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The ₂ Domain of H-2D^d Restricts the Allelic Specificity of the Murine NK Cell Inhibitory Receptor Ly-49A¹

Jonas Sundbäck^2,*, Mary C. Nakamura, Margareta Waldenström^*, Eréne C. Niemi, William E. Seaman^,, James C. Ryan and Klas Kärre^*

^* Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden; Departments of Microbiology and Immunology and Medicine, University of California, San Francisco, CA 94143; and Veterans Administration Medical Center, San Francisco, CA 94121

	Abstract

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Mouse NK lymphocytes express Ly-49 receptors, which inhibit cytotoxicity upon ligation by specific MHC I molecules on targets. Different members of the lectin-like mouse Ly-49 receptor family recognize distinct subsets of murine H-2 molecules, but the molecular basis for the allelic specificity of Ly-49 has not been defined. We analyzed inhibition of natural killing by chimeric MHC I molecules in which the ₁, ₂, or ₃ domains of the Ly-49A-binding allele H-2D^d were exchanged for the corresponding domains of the nonbinding allele H-2D^b. Using the Ly-49A-transfected rat NK cell line, RNK-mLy-49A.9, we demonstrated that the H-2D^d ₂ domain alone accounts for allelic specificity in protection of rat YB2/0 targets in vitro. We also showed that the H-2D^d ₂ domain is sufficient to account for the allele-specific in vivo protection of H-2^b mouse RBL-5 tumors from NK cell-mediated rejection in D8 mice. Thus, in striking contrast to the ₁ specificity of Ig-like killer inhibitory receptors for human HLA, the lectin-like mouse Ly-49A receptor is predominantly restricted by the H-2D^d ₂ domain in vitro and in vivo.

	Introduction

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Cytotoxicity by NK cells is inhibited by the expression of class I MHC molecules on target cells (1, 2). This inhibition is mediated by cell surface receptors that recognize subsets of class I MHC molecules (3, 4). Individual NK cells vary in their inhibitory receptors, but each NK cell bears at least one receptor for a self-MHC I molecule, so that NK cells are prevented from responding against normal host cells (5, 6). If, however, expression of a self-MHC I molecule is lost or if the molecule is altered beyond recognition, the subset of NK cells normally held in check by this molecule can proceed to kill these altered targets (1, 2). Thus, NK cells may provide surveillance for cells with altered or missing self-MHC I molecules.

Two distinct types of inhibitory receptors for MHC I have been identified on NK cells (3, 4). Members of the first type belong to a superfamily of lectin-like, type II integral membrane proteins that are expressed on the surface of NK cells as dimers. These receptors were first described in mice, where they include members of the Ly-49 family (7, 8, 9, 10, 11). Of these receptors, the first and most thoroughly characterized is Ly-49A, which is inhibited by certain alleles of H-2D, including D^d, D^k, and D^p, but not by D^b, L^d, or K^d (7, 12). No homologues for the Ly-49 family of receptors have yet been found in humans, but other inhibitory lectin-like receptors are expressed on human NK cells, including the CD94/NKG2A heterodimeric complex (13).

Members of the second type of inhibitory MHC I receptor for type I integral membrane proteins are the Ig-like killer inhibitory receptors (KIRs)³ that were first identified on human NK cells (14, 15). More than 20 Ig-like KIRs have been cloned, and KIRs specific for subsets of HLA-A, -B, and -C have been characterized (15, 16, 17, 18, 19, 20, 21, 22, 23, 24). The binding specificities of these receptors are influenced in particular by polymorphisms clustered between amino acids 73 and 90 of the ₁ domains of their MHC ligands (15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Although other Ig-like inhibitory receptors are expressed on mouse NK cells, these have not been shown to recognize MHC I, and no direct KIR homologues have been defined on murine NK cells (25, 26).

The marked structural differences between the lectin-like Ly-49 receptors and the Ig-like KIRs suggest that they may differ in their requirements for interaction with class I MHC molecules. To date, MHC I-mediated inhibition of murine NK cells has been mapped to a determinant within the ₁/₂ domains of the MHC I molecule H-2D^d, but the fine specificity of binding by Ly-49 molecules has not been defined (7, 27, 28). We, therefore, constructed chimeric class I molecules between the Ly-49A ligand H-2D^d and H-2D^b (not recognized by Ly-49A) and examined their ability to inhibit rodent NK cell function in vivo and in vitro. Our results demonstrate that, in contrast to the dominant role of the MHC I ₁ domain in determining the binding specificity of KIRs, the ₂ domain of H-2D^d plays a dominant role in defining the class I specificity of Ly-49A.

