesized proteins, most likely MHC class I, on the cell surface prevents the loss of sensitivity to NK lysis of peptide-pulsed target cells. The possibility that BFA is having some other effect on MHC class I expression is explicitly addressed in the following section.

View larger version (12K): [in this window] [in a new window]   FIGURE 5. The NK target structure formed by incubation with peptide is stable in the presence of BFA. A, B6 Con A lymphoblasts were pulsed at 4°C with 1 ng/ml Flu-NP (squares) or no peptide (triangles), washed free of peptide, and incubated in CM at 37°C for varying lengths of time (abscissa) as described in Figure 3A with (filled symbols) or without (open symbols) added BFA (5 µg/ml) before being used as targets for B6 NK cells as described in Figure 1. BFA (0.4 µg/ml) was also included in the cytotoxicity assay for those groups (filled symbols) for which it had been used previously. The figure is representative of six independent experiments. B, Same as in A except that the effector cells were Flu-NP specific CTL generated and analyzed as described in Figure 3B. The figure is representative of four independent experiments. C, Same as in A except that OVAp (1 ng/ml; squares) was used to pulse the B6 Con A lymphoblasts. The figure is representative of two independent experiments.

One explanation for the peptide-induced sensitization to NK lysis is that NK inhibitory receptors recognize empty MHC class I molecules on the target cell surface and that the addition of high affinity peptide fills the empty MHC class I molecules. Therefore, cells become sensitive to NK lysis because the added peptide blocks the NK recognition of inhibitory ligand. In the absence of exogenous peptide, newly synthesized empty MHC class I molecules emerge onto the target cell surface and regenerate the inhibitory signal, thus preventing lysis.

To test directly for a correlation between the absence of empty MHC class I and sensitivity to NK lysis, and the reappearance of empty MHC class I and the loss of sensitivity to lysis, we measured the relative number of full and empty K^b molecules on Con A lymphoblasts pulsed with OVAp (K^b-specific). OVAp-pulsed Con A lymphoblasts were washed free of unbound OVAp and incubated at 37°C for 90 min in the presence or the absence of BFA. The expression of peptide-K^b complex was measured before and after the 90-min incubation using the mAb, 5F1, which recognizes specifically the trimolecular K^b complex (H-ß₂m-p) (34, 41). We found that the level of peptide-K^b complex expression was not affected by the 90-min incubation in the absence of BFA (Fig. 6, a and b), but fell slightly (10–20%) in the presence of BFA (Fig. 6c), perhaps because BFA prevents the transport of newly synthesized trimolecular MHC class I to the cell surface while having no effect on the endocytosis of cell surface proteins. Empty K^b molecules appeared only in the absence of BFA (Fig. 6e). To detect empty K^b molecules, we used an OVAp peptide in which the lysine (K) at position 7 was biotinylated (OVAp_K-bio). This lysine side chain is known to be one of the CTL epitopes in OVAp and is therefore expected to protrude from the peptide binding groove (42, 43). We found that this peptide binds specifically to K^b and can be readily detected by the addition of streptavidin-phycoerythrin (Fig. 6 and data not shown). As a control peptide, we used OVAp to which a biotinylated aminocaproic acid was added to the N-terminus (OVAp_X-bio). This peptide did not bind (Fig. 6 and data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Total peptide-Kb expression was little affected by BFA, but newly synthesized empty Kb molecules emerged only in the absence of BFA during a 90-min incubation. A, FACS profiles for peptide-Kb expression (left panels) and for empty Kb expression (right panels). The left panels (a–c) show peptide-Kb complex expression (measured with mAb 5F1 staining) immediately after peptide pulsing (a) and 90 min later in the absence (b) or the presence (c) of BFA. The right panels (d–f) show binding of biotinylated OVAp immediately after peptide pulsing (d) and 90 min later in the absence (e) or the presence (f) of BFA. OVApX-bio, in which X is an aminocaproic acid serving as a linker between biotin and the peptide (does not bind to MHC class I), and OVApK-bio, which binds to Kb, were used in the staining assay. The binding of biotinylated OVAp was visualized with R-phycoerythrin-conjugated streptavidin (Sigma), which binds to biotin, and were analyzed using LYSIS II program (Becton Dickinson). The figure is representative of six independent experiments. B is the mean channel value ± SD of three replicates of the nine profiles shown in the right panels (d–f).

Clearly, the loss of empty K^b molecules after peptide pulsing correlated with the sensitivity of these target cells to NK lysis, and the reappearance of empty K^b molecules after the 90-min incubation at 37°C coincides with the loss of sensitivity to NK lysis. Thus, these data are fully consistent with our hypothesis that NK inhibitory receptors recognize empty class I molecules.

