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Influence of Xenogeneic ₂-Microglobulin on Functional Recognition of H-2K^b by the NK Cell Inhibitory Receptor Ly49C¹

Loralyn A. Benoit^*,, John Shannon, John W. Chamberlain^*, and Richard G. Miller^2,*,

^* Department of Immunology, University of Toronto, Toronto, Ontario, Canada; Division of Cellular and Molecular Biology, Ontario Cancer Institute, Toronto, Ontario, Canada; and Program in Infection, Immunity, Injury and Repair, Hospital for Sick Children, Toronto, Ontario, Canada

	Abstract

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NK cells maintain self-tolerance through expression of inhibitory receptors that bind MHC class I (MHC-I) molecules. MHC-I can exist on the cell surface in several different forms, including "peptide-receptive" or PR-MHC-I that can bind exogenous peptide. PR-MHC-I molecules are short lived and, for H-2K^b, comprise 10% of total MHC-I. In the present study, we confirm that signaling through the mouse NK inhibitory receptor Ly49C requires the presence of PR-K^b and that this signaling is prevented when PR-K^b is ablated by pulsing with a peptide that can bind to it with high affinity. Although crystallographic data indicate that Ly49C can engage H-2K^b loaded with high-affinity peptide, our data suggest that this interaction does not generate an inhibitory signal. We also show that no signaling occurs when the PR-K^b complex has mouse ₂-microglobulin (₂m) replaced with human ₂m, although replacement with bovine ₂m has no effect. Furthermore, we show that ₂m exchange occurs preferentially in the PR-K^b component of total H-2K^b. These conclusions were reached in studies modulating the sensitivity to lysis of both NK-resistant syngeneic lymphoblasts and NK-sensitive RMA-S tumor cells. We also show, using an in vivo model of lymphocyte recirculation, that engrafted lymphocytes are unable to survive NK attack when otherwise syngeneic lymphocytes express human ₂m. These findings suggest a qualitative extension of the "missing self" hypothesis to include NK inhibitory receptors that are restricted to the recognition of unstable forms of MHC-I, thus enabling NK cells to respond more quickly to events that decrease MHC-I synthesis.

	Introduction

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The reactivity of a NK cell is controlled by the balance of signaling received through its activation and inhibitory receptors (1). Inhibitory receptor engagement of MHC class I (MHC-I)³ or MHC-I like homologues promotes functional quiescence such that a reduction of MHC surface expression can tilt the signaling balance toward NK activation, resulting in elimination of the target cell (2). In the mouse system, NK inhibitory receptors that bind to MHC-I molecules belong to the Ly49 family (3). There is some controversy as to the specific form of MHC-I that is engaged by various Ly49 family members. This study provides new evidence supporting a previous conclusion (4) that the mouse inhibitory receptor Ly49C, which recognizes H-2K^b as a ligand, can generate an inhibitory signal only following engagement of the unstable subset of H-2K^b that is capable of binding exogenous peptide.

MHC-I molecules found on the cell surface are heterogeneous in conformation. The most stable form is a heterotrimeric complex composed of H chain (H) folded with an appropriate short peptide (p) and stabilized by ₂-microglobulin (₂m). Both peptide and ₂m are bound noncovalently to the H chain and can dissociate and/or exchange with peptide or ₂m from the surrounding medium (5, 6). Thus, in principle, MHC-I can exist on the cell surface in several forms, including lacking peptide or "empty" (H-₂m), lacking ₂m (H-p), or lacking both (free H). It is difficult to quantify the relative numbers of these different forms. However, with the exception of H-2L^d (7), truly peptide-empty forms of murine MHC-I appear highly unstable and are likely very few in number. In this regard, radiochemical studies using the Tap-2-deficient cell line RMA-S incubated at 26°C have revealed that the "empty" H-2K^b molecules on the cell surface are actually occupied by low-affinity peptide (8). However, when the temperature is raised to 37°C, there is a rapid loss of this low-affinity peptide and loss of any detectable H chain expression (8). As well, molecular dynamics simulation studies measuring conformational flexibility of the H chain ₁-₂ domain indicate that the truly peptide-empty form of MHC-I rapidly assumes a partially unfolded state consistent with a molten globule-type structure (9, 10), which is no longer capable of binding peptide. Thus, it appears that the dominant form of the "empty" MHC-I molecule that exists on the surface is in fact occupied by low-affinity peptide that can readily be replaced with exogenous peptide. Following the precedent of Day et al. (11), we refer collectively to these molecules and to truly empty molecules as peptide-receptive (PR) MHC-I molecules based on their ability to bind exogenous peptide.

In studies of activated mouse T lymphoblasts, it was found that mouse MHC-I H-2K^b molecules on the cell surface can be classified into two populations of approximately the same size but very different mean half lives of 1 and 20 h, respectively (12). The more stable population contains peptide bound with medium to high affinity. The less stable, which includes PR-K^b as a major component, contains peptide bound with low affinity. Accordingly, PR-MHC-I represents a significant proportion (10–15%) of total MHC-I on the cell surface and thus provides a practical rationale for this species of MHC-I to function as a structural ligand for immune receptors.

B₂m binds to MHC-I H chain and as well is present as a soluble factor in serum. B₂m bound to MHC-I can exchange with ₂m present in the serum. Both human (h-) and bovine ₂m (b-₂m) free forms can bind murine MHC-I H chain with an affinity two to three times higher than the binding of mouse ₂m (m-₂m) (13). Sequence comparisons indicate that h-₂m and b-₂m share 76 and 70% identity with m-₂m (13). The altered binding affinities and sequence differences of the different ₂m moieties could well induce changes in MHC-I structure capable of influencing recognition by Ly49. Indeed, ₂m has already been implicated in Ly49 recognition: MHC-I tetramer-analysis of the Ly49 ligand-binding repertoire indicates that certain Ly49 interactions, including H-2K^b engagement by Ly49C, do not occur when using tetramers complexed with h-₂m (14, 15, 16). Furthermore, functional studies using cultures containing bovine- and human-derived ₂m suggest that the presence of xenogeneic ₂m may interrupt recognition of H-2D^d by Ly49A (17) and that species-specific residues of m-₂m, namely Q29 and K58, when mutated to alanine or human-encoded residues, likewise interfere with Ly49A ligand binding (18, 19). Although it has been shown (20) that one of these ₂m residues, the Q29A point mutation, has only a nominal affect in Ly49C recognition of H-2K^b as determined by surface plasmon resonance analysis, the cocrystal structure analyses of both of these MHC-I/Ly49 interactions underscore the crucial role of ₂m in forming the inhibitory interface (20, 21).