	Materials and Methods

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Mice

C57BL/6 and D8 mice, C57BL/6 mice transgenic for H-2D^d (29), were bred and maintained at the Microbiology and Tumor Biology Center, Karolinska Institute (Stockholm, Sweden).

Cell lines

RBL-5, a Raucher virus-induced T cell lymphoma from C57BL/6 mice, and RBL-5-D^d, a clone of RBL-5 transfected with the vector pD^d-1 encoding genomic H-2D^d, were grown in RPMI 1640, 5% FCS, and penicillin/streptomycin (30). RNK-16, a spontaneous NK cell leukemia from F344 rats, was obtained from Craig Reynolds (National Cancer Institute, Frederick, MD) (31). YB2/0 cells were obtained from the American Type Culture Collection (Rockville, MD). Parental cells were grown in RPMI 1640, 10% FCS, penicillin/streptomycin, L-glutamine, and 5 x 10^-5 M 2-ME (complete RPMI). The mouse Ly-49A transfectant of RNK-16, RNK-mLy-49A.9, has been previously described and was grown in complete RPMI supplemented with 1 mg/ml of G418 (Boehringer Mannheim, Indianapolis, IN) (32). Transfected RNK-16 effectors and transfected YB2/0 targets were maintained in 1 mg/ml G418, but were grown in complete RPMI without G418 for at least 2 days before functional assays.

Vectors and chimeric H-2D^d/b constructs

pD^d-1, an 8-kb EcoRI fragment of the genomic H-2D^d in the plasmid pBR322, was a gift from David H. Margulies (National Institutes of Health, Bethesda, MD) (33, 34). pSV2neoD^d, an 8-kb EcoRI fragment of the genomic H-2D^d in the plasmid pSV2neo, was a gift from Peter Robinson (Clinical Research Center, Harrow, U.K.) (33). pD^bHd-, a pBR327 plasmid with a 10.2-kb HindIII insert containing the genomic H-2D^b, was a gift from Andrew Mellor (National Institute for Medical Research, London, U.K.). pSV2gptD^b a 10.3-kb HindIII genomic H-2D^b insert in the plasmid pSV2gpt was a gift from Alain Townsend (University of Oxford, Oxford, U.K.) (35). pCMVneoBam, a pBR322 plasmid carrying the neomycin resistance and the CMV promoter upstream of a multicloning site, was used for cotransfection with the chimeric molecule D^d_1,2D^b₃ to obtain G418-resistant RBL-5 cells (36). The cloning vector pBluescript II KS⁺ was purchased from Stratagene (La Jolla, CA).

The chimeric molecules were made by exon shuffling between genomic clones of H-2D^d and H-2D^b using standard methods (37). The chimeric molecule that we have termed D^d_1,2D^b₃ encodes the ₁ and ₂ domains of H-2D^d with the ₃ domain of H-2D^b. This was constructed by digesting the vector pSV2neoD^d with NotI and SpeI, and then isolating the fragment containing the ₁ and ₂ exons of H-2D^d. This was then exchanged for the corresponding NotI/SpeI ₁/₂ H-2D^b exon fragment in the vector pD^bHd-, producing the chimeric molecular construct.

The chimeric molecule that we have termed D^b_1,2D^d₃ encodes the ₁ and ₂ domains of H-2D^b with the ₃ domain of H-2D^d. This was constructed by digesting the vector pSV2gptD^b with NotI and SpeI. The resultant H-2D^b ₁/₂ exon fragment was isolated and substituted for the corresponding NotI/SpeI H-2D^d ₁/₂ exon fragment in pSV2neoD^d.

