Mouse Ly-49D Recognizes H-2D^d and Activates Natural Killer Cell Cytotoxicity

RNK-16, a spontaneous NK cell leukemia from F344 rats, was a gift from Craig Reynolds (NCI, Frederick, MD) and was adapted for in vitro growth in cRPMI (RPMI 1640 supplemented with 10% heat-inactivated FCS, 25 μM 2-ME, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) (24). Tumor target cell lines cultured in cRPMI included YAC-1 (mouse lymphoma, H-2^a) and YB2/0 (rat myeloma) from the American Type Culture Collection. The mouse Ly-49A transfectant of RNK-16, RNK.mLy-49A (previously described as RNK-mLy-49A.9; reference 25), was grown in cRPMI supplemented with 1 mg/ml of G418 (Boehringer Mannheim). Transfected RNK-16 effectors and transfected YB2/0 targets were maintained in 1 mg/ml G418, but were grown in cRPMI without G418 for at least 2 d before functional assays.

mAbs to mouse Ly-49D (12A8, rat IgG2a), OVA (2C7, rat IgG2a), or H-2D^d (34-5-8S, mouse IgG2a) were produced from their respective hybridoma lines. Antibodies were partially purified from ascites by ammonium sulfate precipitation. F(ab′)₂ fragments were generated by pepsin digestion (26), and completion of digestion was verified by SDS-PAGE and silver staining. For fluorescence analysis, mAbs were used at a concentration of 1 μg/10⁶ cells. Routine analysis was performed using a FACScan^®.

Specific lysis of NK targets was determined by using a standard 4-h ⁵¹Cr-release assay as previously described (27). In brief, target cells were harvested and labeled for 1 h at 37°C with 200 μCi of [⁵¹Cr]sodium chromate (Amersham) 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 ratios. Plates were incubated at 37°C for 4 h, then centrifuged for 5 min. 100 μl of supernatant was counted in a gamma counter and the specific cytoxicity was calculated as previously described (27). All assays were performed in triplicate. For antibody inhibition studies, effector cells were preincubated for 15 min at room temperature with F(ab′)₂ at a concentration of 25 μg/10⁶ effectors or with intact antibody at a concentration of 10 μg/10⁶ effectors before the addition of targets.

The Ly-49D cDNA was obtained by PCR amplification from cDNA prepared from 7-d–IL-2-activated LAK cells from C57BL/6J mice using the 5′ primer 5′-TCACAGAAATCACTCAAGGACAT-3′ and the 3′ primer 5′-TTTACTTTTAACACTCAC-3′. The Ly-49D cDNA obtained is identical to the Ly-49D cDNA previously published in the extracellular and transmembrane domains (17). There is a 9-nucleotide insertion at the 3′ end of the coding sequence for the cytoplasmic domain in the Ly-49D cDNA obtained and used in these studies, which is identical to the alternately spliced long Ly-49D cDNA sequence published by Silver et al. (28). The protein sequence of the 44-amino acid cytoplasmic domain of the Ly-49D cDNA obtained reads MTEQEDTFSAVRFHKSSGLQNEMRLKETRKPEKARLRVCSVPWQ (the initial published sequence is identical except it lacks the underlined 3-amino acid insertion). This Ly-49D cDNA PCR product was subcloned into the TA cloning vector, pCR2.1 (Invitrogen), shuttled through the vector pSP72 (Promega) using the 5′ Hind III/XbaI 3′ sites, and then ligated into the XhoI site in the expression vector BSRαEN (a gift from A. Shaw and M. Olszowy, Washington University, St. Louis, MO). Constructs were confirmed by sequencing in both directions before transfection. Vectors used to transfect YB2/0 target cells with H-2D^d, H-2D^b, and chimeric H-2D^d/b class I MHC have been described previously (29). The chimeric molecules were made by exon shuffling between genomic clones of H-2D^d and H-2D^b using standard methods (30), and details of the construction have been described previously (29). The chimeric molecule that we have termed D^dα₁α₂D^b encodes the α₁ and α₂ domains of H-2D^d with the α₃ domain of H-2D^b. The chimeric molecule that we have termed D^bα₁α₂D^d encodes the α₁ and α₂ domains of H-2D^b with the α₃ domain of H-2D^d.