There are currently two discordant hypotheses describing the molecular requirements for functional Ly49C/H-2K^b interaction. The first is based on an analysis of NK cell lysis of the H-2^b thymoma, RMA-S. This cell line expresses minimal surface MHC-I and thus behaves as an excellent NK target. Incubating RMA-S cells overnight at a reduced temperature (26°C) in the presence of a H-2K^b-binding peptide partially protected RMA-S from Ly49C⁺ NK-mediated lysis to an extent that varied with the peptide used (22). The authors concluded that recognition of H-2K^b by Ly49C required the presence of a bound peptide. The second model system used syngeneic T lymphoblasts as targets. Lymphoblasts express relatively high levels of MHC-I (both total and PR-MHC-I) and are not lysed readily by NK cells. However, when briefly pulsed with a high-affinity MHC-I-binding peptide, so as to occupy all PR-K^b molecules, the cells became sensitized to syngeneic Ly49C⁺ NK cell lysis. This led to the conclusion that Ly49C engages the PR conformation of H-2K^b (4).

This last study used an in vitro system using FBS-supplemented medium (4). On account of the ability of serum-derived b-₂m present in the medium to bind to mouse MHC-I molecules, thereby potentially altering the conformation of the inhibitory ligand, interpretation of these findings has been questioned. In experiments reported here using both T lymphoblast and RMA-S targets, we confirm that PR-K^b is indeed the natural ligand for Ly49C and moreover show that functional recognition can occur when PR-K^b is complexed with either m- or b-₂m but not when complexed with h-₂m. Importantly, these data imply that the details of ligand conformation are extremely critical for determining the affinity of Ly49 recognition.

	Materials and Methods

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Mice and cell lines

C57BL/6J, TapI^–/–, and FcR^–/– B6 mice (23) were purchased from The Jackson Laboratory and Taconic Farms. H-2D^b–/– and H-2D^bK^b–/– B6 mice (24) were established from breeding pairs donated by Dr. F. Lemonnier (Pasteur Institute, Paris, France). C57BL/6 (B6) mice expressing h-₂m as a transgene either in the absence or in the presence of m-₂m were generated at the Hospital for Sick Children by breeding B6 h-₂m transgenic mice (25) with B6 murine ₂m^–/– mice (26). Mice were maintained in the specific pathogen-free vivarium at the Ontario Cancer Institute according to institutional guidelines. Typically, tissue was obtained from female mice, aged 6–10 wk and cultured in -MEM (Invitrogen Life Technologies) supplemented with serum, 50 µM 2-ME, and 10 mM HEPES. RMA-S was obtained from Dr. P. Ohashi, (Ontario Cancer Institute, Toronto, Ontario, Canada).

Serum

Mouse serum (MS) was bought as pooled stock (Cedarlane Laboratories) or obtained from B6 mice via cardiac puncture. Human serum (HS) was obtained from healthy volunteers. FBS was purchased from Wisent. All sera were heat inactivated before use. For cytotoxicity experiments involving MS, the serum was fractionated at the 50,000 cutoff by size exclusion using an ultra-free 4-ml filter fitted with a Biomax 50K NMWL membrane (Millipore).

Peptides, h-₂m, and tetramer

H-2K^b-binding peptides were pOVA (SIINFEKL), residues 257–264 derived from chicken OVA (27) and pVSV (RGYVYQGL), residues 52–59 from vesicular stomatitis virus nucleoprotein (28). H-2K^d-restricted control peptide was pFNP (TYQRTRALV). Peptides were purchased from the Alberta Peptide Institute and were >95% pure by HPLC. Lyophilized h-₂m was purchased from Sigma-Aldrich Canada (purity >90%). Peptides and h-₂m were dissolved in PBS^–/– at 1 mg/ml and stored at –80°C. pOVA-H-2K^b tetramers were a kind gift from Dr. R. Tan (University of British Columbia, Vancouver, British Columbia, Canada).

Antibodies

The following mAbs were used: 5E6 (BD Biosciences), which reacts with Ly49C and Ly49I (29), Y-3 (American Type Culture Collection), which binds the ₁₂ domain of H-2K^b irrespective of the ₂m moiety present (30, 31, 32), AF6-88.5-88.5 (BD Biosciences), which recognizes H-2K^b when bound with m-₂m but not when bound with h-₂m or b-₂m (30, 33), KH95 (BD Biosciences), which binds H-2D^b irrespective of the ₂m moiety present (34, 35), W6/32 (American Type Culture Collection), which reacts with numerous HLA/MHC determinants, including b- or h-₂m-bound H-2D^b but not m-₂m bound H-2D^b (36), S19.8 (provided by Dr. U. Hammerling, Memorial Sloan-Kettering Cancer Center, New York, NY), which binds to m-₂m (37), and 25-D1.16 (provided by Dr. R. Germain, National Institute of Allergy and Infectious Diseases, Bethesda MD), which binds pOVA complexed with H-2K^b (38). Production of Abs from hybridomas involved isolation on protein G (Sigma-Aldrich Canada) or gammabind plus (Amersham Biosciences) Sepharose-based chromatography and FITC labeling using FITC-CELITE (Calbiochem).

Staining and flow cytometry

For staining, cells were washed in BSA/serum-free PBS^–/– buffer and resuspended at 2 x 10⁵ cells in 100-µl volumes for 30 min on ice. Determination of PR-K^b surface expression was ascertained by pulsing 2 x 10⁵ cells with 1 µg of pOVA or pVSV (control) on ice for 45 min, washing, and subsequently staining with 25-D1.16. In certain instances, cells were treated with brefeldin A (BFA) (5 µg/ml; Sigma-Aldrich Canada) to block intracellular transport. Samples were analyzed on a FACSCalibur (BD Biosciences) in logarithmic amplification of the fluorescent light signals and linear amplification of the forward/side scatter light signals following compensation to remove spectral overlap in the fluorescent channels. Data were analyzed on CellQuest software (BD Biosciences) using the gated, viable population as ascertained by linear scale forward/side scatter.