The chimeric molecule that we have termed D^d₁D^b₂D^d₃ encodes the ₁ and ₃ domains of H-2D^d, with the ₂ domain of H-2D^b. This was constructed by first shuttling the NotI/SpeI ₁/₂ exon fragment of H-2D^d into pBluescript II KS⁺. The H-2D^d ₂ exon was excised from the resulting vector pBluescriptNot/SpeD^d_1,2 using AatII and SpeI and replaced with the corresponding AatII/SpeI H-2D^b ₂ exon fragment from pSV2gptD^b. The H-2D^d₁D^b₂ exon fragment from this intermediate construct, pBluescriptD^d₁D^b₂, was then subcloned into the NotI/SpeI-prepared vector pSV2neoD^d.

The corresponding single domain shuffled chimeric molecule that we have termed D^b₁D^d_2,3 encodes the ₂ and ₃ domains of H-2D^d, with the ₁ domain of H-2D^b. This was constructed in a similar way. A NotI/KpnI fragment consisting of the H-2D^d ₁ and ₂ exons from pD^d-1 was subcloned into pBluescript II KS⁺. The H-2D^d ₁ exon was excised from the resulting vector pBluescriptNot/KpnD^d_1,2 using NotI and AatII and substituted with the NotI/AatII-prepared fragment containing the H-2D^b ₁ exon isolated from pSV2gptD^b. The H-2D^b₁D^d₂ exon fragment was then excised from pBluescript using NotI and KpnI and ligated into the NotI/KpnI-prepared vector pSV2neoD^d.

The expression construct for the transfection of the H-2D^d cDNA into YB2/0 was prepared by subcloning the H-2D^d cDNA, a gift from Wayne Yokoyama (Washington University, St. Louis, MO), into the SalI (5') and blunted BamHI (3') sites of the eukaryotic expression vector pHßAP (38). The H-2D^b cDNA, a gift from Alain Townsend (University of Oxford, Oxford, U.K.), was subcloned by blunt ligation into pHßAP, and the identities of the cDNA inserts were confirmed by DNA sequencing.

Transfection

YB2/0 cells were transfected using cesium-purified genomic plasmids or Qiagen-purified (Qiagen, Chatsworth, CA) cDNA plasmids using standard methods (32). Briefly, YB2/0 cells in exponential growth were transfected with 20 µg of EcoRI-linearized pHßAP-H-2D^d or pHßAP-H-2D^b DNA using a BTX-600 Electro Cell Manipulator (BTX, Genetronics, San Diego, CA). For transfection of YB2/0 cells with H-2D^d/b chimeric genomic plasmids, 50 µg of genomic plasmid was cotransfected with 5 µg of pHßAP vector. Each transfection was performed using 3 to 6 x 10⁶ cells/ml in 2-mm cuvettes in a total volume of 400 µl of complete RPMI at 115 V, 850 µF, and 129 ohms. Cuvettes were incubated on ice for 15 min after electroporation. Cells were then cultured overnight and plated in 96-well plates at a density of 2 to 5 x 10³ cells/well in complete RPMI supplemented with 1 mg/ml active G418. G418-resistant cells grew out in 7 to 12 days in 10 to 40% of wells. Transfectants were subcloned and characterized by flow cytometry.

RBL-5 cells used for in vivo studies were transfected with PvuI-linearized genomic plasmids using either a Bio-Rad Gene Pulser (Bio-Rad, Richmond, CA) or lipofectamine (Life Technologies, Gaithersburg, MD). Electroporation was performed according to the protocol supplied by Bio-Rad. Briefly, 5 x 10⁶ cells were washed and resuspended in 0.8 ml of PBS, and 8 µg of plasmid DNA was added. The transfection mixture was incubated on ice for 10 min, then electroporated in 4-mm cuvettes at 250 V and 960 µF. After electroporation, the cells were incubated at room temperature for 10 min, then transferred to RPMI 1640 with 10% FCS and penicillin/streptomycin. RBL-5 transfection using lipofectamine was performed according to the manufacturer’s protocol. Briefly, 1.5 µg of DNA was mixed with 100 µl Opti-MEM (Life Technologies), and 5 µl of lipofectamine was mixed with 100 µl of Opti-MEM. Both solutions were mixed and incubated for 20 min at room temperature. Cells (10⁶) were washed, then diluted in 0.8 ml of Opti-MEM. Cells were incubated with the DNA/lipofectamine mixture at 37°C for 4 h. After incubation, cells were transferred to 9 ml of RPMI 1640, 5% FCS, and penicillin/streptomycin. Following both transfection strategies, the RBL-5 cells were transferred to RPMI 1640, 5% FCS, penicillin/streptomycin, and 0.6 mg/ml G418 48 h after transfection.

