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iim$$$0407 International Immunology, Vol. 9, No. 4, pp. 533–540 © 1997 Oxford University Press The NK2.1 receptor is encoded by Ly-49C and its expression is regulated by MHC class I alleles Pierre Gosselin1,3, Yvette Lusignan1, Jack Brennan2, Fumio Takei2 and Suzanne Lemieux1 1Centre de Recherche en Immunologie, Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada H7V 1B7 2Terry Fox Laboratory, British Columbia Cancer Agency, University of British Columbia, Vancouver, British Columbia, Canada V5Z 1L3 3Present address: Laboratory of Experimental Immunology, Division of Basic Sciences, NCI-FCRDC, Frederick, MD, 21702-1201, USA Keywords: Ly-49 receptor, murine MHC molecules, NK cell Abstract A dual receptor system composed of activation and inhibitory receptors apparently controls NK cell-mediated lysis. In the C57BL/6 mouse, the NK1.1 molecule acts as an activation receptor whereas Ly-49A, C and G2 can inhibit NK cell lysis of target cells expressing specific MHC class I molecules. We previously reported that NK2.1 is an activation receptor sharing structural properties with members of the NKR-P1 and Ly-49 receptor families. In this study, we have shown that NK2.1 is encoded by the previously described Ly-49C gene. We also found that the expression level of NK2.1/Ly-49C is modulated by H-2-dependent factors and that this regulation differs from that previously described for Ly-49A. Flow cytometry analyses of NK-enriched spleen cells from MHC congenic strains on C57BL/10 and BALB/c backgrounds indeed revealed that the level of NK2.1/Ly-49C expression, but not the number of positive cells, is low in strains expressing H-2b and H-2k haplotypes as compared to H-2d mice. Similar analyses of splenic NK cells from two series of congenic and congenic recombinant strains on the C57BL/10 background indicate that the main regulatory element(s) are most likely the H-2Kb and H-2Dk alleles. Together with our and others previous observations, these results identify the NK2.1/Ly-49C antigen as a receptor for MHC class I molecules whose expression is regulated by host MHC genes. Introduction NK cells can kill tumor cells and virus-infected cells without the need for prior sensitization and without the requirement for MHC molecule expression on target cells (1). Instead, it is of general agreement that MHC class I molecules expressed by target cells may interrupt the lytic process of NK cells, following their interaction with specific inhibitory receptors (2). Ly-49A, which selectively binds H-2Dd and Dk molecules (3), is the first mouse NK cell antigen shown to have such inhibitory properties (4). In addition to inhibition of NK cell lysis, the interaction between membrane-bound H-2Dd or Dk host molecules and Ly-49A was shown to result in downregulation of Ly-49A expression on individual NK cells although the size of the Ly-49A1 subpopulation is unaltered (5). On the other hand, NK cell-mediated killing can be triggered through activation receptors such as NK1.1 (6) which is a member of the NKR-P1 family (7). The physiologic ligand of NK1.1 is still unknown and, contrary to Ly-49A, NK1.1 expression is not influenced by host MHC haplotypes (5). Ly-49A and NK1.1 belong to two families of genes mapping to the distal segment of mouse chromosome 6, named the NK gene complex, and encoding type II integral membrane proteins homologous to C-type animal lectins (8–15). Whereas in positive strains NK1.1 is expressed by most NK cells (16), Ly-49A defines a small subset of splenic NK cells (12). The Ly-49 family also includes the 5E6 and LGL-1 molecules, also expressed by NK cell subpopulations (17,18) and encoded by Ly-49C and Ly-49G2 genes respectively (19–21). As reported for Ly-49A1 cells, Ly-49G21 NK cells are inhibited Correspondence to: S. Lemieux Transmitting editor: H. R. MacDonald Received 26 August 1996, accepted 25 December 1996 534 MHC-dependent regulation of NK2.1/Ly-49C expression by MHC class I molecules expressed on target cells (21). More recently, binding experiments showed that Ly-49C binds to the H-2Kb, Kd and Dd molecules (22). Observations made in hybrid resistance studies further suggested that Ly-49C can inhibit NK cell lysis upon recognition of H-2Kb and H-2d molecules (23). Interestingly, the different members of the Ly49 family appear to have distinct but overlapping class I specificities. Until recently, the nature and function of the mouse NK2.1 molecule, an NK cell antigen initially identified with an NZB anti-BALB/c antiserum (24), were still unknown. Our laboratory has produced an anti-NK2.1 mAb (25) and characterized the NK2.1 antigen as a highly glycosylated disulfide-linked protein dimer of 65 kDa subunits (26). We further showed that immobilized anti-NK2.1 mAb induced granule exocytosis from IL-2-activated cells and that soluble mAb as well as its F(ab9)2 and Fab fragments increased lysis of NK susceptible target cells by resting or