YB2/0 or RNK-16 cells were transfected by electroporation as previously described, using cesium-purified genomic plasmids or Qiagen-purified (Qiagen) cDNA plasmids using standard methods (25, 29).

Strain C57BL/6, BALB/c, BALB.B10 (C.B10-H-2^b/ LiMcdJ), BALB.K (C.B10-H-2^k/LiMcdJ), B10.D2, B10.BR, B10.S, DBA/2, and C3H mice were obtained from The Jackson Laboratory and were used at 6–8 wk of age.

Con A–stimulated blasts were prepared from the spleens of mice from the strains described above, using methods that have been described previously (31). In brief, spleens were harvested aseptically and separated into single cell suspensions. After lysis of red blood cells, cells were washed in cRPMI and placed in culture at a density of 10⁶ cells/ml in cRPMI with 3 μg/ ml Con A (Sigma Chemical Co.). After 48 h of culture at 37°C, cells were harvested, purified over Ficoll-Hypaque, washed twice in cRPMI, and labeled for use as targets in cytotoxicity assays.

To examine Ly-49D–ligand interactions in the absence of mouse inhibitory NK cell receptors, we transfected the mouse Ly-49D receptor into RNK-16, a rat tumor cell line with phenotypic and functional characteristics of rat NK cells (24). Cell-surface staining of a representative RNK.mLy-49D clone with the anti–Ly-49D mAb, 12A8, is shown in Fig. 1 B. In murine NK cells, the Ly-49D receptor has previously been demonstrated to mediate “redirected lysis” of FcR-bearing targets in the presence of the anti–Ly-49D mAb, 12A8 (14). As shown in Fig. 1 D, killing of the FcR-bearing target YB2/0 by the RNK. mLy-49D transfectant was similarly stimulated by anti–Ly-49D mAb, but not by isotype-matched control mAb. Neither F(ab′)₂ anti–Ly-49D nor control F(ab′)₂ 2C7 had any effect on cytotoxicity (panel F), indicating that the binding of the intact mAb to target FcR is required for augmentation of killing of YB2/0 (redirected lysis). Lysis of YB2/0 by wild-type RNK-16 cells was unchanged by mAb anti– Ly-49D, by control mAb (panel E), or by F(ab′)₂ fragments derived from either antibody (panel G). These studies demonstrate that the mouse Ly-49D receptor can function as an activating receptor in RNK-16.

RNK.mLy-49D transfectants were also tested against the standard NK cell tumor target, YAC-1. Interestingly, killing of YAC-1 by RNK.mLy-49D was decreased compared with killing by RNK-16 (Fig. 1, G and H). The anti–Ly-49D mAb, 12A8, had no effect on the lysis of YAC-1 by RNK.mLy-49D transfectants (data not shown).

To examine the MHC restriction of Ly-49D, we tested the RNK.mLy-49D transfectant in cytotoxicity assays against a panel of rat YB2/0 myeloma target cells transfected with one of the murine H-2 alleles D^d, D^b, K^b, or K^k. As seen in Fig. 2, RNK-mLy-49D effectors lysed YB2/0.D^d targets (panel A) more efficiently than YB2/0.D^b (panel B), YB2/ 0.K^b (panel C), or YB2/0.K^k (panel D). Augmented killing of YB2/0.D^d by RNK.mLy-49D effectors was specifically blocked by F(ab′)₂ anti–Ly-49D (12A8), but not by isotype-matched control F(ab′)₂ 2C7 (panel A). Lysis of the YB2/0.D^d, D^b, K^b, and K^k transfectants by wild-type RNK-16 was unchanged in the presence of F(ab′)₂ anti– Ly-49D or control F(ab′)₂ 2C7 (panels E–H). We previously described RNK-16 cells transfected with the mouse NK inhibitory receptor, Ly-49A (RNK.mLy-49A), which also recognizes H-2D^d (25). In contrast to RNK.mLy-49D cells, which were activated by YB2/0.D^d cells, RNK.mLy-49A cells were unable to lyse YB2/0.D^d cells, as they were inhibited through the Ly-49A receptor. Because the 12A8 mAb binds to Ly-49A as well as to Ly-49D (14), we were able examine the effects of 12A8 on lysis by RNK.mLy-49A cells. As shown in Fig. 2 A, F(ab′)₂ 12A8 blocked H-2D^d– specific activation of cytotoxicity by RNK.mLy-49D cells, but reversed H-2D^d–specific inhibition of lysis by RNK. mLy-49A cells (panel I). Thus, the activating Ly-49D receptor and the inhibitory Ly-49A receptor share ligand specificity for H-2D^d on target cells, and the interaction between ligand H-2D^d and either Ly-49A or Ly-49D can be blocked by F(ab′)₂ 12A8. Neither F(ab′)₂ 12A8 nor control F(ab′)₂ 2C7 had any significant effect on the lysis of YB2/0.D^b, K^b, or K^k targets by any effector (panels B–D, F–H, and J–L).

