NK cells become functionally competent to be triggered by their activation receptors through the interaction of NK cell inhibitory receptors with their cognate self-MHC ligands, an MHC-dependent educational process termed “licensing.” For example, Ly49A+ NK cells become licensed by the interaction of the Ly49A inhibitory receptor with its MHC class I ligand, H2Dd, whereas Ly49C+ NK cells are licensed by H2Kb. Structural studies indicate that the Ly49A inhibitory receptor may interact with two sites, termed site 1 and site 2, on its H2Dd ligand. Site 2 encompasses the α1/α2/α3 domains of the H2Dd H chain and β2-microglobulin (β2m) and is the functional binding site for Ly49A in effector inhibition. Ly49C functionally interacts with a similar site in H2Kb. However, it is currently unknown whether this same site is involved in Ly49A- or Ly49C-dependent licensing. In this study, we produced transgenic C57BL/6 mice expressing wild-type or site 2 mutant H2Dd molecules and studied whether Ly49A+ NK cells are licensed. We also investigated Ly49A- and Ly49C-dependent NK licensing in murine β2m-deficient mice that are transgenic for human β2m, which has species-specific amino acid substitutions in β2m. Our data from these transgenic mice indicate that site 2 on self-MHC is critical for Ly49A- and Ly49C-dependent NK cell licensing. Thus, NK cell licensing through Ly49 involves specific interactions with its MHC ligand that are similar to those involved in effector inhibition.

Natural killer cells play a major role in innate immunity against tumors and virally infected cells (1). When NK cells engage cellular targets, they can be activated to kill and secrete immunoregulatory cytokines such as IFN-γ (2). NK cells express two functional types of cell surface receptors that modulate these effects: activation and inhibitory receptors (3). Engagement of activation NK cell receptors with induced endogenous and virally encoded ligands results in stimulation of NK cell responses. By contrast, inhibitory NK cell receptors recognize MHC class I molecules and block NK cell stimulation. The recognition of self-MHC class I molecules by inhibitory receptors in part explains the “missing-self” hypothesis and prevents dysregulation of NK cells in the normal host.

According to the “missing-self” hypothesis, NK cells survey the cell surfaces of tissues for the presence of MHC class I molecules (“self”) that are normally ubiquitously expressed and prevent NK cell attack (4). Downregulation of MHC class I, such as in virus-infected or tumor cells, permits NK cell stimulation via activation receptors. However, one prediction of the missing-self hypothesis is that NK cells should be overactive or autoreactive in MHC class I-deficient hosts. Yet, NK cells from MHC class I-deficient humans and mice generally have poor cytotoxic capacity (5, 6), indicating that another tolerance mechanism must also be operative.

Studies from several laboratories on mouse and human NK cells indicate that the inhibitory receptors for MHC class I have a second function in “licensing” or education of NK cells (711). The licensing model predicts that engagement of inhibitory receptors by self-MHC class I confers NK cells with the capacity to be subsequently stimulated through their activation receptors (i.e., NK cells require licensing to attain functional competence). NK cells that do not express the appropriate inhibitory receptors for self-MHC class I do not attain full functional competence and are thus self-tolerant. For example, in vitro stimulation of murine NK cells via Ab cross-linking of the Nkrp1c (NK1.1, Klrb1) receptor resulted in IFN-γ production primarily from NK cells that expressed an inhibitory receptor for self-MHC, such as Ly49A in a mouse expressing H2Dd, the cognate MHC class I ligand for Ly49A (7). Similarly, Ly49C+ NK cells are licensed by the cognate ligand (H2Kb) for Ly49C. Thus, Ly49A–H2Dd and Ly49C–H2Kb interactions have provided support for a licensing or an MHC-dependent education effect.