mAbs and flow cytometry

The anti-H-2 mAbs used in these studies as well as their reported domain specificities are presented in Table I (33, 39, 40, 41). The mAb directed against the ₃ domain of H-2D^b (28-14-8) was purchased from PharMingen (San Diego, CA). The anti-Ly-49A (A1) hybridoma was a gift from Wayne Yokoyama (Washington University). The anti-NK1.1 hybridoma (PK.136) was a gift from Gloria Koo (Merck, Piscataway, NJ). The anti-₁ H-2D^b hybridoma (22.249) was obtained from David Raulet (University of California, Berkeley, CA). The anti-_1,2 H-2D^d hybridoma (34-5-8S), the anti-_1,2 H-2D^d (34-4-21S) hybridoma, and the anti-₃ H-2D^d (34-2-12S) were obtained from the American Type Culture Collection.

Cytotoxicity assays

Specific lysis of NK targets was determined by using a standard 4-h ⁵¹Cr release assay as previously described (32). Briefly, targets cells were harvested and labeled for 1 h at 37°C with 200 µCi of sodium ⁵¹Cr (Amersham, Arlington Heights, IL) in complete RPMI. Labeled target cells were washed and resuspended at 10⁵ cells/ml, and 0.1 ml of this cell suspension was added to each well of 96-well plates containing 0.1 ml of effector cells at the indicated E:T cell ratios. Plates were incubated at 37°C for 4 h, then centrifuged for 5 min. One hundred microliters of supernatant was counted in a gamma counter, and the percent cytotoxicity was calculated as previously described (32). All assays were performed in triplicate. SEs for each point were always <2% (not shown). For Ab inhibition studies, effector cells were preincubated for 15 min at room temperature with mAb at a concentration of 20 µg/10⁶ effectors before the addition of targets.

In vivo tumor outgrowth

Tumor cell lines used were grown as ascites before inoculation into D8 transgenic mice. Mice were inoculated s.c. with 10³ or 10⁴ cells as described previously (42). Tumor growth was followed by weekly palpation for up to 6 wk. NK1.1-treated D8 mice were injected i.p. with 0.2 ml (200 µg) of anti-NK1.1 mAb (PK.136) ascites 1 day before inoculation.

	Results

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Construction and expression of H-2D^d/b chimeric molecules

To investigate the structural motif on H-2D^d involved in mediating inhibitory signals to murine NK cells, we constructed exon-shuffled H-2D^d/b chimeric molecules as described in Materials and Methods. The single domain swap chimeric molecules D^b₁D^d_2,3 and D^d₁D^b₂D^d₃ were produced to determine the specific NK cell inhibitory domain of H-2D^d. The D^d_1,2D^b₃ and D^b_1,2D^d₃ chimeric molecules were made as control constructs, since previous studies have shown that the ₃ domain of H-2 does not influence the specificity in target cell protection against lysis by murine NK cells (27, 28).