We next extended our observations regarding the lysis of transfected cells by examining the lysis of Con A blasts derived from mice of various H-2 haplotypes. For these studies we tested RNK.mLy-49D effectors in cytotoxicity assays against Con A blasts prepared from MHC congenic resistant strains of BALB/c and C57BL/10 mice.

As shown in Fig. 3, RNK.mLy-49D cells effectively lysed Con A blasts prepared from BALB/c (H-2^d; panel A) and B10.D2 (H-2^d; panel B) mice, but not Con A blasts prepared from BALB.B10 (H-2^b; panel C), C57BL/6 (H-2^b; panel D), BALB.K (H-2^k; panel E), or B10.BR (H-2^k; panel F) mice. In experiments not shown, RNK.mLy-49D effectors also lysed Con A blasts from DBA/2 (H-2^d) mice but not blasts from C3H (H-2^k) or B10.S (H-2^s) mice. Lysis of all H-2^d blasts by RNK.mLy-49D effectors was inhibited by anti–Ly-49D mAb 12A8, but not by isotype-matched control mAb 2C7 (panels A and B).

These studies fully supported our experiments with transfected tumor targets, identifying an H-2^d–encoded structure or structures as ligands for Ly-49D. Notably, Con A blasts expressing alleles of H-2^k failed to activate Ly-49D transfectants. Thus, in constrast to the Ly-49A receptor, Ly-49D does not recognize H-2D^k.

We next examined the domains of H-2D^d that are recognized by Ly-49D. To this end, we examined the lysis by RNK.mLy-49D effectors of YB2/0 cells transfected with genes in which exons for the α₁, α₂, and α₃ domains of H-2D^d and H-2D^b were recombined to create chimeric class I molecules. As shown in Fig. 4 C, the chimeric molecule D^bα_1,2D^d, containing only the α₃ domain of D^d, failed to activate cytotoxicity by RNK.mLy-49D cells, whereas the chimeric H-2 molecule D^dα_1,2D^b, which contains the α_1,2 domains of D^d and the α₃ domain of D^b, activated cytotoxicity by these cells (panel F). Augmentation of RNK.mLy-49D lysis of D^dα_1,2D^b was inhibited by F(ab′)₂ anti–Ly-49D, but not by control F(ab′)₂ (panel F). As controls for the specific effect of the mAb on the Ly-49D receptor, lysis of targets expressing D^bα_1,2D^d by either RNK-16 or RNK.mLy-49A cells was unchanged by F(ab′)₂ 12A8 or control F(ab′)₂ (panels A and B). As we have previously shown, RNK.mLy-49A effectors were unable to lyse YB2/0 cells expressing D^dα_1,2D^b (29). However, lysis of this target by RNK.mLy-49A could be restored by the addition of blocking F(ab′)₂ 12A8 (panel E). Thus, both Ly-49D and Ly-49A bind to the α₁ and α₂ domains, but not the α₃ domain, of H-2D^d.

Previous studies have shown that the mLy-49A/H-2D^d interaction can be blocked by one anti–H-2D^d antibody (34-5-8S), which is specific for a conformationally dependent epitope in the H-2D^d α₁/α₂ domains. Other mAbs to H-2D^d do not interrupt its interaction with Ly-49A (32). We, therefore, determined whether F(ab′)₂ 34-5-8S could inhibit the Ly-49D/H-2D^d interaction as well. As shown in Fig. 5, activation of RNK.mLy-49D effectors by YB2/ 0.D^d was inhibited by F(ab′)₂ 34-5-8S (panel C), whereas lysis of YB2/0.D^d by RNK.mLy-49A effectors was restored in the presence of F(ab′)₂ 34-5-8S. RNK-16 lysis of YB2/0.D^d was unaffected by F(ab′)₂ 34-5-8S.