The interactions between Ly49 receptors and their MHC ligands have been analyzed at the crystallographic level and in assays of effector inhibition. For example, the structure of Ly49A in complex with H2Dd revealed two potential interaction sites on H2Dd (12). Site 1 consists of the “left” side of the peptide-binding cleft, as viewed from above with the α1 helix at the top, whereas site 2 consists of all three domains of H2Dd and β2-microglobulin (β2m) underneath the peptide-binding cleft. In vitro mutagenesis studies of H2Dd showed that site 2 is the key binding site for Ly49A receptors in trans interactions (i.e., when Ly49A engages H2Dd on a target cell and inhibits natural killing of the target) (13, 14). For example, a point mutation (Arg to Ala) at residue 6 (R6A) in site 2 of H2Dd completely prevented Ly49A-dependent inhibition of natural killing of the T cell tumor C1498 (13). Moreover, Ly49A-dependent interactions with H2Dd require species-specific residues in β2m, such that H2Dd associated with human β2m does not interact with Ly49A (1316). A similar site on H2Kb involving β2m is involved in interaction with Ly49C (17). In addition, Ly49A can also use site 2 to interact in cis with H2Dd expressed on the NK cell itself (18). Cis interactions can be detected by decreased binding of an anti-Ly49A mAb, such as diminished mean fluorescence intensity (MFI) during flow cytometry (19). Thus, although a role for binding at site 1 has not yet been described, Ly49 receptors can interact with site 2 on their MHC ligands in both trans and cis.

It is not known, however, if site 2 is critical for Ly49-dependent NK cell licensing, the topic of the current study. In this study, we examined licensing of Ly49A+ NK cells with novel transgenic animal models expressing either wild-type (WT) H2Dd or site 2 mutant H2Dd molecules. We also investigated whether substitution of mouse with human β2m influences NK licensing with respect to Ly49A and Ly49C.

C57BL/6 (B6) mice were purchased from the National Cancer Institute. H2Kb−/− Db−/− (KODO) mice were described previously (20) and were purchased from the National Institute of Allergy and Infectious Diseases repository through Taconic (Hudson, NY). The D8 mouse is an H2Dd-transgenic line produced with an H2Dd genomic construct on B6 background and has been described previously (21). Human β2m transgenic mice (22) were kindly provided by Dr. Chella David (Mayo Clinic, Rochester, MN) and bred to D8 KODO mice deficient in mouse β2m such that H2Dd is expressed with human β2m. Triple knockout (TKO; murine β2m−/− H2Kb−/− Db−/−) mice, which lack H2KbDb and murine β2m, were obtained from Dr. Ted Hansen (Washington University, St. Louis, MO). All mice were used in accordance with institutional guidelines for animal experimentation.

H2Dd WT (WT Dd) and site 2 mutant (R6A) constructs were described previously (13). These H2Dd transgene inserts were subcloned into the expression vector pHSE3′, which contains the H2Kb promoter and Ig enhancer sequences for transgene expression (23). For the R6A-1 mouse, we subcloned the R6A transgene insert into the expression vector pHβ-Apr-1-neo, which contains a human β-globin promoter sequence for transgene expression (24). Linearized transgene constructs were purified by electroelution, resuspended in microinjection buffer, and microinjected into B6 mouse embryos at the Transgenic Knockout Microinjection Core in the Department of Pathology and Immunology (Washington University, St. Louis, MO). Transgenic founder mice were screened by Southern blot and PCR. Candidate founder mice were bred to B6 mice to generate H2Dd transgenic mice on B6 background. Each H2Dd transgenic line was then crossed to KODO mice to yield H2Dd transgenic mice on KODO background (R6A-1 KODO, R6A-2 KODO, and WT Dd KODO, respectively). The genetic backgrounds of all mice were double checked for genotype by the Speed Congenics Core Facility at Washington University. The characteristics of each H2Dd transgenic line are summarized in Table I.