Target cell generation

B6 Con A blasts were generated by incubating 10⁷ splenocytes for 2 days in complete medium supplemented with 2 µg/ml Con A (ICN Pharmaceuticals Canada). Upon harvesting, viable cells were enriched on Lympholyte-M (Cedarlane Laboratories), washed, and incubated for 2 min in 200 mM methyl -D-mannoside/methyl -D-glucoside (Sigma-Aldrich Canada) to remove residual lectin.

Lymphokine-activated killer (LAK) effector generation

LAK cells were generated as described previously (4). Briefly, splenocytes were red cell depleted using Lympholyte-M, and adherent cells were depleted using nylon wool columns. The nonadherent fraction was NK enriched using anti-CD4, CD8, and B220-conjugated magnetic beads (Dynal). Remaining cells were cultured for 6–11 days in 5.0 ml of -MEM supplemented with 10% FBS, 50 µM 2-ME, and 10,000 U/ml human rIL-2 (Chiron). Ly49C⁺ and Ly49C^– LAK populations were stained using mAbs 5E6 and NK1.1 (PK136) and sorted using a BD FACSVantage. Cells were recultured for at least 48 h before use. Note that a previous study (4) had shown that Ly49I cannot recognize either H-2K^b or H-2D^b.

Cytotoxicity assay

RMA-S or Con A blasts were labeled for 90 min with 100 µCi Na⁵¹CrO₄ (NEN Life Science) in 100 µl of serum (Con A blasts) or 1% FCS in complete medium (RMA-S). Targets were then washed. In certain instances, labeled targets were then incubated with 1 µg/ml pOVA or 10 µg/ml h-₂m for 45 min at 26°C. Anti-Ly49C (5E6) was directly added at 1–2 µg/well to the plated effectors, followed by mixing. Targets were plated at 20,000 viable cells/ml in 100-µl aliquots. Plates were incubated at 26°C (for RMA-S targets) or 37°C (for Con A targets) for 4 h, after which 100 µl of cell-free supernatant were harvested. Percent-specific lysis at each E:T ratio was determined by averaging the counts per minute from five replicate wells and calculated as previously (4). All experiments reported here were repeated a minimum of three times.

Preparation and injection of donor cells

Splenocytes from h-₂m Tg, wild-type (Wt), and ₂m^–/– mice were depleted of red cells and labeled with 1 µM CFSE (BioCan Scientific) for 10 min at 37°C in serum-free PBS. Cells were washed twice in complete medium. Then 3.0 x 10⁷ cells were injected into the lateral tail vein of recipient mice. In vivo NK activation was induced by i.p. administration of 100 µg of polyinosine-polycytidylic acid (poly I:C) (Sigma-Aldrich Canada) at 1 and 24 h postengraftment. At 48 h postengraftment, inguinal and axillary lymph nodes were harvested and pooled. Single-cell suspensions were analyzed by flow cytometry to determine the relative percentage of CFSE-labeled leukocytes. The experiment was repeated three times. Error refers to SEM generated by quadruplicate measurements from one experiment.

	Results

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Effect of xenogeneic serum on lymphoblast lysis by syngeneic LAK

To test whether the source of serum used during the in vitro generation of mouse Con A blasts had an effect on sensitivity to lysis by syngeneic NK cells, B6 splenocytes were cultured for 2 days in complete medium containing 2 µg/ml Con A and either FBS, MS, or HS. The cells were then assessed for sensitivity to lysis by syngeneic lymphokine-activated NK cells (LAK). As demonstrated in Fig. 1.1.A and in accord with previous findings (4), Con A blasts that had been cultured in medium supplemented with 10% FBS and then pulsed with the H-2K^b high-affinity binding peptide, pOVA, before the killing assay, were 5.6-fold more sensitive to lysis (based on LU analysis of the titration data) than Con A blasts that had been left unpulsed or pulsed with a control peptide. Target cells grown in 3% (syngeneic) MS behaved similarly, becoming 6.8-fold more sensitive to lysis when pulsed with pOVA relative to unpulsed or control pulsed targets (Fig. 1.1.B). However, Con A blasts grown in 3% HS were not further sensitized to lysis following addition of peptide. Indeed, the background lysis of (unpulsed) targets grown in HS was much higher than for cells grown in FBS or MS and was comparable to that seen when FBS- or MS-cultivated Con A blasts were pulsed with pOVA (Fig. 1.1.C). Control growth kinetic experiments titrating the different sera indicated that 10% FBS, 3% MS, and 3% HS produced comparable growth of Con A blasts (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Cells grown using different sera compared for sensitivity to lysis by syngeneic LAK and for expression of different forms of MHC-I. 1.1) Day 2 B6 Con A blasts cultured in 10% FBS (A), 3% MS (B), or 3% HS (C) were assessed for sensitivity to lysis by syngeneic LAK (35% Ly49C+). Assay cultures contained 1% of the respective serum. Target cells were either untreated or pulsed with pFNP (pCTRL, does not bind) or with pOVA, as indicated. 1.2) Day 2 B6 Con A blasts cultured in 10% FBS were assessed for sensitivity to lysis by syngeneic LAK. The effects of pulsing with pOVA and blockading Ly49C are compared either performed separately or together. 1.3) Flow cytometric analysis of MHC-I and 2m expression levels on cells from Fig. 1.1.

Modulation of LAK-mediated lysis of a resistant target

We hypothesized that binding of h-₂m to H-2K^b interferes with the ability of Ly49C to engage its ligand. To assess directly whether the effect is due to h-₂m and not some other serum component, lymphoblast targets were cultured and assayed in medium containing reduced (5%) FBS with or without being pulsed with exogenous-purified h-₂m (10 µg/ml) immediately before being used as targets for Ly49C⁺ and Ly49C^– LAK in the presence of anti-Ly49C mAb, pOVA (1 µg/ml), or mock treatment. The results are summarized in Table II. Pulsing target cells with h-₂m resulted in enhanced Ly49C⁺ LAK-mediated killing that was comparable to the lysis achieved through either pOVA pulsing or Ly49C blockade. We conclude that inclusion of h-₂m is altering the PR-K^b structure such that it is no longer recognizable by Ly49C. However, h-₂m appeared to exert an additional effect: although killing by Ly49C^– LAK was unaffected by either peptide pulsing or Ly49C blockade, it was enhanced by addition of h-₂m to the same extent as the Ly49C⁺ LAK-mediated killing. Note that this same effect was seen previously for target cells cultured in HS (Table I). One explanation is that there is an inhibitory receptor (not Ly49C) that is specific for H-2D^b but which cannot recognize H-2D^b complexed with h-₂m. To test this, the experiment was repeated using H-2D^b–/– B6 mice for the generation of both target cells and LAK (Table II). As before, inclusion of h-₂m in the assay made H-2D^b–/– Con A blasts sensitive to Ly49C⁺ LAK-mediated killing. However, now inclusion of h-₂m did not augment Ly49C^– LAK-mediated killing, supporting the notion of a novel H-2D^b-specific inhibitory receptor (see Discussion).