For in vitro cytotoxicity studies, the chimeric constructs were transfected into rat YB2/0 targets, which are devoid of mouse H-2 molecules and are susceptible to lysis by rodent NK cells and by the RNK-16 rat NK cell line (J. Ryan, unpublished preliminary results). G418-resistant clones expressing high levels of the chimeric H-2 molecules were selected using flow cytometry. Staining with a panel of mAbs reported to react against different domains of H-2D^d and H-2D^b confirmed the expression of chimeras on the surface of YB2/0 as shown in Figure 1. YB2/0 cells transfected with H-2D^d (Fig. 1, row A) stained with anti-_1,2 H-2D^d (34-5-8S) and with anti-₃ H-2D^d (34-2-12S), but with neither of the anti-H-2D^b mAbs. Similarly, YB2/0-H-2D^b (Fig. 1, row B) stained with anti-₁ H-2D^b (22.249) and with anti-₃ H-2D^b (28-14-8), but not with the anti-H-2D^d mAbs. YB2/0 cells transfected with the D^d_1,2D^b₃ chimera (Fig. 1, row C) stained with the anti-_1,2 H-2D^d (34-5-8S) and with the anti-₃ H-2D^b mAb (28-14-8), but not with the anti-₁ H-2D^b mAb (22.249) or with the anti-₃ H-2D^d mAb (34-2-12S). YB2/0 cells transfected with the D^b_1,2D^d₃ chimera (Fig. 1, row D) stained with the anti-₁ H-2D^b mAb (22.249) and with the anti-₃ H-2D^d mAb (34-2-12S), but not with the anti-_1,2 H-2D^d (34-5-8S) or with the anti-₃ H-2D^b mAb (28-14-8). YB2/0 cells transfected with the D^b₁D^d_2,3 chimera (Fig. 1, row E) stained with the anti-₁ H-2D^b mAb (22.249) as well as with the anti-₃ H-2D^d mAb (34-2-12S) and anti-_1,2 H-2D^d mAb (34-5-8S). Finally, the YB2/0 cells transfected with the D^d₁D^b₂D^d₃ chimera (Fig. 1, row F) stained with the anti-₃ H-2D^d mAb (34-2-12S), but not with the anti-₁ H-2D^b (22.249) or with the anti-₃ H-2D^b mAb (28-14-8). Notably, mAb 34-5-8S, reported to be specific for both the ₁ and ₂ domains of H-2D^d, did not stain the D^d₁D^b₂D^d₃ chimera, indicating that this mAb preferentially recognizes an ₂ domain-encoded epitope on H-2D^d.

View larger version (52K): [in this window] [in a new window]   FIGURE 1. Expression of H-2Dd, H-2Db, and H-2Dd/b chimeric molecules on rat YB2/0 transfectants. YB2/0 cells were transfected with H-2Dd (row A) and with H-2Db (row B). In addition, YB2/0 cells were transfected with chimeric molecules Dd1,2Db3 (row C), Db1,2Dd3 (row D), Db1Dd2,3 (row E), and Dd1Db2Dd3 (row F). Untransfected (dotted histograms) and transfected (solid histograms) YB2/0 were stained with mAbs specific for anti-1,2 H-2Dd (34-5-8S), anti-3 H-2Dd (34-2-12S), anti-1 H-2Db (22.249), and anti-3 H-2Db (28-14-8) as indicated. The mAb reactivities confirmed the expression and the identity of each chimeric receptor on the indicated YB2/0 transfectants.

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Expression of the H-2Dd and H-2Dd/b chimeric molecules on RBL-5 transfectants. The transfected RBL-5 cells were stained with mAbs specific for anti-1,2 H-2Dd (34-4-21S), anti-1,2 H-2Dd (34-5-8S), or anti-3 H-2Dd (34-2-12S) and analyzed by flow cytometry. Open histograms show FITC-conjugated secondary mAb only. Filled histograms show specific mAb and secondary mAb.

In vitro cytotoxicity by RNK-16 and RNK-mLy-49A.9

We analyzed the protective capacities of our chimeric molecules in vitro, using NK cell effectors that are uniform in murine Ly-49A expression. Mouse NK cells express overlapping subsets of diverse Ly-49 receptors (6). Since these Ly-49 receptors have different allelic specificities for MHC class I, nonclonal populations of mouse NK cells may be inhibited through multiple Ly-49 receptors by different target MHC I molecules (6). Thus, to analyze the selective effects of our chimeric H-2D^d/b molecules on Ly-49A-mediated inhibition, we used the rat NK-like cell line RNK-16, which has phenotypic and functional characteristics of rat NK cells and, naturally, expresses no mouse Ly-49 molecules (31). We have previously shown that mouse Ly-49A is fully functional when transfected into RNK-16, inhibiting lysis of targets expressing H-2D^d (32). Our clonal Ly-49A transfectant RNK-mLy-49A.9 fails to kill H-2D^d targets unless blocking quantities of mAb anti-Ly-49A are added to the killing assay (32). Since parental RNK-16 cells do not lyse RBL-5 targets, we used the rat myeloma YB2/0 target line for these in vitro studies.