A potential problem with this model is the observation (Fig. 5) that Con A lymphoblasts not pulsed with peptide and incubated with BFA remained resistant to lysis. Empty MHC class I molecules are known to be unstable, and if BFA blocks the expression of new empty MHC class I, one might expect all empty MHC class I to disappear over the time of the assay, thus rendering the Con A lymphoblasts sensitive to lysis. To address this possibility, we measured the relative number of empty K^b molecules on Con A lymphoblasts not pulsed with peptide and incubated with or without BFA for 0 and 4 h using OVAp_K-bio as shown in Figure 6. The relative number of empty K^b fell 31 ± 7% in the presence of BFA and 11 ± 12% in the absence of BFA over the 4-h incubation. We hypothesize that the remaining empty MHC class I molecules are sufficient to provide protection from NK lysis (see Discussion).

The Ly49A molecule does not recognize empty D^d molecules

In contrast to our observations, other groups have shown that under appropriate conditions, addition of H-2D^d specific peptide creates an inhibitory signal that protects NK-susceptible target cells expressing H-2D^d from being lysed by Ly49A⁺ B6 NK cells (12, 13). To attempt to reconcile this difference, we studied the recognition of D^d molecules by Ly49A⁺ and Ly49A^- NK cells in our syngeneic experimental system. The Con A lymphoblasts and NK cells used were derived, respectively, from normal and athymic nude BALB/c (H-2^d) mice. Day 3, rIL-2-activated NK cells were sorted into Ly49A⁺ and Ly49A^- subsets using the mAb JR9-318, which recognizes Ly49A molecules on both B6 and BALB/c NK cells (35, 44). BALB/c Con A lymphoblasts, both pulsed and not pulsed with a D^d-specific peptide, HIVgp160_318–327 (HIVp) (27), were examined for sensitivity to lysis mediated by either Ly49A⁺ NK cells or Ly49A^- NK cells. The results show that BALB/c lymphoblasts, whether pulsed or not with HIVp, were resistant to lysis mediated by the Ly49A⁺ NK cells, but when F(ab')₂ anti-Ly49A mAb (JR9-318) was included in the assay, both normal and HIVp-pulsed lymphoblasts were lysed by Ly49A⁺ NK cells (Fig. 7A; see Discussion). In contrast, when Ly49A^- NK cells were used, they lysed HIVp-pulsed lymphoblasts and spared unpulsed lymphoblasts regardless of whether F(ab')₂ anti-Ly49A mAb was present (Fig. 7B). When a K^d-specific peptide (Flu-NP-K^d) was used for pulsing BALB/c lymphoblasts, both Ly49A⁺ and Ly49A^- NK populations could produce lysis. Interestingly, a mixture of both Flu-NP-K^d and HIVp peptide in the absence of F(ab')₂ anti-Ly49A mAb (JR9-318) enabled lysis by Ly49A⁺ as well as Ly49A^- NK cells (Fig. 7, C and D; see Discussion).

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Ly49A+ NK cells lyse Flu-NP (Kd-specific)-pulsed Con A lymphoblasts, but not HIVp (Dd-specific)-pulsed Con A lymphoblasts. A, BALB/c Con A lymphoblasts, whether pulsed with HIVp (filled square) or with CM alone (open square), were resistant to lysis mediated by Ly49A+ NK cells. The addition of F(ab')2 anti-Ly49A mAb (JR9-318) in the assay made both HIVp-pulsed lymphoblasts (closed triangles) and CM-pulsed lymphoblasts (open triangles) susceptible to lysis by Ly49A+ NK cells. The figure is representative of three independent experiments. B, Normal lymphoblasts pulsed with CM alone (open symbols) were resistant to lysis by Ly49A- NK cells in either the presence (triangles) or the absence (squares) of F(ab')2 anti-Ly49A mAb (JR9-318), while HIVp-pulsed lymphoblasts (closed symbols) were lysed by Ly49A- NK cells regardless of whether the F(ab')2 anti-Ly49A mAb (JR9-318) was added. C, Both CM-pulsed (open circle) and HIVp-pulsed (filled squares) lymphoblasts were resistant to lysis by Ly49A+ NK cells, while lymphoblasts pulsed either with Flu-NP-Kd peptide (Kd-specific, closed circles) alone or with a mixture of both Flu-NP peptide and HIVp (closed diamonds) were lysed. D, Lymphoblasts pulsed with Flu-NP-Kd (closed circles), HIVp (closed squares), or both (closed diamonds) were lysed by Ly49A-NK cells, while normal lymphoblasts (open circles) remained resistant to lysis. The figure is representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We here confirm previous results (8, 9) that normal Con A-induced lymphoblasts become sensitive to lysis by syngeneic NK cells when incubated with peptide that can bind to their MHC class I molecules (Fig. 1). The process does not seem to be peptide sequence specific, in that all peptides tested sensitized targets for NK lysis provided that they could bind to MHC class I in the peptide-pulsing procedure. Concentrations of 1 to 10 pg/ml of the peptide tested (all of which bind with high affinity) were sufficient to produce significant sensitization (Fig. 1). For comparison, concentrations 100-fold lower are sufficient to sensitize lymphoblasts to lysis by CTL lines specific for the same or similar high affinity peptides (36).