Table I.
H2Dd transgenic mice
MouseH2Dd InsertExpression VectorH2Dd (%)Reference
D8 WT Dd gene Genomic construct 99 Bieberich et al. (21
WT Dd WT Dd cDNA pHSE3′ 50–60 This study 
R6A-1 R6A (site 2 mutant) cDNA pHβ-Apr-1-neo 50–60 This study 
R6A-2 R6A (site 2 mutant) cDNA pHSE3′ 99 This study 
MouseH2Dd InsertExpression VectorH2Dd (%)Reference
D8 WT Dd gene Genomic construct 99 Bieberich et al. (21
WT Dd WT Dd cDNA pHSE3′ 50–60 This study 
R6A-1 R6A (site 2 mutant) cDNA pHβ-Apr-1-neo 50–60 This study 
R6A-2 R6A (site 2 mutant) cDNA pHSE3′ 99 This study 

The mAbs 145-2C11 (anti-CD3), 1D3 (anti-CD19), PK136 (anti-NK1.1), A1 (anti-Ly49A), 34-2-12 (anti-H2Dd α3), 34-5-8 (anti-H2Dd α1/α2), 28-8-6 (anti-H2Kb/Db), and XMG1.2 (anti–IFN-γ) were purchased from BD Biosciences (San Jose, CA). Culture supernatants were produced from hybridoma 2.4G2 (anti-FcγRII/III) (American Type Culture Collection, Manassas, VA). JR9 (anti-Ly49A) (25) was purified from culture supernatants derived from hybridomas kindly provided by Jacques Roland (Pasteur Institute, Paris, France) and then conjugated to FITC. The mAb 4LO (anti-Ly49C) was purified from culture supernatants derived from the hybridoma kindly provided by Dr. Suzanne Lemieux (Institut Armand-Frappier, Montreal, QC, Canada) and then biotinylated in the laboratory.

Splenocytes were suspended in RPMI 1640 media supplemented with 10% FBS, l-glutamine, penicillin, streptomycin, and 2-mercaptoethanol (R10 media). After RBC lysis, all cells were adjusted to the concentration of 107 cells per milliliter R10 media. For most staining, 106 cells were used for staining. After mAb staining for 30 min, cells were washed with and resuspended in ice-cold sorter buffer (PBS containing 2% FCS and 0.1% sodium azide), and 2.4G2 supernatant (anti-FcγRII/III) was added to all staining to prevent nonspecific Fc receptor effects. All flow cytometry data were collected using FACS Canto/Calibur/Scan flow cytometers (BD Biosciences) and then analyzed using FlowJo software (Tree Star, Ashland, OR).

Wells in 6-well culture plates were coated with 1 ml purified PK136 (anti-NK1.1) Ab diluted in PBS at concentrations of 2 μg/ml or 5 μg/ml for 90 min, then washed three times with PBS. Splenocytes (107) in 1 ml R10 media were placed in each well and incubated for 1 h at 37°C and 5% CO2. Subsequently, Golgi-Plug (BD Biosciences) containing brefeldin A was added to each well. Three milliliters ice-cold sorter buffer was added to each well after 6 h of additional incubation, and cells were then harvested. Cells were stained for 30 min with mAbs for cell surface Ags, fixed with Cytofix (BD Biosciences) and permeabilized by Perm/Wash buffer (BD Biosciences) containing saponin. The cells were washed twice with Perm/Wash buffer and stained with XMG1.2 (anti–IFN-γ) Ab.

The licensing ratio for a given Ly49 receptor was determined by assessing the percentage of IFN-γ–producing cells in receptor-positive and receptor-negative NK cell populations. For example, Ly49A-dependent licensing ratio = (% IFN-γ–producing Ly49A+ cells)/(% IFN-γ–producing Ly49A cells), whereas Ly49C-dependent licensing ratio = (% IFN-γ–producing Ly49C+ cells)/(% IFN-γ–producing Ly49C cells). These ratios were calculated from the dot plots where Ly49 is on the x-axis and IFN-γ staining is on the y-axis, using the following formula: [(UR)/(UR + LR)]/[(UL)/(UL + LL)], where UR is upper right, LR is lower right, UL is upper left, and LL is lower left.