These data show that the activating Ly-49D receptor shares some aspects of ligand recognition with the inhibitory Ly-49A receptor. Both receptors recognize the α₁/α₂ domains of H-2D^d, and F(ab′)₂ anti–H-2D^d 34-5-8S can block the interaction of H-2D^d with either Ly-49A or Ly-49D. However, the ligand specificity of Ly-49D does not completely mirror that of Ly-49A, as Ly-49D–mediated cytotoxicity is not activated by H-2^k blasts, which express the Ly-49A ligand H-2D^k (33–35). These data suggest that the structural features that restrict the allelic specificities of Ly-49D and Ly-49A are similar, but not identical.

Cytotoxicity by NK cells is generally inhibited by the expression of class I MHC antigens on target cells. The “missing self  ” hypothesis proposes that loss of target cell MHC class I may lead to the unopposed activation of natural killing and eventually to target cell lysis (5). However, several previous lines of evidence suggest that rodent NK cells may possess activating receptors in addition to inhibitory receptors for MHC-encoded target structures. First, irradiated C57BL/6 (H-2^b) mice rapidly reject bone marrow from H-2D^d transgenic C57BL/6 mice, unless recipient mice are depleted of NK cells before transplantation (19, 20). Thus, the transgenic expression of H-2D^d on C57BL/6 blasts is associated with the selective acquisition of susceptibility to lysis by mouse NK cells. Second, genetic studies in rats have shown that NK cells are activated by structures encoded within the MHC locus, and NK cells from the PVG (RT1^c) rat strain efficiently lyse blast targets from LEW (RT1^l) rats, but not from mutant LEW.LM1 (RT1^lm1) rats, which have a homozygous 100-kb deletion in the nonclassical RT1.C^l region (21). These data suggest that the ability of NK cells to lyse allogeneic blast targets may directly involve activating receptors for target MHC. A gene controlling NK alloactivating responses in rats has been linked to the Ly-49 gene family in NK alloresponder PVG rats backcrossed to DA rats, which are selectively deficient in NK allorecognition. These studies implicate Ly-49–like molecules in the activation of cytotoxicity by target MHC antigens (22).

Mason et al. previously demonstrated that Ly-49D can activate NK cell cytotoxicity, but the specific activation of Ly-49D⁺ NK cells by target MHC class I antigens was not demonstrated in vitro (14). Subsequent in vivo studies revealed that depletion of Ly-49D⁺ cells from C57BL/6 mice prevented their ability to reject H-2D^d+ bone marrow grafts (23), consistent with the hypothesis that these cells are functionally activated by MHC-encoded structures. We examined target-induced Ly-49D activation using RNK.mLy-49D transfectants, which can activate NK cell cytotoxicity through the Ly-49D receptor. Interestingly, when compared with wild-type RNK-16, RNK. mLy-49D effectors demonstrated diminished lysis of YAC-1 and YB2/0, as well as diminished antibody-dependent cellular cytotoxicity and diminished redirected lysis through the rat NKR-P1A receptor (data not shown). This change was not associated with changes in expression of NKR-P1A on the RNK-16 transfectants (data not shown). Because these changes in cytolytic specificity were seen in three different RNK.mLy-49D clones, it seems unlikely that they were unique to Ly-49D integration sites in stable transfectants, although this possibility cannot be ruled out completely. It is also possible that the overexpression of the Ly-49D activating receptor leads to sequestration of signaling intermediates required for activation, which, in turn, leads to a decrease in lysis through other activating receptors. However, we were easily able to observe specific activation through Ly-49D, demonstrating that the cytolytic capacity of RNK.mLy-49D cells was intact.