It is now widely accepted that if an activating receptor on an NK cell recognizes a cell, that cell will be killed unless an inhibitory receptor on the NK cell also recognizes the cell. According to this model, the added peptide in our system must be altering the target cell either by creating a new ligand recognized by an activating receptor or by altering or masking a ligand recognized by an inhibitory receptor (or possibly both). To distinguish between these two possibilities, the most powerful approach is to use specific mAb F(ab')₂ fragments against the ligand as has been done to block the inhibitory MHC class I interaction (10, 45). However, this approach cannot yet be used in this study because the putative receptor involved in our system has yet to be identified (except that Ly49A is not involved). We have relied on cold target competition as an alternative for providing insight into the nature of the ligand affected by peptide pulsing and found that unlabeled Con A lymphoblast targets, regardless of whether peptide pulsed, were equally effective competitors for NK-mediated lysis of labeled peptide-pulsed targets (Fig. 2). We also found, using flow cytometry, that lymphoblasts pulsed or not pulsed with peptide were equally effective in forming conjugates with FITC-labeled NK cells (Fig. 3). Similarly, Ljünggren et al. found that the cell line RMA, which is moderately resistant to NK lysis, and RMA-S, a mutant cell line derived from it and highly sensitive to NK lysis, were equivalent in the ability to bind NK cells (46). Taken together with the fact that only peptide-pulsed targets are lysed, we conclude that the added peptide is most likely altering or masking the ligand recognized by an NK inhibitory receptor. The BFA experiments support this conclusion. Preventing surface arrival of proteins should not affect an activating ligand that is already there.

The observation that the ligand recognized by the NK inhibitory receptor operative in this system and the CTL receptor are affected differently by BFA leads to the hypothesis that they are recognizing different ligands and, in particular, that the inhibitory NK receptor recognizes the empty form of MHC class I molecules on syngeneic lymphoblasts. The data presented in Figures 1 to 5 can be explained by and support this hypothesis. Thus, normal lymphoblasts can be recognized by syngeneic NK cells, but their lysis is normally prevented when negative inhibitory signals generated by recognition of empty MHC class I molecules are above some threshold level and override the activation signal in the NK cell. Pulsing normal lymphoblasts with peptide of high binding affinity fills most (if not all) empty MHC class I molecules and thus reduces the negative inhibitory signal below the threshold level in some NK cells and renders the lymphoblasts susceptible to lysis (Fig. 1). When peptide-pulsed lymphoblasts are incubated in the absence of exogenous peptide (preincubation experiment, Fig. 3A), newly synthesized empty MHC class I molecules are transported to and expressed on the cell surface, where they regenerate the inhibitory structure that increases the inhibitory signal above the threshold level and thus prevents NK lysis. Furthermore, if MHC class I-specific peptide is added to the target cells again, lysis can be restored (Table I). If the "regeneration" of the inhibitory structure is prevented by blocking the expression of newly synthesized MHC class I in the preincubation step (BFA experiment, Fig. 4), the targets remain sensitive to lysis.