View this table: [in this window] [in a new window]   Table II. Effect of exogenous h-2m on B6 lymphoblast sensitivity to lysis by Ly49C+ or Ly49C– LAKa

Dynamics of PR-K^b on the cell surface

Previous work (12, 39) has shown that PR-MHC-I on the cell surface has a short half-life but is being continuously replaced from the cell interior. Cells made sensitive to lysis by being pulsed briefly with peptide (thus ablating PR-MHC-I) regain resistance to lysis after an incubation of 90 min in the absence of peptide through replacement of the inhibitory ligand by de novo export of PR-MHC-I from the cell interior. However, cells remained sensitive to lysis when export of protein from the interior was blocked by addition of BFA, a blocker of protein export. The converse of this is that cells resistant to lysis should become sensitive after being incubated with BFA for a time sufficiently long (at least 3 h) for the number of PR-MHC-I on the cell surface to decay below the critical number required for the generation of an inhibitory signal. The experiment of Fig. 2 demonstrates this to be the case. Normal B6 Con A blasts either pulsed with pOVA or pulsed with h-₂m or incubated with BFA for 3.5 h all became approximately four times more sensitive to lysis (LU analysis) than control untreated cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Effect on sensitivity to lysis of long incubation in BFA vs short pulse with pOVA or h-2m. Day 2 B6 Con A blasts were pulsed with pOVA or h-2m as in Table II or were cultured for 4 h in BFA before being compared for sensitivity to lysis by syngeneic LAK.

Relationship between H-2K^b molecules that bind peptide and that exchange ₂m

When Con A blasts were incubated in vitro with HS, h-₂m appeared to bind to only a small fraction of total H-2K^b molecules (compare Fig. 1.3, D and E). Similarly, when Con A blasts are pulsed with pOVA, peptide binds to only a small fraction (10%) of H-2K^b molecules (see e.g., Ref.12). In either case, this low level of binding is sufficient to alter H-2K^b such that it was no longer functionally recognizable by Ly49C. As addition of either h-₂m or pOVA produced equivalent enhancement of lysis by Ly49C⁺ NK cells (e.g., Tables I and II), we assessed whether h-₂m might have a preferential ability to complex with PR-K^b molecules. B6 Wt Con A blasts were either untreated or pretreated with pOVA (45 min at 37°C) to ablate PR-K^b molecules and then assessed by flow cytometry for the ability of H-2K^b molecules to bind exogenous h-₂m. Because nascent PR-K^b molecules are being continuously exported to the cell surface, the experiments were done in the presence of BFA. Binding of h-₂m was then assessed by comparing flow cytometric MFI values for mAb Y-3 (recognizes H-2K^b bound by either h-₂m or m-₂m; see Fig. 3) with MFI values for mAb AF6-88.5 (recognizes H-2K^b bound by m-₂m but not h-₂m; again see Fig. 3) for cells exposed to h-₂m that had or had not been pretreated with pOVA. Results are shown in Fig. 4.A and are normalized to the MFI for cells treated with neither pOVA nor h-₂m (Fig. 4.A, line 1). For cells not exposed to pOVA, incubation with h-₂m (45 min at 37°C) reduced m-₂m specific AF6-88.5 binding by 13%, while slightly increasing Y-3 staining (Fig. 4.A, lines 2 vs 1), suggesting that h-₂m was binding to 13% of total H-2K^b. When cells were pretreated with pOVA before addition of h-₂m, total H-2K^b expression was elevated 12–15% (Fig. 4.A, compare lines 1 and 3), but there was little difference in AF6-88.5 staining (line 3), which is consistent with there being no sites available for h-₂m to bind following pOVA addition. Thus, binding of pOVA to PR-K^b appears to prevent ₂m exchange. The elevated H-2K^b expression seen in Fig. 4.A, line 3, was likely due to the stabilizing effect of peptide binding to PR-K^b molecules that otherwise would have denatured. Consistent with this reasoning, pulsing with pOVA alone increased the staining of both mAbs (Fig. 4.A, line 4). We conclude that ablation of PR-K^b greatly reduces the ability of H-2K^b to bind exogenous h-₂m.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Flow cytometric analysis of the binding of the anti-H2-Kb mAbs AF6-88.5 (A) and Y-3 (B) to spleen cells from either Wt or transgenic h-2m mice.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Alterations in H-2Kb epitopes produced by addition of peptide and/or h-2m. Con A blasts cultured in 5% FBS were treated with BFA (5 µg/ml) and then given two consecutive 45-min incubations with nothing or pOVA (1.0 µg/ml) in the first and nothing or h-2m (10 µg/ml) in the second (A) or nothing, h-2m (1.0 µg/ml), or pVSV (1.0 µg/ml) in the first and nothing or pOVA (1.0 µg/ml) in the second (B). Cells in each group in A were stained with either mAb Y-3 (recognizes H-2Kb containing h- or m-2m) or with mAb AF6-88.5 (recognizes only H-2Kb containing m-2m). Cells in each group in B were stained with mAb 25D-1.16 (recognizes PR-Kb).

Influence of transgenic h-₂m on target cell susceptibility in vitro

In all the above experiments, ₂m exchange has been limited to a small subset of all MHC-I molecules. We sought to confirm our findings using cells in which all MHC-I molecules contained h-₂m. To this end, B6 m-₂m^–/– mice were bred with h-₂m transgenic B6 mice (see the Materials and Methods). Con A blasts derived from Wt (only m-₂m), heterocompound (both h-₂m and m-₂m) and h-₂m transgenic (only h-₂m) B6 mice were tested as targets for Wt B6 LAK (Fig. 5.1.A–C). Both heterozygous and h-₂m transgenic targets demonstrated elevated sensitivity to Wt LAK. Peptide pulsing increased the lysis of Wt control targets but caused no additional increase in lysis of targets containing h-₂m.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Effect of replacing m-2m with h-2m on sensitivity to LAK-mediated lysis and on expression of MHC-I. 5.1) Cells from Wt B6 (A), heterozygous B6 (both h- and m-2m; B), and transgenic B6 (only h-2m; C) were cultured in medium containing FBS and tested for sensitivity to lysis by Wt B6 LAK. Target cells were either untreated or pOVA pulsed before assay. 5.2) Flow cytometry analysis of H-2Db, H-2Kb, PR-Kb, h-2m, and m-2m on Wt, heterozygous, and transgenic Con A target cells.