The general protocol for chromium release assay by Ly49A+ IL-2–activated NK cells was described previously (26). In brief, splenocytes from D8 KODO mice were harvested and cultured in 800 IU/ml concentration of IL-2 for 9 d. Panning by JR9 (anti-Ly49A) mAb was performed on day 7 to isolate Ly49A+ and Ly49A cells. The percentage of Ly49A+ IL-2–activated NK cells in effector cell was 90–95% by FACS verification. For target cells, splenocytes were harvested from each mouse (KODO, R6A-2 KODO, WT Dd KODO, D8 KODO) and cultured with Con A (6 μg/ml) for 2 d then washed and labeled with 51Cr. YAC-1 tumor cells were used as control target cells. Percentage specific lysis was calculated by standard equation (26).

To assess specifically the role of site 2 on H2Dd in Ly49A-mediated licensing, we produced transgenic mice expressing WT and site 2 mutant H2Dd (R6A) molecules (13) directly in B6 mice. Initial screening of founder mice was performed by PCR and Southern blot. Candidate founder mice showing high levels of H2Dd transgene expression were bred to B6 mice and then crossed to KODO mice for further analysis. In this manner, we obtained one new WT H2Dd and two new R6A H2Dd mutant (R6A-1 and R6A-2) transgenic lines on the KODO background (Table I). Similar H2Dd mRNA expression by RT-PCR and H2Dd alloreactivity by MLR were obtained with all WT and mutant H2Dd mice (data not shown), indicating that all of the lines carry functionally expressed transgenes. Moreover, H2Dd transgenic protein expression on the cell surface of splenocytes was assessed by FACS staining with mAbs that detect different portions of the H2Dd molecule. When stained with mAb 34-5-8, which binds the α1/α2 domain of H2Dd, splenocytes from R6A-1 mice express mutant H2Dd at intermediate levels in a monophasic manner (Fig. 1A). These levels were somewhat lower than H2Dd in D8 mice (21), a previously produced WT H2Dd transgenic line that expresses H2Dd at levels comparable with those of non-transgenic H2d mice (data not shown). By contrast, R6A-2 splenocytes have biphasic expression of mutant H2Dd, with both populations expressing mutant H2Dd at levels equal to or higher than those of splenocytes in D8 mice. Finally, splenocytes from our new WT Dd transgenic mice also have biphasic expression of H2Dd at levels equal to or lower than those of splenocytes in R6A-2 mice. Similar results were obtained with mAb 34-2-12, which binds the α3 domain of H2Dd (Fig. 1B). Thus, expression of R6A mutant and WT H2Dd was readily detectable with mAbs specific for all three extracellular domains in the transgenic mice.

FIGURE 1.

Surface expression of transgenic H2Dd. Two mAbs that detect different portions of H2Dd were used to assess transgene expression on splenocytes. A, Histograms of splenocytes from the indicated H2Dd transgenic mice (solid lines) stained with PE-conjugated mAb 34-5-8, which detects the α1/α2 domain of H2Dd. B, Histograms of splenocytes from the indicated H2Dd transgenic mice (solid lines) stained with FITC-conjugated mAb 34-2-12, which detects α3 domain of H2Dd. These histograms are representative of six independent experiments. In all histograms, the profiles of splenocytes from KODO (dotted line) and D8 KODO (dashed line) mice are shown. The percentage of splenocytes expressing transgenic H2Dd is shown in each histogram.

FIGURE 1.

Surface expression of transgenic H2Dd. Two mAbs that detect different portions of H2Dd were used to assess transgene expression on splenocytes. A, Histograms of splenocytes from the indicated H2Dd transgenic mice (solid lines) stained with PE-conjugated mAb 34-5-8, which detects the α1/α2 domain of H2Dd. B, Histograms of splenocytes from the indicated H2Dd transgenic mice (solid lines) stained with FITC-conjugated mAb 34-2-12, which detects α3 domain of H2Dd. These histograms are representative of six independent experiments. In all histograms, the profiles of splenocytes from KODO (dotted line) and D8 KODO (dashed line) mice are shown. The percentage of splenocytes expressing transgenic H2Dd is shown in each histogram.