An understanding of the number of "empty" MHC class I molecules on the cell surface under varying conditions is central to our model. New empty MHC class I molecules might arise on the cell surface through loss of peptide from the trimolecular complex on the cell surface, as both peptide and ß₂m can freely and independently disassociate from the trimolecular complex (19, 47, 48). Alternatively, newly synthesized empty MHC class I molecules can also arrive at the cell surface (14, 15). Whether MHC class I molecules are truly empty or contain peptide binding with low affinity (p_L) that is readily lost is not clear. To distinguish between these possibilities, we directly examined the relative frequency of full and empty K^b MHC class I on B6 Con A lymphoblasts pulsed with a high affinity binding peptide (OVAp) and then incubated for 90 min in the presence or the absence of BFA (Fig. 6). In the presence of BFA, which should prevent the emergence of new empties from the cell interior, no new empties were detected after a 90-min incubation, implying that the trimolecular complex has a half-life much greater than 90 min and that little peptide was lost during this 90-min incubation. There is no direct measurement of the half-life of this trimolecular complex. However, a possibly similar Flu-NP-D^b trimolecular complex (the same as studied here, see Fig. 1) is known to have a half-life of 10 h (48). In the absence of BFA, a significant number of new empties appeared on the cell surface (Fig. 6). We assume that these are newly synthesized molecules exported from the cell interior. Their appearance correlates with the disappearance of sensitivity to NK lysis (Figs. 4 and 5). In examining the effect of BFA on normal B6 lymphoblasts, we found that empty MHC class I were still detected even after the cells were incubated for 4 h in the presence of BFA at 37°C, although a decrease in the level of empty MHC class I expression was observed. Given that the H-ß₂m forms of D^b and K^b molecules have been reported to have half-lives much less than 2 h (15, 19, 20), one might have expected unpulsed Con A lymphoblasts to have lost all their empty MHC class I molecules during the incubation with BFA. That they did not has two possible explanations: 1) new empty MHC class I molecules are continuously formed through loss of low affinity peptide from trimolecular MHC class I complexes (p_L-H-ß₂m) already on the cell surface; and 2) measurements of H-ß₂m half-lives have been made by extracting H-ß₂m complexes from the cell surface with mAb. Molecules embedded in the membrane of a normal, viable cell may be more stable. We conclude that the ligand for the inhibitory receptor operative in our system is most likely to be empty MHC class I.

The model that added peptides might be displacing protective "self" peptides is rendered unlikely by the current data

A 45-min pulse with a high affinity peptide produced a state of sensitization (Fig. 1). If the high affinity peptide is displacing particular protective self peptides, then they must be bound with low affinity to be displaced in such a short time pulse (19). This, then, implies that control target cells not pulsed with high affinity peptide and then incubated with BFA should have become sensitive to lysis as the protective self peptide was lost. This was not seen (Fig. 5).

An as yet unidentified inhibitory receptor, differing from Ly49A, recognizes the bimolecular form of the D^d molecule

As described in the introduction, three groups have shown that, under appropriate conditions, addition of MHC class I binding peptides to a target can prevent NK lysis (12, 13, 16). To reconcile the difference between these published and our experimental data, we studied the recognition of the D^d molecule by Ly49A⁺ and Ly49A^- NK subsets in our syngeneic experimental system. We found that the Ly49A⁺ subset of NK cells could not lyse syngeneic D^d-bearing lymphoblasts pulsed with D^d-binding peptide (Fig. 7), consistent with the Ly49A inhibitory receptor recognizing the D^d trimolecular complex and providing a dominant negative signal (Fig. 7). In agreement with this, the targets were lysed when Ly49A was covered up by F(ab')₂ anti-Ly49A mAb. The Ly49A^- subset of NK cells killed the same syngeneic D^d-bearing lymphoblasts pulsed with D^d-binding peptide, consistent with our hypothesis that there might be an as yet unidentified inhibitory receptor (which may or may not be a member of the Ly49 family) that recognizes the empty form of the D^d molecule.

In support of our hypothesis that there are inhibitory receptors recognizing empty MHC class I molecules, a recent report (49) concluded that cell surface expression of human MHC class I molecules, in the absence of peptide, was both necessary and sufficient to inhibit HLA-specific human NK lines and clones. They transfected RMA-S cells with human HLA-C of two different allotypes along with human ß₂m. Culture of the cells at 26°C without exogenous peptide allowed for high expression of the transfected class I, and this persisted for at least 2 h after the cells were transferred to 37°C. The presence of a particular empty HLA-C allotype was sufficient to inhibit lysis by an NK clone specifically inhibited by that allotype. Note that the inhibitory receptors involved in this study are most likely the members of the NK inhibitory receptor family, structurally unrelated to the Ly49 family (50, 51).

A teeter-totter model for resistance vs sensitivity to NK lysis

Correa et al. have shown that the Ly49A-D^d interaction is sufficient to inhibit all types of NK cell activation pathways that have been examined, but the contrary has been observed in this study (52). Our data showed that Ly49A⁺ NK cells could lyse D^d-expressing lymphoblasts pulsed with Flu-NP-K^d peptide even if they were also pulsed with D^d-binding peptide (Fig. 6C). This apparent discrepancy can be explained by the following model. 1) Individual NK cells have different inhibitory receptors that can recognize either empty or full MHC class I molecules. 2) The strength of the inhibitory signal generated by a particular receptor is proportional to the number of MHC class I molecules it can recognize. 3) For inhibition of lysis to occur, the summation of all inhibitory signals must exceed some critical threshold value.