IL-2-activated cells (27). These results are thus consistent with NK2.1 being a relevant activation receptor for natural killing. Similar to Ly-49 receptors characterized thus far, NK2.1 is expressed by a subpopulation rather than all NK cells (25–28). In addition, the percentage of NK2.11 cells in nylon-wool non-adherent spleen cells as well as the level of NK2.1 expression are variable among positive mouse strains (28). In the present study, we report that NK2.1 is encoded by the Ly-49C gene and that its cell surface expression is down-regulated by host MHC genes of H-2b and H-2k haplotypes. Methods Mice AKR/N, BALB/cAnN, C57BL/6N, C3H/HeN, DBA/2N and (BALB/cAnN3C57BL/6N)F1 mice were purchased from Charles River Canada (St Constant, Québec, Canada). 129/ Sv, A/J, C57BL/10SnJ, CBA/J, LP/J, B10.A/SgSnJ, B10.A(2R)/ SgSnJ, B10.A(5R)/SgSnJ, B10.BR/SgSnJ, B10.D2/nSnJ, B10.D2(R103)/EgDvEgJ, B10.D2(R107)/EgDvEgJ, BALB.B/ LiMcdJ, BALB.K/LiMcdJ and NZB/BINJ mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Antibodies The 4LO3311 anti-NK2.1 mAb (mouse IgG3) was generated in our laboratory (25). The hybridoma PK136 producing antiNK1.1 mAb (mouse IgG2a) (29) was purchased from ATCC (Rockville, MD). These mAb were purified by affinity chromatography and biotinylated using standard methods. Biotinylated 5E6 mAb (mouse IgG2a) (17) was obtained from PharMingen (San Diego, CA). Phycoerythrin (PE)-labeled A1 mAb (mouse IgG2a) (8) was kindly provided by Dr K. P. Kane (University of Alberta, Edmonton, Alberta, Canada). Irrelevant biotinylated mouse IgG3 mAb, purchased from PharMingen, and mouse IgG2a mAb, generously provided by Dr P. J. Talbot (Institut Armand-Frappier), were used as isotype controls. Enrichment of splenic NK cells Splenic NK cells were enriched by selective depletion of T lymphocytes from nylon-wool non-adherent cells using antiCD4 and anti-CD8 rat mAb and sheep anti-rat IgG-coated magnetic beads (Dynal, Great Neck, NY) as previously described (26). We reported elsewhere that .90% of these cells react with anti-asialo GM1 antiserum (27). NK cell staining and flow cytometry analysis Splenic NK cells were incubated for 30 min on ice with optimal concentrations of PE-labeled A1 mAb or biotinylated mAb. Binding of biotinylated mAb was detected with PE-labeled streptavidin (Becton Dickinson, Mountain View, CA). Control samples were incubated with biotinylated isotype control mAb and streptavidin–PE. In the phenotypic study of (1293C57BL/ 6)F13129 backcross animals, samples from individual mice were analyzed on a Coulter Epics C flow cytometer (Coulter Electronics, Hialeah, FL) equipped with a water-cooled 5 W argon laser of 488 nm. The analysis of NK2.1 expression in inbred, congenic and hybrid mice was performed with a Coulter Epics XL equipped with an air-cooled 15 mW argon laser of 488 nm. Results are expressed as the percentage of lymphocytes, defined by forward and side scatter gates, which reacted with anti-NK mAb. Data analysis based on collection of 10,000 events/sample was done with XL software. The cytometers used for NK cell analysis were calibrated daily with 10 µm Coulter DNA-check beads (Coulter, Hialeah, FL) using a standard protocol. COS cell transfections and analyses cDNAs encoding individual members of the Ly-49 family (Ly-49AB6, B, CBALB/CBA, DB6, EB6, FB6, G4B6 and H) were subcloned into the expression vector pAX142 and COS cells were transfected by the DEAE–dextran method as previously described (19). The Ly-49EB6 and Ly-49FB6 cDNAs (15) were obtained from Dr W. M. Yokoyama (Washington University, St Louis, MO). Cells were analyzed for cell surface expression by flow cytometry 72 h post-transfection. R-PE-conjugated 5E6 and FITC-labeled A1 were purchased from PharMingen (San Diego, CA). Cell staining procedures were carried out for 30 min at 4°C at concentrations of 107 cells/ml, followed by two washes in PBS containing 2% FCS. Those samples stained with biotinylated 4LO3311 required a secondary step with streptavidin–FITC. Analysis was performed on a FACSort (Becton Dickinson), and dead cells were stained with propidium iodide (1 µg/ml in final wash) and gated out. Results Linkage of the NK2.1 gene to the NK gene complex To investigate whether the NK2.1 gene is associated with those of the NK gene complex, the expression of NK1.1 (musNKR-P1C), 5E6 (Ly-49C) and NK2.1 on NK-enriched spleen cells from 80 individual mice of the (1293C57BL/ 6)F13129 backcross progeny was determined by flow cytometry analysis. This strain combination was suitable for a linkage study since 129 mice are NK1.1–5E6–NK2.1– whereas all three antigens are expressed in C57BL/6 (17,24,25,30). Because only a low number of dimly fluorescent cells was detected after incubation of C57BL/6 nylon-wool non-adherent spleen cells with 4LO3311 anti-NK2.1 mAb (28), the backcross analysis was performed on NK-enriched spleen cells in order to get more reliable results. Our data showed that musNKR- MHC-dependent regulation of NK2.1/Ly-49C expression 535 Table 1. Expression of NK1.1, 5E6 and NK2.1 antigens in the (1293C57BL/6)F13129 backcross progenya NK2.11 NK2.1– Total NK1.11/5E61 NK1.1–/5E6– Total 36 0 36 0 44 44 36 44 80 aExpression of NK1.1, NK2.1 and 5E6 was determined by flow cytometry analysis of NK-enriched spleen cells from individual animals as described in Methods. Results shown correspond to the number of mice expressing a given phenotype. P1C, Ly-49C and NK2.1 genes never segregated in backcross mice (Table 1). In agreement with these results, a separate study from our laboratory revealed that none of the recombinant inbred mice of the 129XB6 series, kindly provided by Dr J.