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Previous studies indicated that transfected WT H2Dd expressed on C1498 tumor cells inhibited killing by Ly49A+ IL-2–activated NK cells, whereas tumor cells expressing transfected R6A mutants were killed (13, 26). We performed chromium release cytotoxicity assays using Con A blast targets derived from splenocytes of the H2Dd transgenic mice on KODO background. Con A blasts retained a similar level of H2Dd expression on cell surface as that in primary cells (data not shown). As expected, similar to Con A blasts from D8 KODO mice, WT Dd KODO Con A blasts inhibited natural killing by Ly49A+ IL-2–activated NK cells (Fig. 2). However, Con A blasts from R6A-2 KODO did not inhibit killing despite expression of H2Dd on R6A-2 at levels equal to or higher than those on WT Dd cells. Of note, specific lysis of Con A blasts from R6A-2 KODO mice was similar to that of KODO targets, indicating that WT but not R6A mutant H2Dd molecules on primary cell targets were recognized in effector responses by the inhibitory Ly49A receptor on NK cells in trans.

FIGURE 2.

Primary cells from WT and mutant H2Dd transgenic mice as targets for natural killing. Effector cells were Ly49A+ IL-2–activated NK cells from D8 KODO mice, and target cells were Con A blasts from the indicated H2Dd transgenic mice on KODO background. YAC-1 tumor cells were used as a positive control. E:T ratios ranging from 1:1 to 20:1 are shown. Results are representative of three independent experiments.

FIGURE 2.

Primary cells from WT and mutant H2Dd transgenic mice as targets for natural killing. Effector cells were Ly49A+ IL-2–activated NK cells from D8 KODO mice, and target cells were Con A blasts from the indicated H2Dd transgenic mice on KODO background. YAC-1 tumor cells were used as a positive control. E:T ratios ranging from 1:1 to 20:1 are shown. Results are representative of three independent experiments.

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Ly49A molecules can also bind site 2 on H2Dd molecules when expressed on the NK cell itself, a cis interaction, as indicated by decreased MFI of anti-Ly49A staining by flow cytometry (19). We compared the MFI of FITC-conjugated JR9 (anti-Ly49A) on Ly49A+ NK cells from the various H2Dd transgenic mice on KODO background with the MFI of FITC-JR9 in KODO mice, an environment lacking H2Dd (Fig. 3A). The Ly49A MFI in KODO mice was set as 100% for ease of comparison (Fig. 3B). NK cells from D8 KODO and WT Dd KODO mice had relative Ly49A MFI values of 41 and 31%, respectively, whereas NK cells from R6A-1 KODO and R6A-2 KODO mice had relative Ly49A MFI values of 97 and 92%, respectively. Similar results were obtained when mAb A1 (anti-Ly49A) was used to stain Ly49A+ NK cells from H2Dd transgenic mice on the B6 background (Supplemental Fig. 1), indicating that Ly49A down-modulation was not observed in R6A-1 and R6A-2 KODO mice. Thus, the site 2 mutation on H2Dd also affected cis recognition of transgenic H2Dd by Ly49A receptors.

FIGURE 3.

JR9 (anti-Ly49A) MFI in WT and mutant H2Dd transgenic mice. A, Representative histograms of NK cells from individual spleens of indicated H2Dd transgenic mice stained with FITC-conjugated JR9 (anti-Ly49A) mAb. In all histograms, the dotted line is the histogram of a KODO mouse, the solid line is that of a D8 KODO mouse, and the shaded histogram is from a representative mouse of the indicated H2Dd transgenic line. In each histogram, the percentage of NK cells expressing Ly49A is shown. B, For ease of comparison, MFIs of Ly49A from the indicated H2Dd transgenic mice are compared with the MFI of Ly49A in KODO mice, which is set as 100%. MFIs of Ly49A from KODO, R6A-1 KODO, R6A-2 KODO, D8 KODO, and Dd WT KODO mice were 2346, 2182, 2158, 727, and 962, respectively. These results are pooled from data from six independent experiments and are shown as mean ± SD.