View this table: [in this window] [in a new window]   Table III. Effect of endogenous h-2m on B6 lymphoblast sensitivity to Wt B6 Ly49C+ or Ly49C– LAKa

Influence of transgenic human-₂m on target cell survival in vivo

MHC-I incompatible lymphocytes i.v. injected into a mouse are rapidly eliminated by host NK cells, whereas syngeneic cells survive and enter the recirculating lymphocyte pool (40). Survival can be easily monitored by fluorescence labeling of the grafted cells before injection. We applied this model to assess whether injected leukocytes, syngeneic to the host except for replacement of m-₂m with h-₂m, would persist in vivo. In this regard, splenocytes obtained from Wt (syngeneic MHC-I, should survive in vivo), ₂m^–/– mice (insignificant MHC-I expression, should be rejected in vivo), or h-₂m transgenic mice were labeled with CFSE and injected (3.0 x 10⁷/recipient) into Wt B6 mice. In addition, a portion of recipients were treated with poly I:C via i.p. injections, as this is known to induce activation of NK cells in vivo (41). At 48 h posttransplantation, rejection of both h-₂m⁺ cells and ₂m-deficient cells was moderate in control animals but near total in animals that were treated with poly I:C (Fig. 6). As expected, the Wt graft was retained, regardless of treatment. As H-2K^b and H-2D^b expression levels were similar for both the Wt and h-₂m transgenic tissue (Fig. 5.2, A and B), rejection of h-₂m transgenic leukocytes coincided with altered MHC-I conformation and not altered MHC-I frequency.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Effect of replacing m-2m with h-2m on otherwise syngeneic leukocyte engraftment: CFSE-labeled splenocytes (3.0 x 107/recipient) from Wt, h-2m-transgenic, and 2m-deficient B6 mice were infused into Wt B6 mice. Recipients were either untreated () or treated with poly I:C (). Inguinal and axillary lymph nodes were harvested at 48 h, and pooled leukocytes were assessed for percentage of CFSE+ cells via flow cytometry. Error refers to SEM generated by quadruplicate measurements of the same experiment. Results are representative of three experiments.

RMA-S tumor cells have greatly reduced MHC-I expression. This makes the cells very sensitive NK targets. Expression of MHC-I can be partially rescued by incubating the cells overnight at 26°C (42, 43). RMA-S cells were incubated overnight at 26°C and then also incubated for an additional 1.5 h (still at 26°C) with nothing, with pOVA, or with h-₂m. Cells were then immediately tested for sensitivity to lysis by Ly49C⁺ LAK effectors at 26°C (Fig. 7.A). Both the h-₂m-pulsed and pOVA-pulsed targets were substantially more sensitive to lysis than unpulsed targets. Based on the results presented above using Con A blasts, we hypothesize that there were appreciable numbers of PR-K^b molecules on the RMA-S cell surface after overnight incubation at 26°C. The h-₂m pulsing rendered these PR-K^b molecules unrecognizable by Ly49C, and the pOVA pulsing converted them to equally unrecognizable stable H2-K^b molecules. As a first test of this, MHC-I levels were also measured for the three conditions (Table IV). H-2K^b expression was increased >5-fold by pulsing with pOVA. The implication is that this increase in total H-2K^b expression was due to binding of pOVA to unstable PR-K^b molecules present during the pOVA pulse. Indeed, staining with the mAb against the pOVA-K^b complex (indicated in the pOVA-K^b column) was up 20-fold over background. In the present study, this is a measure of the amount of PR-K^b present at the time of pulsing. It is clear that the pOVA pulsing produced not only an increased level of H-2K^b expression but also an increase in lysis, clearly incompatible with peptide-loaded H-2K^b being the ligand for Ly49C but in accord with the PR-MHC-I model of Ly49C/H-2K^b recognition. Note that Table IV contains a quantitative analysis of target cell sensitivity for the different treatments and effector populations (E:T ratio required for 15% lysis), as well as the MHC-I expression level data.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. Lysis of a NK-sensitive tumor target. RMA-S was incubated overnight at 26°C with or without pOVA (10 µg/ml). Target cells were labeled and then some pulsed with h-2m or pOVA at 26°C as indicated and assessed for sensitivity to lysis, also at 26°C, by Ly49C+ (A) or Ly49C– (B) LAK effectors. C, RMA-S cells treated with h2m, pVSV, or pOVA as indicated were assessed for sensitivity to lysis at 37°C by unfractionated B6 LAK.

View this table: [in this window] [in a new window]   Table IV. Effect of exogenous peptide and h-2m on RMA-S sensitivity to Wt Ly49C+ LAK and on MHC-I expressiona

Lysis of RMA-S cells by Ly49C^– NK cells was similarly tested (Table IV, Fig. 7.B). Exposure to pOVA overnight produced a >10-fold decrease in sensitivity to lysis (LU analysis) as compared with cells unexposed to pOVA. In contrast, RMA-S cells exposed to pOVA overnight and then prepulsed with pOVA again immediately before assay produced a very similar increase in susceptibility to lysis (>5-fold) as only prepulsing before assay with peptide. It is known that pOVA can bind to H-2D^b, although it does so with very low affinity (44), consistent with the small increase in H-2D^b expression seen in Table IV. The results are consistent with a novel inhibitory receptor that is specific for peptide-loaded H-2D^b but one that cannot recognize H-2D^b that contains h-₂m. It is most likely the same receptor as implicated above (Tables I–III) in the lysis of B6 Con A blasts by Ly49C^– LAK following pulsing with h-₂m (see Discussion).