-L. Guenet (Institut Pasteur, Paris, France), were NK1.1 or NK2.1 single positive (unpublished observations). It thus appears that the NK2.1 gene maps to chromosome 6 either near or within the NK gene complex, in association with the NKR-P1 and Ly-49 gene families. Identification of NK2.1 as Ly-49C Several features of NK2.1 initially suggested that it might be encoded by a member of the NKR-P1 or Ly-49 gene families. In addition to its chromosomal localization to the NK gene complex, NK2.1 has been shown to be a dimeric cell surface antigen expressed by a subset of NK cells (26). The fact that NK2.1 is expressed in strains that lack NKR-P1 transcripts (31) led us to first investigate the Ly-49 gene family. Individual Ly-49 cDNAs were transiently expressed in COS cells which were subsequently tested for reactivity with the anti-NK2.1 mAb. 4LO3311 was found to react specifically with Ly-49C (Fig. 1) but none of the other Ly-49s tested (data not shown for Ly-49B, D, E, F, G4 and H). As reported previously (10,19), the A1 mAb specifically bound Ly-49A and the 5E6 mAb also recognized Ly-49C (Fig. 1). Although both 5E6 and 4LO3311 mAb apparently recognize the same Ly-49 molecule, they likely detect different epitopes of that polymorphic receptor. This is suggested by the existence of 5E614LO3311– cells in certain strains of mice (17,25) and the absence of competition between 4LO3311 and 5E6 mAb for staining BALB/c NK cells (data not shown). Variations in NK2.1/Ly-49C expression among inbred mouse strains Our earlier flow cytometry analyses of nylon-wool non-adherent spleen cells from selected mouse strains have suggested a strain variation in the level of NK2.1 expression (28). Since expression of Ly-49A has been reported to be regulated by host MHC class I alleles (5,32) and Ly-49C has recently been shown to also be a receptor for MHC class I molecules (19,22,23), it was of interest to determine whether NK2.1/Ly49C expression may be subject to a similar regulation. In order to answer that question, we extended the flow cytometry analysis of NK2.1/Ly-49C expression on NK-enriched spleen cells to a number of mouse strains of different H-2 haplotypes. As presented in Fig. 2 and Table 2, the percentage of NK2.1/ Fig. 1. Expression of NK2.1 on COS cells transfected with Ly-49C. Ly-49 cDNAs were expressed in COS cells and stained with the antibodies A1, 4LO3311 or 5E6. Solid histograms represent COS cells transfected with Ly-49A or C and empty histograms are cells transfected with vector alone. Ly-49C1 cells and/or their mean fluorescence intensity (MFI) was found to be low in C57BL/6 and C57BL/10 (H-2b) mice, intermediate in AKR, C3H and CBA (H-2k) mice, and high in BALB/c and DBA/2 (H-2d) mice. Interestingly, NK2.1/Ly-49C1 cells of A/J (H-2a) mice, which express H-2Kk and Dd molecules, showed a MFI in the range of H-2d mice. With an NK2.1/ Ly-49C1 cell population with a higher MFI than other H-2b strains as well as a second peak of low intensity, LP mice showed a unique phenotype compared to all other mouse strains. Altogether, these results indicated that H-2-linked factors might influence NK2.1/Ly-49C expression but also strongly suggest that H-2-independent factors are involved. Variations in NK2.1/Ly-49C expression in H-2-congenic mice To better evaluate the putative modulation of NK2.1/Ly-49C by H-2 haplotypes, we carried out a series of flow cytometry analyses of NK2.1/Ly-49C expression on NK-enriched spleen cells from selected MHC-congenic strains. Since C57BL/10 and BALB/c mice showed respectively the NK2.1/Ly-49C NK cell populations with the lowest and highest MFI, we reasoned that if NK2.1/Ly-49C expression is indeed H-2-dependent, the NK2.1/Ly-49C phenotype of congenic mice would tend towards the phenotype of the mouse strain contributing the H-2 allele, and thus vary in opposite directions in B10 and BALB/c congenic mice expressing a given haplotype. With MHC-congenic strains on B10 background, the MFI of NK2.1/ Ly-49C1 cells was enhanced three to four times in B10.D2 (H-2d) mice (P , 0.0001 as calculated by the two tailed Student’s t-test from three to four mice per group) but remained unchanged in B10.BR (H-2k) mice (Fig. 3A). No significant variation in the percentage of NK2.1/Ly-49C1 cells was detected in these congenic mice. As reported by others (5), we found no alteration of NK1.1 expression in B10 congenic strains expressing H-2d or H-2k haplotypes, whereas the Ly49A expression detected by the A1 mAb was significantly reduced (data not shown). As expected, when similar analyses were done with MHC-congenic strains on the BALB/c background, a significant down-regulation of NK2.1/Ly-49C expression level was observed in BALB.K (H-2k) and BALB.B (H-2b) mice (P , 0.0001) (Fig. 3B). An increase in the percentage of NK2.1/Ly-49C1 cells was detected in BALB.K (H-2k) mice (P , 0.0001) but not in BALB.B (H-2b) mice. These results strongly support the hypothesis that the expression of NK2.1/Ly-49C is regulated by the host H-2 haplotype. 