FIGURE 3.

JR9 (anti-Ly49A) MFI in WT and mutant H2Dd transgenic mice. A, Representative histograms of NK cells from individual spleens of indicated H2Dd transgenic mice stained with FITC-conjugated JR9 (anti-Ly49A) mAb. In all histograms, the dotted line is the histogram of a KODO mouse, the solid line is that of a D8 KODO mouse, and the shaded histogram is from a representative mouse of the indicated H2Dd transgenic line. In each histogram, the percentage of NK cells expressing Ly49A is shown. B, For ease of comparison, MFIs of Ly49A from the indicated H2Dd transgenic mice are compared with the MFI of Ly49A in KODO mice, which is set as 100%. MFIs of Ly49A from KODO, R6A-1 KODO, R6A-2 KODO, D8 KODO, and Dd WT KODO mice were 2346, 2182, 2158, 727, and 962, respectively. These results are pooled from data from six independent experiments and are shown as mean ± SD.

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To assess licensing of NK cells from mutant mice, NK cells were stimulated with immobilized PK136 (anti-NK1.1), which has previously been shown to more robustly stimulate IFN-γ production by licensed compared with unlicensed NK cells (7). The Ly49A-dependent licensing ratio is calculated by dividing the percentage of IFN-γ–producing Ly49A+ NK cells by the corresponding percentage among Ly49A NK cells, and it indicates whether the Ly49A+ NK cells are more reactive or licensed than the counterpart Ly49A population from the same mouse (Fig. 4A). The KODO mice and D8 KODO mice were used as a negative and a positive control, respectively. At a low concentration (2 μg/ml) of immobilized PK136 (anti-NK1.1), the licensing ratio in WT Dd KODO mice was greater than 1, indicating a positive licensing effect of transgenic H2Dd on Ly49A+ NK cells. However, there was a significantly lower effect on NK cells from R6A-1 and R6A-2 mice, which exhibited a Ly49A-dependent licensing ratio similar to KODO mice. Similar results were obtained when we used a higher concentration (5 μg/ml) of PK136 to stimulate the NK cells (Fig. 4B, 4C). These results indicate that R6A mutants do not have the capacity to license NK cells via Ly49A and that preservation of site 2 in H2Dd is critical for licensing of NK cells via Ly49A and H2Dd interactions.

FIGURE 4.

NK cell licensing in WT and mutant H2Dd transgenic mice. A, A representative experiment is shown when cells from an individual mouse of the indicated genotype were stimulated by plate-bound PK136 (anti-NK1.1) Ab. In this experiment, PK136 Ab at the concentration of 2 μg/ml was used for coating the culture well. The Ly49A licensing ratio (R) is shown in each plot. See 1Materials and Methods for calculation of licensing ratio. Cells shown are gated on CD3, CD19, NK1.1+ splenocytes. B and C, The licensing ratios (mean ± SD) are shown for pooled results from six independent experiments with PK136 coating concentration of 2 μg/ml (B) and 5 μg/ml (C). The p value for Student t test is shown.

FIGURE 4.

NK cell licensing in WT and mutant H2Dd transgenic mice. A, A representative experiment is shown when cells from an individual mouse of the indicated genotype were stimulated by plate-bound PK136 (anti-NK1.1) Ab. In this experiment, PK136 Ab at the concentration of 2 μg/ml was used for coating the culture well. The Ly49A licensing ratio (R) is shown in each plot. See 1Materials and Methods for calculation of licensing ratio. Cells shown are gated on CD3, CD19, NK1.1+ splenocytes. B and C, The licensing ratios (mean ± SD) are shown for pooled results from six independent experiments with PK136 coating concentration of 2 μg/ml (B) and 5 μg/ml (C). The p value for Student t test is shown.