The lysis values indicated in Fig. 7, A and B, are low as the cytotoxicity assay was done at 26°C. To verify that the same conclusions apply at 37°C and also to test directly the effect of overnight pulsing with a peptide of lower affinity, we did the experiment of Fig. 7.C in which we also tested pVSV, a peptide that binds H-2K^b but with much less affinity than pOVA. Thus, the K_D, defined here as the concentration of peptide present during pulsing required to sensitize B6 Con A blasts to half-maximum values of NK lysability, was 1000-fold higher for pVSV than for pOVA (100 vs 0.1 pM) (4). Cells incubated overnight with either pOVA or pVSV were protected compared with the no peptide control, but the protection conferred by pVSV was substantially less (Fig. 7.C, compare with and ). Cells incubated overnight without peptide and then pulsed immediately with either pVSV or pOVA before assessment were also tested. Lysis of both pVSV- and pOVA-pulsed targets increased relative to unpulsed control, with pOVA-pulsed target cells demonstrating greater sensitization (Fig. 7.C, compare with and ). Thus, although the lysis levels are greater at 37°C, the results are qualitatively similar at the two temperatures. Furthermore, pVSV, which binds with lower affinity, conferred less effective protection, thus suggesting that peptides with different MHC-I-binding affinities can selectively affect ligand recognition by Ly49C⁺ LAK.

Modulation of NK-mediated lysis of a NK-sensitive nontransformed target

As a final test of the PR-MHC-I concept, we assessed the sensitivity to LAK lysis of two untransformed cell sources that have either very low or no MHC-I expression and which are thus very sensitive to NK lysis. Specifically, target cells were Con A blasts derived from B6 Tap-1^–/– and B6 K^bD^b–/– mice. Tap-1-deficient tissue, as with RMA-S, is capable of exporting and presenting PR-K^b molecules on the cell surface. High-affinity peptide-filled MHC-I does not occur in these cells due to the absence of an intact peptide transporter complex. In contrast, K^bD^b–/– mice completely lack PR-K^b as the genes for both H-2K^b and H-2D^b are deleted in these animals. Results of measuring their respective sensitivity to lysis by Wt B6 LAK are shown in Fig. 8.A. Both knockout targets were more sensitive to lysis than Wt controls, but K^bD^b–/– target cells were 5-fold more sensitive (LU analysis) than the Tap-1^–/– Con A blasts. Pulsing with either pOVA or h-₂m did not change the sensitivity to lysis of the K^bD^b–/– targets but increased the lysis of the Tap-1^–/– 5-fold to a level comparable to that seen for the K^bD^b–/– targets. This is consistent with the fact that Tap-1^–/– but not the K^bD^b–/– targets bear PR-K^b on the cell surface and are thus partially spared from B6 LAK-mediated cytolysis. To functionally map this observation, Ly 49C⁺ and Ly 49C^– B6 LAK were tested separately for their ability to kill the Tap-1^–/– and K^bD^b–/– B6 targets (Fig. 8, B and C). Ly49C^– effectors killed both types of targets equally well, but Ly49C⁺ were significantly less effective at killing Tap-1^–/– than K^bD^b–/– target cells. This finding is consistent with the model that Ly49C recognizes PR-K^b, which is present on Tap-1^–/– targets but not on K^bD^b–/– targets.

View larger version (28K): [in this window] [in a new window]   FIGURE 8. Lysis of two NK-sensitive nontumor targets. Con A blasts were generated from B6 Tap-1–/– or B6 DbKb–/– mice and tested as target cells for B6 LAK effector cells, either unfractionated (A) or sorted into Ly49C+ and Ly49C– fractions (B and C). A, Targets were tested with or without added pOVA as in Fig. 1.

	Discussion

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We found that B6 Con A blasts pulsed briefly with a peptide (pOVA) that binds to H-2K^b with high affinity became sensitive to lysis by syngeneic NK cells. When the NK cells were fractionated on the basis of Ly49C expression, only the Ly49C⁺ NK cells lysed pOVA-pulsed syngeneic blasts. Blockading effector cell Ly49C with anti-Ly49C mAb instead of pulsing the target cell with pOVA also enabled lysis (Fig. 1.1, Tables I–III, confirming Ref.4). Simultaneously, pulsing with peptide and blockading Ly49C produced no additional lysis (Fig. 1.2). Our interpretation of these data is that Ly49C binds to PR-K^b with sufficient affinity to generate an inhibitory signal and that binding of pOVA to PR-K^b is altering the configuration of the PR-K^b molecule such that, even if weak Ly49C binding can still occur, it can no longer generate an inhibitory signal.

It is important to note that the mAb 5E6, used here to separate Ly49C⁺ and Ly49C^– NK cells, can also recognizes Ly49I (29, 45). In a previous study (4), we concluded that Ly49I was not involved in the phenomena described here. 5E6⁺ cells were sorted into Ly49C⁺ and Ly49I⁺ fractions. Neither fraction lysed normal B6 Con A blasts, whereas B6 Con A blasts pulsed with K^b-binding peptide were lysed by the Ly49C⁺ fraction but not by the Ly49I⁺ fraction. It was further shown that H-2D^b–/– Con A blasts (but not H-2K^b–/– Con A blasts) could bind to COS-7 cells transiently expressing Ly49C and that this binding did not occur when pOVA was added to the medium. COS-7 cells transiently expressing Ly49I could bind neither B6 H-2D^b–/– nor B6 H-2K^b–/– Con A blasts in either the presence or the absence of pOVA. Therefore, it seems unlikely that Ly49I is contributing to the effects mapped out in these experiments.

We found that the above results were equally valid for Con A blasts generated in medium containing either FBS or MS. Although when cells were cultured with FBS, exchange of b-₂m for m-₂m could occur on a small fraction of MHC-I molecules (Fig. 1.3, D and E); this appeared to have no effect on the recognition processes (Fig. 1.1, A and B). However, the situation was different for cells cultured in HS. Again, ₂m exchange occurred for only a small fraction of all MHC-I molecules. For H-2K^b, exchange occurred predominantly for the 10% of all molecules that were peptide receptive (Fig. 4.A). These PR-K^b molecules remained peptide receptive even after binding h-₂m (Fig. 4.B). However, binding of h-₂m made the B6 Con A blast targets equally as sensitive to lysis by Ly49C⁺ NK cells as either the ablation of PR-K^b by pulsing with pOVA or the blockade of Ly49C with anti-Ly49C mAb (Table II). Our interpretation of these data is that exchange of h-₂m for m-₂m on PR-K^b is altering the conformation of the H-2K^b molecule such that it is no longer recognizable by Ly49C.