536 MHC-dependent regulation of NK2.1/Ly-49C expression Fig. 2. NK2.1/Ly-49C expression on NK-enriched spleen cells from different inbred mouse strains. Nylon-wool non-adherent CD4–CD8– spleen cells were stained with biotinylated anti-NK2.1 mAb (solid lines) or isotype control mAb (dotted lines) and streptavidin–PE, and then analyzed by flow cytometry. The histograms are representative of three to five experiments. NK2.1/Ly-49C expression is mainly modulated by H-2Kb and H-2Dk To clarify further the contribution of H-2 loci in regulating NK2.1/Ly-49C expression, a series of B10 congenic strains carrying a chromosome 17 differential segment of variable length inherited from DBA/2 or A mice were included in the study. Comparative flow cytometry analysis of NK-enriched spleen cells revealed that the percentage of NK2.1/Ly-49C1 cells was almost the same in all MHC-congenic and recombinant lines tested (Table 3). However, it appeared that the MFI of the NK2.1/Ly-49C1 cell population was markedly lowered in all mice expressing H-2Kb as compared to H-2Kd. In mice expressing H-2Kb, an haplotype change from b to d at the D locus, as in B10.D2(R107) and B10.A(5R), did not further change the level of NK2.1/Ly-49C expression. As NK2.1/Ly49C expression is low in B10.BR (Table 3), it is likely that the H-2k haplotype also contributes to the down-regulation observed. In this case, however, the H-2D rather than H-2K locus appears to be determinant. Consistent with this hypothesis, the level of NK2.1/Ly-49C expression in B10.A was only slightly reduced compared to B10.D2, whereas it is low in B10.BR which differs from B10.A by expressing H-2Dk rather than H-2Dd. These results suggest that as for Ly-49A, the host MHC class I molecules which are the natural ligands of NK2.1/Ly-49C are involved in regulating the level of expression of this NK cell receptor. Two NK cell populations expressing different levels of NK2.1/ Ly-49C are present in (BALB/c3C57BL/6)F1 mice Since NK2.1/Ly-49C is highly expressed in H-2d mice and down-regulated in H-2b mice, it was of interest to determine its expression level in H-2d/b heterozygous mice. As shown in Fig. 4, two slightly overlapping NK2.1/Ly-49C1 cell populations were observed in (BALB/c3C57BL/6)F1 mice. This detection of a smaller C57BL/6- and a larger BALB/c-like population in the hybrid mice with the 4LO3311 mAb further supports the recent observation that Ly-49C is subject to allelic exclusion (37). Interestingly, the expression level of NK2.1/Ly-49C on the BALB/c-like population was half the one found in BALB/c (H-2d) mice (MFI: 49 and 104 respectively). It was however about twice the expression level found on BALB.B (H-2b) NK cells (MFI: 25 as shown in Fig. 3). A similar pattern was observed for the C57BL/6-like population. Whereas NK2.1/ Ly-49C was expressed on these cells at about twice the level found in C57BL/6 (H-2b) mice (MFI: 18 and 10 respectively) it was half the one found in B10.D2 (H-2d) mice (MFI: 38 as shown in Table 3). The cell surface expression level of NK2.1/ Ly-49C thus appears to be down-regulated to a lower extent MHC-dependent regulation of NK2.1/Ly-49C expression 537 Table 2. Expression of NK2.1/Ly-49C on NK-enriched spleen cells from various inbred mouse strainsa Strain C57BL/6 C57BL/10 LPb AKR C3H CBA A BALB/c DBA/2 129 NZB H-2 haplotype b b b k k k a d d b d Percent positive cells 6 SD MFI 6 SD 28 6 25 6 33 6 40 6 39 6 33 6 33 6 52 6 28 6 ,1 ,1 10 6 1 11 6 1 27 6 4 20 6 2 24 6 3 17 6 2 78 6 10 115 6 15 66 6 8 NAc NA 2 6 8 5 3 2 2 5 1 aExpression of NK2.1 on NK-enriched spleen cells from three to seven individual mice of each strain was analyzed by flow cytometry as described in Methods. bIn this mouse strain, an NK2.1int cell populations representing 13 6 2 % of the cells with a MFI of 4 6 1 was also clearly detectable in every mouse tested. cNot applicable. when the regulatory MHC gene is expressed co-dominantly with another allele. Discussion Our previous structural and functional studies of the NK2.1 antigen indicated that this molecule was a disulfide-linked dimer, activation receptor specifically expressed by NK cells (26,27). An activating function for NK2.1 was suggested by the ability of anti-NK2.1 to augment the lysis of NK-sensitive targets by fresh