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To corroborate the studies of mutant H2Dd mice, we next sought to assess the role of β2m subunit in Ly49-dependent licensing because Ly49 interactions with MHC class I ligands is dependent on species-specific residues in mouse β2m (1316). For this analysis, we used human β2m transgenic mice that had been well studied previously for T cell responses (22, 27). These mice were then back-crossed to the D8 TKO mice (Table II). When stained with mAb 34-5-8, which detects α1/α2 domain of H2Dd molecule, human β2m (huβ2m) D8 TKO mice showed levels of H2Dd transgene expression comparable with those of D8 KODO mice (Fig. 5A). Similar results were obtained with mAb 34-2-12, which detects the α3 domain of H2Dd. Thus, H2Dd transgenic (Tg) protein complexed with human β2m was normally expressed on splenocytes from these mice as assessed by FACS staining with mAbs that detect different portions of the H2Dd molecule.

Table II.
H2Dd and human β2m double transgenic mice
MiceH2Dd TgHuman β2m TgKbDbMurine β2m
D8 KODO − −/− +/+ 
huβ2m D8 TKO −/− −/− 
TKO − − −/− −/− 
MiceH2Dd TgHuman β2m TgKbDbMurine β2m
D8 KODO − −/− +/+ 
huβ2m D8 TKO −/− −/− 
TKO − − −/− −/− 

+, present; −, absent on one or both alleles, or Tg as indicated.

FIGURE 5.

Ly49A-dependent NK cell licensing in human β2m transgenic mice. A, Expression of H2Dd is shown. Splenocytes from each transgenic mouse were stained with mAb 34-5-8, which detects α1/α2 domain of H2Dd, and 34-2-12, which detects α3 domain of H2Dd. Dotted line, dashed line, and solid line are the histograms of TKO, D8 KODO, and huβ2m D8 TKO mice, respectively. The huβ2m D8 TKO mice showed comparable level of H2Dd expression on cell surface compared with that of D8 KODO mice, as indicated by two different mAbs that detect different domains of H2Dd. B, Ly49A MFI is shown. For ease of comparison, MFIs of Ly49A from the indicated transgenic mice were compared with the MFI of Ly49A in TKO mice, which was set as 100%. The huβ2m D8 TKO mouse showed a comparable level of Ly49A MFI (97%), whereas D8 KODO mouse showed a much lower level (43%). MFIs of Ly49A from TKO, huβ2m D8 TKO, and D8 KODO mice were 92, 89, and 40, respectively. C and D, Ly49A-dependent licensing ratios at PK136 (anti-NK1.1) concentration of 2 μg/ml (C) and 5 μg/ml (D) are shown. These results are shown as mean ± SD. Mice compared in this figure are listed in Table II.

FIGURE 5.

Ly49A-dependent NK cell licensing in human β2m transgenic mice. A, Expression of H2Dd is shown. Splenocytes from each transgenic mouse were stained with mAb 34-5-8, which detects α1/α2 domain of H2Dd, and 34-2-12, which detects α3 domain of H2Dd. Dotted line, dashed line, and solid line are the histograms of TKO, D8 KODO, and huβ2m D8 TKO mice, respectively. The huβ2m D8 TKO mice showed comparable level of H2Dd expression on cell surface compared with that of D8 KODO mice, as indicated by two different mAbs that detect different domains of H2Dd. B, Ly49A MFI is shown. For ease of comparison, MFIs of Ly49A from the indicated transgenic mice were compared with the MFI of Ly49A in TKO mice, which was set as 100%. The huβ2m D8 TKO mouse showed a comparable level of Ly49A MFI (97%), whereas D8 KODO mouse showed a much lower level (43%). MFIs of Ly49A from TKO, huβ2m D8 TKO, and D8 KODO mice were 92, 89, and 40, respectively. C and D, Ly49A-dependent licensing ratios at PK136 (anti-NK1.1) concentration of 2 μg/ml (C) and 5 μg/ml (D) are shown. These results are shown as mean ± SD. Mice compared in this figure are listed in Table II.