The panel of YB2/0 transfectants was examined in cytotoxicity assays using wild-type RNK-16 and RNK-mLy-49A.9 effectors (Fig. 3). As expected, all YB2/0 transfectants were readily killed by wild-type RNK-16 (Fig. 3, A–F,bottom). YB2/0-H-2D^d was not killed by RNK-mLy-49A.9 unless anti-Ly-49A mAb was added to the killing assay (Fig. 3A, top). YB2/0-H-2D^b was readily killed by this effector (Fig. 3B,top). Ly-49A expression on RNK-mLy-49A.9 inhibited lysis of YB2/0-D^d_1,2D^b₃ (Fig. 3C, top) and of YB2/0-D^b₁D^d_2,3 transfectants (Fig. 3E, top), indicating that Ly-49A recognizes an ₂-dependent determinant on this chimeric molecule. This interpretation is further supported by the observation that RNK-mLy-49A.9 cytotoxicity is not inhibited by the chimeric molecule D^b_1,2D^d₃ (Fig. 3D, top) or by D^d₁D^b₂D^d₃ (Fig. 3F, top). Taken together, these data indicate that the expression of at least one determinant contributing to protection through Ly-49A is dependent on the ₂ domain, but not the ₁ domain, of H-2D^d in these chimeric MHC I molecules.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Ly-49A recognizes a determinant that is contributed by the 2 domain of H-2Dd. RNK-mLy-49A.9 (top) and wild-type RNK-16 (bottom) effectors were tested in 4-h cytotoxicity assays alone (open circles), in the presence of blocking anti-Ly-49A mAb (closed diamonds), or in the presence of isotype-matched control anti-NK1.1 mAb (open diamonds). Targets were YB2/0 cells transfected with H-2Dd (A), H-2Db (B), H-2Dd/b chimeric molecules Dd1,2Db3 (C), Db1,2Dd3 (D), Db1Dd2,3 (E), and Dd1Db2Dd3 (F). RNK-mLy-49A.9 effectors did not lyse cells expressing the 2 domain of H-2Dd (open circles; A, C, and E,top) unless blocking anti-Ly-49A mAb was added (closed diamonds; A, C, and E,top). Targets lacking the 2 domain of H-2Dd, however, were readily killed by RNK-mLy-49A.9 effectors (B, D, and F,top). All YB2/0 target cell lines were killed by untransfected RNK-16 cells (A–F,bottom). Both RNK-16 and RNK-mLy-49A.9 effectors killed untransfected YB2/0 cells equally well, and cytotoxicity was unaffected by the addition of anti-Ly-49A mAb (not shown).

To investigate the ability of H-2D^d/b chimeric molecules to protect transfected RBL-5 cells from rejection by NK cells in vivo, we inoculated the tumors into D8 mice, which are transgenic for H-2D^d on a C57BL/6 (H-2^b) background (29). NK cells of D8 mice are known to reject H-2^b tumor cells, but not their H-2D^d transfectants (42). Challenging doses of tumor cells were chosen on the basis of previous experiments so that tumor outgrowth of H-2D^d negative cells would be prevented by NK cells in the majority of mice (42). We focused on early rejection vs outgrowth, rather than on late regression, which is usually mediated by T cells. Such T cell-mediated rejection was an obvious possibility, since the chimeric molecules must be regarded as allogeneic. Tumor growth of the transfectants in D8 mice is presented in Table II.

Both RBL-5-D^d and RBL-5-D^b₁D^d_2,3 tumors eventually regressed in many mice. We interpret these late rejections as a T cell response, since the RBL-5-D^b₁D^d_2,3 grew progressively in BALB/c nu/nu mice (data not shown).