and IL-2-activated NK cells and to induce granule exocytosis from IL-2-activated BALB/c NK cells (27). It is therefore of interest that NK2.1 was found to be encoded by Ly-49C because recent studies of this molecule have suggested that it (23), like Ly-49A (4) and G2 (21), functions as an inhibitory receptor. Indeed, Ly-49C1 NK cells, as recognized by the mAb 5E6, acquire the ability to lyse otherwise resistant H-2Kb bearing targets following the addition of either F(ab9)2 5E6 or anti-Kb antibodies (23). This observation, together with the binding specificity of Ly-49C for Kb (22) strongly suggests that the recognition of MHC class I by this receptor results in the delivery of a negative Fig. 3. NK2.1/Ly-49C expression on NK-enriched spleen cells from H-2 congenic mice on the B10 and BALB/c backgrounds. NK-enriched spleen cells from inbred and congenic mice on the B10 (A) and BALB/c (B) backgrounds were stained with biotinylated 4LO3311 mAb (solid lines) or isotype control mAb (dotted lines) and streptavidin–PE before being analyzed by flow cytometry. The histograms are representative of three to five experiments. The percentage of NK2.1/Ly-49C1 cells and the corresponding MFI are indicated (% cells/MFI). These are mean values from three to five mice. 538 MHC-dependent regulation of NK2.1/Ly-49C expression Table 3. Expression of NK2.1/Ly-49C on NK-enriched spleen cells from MHC congenic and congenic recombinant micea Strain B10.D2 C57BL/10 B10.BR B10.D2(R103) B10.D2(R107) B10.A B10.A(2R) B10.A(5R) H-2 haplotype d b k g3 i7 a h2 i5 Positive cells 6 SD (%) H-2 allelesb for K A E D d b k d b k k b d b k d b k k b d b k d b k k / k d b k / b / d d / b d 24 27 33 22 24 25 27 31 6 6 6 6 6 6 6 6 2 4 8 9 8 3 6 8 MFI 6 SD MFI reduction (relative to H-2d) (%) 38 6 3 11 6 3 863 24 6 7 963 28 6 7 18 6 2 13 6 3 N/Ac 71 79 37 76 26 53 66 a b See legend to Table 2. For each strain, the haplotype of each MHC subregion is shown with a slash indicating the site of recombination when applicable. c Not applicable signal to an NK cell. The stimulatory effects that we have seen with the anti-NK2.1 mAb are therefore provocative and suggest that this receptor may in some cases have an activating function. Although the enhanced lysis which follows the addition of anti-NK2.1 mAb may also be interpreted in terms of a blockage of negative signalling, the induced granule exocytosis appears to be the result of an actual triggering effect because immobilized isotype control antibodies (IgG3) had no such effect (27). Moreover, it was previously reported that 5E6 anti-Ly-49C mAb was able to trigger redirected lysis of FcR1 targets (33), an observation consistent with activating properties. Studies of human NK cell recognition have found that activating and inhibitory functions may be shared by the same type of receptor. The first such example described was that of CD941 NK cell clones, some of which are activated and some of which are inhibited in the presence of an anti-CD94 mAb (34). Although the mechanisms responsible for this ambivalence remain unclear, high-level CD94 expression on a NK cell appears to be required for inhibition. The p58 family of molecules, members of which bind HLA-C and inhibit NK cell killing, has also been found to encode receptors (p50) which activate NK cells upon class I recognition. These activation structures differ from the inhibitory receptors in the transmembrane region and in their truncated cytoplasmic domains (35). The occurrence of either activating or inhibitory effects upon binding of NK cells to target MHC molecules has also been documented in the rat (36), thus supporting the idea that dual MHC antigen-induced signaling functions may be more common than originally anticipated. In the case of Ly-49, however, it remains unknown what factors or experimental conditions may result in activation or inhibitory signals. Original studies of the Ly-49 gene family showed significant restriction fragment length polymorphism among various inbred strains of mice, suggesting that these genes are highly polymorphic (12). In the case of Ly-49C, it was reported that C57BL/6, NZB and 129 mice each express distinct alleles that are different from the one shared by BALB/c, CBA and A/Sn mice (20,37,38). The 4LO3311 and 5E6 mAb were both derived from 129 mice immunized with C57BL/6 NK cells (17,25) and they both recognize Ly-49C (20 and this study). Fig. 4. NK2.1/Ly-49C expression in H-2d/b hybrid mice. NK-enriched spleen cells from BALB/c (H-2d) (dotted line), C57BL/6 (H-2b) (thin solid line) and (BALB/c3C57BL/6)F1 (H-2d/b) (thick solid line) mice were stained with biotin-conjugated 4LO3311 mAb and streptavidin– PE and then analyzed by flow cytometry. The histogram shown is representative of four similar experiments. Since 5E614LO3311– splenic NK cells were detected in certain strains of mice (17, 25), it is likely that 4LO3311 and 5E6 mAb bind different epitopes of this polymorphic molecule. Allelic variations may