Our in vitro studies demonstrate that Ly-49D is an activating NK cell receptor specific for H-2D^d. The acquisition of enhanced cytotoxicity against H-2D^d–transfected YB2/0 targets was specifically blocked by F(ab′)₂ anti–Ly-49D or by F(ab′)₂ anti–H-2D^d. Activation of NK cells by Ly-49D was not unique to transfected targets, as Ly-49D also stimulated lysis against blasts from H-2^d mice, but not against blasts from H-2^b or H-2^k mice. We considered the possibility that these results might be obtained if Ly-49D were not the activation receptor itself but instead, through its interaction with H-2D^d, facilitated activation through a separate activating receptor. However, it is unlikely that adhesion alone between Ly-49D and H-2D^d accounts for the observed activation of cytotoxicity. First, stimulation of the Ly-49D receptor with antibody is known to induce activation of cytotoxicity by NK cells, as shown here and by others (14). Second, we have previously demonstrated that adhesion between another Ly-49 receptor and H-2D^d is not sufficient to activate NK cell lysis. Specifically, we have previously demonstrated that interaction of an inactive Ly-49A receptor with H-2D^d does not activate NK cell lysis through other receptors on RNK-16 (25). The Ly-49A receptor normally binds to H-2D^d and thereby leads to inhibition of NK cell lysis. Our previous studies showed inhibition of lysis of H-2D^d expressing targets by RNK-16 cells transfected with Ly-49A. We also studied a mutated Ly-49A receptor, containing a point mutation in the cytoplasmic domain of the receptor which disrupts the ITIM motif required for inhibitory function. This mutated Ly-49A receptor expressed on RNK-16 cells failed to inhibit or to augment lysis of H-2D^d–expressing targets. This mutated Ly-49A receptor is unaltered in the extracellular or transmembrane domains so its binding to H-2D^d is presumably unaltered. Thus, engagement of H-2D^d by the mutant Ly-49A receptor does not facilitate the activation of lytic pathways through other receptors on RNK-16.

It is of considerable interest whether the inhibitory Ly-49A receptor and the activating Ly-49D receptor recognize the same or distinct domains of the H-2D^d antigen. As with Ly-49A, Ly-49D–dependent effects required the α₁/α₂ domains of H-2D^d and could be blocked by an α₁/α₂-specific F(ab′)₂ anti–H-2D^d mAb (34-5-8S) (references 32–34, 36). The extracellular domains of Ly-49A and Ly-49D may recognize similar regions of H-2D^d, but only Ly-49A recognizes H-2D^k (33–35). Thus, Ly-49A and Ly-49D appear to exhibit overlapping but distinct allelic specificities for murine class I MHC antigens. Studies by several groups have indicated that Ly-49A does not discriminate between different peptides presented by H-2D^d (32, 37). The role of antigenic peptides in the specificity of Ly-49D–H-2D^d interaction remains to be examined.

It has been speculated that activating Ly-49–like receptors may participate in the recognition of abnormal or virally-encoded MHC-like molecules by NK cells, although this has not been proven (6, 38). However, the physiologic significance of alloactivating NK receptors is unknown. Although their effects may be partially masked by inhibitory NK cell receptors, the identification of in vivo NK alloactivating functions indicates that alloactivation is not completely abrogated by inhibitory MHC receptors in vivo (19–22). A delicate balance between activating and inhibitory alloreceptors may be influenced by different affinities or structural requirements for ligand binding. Alternatively, expression of opposing alloreceptors on different NK cell subsets may permit NK alloresponses in vivo. Alloactivating NK cell receptors, in addition to allospecific T cell receptors, likely play a role in the physiologic rejection of foreign cells (6, 7, 23, 39).

In conclusion, our experiments using gene transfer provide the first direct evidence that an activating receptor on murine NK cells specifically directs cytotoxicity against a classical class I MHC ligand. They also provide a model for dissecting the molecular pathways involved in this interaction. Other candidate alloactivating receptors on human NK cells include the Ig-like short domain KIR (killer cell inhibitory receptor) receptors, which may recognize alleles of HLA-C (8, 15, 16), and the activating CD94/NKG2C receptor, which can bind to ligand HLA-E (11). Future studies to define the distribution and activation requirements for these receptors will help us to understand the physiologic functions of alloactivating receptors in NK cell biology.

This work was supported by the Veterans Administration and by National Institutes of Health (NIH) grant RO1 CA69299 (to W.E. Seaman). J.C. Ryan is the recipient of NIH grant R29 CA60944 and is supported by the International Human Frontiers in Science Program. M.C. Nakamura is supported by NIH grant K11 AR01927, the Rosalind Russell Arthritis Foundation, and NIH Multipurpose Arthritis Center grant P60 AR20684.

ITIM

immunoreceptor tyrosine based inhibitory motif