Let us apply this model to all the data in Figure 7 using Ly49A⁺ NK cells (Table II). We assume that 10% (0.1) of K^d and D^d molecules on the lymphoblasts used are empty, as has been reported (14) for D^b MHC class I molecules, but would reach the same conclusions for any value >0 and <1. For normal lymphoblasts (Table II, line 1), there is a total inhibitory signal of 1.1 (0.9 (from Ly49A recognizing peptide-bound D^d) plus 0.1 (from a new receptor recognizing empty D^d) plus 0.1 (from a second new receptor recognizing empty K^d); no lysis is seen. When the lymphoblasts are pulsed with D^d-specific peptide (Table II, line 2), the total inhibitory signal remains 1.1, because as the Ly49A signal goes up by 0.1, the inhibitory signal generated by the receptor recognizing empty D^d goes down by the same amount, 0.1, and again no lysis is seen. However, when they were pulsed with K^d-specific peptide (line 3) or with both K^d- and D^d- specific peptide (line 4), the total inhibitory signal falls to 1.0; lysis is now seen. In going down Table II, one sees that lysis was observed whenever the summation of inhibitory signals was 1.0 or less. Comparison of lines 2 and 4 is particularly interesting, in that pulsing lymphoblasts with D^d-binding peptide alone does not block inhibition (line 2), but pulsing lymphoblasts with both K^d- and D^d-binding peptide does (line 4).

The model implies that there is a critical balancing of activating and inhibitory signals leading either to sensitivity or resistance to lysis. It is much like a teeter-totter in a children’s playground, in which a given end is either fully up or fully down depending upon the balance of the forces last acting on the two ends. Whether there is a subset of B6 NK cells with an inhibitory receptor that recognizes peptide-bound K^d molecules cannot be determined from these data, as inhibition or activation of such a subset is difficult to detect unless the subset is relatively pure. We could detect the effect of the Ly49A inhibitory receptor in our system only after purifying Ly49A⁺ cells.

Most previous studies (for an exception, see 53 supporting the existence of negative-signaling NK receptors have involved the protection from lysis of allogeneic target cells recognized by inhibitory receptors. The data presented here provide direct evidence that negative-signaling receptors can also protect normal syngeneic target cells from lysis. They also suggest a possible explanation for why some virus-infected cells become targets for syngeneic NK cells: as a result of the virus infection, very few empty MHC class I molecules are exported to the cell surface either because very large quantities of viral peptide inside the cell saturate MHC class I or because the virus greatly reduces overall MHC class I production such that few empties (albeit a higher percentage of all class I) reach the cell surface.

	Acknowledgments

 
We thank Dr. D. Raulet (University of California, Berkeley) for the kind gift of hybridoma JR9-318 with permission from Dr. J. Roland (Pasteur Institute, Paris, France), and J. Ferguson for help in HPLC purification of peptides and synthesizing the biotinylated peptides.

	Footnotes

 
¹ This work was supported by a Connaught-University of Toronto Research Fund grant and a Human Frontiers of Science grant (both to R.G.M.). S.K.-P. Kung is a research student of the National Cancer Institute of Canada supported by funds provided by the Canadian Cancer Society.

² Address correspondence and reprint requests to Dr. Richard G. Miller, Department of Medical Biophysics, Ontario Cancer Institute, 610 University Ave., Toronto, Ontario, Canada M5G 2M9.

³ Abbreviations used in this paper: KIR, human killer cell inhibitory receptor (e.g., p58.1, p58.2, or p70); H, heavy chain of MHC class I; p_H, MHC type I-specific peptide with high binding affinity; Flu-NP-D^b, restricted epitope of influenza nucleoprotein, ASNENMETM; Flu-NP-K^d, restricted epitope of influenza nucleoprotein, TYQRTRALV; Tum^-, L^d-restricted epitope, ISTQNHRALDLVAAK; HIVp-D^d, restricted epitope of human immunodeficiency virus gp160, RGPGRAFVTI; BFA, brefeldin A; OVAp_X-bio, biotinylated OVAp, bio-XSIINFEKL; OVAp_K-bio, biotinylated OVAp, SIINFEK(bio)L; GAD-Flu-NP, Flu-NP with three additional amino acids added to the N-terminus, GADASNENMETM; OVAp-K^b, restricted epitope of chicken ovalbumin, SIINFEKL; p_L, MHC class I-specific peptide with low binding affinity.

Received for publication December 19, 1997. Accepted for publication March 24, 1998.

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