also result in some forms of Ly-49C that are recognized by neither, both or only one of these antibodies. Finally, it is also possible that in addition to react with Ly-49C, one or both of these antibodies recognize other unidentified Ly-49 molecules. We confirmed by competition studies that 5E6 and 4LO3311 mAb bind different epitopes of Ly-49C. In a concurrent study, we demonstrated that in C57BL/6 mice, 5E6 but not 4LO3311 mAb detects two distinct but highly related molecules (39). The one reacting with 5E6 but not with 4LO3311 mAb was identified as the product of a gene previously thought to be the B6 allele of Ly-49C, which was renamed Ly-49I. From the comparison of the deduced amino acid sequences of the cDNAs, it appeared that the BALB/c and C57BL/6 Ly-49C gene products differ at only four residues. While our paper was in press, the cloning of an identical C57BL/6 Ly-49C cDNA and the recognition of its gene product by the 5E6 mAb were reported by Sundbäck et al. (38). Their observations and those of previous reports MHC-dependent regulation of NK2.1/Ly-49C expression 539 (20,37) confirm that in C57BL/6 mice, the 5E6 mAb reacts with two molecules. The 4LO3311 mAb thus appears to be the only known antibody reacting only with Ly-49C. The reactivity of the 5E6 mAb for Ly-49C and Ly-49I could therefore certainly account for the recently reported inconsistencies regarding Ly-49C expression in relation to host MHC class I haplotypes (40). The variation in NK2.1/Ly-49C expression on NK cells from several inbred, MHC-congenic, and congenic recombinant mouse strains raised the possibility that NK2.1/Ly-49C expression is regulated in a way similar to Ly-49A and Ly-49G2 in hosts expressing their class I ligands (5,40). The enhanced NK2.1/Ly-49C expression in B10.D2 (H-2d) compared to B10 (H-2b) and B10.BR (H-2k) and, conversely, the reduced NK2.1/ Ly-49C expression in BALB.B (H-2b) and BALB.K (H-2k) compared to the BALB/c (H-2d) inbred partner strongly support an H-2-dependent regulation of this receptor. The low MFI of NK2.1/Ly-49C1 cells in B10 (H-2Kb, H-2Db), B10.D2 (R107) (H-2Kb, H-2Dd) and B10.A (5R) (H-2Kb, H-2Dd) mice are consistent with NK2.1/Ly-49C expression being downregulated by H-2Kb. In view of the recently described interactions between Ly-49C and H-2Kb (22), the expression of NK2.1/Ly-49C on NK cells, like that of Ly-49A, appears to be down-regulated in the presence of its MHC class I ligand. Although the occurrence of NK2.1/Ly-49C1 cells with intermediate fluorescence intensity in LP mice appears inconsistent with the hypothesis that H-2Kb molecules down-regulate NK2.1/Ly-49C expression, this is most probably explained by allelic variation in Ly-49C which has resulted in a receptor with reduced affinity for Kb and therefore shows little or no down-regulation by it. In our experiments, the highest NK2.1/Ly-49C expression was seen in H-2d mice, and the lowest was in H-2b and H-2k mice. All observations concerning NK2.1/Ly-49C calibration are therefore made relative to these two levels. H-2 recombinant analysis has in fact shown that the level of NK2.1/Ly-49C expression falls along a continuum between these high (H-2d) and low (H-2b and H-2k) standards. In addition to the maximal down-regulation associated with the K locus of the b haplotype (66–76% MFI reduction), the D locus of this same haplotype resulted in a moderate down-regulation (37%). With regards to H-2k down-regulation (79%), the K locus alone was shown to have only a modest effect (26%). It is therefore thought that Dk may have a more significant role in NK2.1/Ly-49C receptor modulation. This variability in the level of receptor calibration is likely influenced by the affinity of an Ly-49 for a given class I molecule, such that stronger interactions bring about greater down-regulation. Because the H-2d haplotype was associated with the highest NK2.1/Ly-49C expression relative to those tested, it is unknown to what degree these H-2 antigens affect NK2.1/Ly-49C expression. In light of binding studies which have shown Ly-49C to bind to H-2b, H-2k, as well as H-2d antigens (including Kd and Dd), it is expected that expression of this receptor is affected by these molecules, although weaker affinity interactions may result in a relatively low degree of down-regulation. The use of congenic strains has limitations since congenic mice generally differ from their inbred partners at hundreds of genes inherited from the donor strain, the exact length of the differential segment often being unknown. Our data at least clearly indicate that the main H-2-associated element down-regulating NK2.1/Ly-49C expression is centromeric to H-2D in H-2b mice, and telomeric to H-2K in H-2k mice, although multiple H-2 regions appear to be involved and their relative importance may be haplotype-dependent. It is noteworthy that although NK2.1/Ly-49C expression in B10.D2 mice is enhanced compared to B10 mice, it does not reach the expression level observed in