As indicated in the introduction, MHC-I molecules on the surface of a B6 Con A lymphoblast can be evenly partitioned on the basis of stability into a long-lived group containing peptide bound with medium to high affinity and a short-lived population, to which PR-MHC-I belongs (12). To maintain the 50:50 steady-state ratio between the two populations, it appears that 20 short-lived molecules must be exported to the surface for each long-lived molecule. Biochemical evidence indicates that a newly synthesized MHC-I molecule can be exported from the endoplasmic reticulum (ER) only after the H chain has been properly folded and has associated with both ₂m and peptide (46). Only peptides with a correct binding motif can bind MHC-I with high affinity. In general, high-affinity peptides are supplied in the ER via Tap, a pore complex required for the export of peptides from the cytoplasm into the ER. The best evidence for this model comes from studies using RMA-S, which, as a result of a lesion in the gene encoding for Tap-2, lacks functional Tap complexes and therefore exports few stable MHC-I molecules to the cell surface (47). Day et al. (11) have made a detailed comparison of the properties of PR-K^b on RMA-S and its parent line, RMA, which expresses customary levels of MHC-I on the surface. They found that parental RMA expresses three to five times more PR-K^b on the cell surface than RMA-S but that the total rate of production and transport of H-2K^b was approximately the same in the two cell lines. These seemingly paradoxical results can be explained as follows. Both RMA and RMA-S cell lines are assembling MHC-I in the ER at the same rate (11), and these molecules require a peptide to complete assembly. Assuming RMA are similar to Con A blasts (12), only 1 in 20 H-2K^b molecules in RMA will bind a peptide with high affinity (this peptide being provided via Tap). The remainder will bind peptides with lower affinity, provided either via Tap or via a Tap-independent pathway. PR-MHC-I molecules denature rapidly upon reaching the cell surface but some will transiently function as PR-K^b molecules before doing so. RMA-S cells only have access to peptides that reach the ER via a Tap-independent pathway. Given that the total export rate is the same as for RMA, the availability of peptides that can bind is therefore not limiting. However, the average binding affinity of these imperfectly derived peptides is likely even lower than the low-affinity peptides loaded by RMA and, accordingly, the lifetime of these PR-MHC-I on the cell surface even shorter than for RMA. This would explain why Day et al. (11) detected three to five times more PR-K^b on the surface of RMA than on RMA-S.

The effects of peptide pulsing on the lysis of RMA-S (Table IV, Fig. 7) have the same pattern as observed for peptide pulsing of Con A blasts. RMA-S cells were used after overnight culture at 26°C to allow for maximal expression of MHC-I on the cell surface. In accordance with Day et al. (11), we hypothesize that a major portion of this MHC-I will be in the peptide-receptive state. Despite the overnight incubation, these cells were still strongly lysed by NK cells. However, they became 5-fold more sensitive to lysis by Ly49C⁺ NK cells when pulsed with either pOVA or h-₂m immediately before the cytotoxicity assay (Fig. 7.A). Our interpretation is that peptide pulsing is ablating the PR-K^b ligand by converting it into stable H-2K^b bound with high-affinity peptide and that addition of h-₂m is making the PR-K^b ligand unrecognizable by Ly 49C. In support of this, the pOVA pulsing increased the total amount of detectable H-2K^b by 5-fold, whereas the h-₂m pulsing produced only a slight increase (Table IV, compare lines 1–3). Note that if Ly49C were recognizing H-2K^b loaded with peptide, then, because by flow cytometric measurement the level of H-2K^b expression went up substantially, the peptide pulsing should have decreased not increased sensitivity to lysis.

RMA-S cells were also tested after overnight culture at 26°C in the presence of pOVA peptide. These cells were significantly less sensitive to lysis by Ly49C⁺ NK cells than cells incubated overnight without peptide (Fig. 7.A). The incubation produced large increases in both H-2K^b and PR-K^b expression (Table IV). We propose that it is the increased expression of PR-K^b that is responsible for protection because, when these cells were pulsed with pOVA immediately before the cytotoxicity assay, their sensitivity to lysis increased substantially and was now nearly identical to the lysis values of cells not incubated with pOVA overnight but pulsed with pOVA immediately before the cytotoxicity assay (Fig. 7.A). The observations are fully compatible with the PR-K^b model but cannot be explained with the peptide-loaded H-2K^b model. We offer the following possible explanation as to why overnight exposure to pOVA favors generation of PR-K^b. From the data of Day et al. (11), PR-K^b on RMA-S probably has a shorter mean lifetime than on RMA. In the overnight cultures, it is clearly possible that much of the added pOVA will degrade into pieces that will bind with only low affinity but perhaps with an affinity higher than the very poor peptides that can be found in the ER in the absence of Tap. These might well increase the size of the PR population by increasing its mean lifetime.

The above arguments provide an explanation for how cells lacking functional Tap can still express PR-MHC-I. However, cells completely lacking the genes for MHC-I should also completely lack expression of PR-MHC-I. We compared cells from Tap-1^–/– and K^bD^b–/– mice and found that this was indeed true (Fig. 8).

In the B6 Con A lymphoblast system, we found (Table II) that Wt cells pulsed with h-₂m became targets for Ly49C^– NK cells but that cells from a H-2D^b knockout mouse did not. Even though pOVA can bind (but with very low affinity (44)) to H-2D^b, pulsing with pOVA had little effect. We conclude that a NK inhibitory receptor that recognizes H-2D^b loaded with peptide is likely involved. The same receptor appears to be relevant in the RMS-S system. In the present study, either overnight incubation or brief pulsing with pOVA greatly reduced sensitivity to lysis (Table IV, Fig. 7.B), perhaps by stabilizing enough peptide-loaded H-2D^b to serve as ligand for this receptor. In both the RMA-S and Con A blast systems, it appears that pulsing with h-₂m converted H-2D^b into an unrecognizable conformation as lysis went up markedly following h-₂m pulsing.