These results indicate that the ₂ domain of H-2D^d is sufficient for protection when expressed in the context of the ₁ domain of a nonprotective H-2D^b allele. The ₁ domain of H-2D^d did not protect RBL-5-D^d₁D^b₂D^d₃ cells from rejection. These cells behaved as parental RBL-5 after in vivo transfer. The level of chimeric expression on unprotected RBL-5-D^d₁D^b₂D^d₃ cells was lower than that on the protected RBL-5-D^b₁D^d_2,3 cells, however, making it difficult to completely exclude a role for the D^d₁D^b₂D^d₃ chimera in protection against natural resistance.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
By constructing mouse class I MHC molecules that are chimeric between H-2D^d and H-2D^b, we have demonstrated an essential role for the ₂ domain of H-2D^d in the inhibition of natural killing in vitro and in vivo. Specifically, the ₂ domain of H-2D^d is required for inhibition through the Ly-49A receptor. Our results from two different experimental systems complement each other. The in vitro, transfectant-based system allowed reductionistic analysis of the role of a single receptor molecule. The tumor rejection system, on the other hand, confirmed that the differences between different chimeric molecules were maintained under in vivo conditions, excluding that the specificity in the protective effect was merely the result of overexpressed receptors or suboptimal activation levels in the RNK line used in the in vitro assay.

Our studies failed to reveal a specific requirement for the H-2D^d ₁ domain in functional recognition by Ly-49A. The simplest explanation for our results is that Ly-49A binds to the ₂ domain, but not the ₁ domain, of H-2D^d. There are, however, other possible interpretations. The Ly-49A receptor may require binding to motifs in both domains: one shared by H-2D^d and D^b in the ₁ domain, and one unique to H-2D^d in the ₂ domain. The first motif would thus be present in all our chimeric molecules, but it would not be sufficient for a functional interaction in the absence of the second motif. Secondly, it is possible that a unique putative Ly-49A binding determinant on the H-2D^d ₁ domain may be dependent upon pairing with the H-2D^d ₂ domain or be prevented by pairing with the H-2D^b ₂ domain. An additional consideration is the possible role of a peptide associated with the class I MHC molecules in recognition by Ly-49A. Studies by Raulet and by Yokoyama have demonstrated that Ly-49A does not discriminate between specific peptides containing H-2D^d anchor residues (44, 45). This may not, however, be true for peptides containing anchor residues specific for our D^d₁D^b₂D^d₃ chimera.

For these reasons, our results do not strictly exclude a role for the H-2D^d ₁ domain in binding by Ly-49A. They do, however, establish the importance of the ₂ domain, which alone can account for the ability of the receptor to discriminate between H-2D^d and H-2D^b. Previous studies of Ly-49A binding to H-2D^d have primarily involved anti-H-2D^d mAb blockade. Yokoyama and colleagues have tested a series of anti-H-2D^d-specific mAb (7, 45). Although several of these mAbs have been previously described as specific for the ₁/₂ domains of H-2D^d, only mAb 34-5-8S blocks Ly-49A-mediated inhibition of H-2D^d targets (45). In addition, Ly-49A-dependent inhibition of NK cell activation as well as efficient expression of the 34-5-8S epitope on H-2D^d are dependent upon occupancy of the peptide binding groove (45, 46). These data suggest that both Ly-49A and mAb 34-5-8S may recognize a shared, peptide-dependent, conformational determinant on H-2D^d (45). McCluskey and colleagues, using H-2D^d/H-2L^d chimeric molecules, found that 34-5-8S recognizes a predominantly ₂ domain-specific epitope, but that this mAb also reacts very weakly with an ₁ domain-encoded structure (47). Our finding that mAb 34-5-8S binds to the ₂, but not the ₁, domain of H-2D^d is consistent with epitope analyses of Stroynowski using interdomain chimeric H-2D^d/Qa-6 molecules (48). Our findings also support studies by Abastado, who used a series of H-2K^d/D^d intradomain recombinants to map the mAb 34-5-8S epitope to a region contained between amino acids 92 and 116 in the NH₂-terminal ₂ domain of H-2D^d (49). These studies also mapped the 34-4-21S epitope within residues 57 and 91 in the carboxyl-terminal ₁ domain of H-2D^d (49). Cells from the H-2D^dm1 mouse, which express a recombinant H-2 ₂ domain containing the NH₂-terminal H-2D^d and the COOH-terminal H-2L^d, are rejected in a pattern similar to cells expressing H-2D^d, rather than to that of cells expressing H-2L^d (50). Thus, our H-2D^d/b chimeric experiments, when considered in the context of mAb blocking/epitope and H-2D^dm1 mouse studies, firmly implicate the NH₂-terminal part of the ₂ domain, but not the ₁ domain, of H-2D^d in Ly-49-mediated NK cell inhibition.