the donor DBA/2 mice. Similarly, the introduction of a B10 differential segment including the H-2 complex into BALB/c mice did not reduce NK2.1/ Ly-49C expression to the level found in B10 mice, thus indicating that as suggested for other Ly-49 receptors (40) non-H-2-dependent factors may also contribute to the regulation of NK2.1/Ly-49C expression and influence the Ly-49 repertoire. It is expected that polymorphism of an individual Ly-49 is one such factor that will affect its regulation by the H-2 complex. Non-MHC-linked genes putatively influencing the expression level of NK2.1/Ly-49C may have contributed to the particular pattern observed in (C57BL/63BALB/c)F1 mice. However, the enhanced expression of NK2.1/Ly-49C on the C57BL/6-like cell population detected in the hybrid mice and its reduced expression on the BALB/c-like cell population, relative to those found in the parental strains, may also indicate that the C57BL/6 and BALB/c Ly-49C alleles are similarly down-regulated by H-2b. This is further supported by our recent observation that these two Ly-49C alleles have indistinguishable MHC class I binding specificity (39). The 10-fold difference observed between BALB/c (H-2d) and C57BL/6 (H-2b) mice, regarding the MFI of NK2.1/Ly-49C1 cells, has certainly facilitated the detection of intermediate cell surface expression levels of this receptor in hybrid mice expressing lower levels of H-2b. Although it was initially reported that the decreased expression of Ly-49A was inherited as a dominant trait (32), it is conceivable that several Ly-49 molecules including Ly-49A may be subject to a H-2dependent fine tuning of cell surface receptor calibration as shown here for Ly-49C. However, subtle variations in the expression level of a given receptor might be difficult to see in hybrid mice when differences in parental strains are ,2fold, as observed recently for Ly-49A and Ly-49G2 (40). Abbreviations MFI PE mean fluorescence intensity Phycoerythrin References 1 Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187. 2 Ljunggren, H.-G. and Kärre, K. 1990. In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol. Today 11:7. 3 Kane, K. P. 1994. Ly-49 mediates EL4 lymphoma adhesion to isolated class I major histocompatibility complex molecules. J. Exp. Med. 179:1011. 4 Karlhofer, F. M., Ribaudo, R. K. and Yokoyama, W. M. 1992. MHC class I alloantigen specificity of Ly-491 IL-2-activated natural killer cells. Nature 358:66. 540 MHC-dependent regulation of NK2.1/Ly-49C expression 5 Karlhofer, F. M., Hunziker, R., Reichlin, A., Margulies, D. H. and Yokoyama, W. M. 1994. Host MHC class I molecules modulate in vivo expression of a NK cell receptor. J. Immunol. 153:2407. 6 Karlhofer, F. M. and Yokoyama, W. M. 1991. Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL-2-activated NK cells possess additional specific stimulation pathways. J. Immunol. 146:3662. 7 Ryan, J. C., Turck, J., Niemi, E. C., Yokoyama, W. M. and Seaman, W. E. 1992. Molecular cloning of the NK1.1 antigen, a member of the NKR-P1 family of natural killer cell activation molecules. J. Immunol. 149:1631. 8 Nagasawa, R., Gross, J., Kanagawa, O., Townsend, K., Lanier, L. L., Chiller, J. and Allison, J. P. 1987. Identification of a novel T cell surface disulfide-bonded dimer distinct from the α/β antigen receptor. J. Immunol. 138:815. 9 Chan, P.-Y. and Takei, F. 1989. Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48. J. Immunol. 142:1727. 10 Yokoyama, W. M., Jacobs, L. B., Kanagawa, O., Shevach, E. M. and Cohen, D. I. 1989. A murine T lymphocyte antigen belongs to a supergene family of type II integral membrane proteins. J. Immunol. 143:1379. 11 Giorda, R. and Trucco, M. 1991. Mouse NKR-P1. A family of genes selectively coexpressed in adherent lymphokine-activated killer cells. J. Immunol. 147:1701. 12 Yokoyama, W. M., Kehn, P. J., Cohen, D. I. and Shevach, E. M. 1990. Chromosomal location of the Ly-49 (A1, YE1/48) multigene family: genetic association with the NK1.1 antigen. J. Immunol. 145:2353. 13 Yokoyama, W. M., Ryan, J. C., Hunter, J. J., Smith, H. R. C., Stark, M. and Seaman, W. E. 1991. cDNA cloning of mouse NKR-P1 and genetic linkage with Ly-49. Identification of a natural killer cell gene complex on mouse chromosome 6. J. Immunol. 147:3229. 14 Wong, S., Freeman, J. D., Kelleher, C., Mager, D. and Takei, F. 1991. Ly-49 multigene family: new members of a superfamily of type II membrane proteins with lectin-like domains. J. Immunol. 147:1417. 15 Smith, H. R. C., Karlhofer, F. M. and Yokoyama, W. M. 1994. Ly-49 multigene family expressed by IL-2-activated NK cells. J. Immunol. 153:1068. 16 Hackett, J., Jr, Tutt, M., Lipscomb, M., Bennett, M., Koo, G. and Kumar, V. 1986. Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.11 cells. J. Immunol. 136:3124. 17 Sentman, C. L., Hackett, J., Jr, Kumar, V. and Bennett, M. 1989. Identification of a subset of murine natural killer cells that mediate rejection of Hh-1d but not Hh-1b bone marrow grafts. J. Exp. Med. 170:191. 