MHC-I tetramer technology has been applied to the visualization of the Ly49-binding repertoire (14, 15, 16). In particular, the H-2K^b-pOVA tetramer has been shown to bind to Ly49C if the ₂m component is of mouse origin but not if it is of human origin (16). To test whether such tetramers can contain PR-MHC, we obtained H-2K^b tetramers containing pOVA and h-₂m. We then assessed whether these tetramers contained PR-K^b subcomponents by raising bulk CTL lines specific for H-2K^b-pOVA and H-2K^b-pVSV. As expected, H-2K^b-pOVA tetramer stained the H-2K^b-pOVA-specific CTL but not the H-2K^b-pVSV CTL. However, when an aliquot of the tetramer was incubated with pVSV, it acquired specificity for the H-2K^b-pVSV CTL line. The degree of staining was almost undetectable for a short pulse but increased linearly with time for longer incubation periods (unpublished data). These results imply that the monomer components of the tetramer can form short-lived PR-MHC molecules that can be rescued from decay by binding another peptide such as pVSV. This pVSV is most likely binding to a truly "empty" H-2K^b molecule. The fact that there was almost no binding of pVSV in an initial pulse implies that the truly empty H-2K^b molecule has an extremely short half-life. When these semi-PR tetramers were used to stain Ly49C⁺ LAK, we found, in agreement with others (16), that they did not bind Ly49C (unpublished data). This observation is consistent with our earlier observation that, although h-₂m can contribute to the formation of PR-MHC-I, the structure of the PR heterodimer is altered such that is it unable to be recognized by Ly49C.

Complexes of Ly49C and H-2K^b containing high-affinity peptide (the same pOVA peptide as we have used here) have been crystallized and characterized (20). This observation is not necessarily incompatible with our concept of PR-K^b being the ligand that triggers Ly49C signaling. Our argument hinges on the possibility that Ly49C can bind to a number of different ligands with a range of different affinities but with only binding events of higher affinity being able to trigger a signaling event. In the same study (20), surface plasmon resonance analysis was used to compare the binding of Ly49C to H-2K^b loaded with pOVA or pVSV, the pVSV also being the same peptide as we have used here. It was found that Ly49C binds much more strongly to pOVA-K^b complexes than to pVSV-K^b complexes. Yet, the interaction of pOVA-K^b with Ly49C was measured to have a K_D of only 80 µM. In an interaction between cells, the total Ly49C-K^b interaction strength may be markedly strengthened by the interaction of the Ly49C homodimer on the NK cell with two peptide-loaded H-2K^b molecules on the target cell (20). However, this particular configuration may still prove to have too low an affinity to trigger an inhibitory signal.

It is known that Ly49C possesses a remarkably promiscuous H-2 ligand-binding repertoire, including H-2^{b, d, f, k, q, r, s, v} elements (14, 15, 29, 48), although a functional consequence of Ly49C engagement has only been demonstrated for H-2K^b (4, 22, 48, 49). It is possible that this functional consequence only occurs after binding to PR-K^b. However, "PR-K^b" is an operational definition that likely includes two very different structures, these being H-2K^b complexed with a peptide bound with low affinity and a transitory true "empty" that has lost the low-affinity peptide and will rapidly denature unless stabilized by recapture of another peptide. As argued above, this truly empty form is probably present in extremely low levels such that almost all PR-K^b is likely to be H-2K^b loaded with low-affinity peptide. It is possible that some of these molecules, which may only be slightly different from H-2K^b loaded with high-affinity peptide, will bind to Ly49C with much higher affinity.

PR-K^b is likely to be very heterogeneous. Binding of peptide to H-2K^b with high affinity requires that the peptide be the right length (8 or 9 aa) and have two critical anchor residues (50). Loss of either anchor and/or an improper length could lead to a low-affinity binding event, and the details of how this peptide is bound could affect overall structure. By our measurements, only a subset (20%) of all unstable H-2K^b complexes can bind to Ly49C with sufficient affinity to trigger a signaling event (4, 12). Because these MHC-I structures are all unstable, they are unlikely to be caught in a crystal structure.

Mouse NK cells appear to have several inhibitory receptors with specificity for PR-MHC-I. Con A blasts from either B6 or BALB/c become sensitive to lysis after pulsing with a peptide that can bind to H-2K^b, H-2D^b, H-2K^d, H-2D^d, or H-2L^d (51). However, to date, Ly49C is the only characterized inhibitory receptor that has been identified as having PR-MHC-I as ligand (4). In contrast, Ly49A, which is expressed on BALB/c NK cells, has been shown to engage H-2D^d complexed with high-affinity peptide (39), although this same study provided evidence that BALB/c NK cells have inhibitory receptors with specificity for both PR-K^d and PR-D^d and that these receptors are present on both Ly49A⁺ and Ly49A^– NK cells. Humans may also have NK inhibitory receptors that respond to PR-MHC-I (52). Moreover, Mandelboim et al. (53) showed that RMA-S cells transfected with different alleles of HLA-C were partly protected from lysis by human NK clones that could recognize the particular allele transfected. These authors emphasized that these HLA-C constructs were being recognized in the absence of high-affinity peptide binding.

The PR-MHC model of Ly49C recognition suggests a mechanism whereby NK cells may be more effective in host defense. PR-MHC is present on the cell surface under normal physiological conditions, and moreover, its expression is highly responsive to cellular fitness (54). Accordingly, following disturbances in normal cell physiology, such as viral infection or neoplastic transformation, that lead to a reduction in protein transport, PR-MHC-I will decrease much more rapidly than total MHC-I. A NK inhibitory receptor that is specific for this ligand rather than a more stable form of MHC-I loaded with high-affinity peptide will lose inhibitory ligand much more rapidly, thus leading to reactivity against the affected cell. Therefore, the PR-MHC-I model represents an extension of the "missing self" hypothesis in which it is a particular conformation of MHC-I and not just total MHC-I that becomes the self-ligand of importance.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by funding to R.G.M from the National Cancer Institute of Canada and to L.A.B. from the Ontario Graduate Scholarship Program.

² Address correspondence and reprint requests to Dr. Richard G. Miller, Department of Cellular and Molecular Biology, Ontario Cancer Institute, 610 University Avenue, Room 9-305, Toronto, Ontario, M5G 2M9 Canada. E-mail address: miller{at}oci.utoronto.ca

³ Abbreviations used in this paper: MHC-I, MHC class I; ₂m, ₂-microglobulin; PR, peptide receptive; h-₂m, human ₂m; b-₂m, bovine ₂m; m-₂m, mouse ₂m; MS, mouse serum; HS, human serum; BFA, brefeldin A; LAK, lymphokine-activated killer; Wt, wild type; poly I:C, polyinosine-polycytidylic acid; MFI, mean fluorescent intensity; ER, endoplasmic reticulum.

Received for publication August 12, 2004. Accepted for publication June 22, 2005.

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