The importance of the ₂ domain in binding by Ly-49A contrasts with the critical role of the ₁ domain in allelic specificity in recognition by the Ig-like KIRs on human NK cells. It has been proposed that the lectin-like receptors preceded KIRs in evolution, and that the rapid evolution of human MHC class I molecules has forced the coevolution of KIRs (51, 52). The specificity of binding by KIRs to human class I MHC molecules is dependent on residues in the carboxyl-terminal ₁ domain. The specific residues vary with individual KIRs, but they include amino acids at positions 73, 74, 76, 77, 80, 83, and 90 (15, 16, 17, 18, 19, 20, 21, 22, 23, 24). These residues are conserved across multiple (but not all) alleles and are thus expressed as "public" specificities. In mouse class I MHC molecules, the corresponding sites are highly polymorphic, suggesting that they may not be subject to the same selection pressures (53). Consistent with this hypothesis, no MHC-binding KIR homologues have been identified in rodents. Similarly, although NK cells from both humans and rodents share the expression of certain lectin-like inhibitory receptors, notably CD94/NKG2 heterodimers, no human homologues for the Ly-49 receptors have been identified (13, 54, 55).

Previous studies have demonstrated additional differences between MHC I recognition by human KIR and by rodent Ly-49. Some KIRs are sensitive to alterations at certain positions of the peptide bound to MHC I, mainly side chains at positions 7 and 8 (56). Although Ly-49A-mediated inhibition requires occupancy of the H-2D^d peptide binding groove, Ly-49A does not discriminate between individual H-2D^d peptides (44, 45). KIR binding does not requireN-linked glycosylation of MHC I (16). In contrast, studies from our laboratory and others have demonstrated a role for carbohydrate in Ly-49 recognition (57, 58). Unlike human class I MHC molecules, each mouse allelic variant contains an N-linked glycosylation site at position 176 in the carboxyl-terminal ₂ domain (52, 53) in addition to the one at position 86 in the ₁ domain. Our observations on a role for the ₂ domain may be related to the carbohydrate side chain in this domain. The carbohydrate chain important for protection via Ly-49A is not known, and it cannot be excluded that critical carbohydrate and protein structures are separated in primary sequence, yet interact in the tertiary structure. Studies by Takei and his colleagues have shown that the divergent specificities of Ly-49A and Ly-49C are not determined by polymorphisms within the carbohydrate recognition domain of Ly-49, but, rather, in a membrane proximal stalk region of the extracellular domain (59, 60). It is thus possible that Ly-49A recognizes both carbohydrate and protein components of the H-2D^d ₂ domain. Thus, although human KIR and rodent Ly-49 receptors share similar cytoplasmic motifs and use similar inhibitory signaling pathways in NK cells, it appears that these structurally divergent receptors differ markedly in their requirements for ligand recognition (32, 61).

These results are the first to directly implicate the ₂ domain in Ly-49-dependent inhibition of NK cell cytotoxicity. As such, they form the basis for future mutational and structural studies of murine MHC I, ultimately defining the molecular basis for the allelic specificities of Ly-49 receptors. They also support previous studies indicating that Ly-49A indeed screens for global changes in the conformation or expression of the H-2D^d heavy chain, in accordance with the "missing self" hypothesis.

	Footnotes

 
¹ This work was supported by the Swedish Society for Medical Research, the Swedish Medical Research Council, the Swedish Cancer Society, the Foundation in Memory of Lars Hierta, and the Karolinska Institute (to J.S., M.W., and K.K.); by the U.S. Veterans Administration and National Institutes of Health Grants RO1CA69299 (to W.E.S.), R29CA60944 (to J.C.R.), and K11AR01927 (to M.C.N.); by the International Human Frontiers in Science Program (to J.C.R.); and by the Rosalind Russell Foundation and Multipurpose Arthritis Center Grant P60AR20684 (to M.C.N.).

² Address correspondence and reprint requests to Jonas Sundbäck, Microbiology and Tumor Biology Center, Box 280, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail address:

³ Abbreviations used in this paper: KIR, killer inhibitory receptor.

Received for publication September 8, 1997. Accepted for publication February 19, 1998.

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