18 Mason, L. H., Mathieson, B. J. and Ortaldo, J. R. 1990. Natural killer (NK) subsets in the mouse. NK1.11/LGL-11 cells restricted to lysing NK targets, whereas NK-1.11/LGL-1– cells generate lymphokine-activated killer cells. J. Immunol. 145:751. 19 Brennan, J., Mager, D., Jefferies, W. and Takei, F. 1994. Expression of different members of the Ly-49 gene family defines distinct natural killer cell subsets and cell adhesion properties. J. Exp. Med. 180:2287. 20 Stoneman, E. R., Bennett, M., An, J., Chesnut, K. A., Wakeland, E. K., Scheerer, J. B., Siciliano, M. J., Kumar, V. and Matthew, P. A. 1995. Cloning and characterization of 5E6 (Ly-49C), a receptor molecule expressed on a subset of murine natural killer cells. J. Exp. Med. 182:305. 21 Mason, L. H., Ortaldo, J. R., Young, H. A., Kumar, V., Bennett, M. and Anderson, S. K. 1995. Cloning and functional characteristics of murine large granular lymphocyte-1: a member of the Ly-49 gene family (Ly-49G2). J. Exp. Med. 182:293. 22 Brennan, J., Mahon, G., Mager, D. L., Jefferies, W. A. and Talei, 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 F. 1996. Recognition of class I major histocompatibility complex molecules by Ly-49: specificities and domain interactions. J. Exp. Med. 183:1553. Yu, Y. Y. L., George, T., Dorfman, J. R., Roland, J., Kumar, V. and Bennett, M. 1996. The role of Ly-49A and 5E6 (Ly-49C) molecules in hybrid resistance mediated by murine natural killer cells against normal T cell blasts. Immunity 4:67. Pollack, S. B. and Emmons, S. L. 1982. NK-2.1: an NK-associated antigen detected with NZB anti-BALB/c serum. J. Immunol. 129:2277. Lemieux, S., Ouellet-Talbot, F., Lusignan, Y., Morelli, L., Labrèche, N., Gosselin, P. and Lecomte, J. 1991. Identification of murine natural killer cell subsets with monoclonal antibodies derived from 129 anti-C57BL/6 immune spleen cells. Cell. Immunol. 134:191. Gosselin, P., Lusignan, Y. and Lemieux, S. 1993. The murine NK2.1 antigen: a 130 kD glycoprotein dimer expressed by a natural killer cell subset of the spleen, thymus, and lymph nodes. Mol. Immunol. 30:1185. Morelli, L. and Lemieux, S. 1993. Triggering of the cytotoxic activity of murine natural killer and lymphokine-activated killer cells through the NK2.1 antigen. J. Immunol. 151:6783. Morelli, L., Lusignan, Y. and Lemieux, S. 1992. Heterogeneity of natural killer cell subsets in NK-1.11 and NK-1.1– inbred mouse strains and their progeny. Cell. Immunol. 141:148. Koo, G. C. and Peppard, J. R. 1984. Establishment of monoclonal anti-NK-1.1 antibody. Hybridoma 3:301. Sentman, C. L., Kumar, V., Koo, G. and Bennett, M. 1989. Effector cell expression of NK1.1, a murine natural killer cell-specific molecule, and ability of mice to reject bone marrow allografts. J. Immunol. 142:1847. Giorda, R., Weisberg, E. P., Ip, T. K. and Trucco, M. 1992. Genomic structure and strain-specific expression of the natural killer cell receptor NKR-P1. J. Immunol. 149:1957. Olsson, M. Y., Kärre, K. and Sentman, C. L. 1995. Altered phenotype and function of natural killer cells expressing the major histocompatibility complex receptor Ly-49 in mice transgenic for its ligand. Proc. Natl Acad. Sci. USA 92:1649. Sentman, C. L., Kumar, V. and Bennett, M. 1991. Rejection of bone marrow cell allografts by natural killer cell subsets: 5E61 cell specificity for Hh-1 determinant 2 shared by H-2d and H-2f. Eur. J. Immunol. 21:2821. Perez-Villar, J. J., Morello, I., Rodriguez, A., Carretero, M., Aramburu, J., Sivori, S., Orengo, A. M., Moretta, A. and LopezBotet, M. 1995. Functional ambivalence of the Kp43 (CD94) NK cell-associated surface antigen. J. Immunol. 154:5779. Moretta, A., Sivori, S., Vitale, M., Pende, D., Morelli, L., Augugliaro, R., Bottino, C. and Moretta, L. 1995. Existence of both inhibitory (p58) and activatory (p50) receptor for HLA-C molecules in human natural killer cells. J. Exp. Med. 182:875. Naper, C., Vaage, J. T., Lambracht, D., Lovik, G., Butcher, G. W., Wonigeit, K. and Rolstad, B. 1995. Alloreactive natural killer cells in the rat: complex genetics of major histocompatibility complex control. Eur. J. Immunol. 25:1249. Held, W., Roland, J. and Raulet, D. H. 1995. Allelic exclusion of Ly-49-family genes encoding class I MHC-specific receptors on NK cells. Nature 376:355. Sundbäck, J., Kärre, K, and Sentman, C. L. 1996. Cloning of minimally divergent allelic forms of the natural killer (NK) receptor Ly-49C, differentially controlled by host genes in the MHC and NK gene complexes. J. Immunol. 157: 3936. Brennan, J., Lemieux, S., Freeman, J. D., Mager, D. L. and Takei, F. 1996. Heterogeneity among Ly-49C natural killer (NK) cells: characterization of highly related receptors with differing functions and expression patterns. J. Exp. Med. 184:2085. Held, W., Dorfman, J. R., Wu, M.-F. and Raulet, D. 1996. Major histocompatibility class I-dependent skewing of the natural killer cell Ly49 receptor repertoire. Eur. J. Immunol. 26:2286.