Close modal

Then we compared the MFI of FITC-conjugated JR9 (anti-Ly49A) on Ly49A+ NK cells from TKO, huβ2m D8 TKO, and D8 KODO mice. The Ly49A MFI in TKO mice was set as 100% for ease of comparison (Fig. 5B). The huβ2m D8 TKO and D8 KODO mice had relative Ly49A MFI values of 97 and 43%, respectively. Thus, Ly49A down-modulation was not observed in huβ2m D8 TKO mice where mouse β2m was replaced by human β2m.

Lastly, we compared the Ly49A-dependent licensing ratio of huβ2m D8 TKO mouse to that of TKO and D8 KODO mice. At a low concentration (2 μg/ml) of immobilized PK136 (anti-NK1.1), the licensing ratio in huβ2m D8 TKO mouse was similar to that of TKO mouse, which was used as a negative control, whereas the licensing ratio in D8 KODO was close to 2 (Fig. 5C). Similar results were obtained at a higher concentration (5 μg/ml) of immobilized PK136 (Fig. 5D). These results indicate that H2Dd complexed with human β2m does not have the capacity to license NK cells via Ly49A. Thus, the β2m subunit provides species-specific sites crucial for licensing of NK cells via Ly49A and H2Dd interactions, consistent with a role for site 2 because mouse but not human β2m provides all critical β2m residues in site 2 (1316).

The interaction of Ly49C with H2Kb is also dependent on species-specific residues in β2m (15, 17). To investigate the role of human β2m in Ly49C-dependent NK licensing, we produced human β2m Tg, murine β2m knockout mice [huβ2mTg murine (mu) β2mKO] on the B6 (H2b) background (Table III). When we compared MHC expression on cell surface of B6 versus huβ2mTg muβ2mKO splenocytes with mAb 28-8-6, which detects H2Kb/Db, both mice showed comparable levels (Fig. 6A). Human β2m transgene protein expression was directly assessed by FACS staining with mAb FITC-TU99 (Fig. 6A). Also, the MFI of Ly49C was compared by staining by mAb 4LO (anti-Ly49C). The Ly49C MFI of β2mKO mice was set as 100% for ease of comparison. The relative values of Ly49C MFI of huβ2mTg muβ2mKO mice and B6 mice were 56 and 25%, respectively (Fig. 6B), showing that Ly49C staining was susceptible to species-specific changes in β2m but less than expected compared with Ly49A (Fig. 5B).

Table III.
Human β2m Tg mice on H2b background
MiceKbDbMurine β2mHuman β2m Tg
B6 (H2b+/+ +/+ − 
huβ2mTg μβ2mKO +/+ −/− 
β2mKO +/+ −/− − 
MiceKbDbMurine β2mHuman β2m Tg
B6 (H2b+/+ +/+ − 
huβ2mTg μβ2mKO +/+ −/− 
β2mKO +/+ −/− − 

+, present; −, absent on one or both alleles, or Tg as indicated.

FIGURE 6.

Ly49C-dependent licensing in human β2m transgenic mice. A, H2KbDb and human β2m expression is shown. Splenocytes from indicated mice were stained with mAb 28-8-6 (anti-H2KbDb) and with mAb TU99 (anti-human β2m), respectively. In all histograms, the dotted line is the β2mKO mouse, the dashed line is the B6 mouse, and the solid line is the huβ2mTg μβ2mKO mouse. B, Ly49C MFI is shown. Relative Ly49C MFIs of Ly49C+ NK cells from indicated mice are compared, where Ly49C MFI of the β2mKO mouse is set as 100%. C, Ly49C-dependent licensing ratios are shown at a lower PK136 concentration (2 μg/ml). D, Ly49C-dependent licensing ratios are shown at a higher PK136 concentration (5 μg/ml) These results are shown as mean ± SD. Mice compared in this figure are listed in Table III.

FIGURE 6.

Ly49C-dependent licensing in human β2m transgenic mice. A, H2KbDb and human β2m expression is shown. Splenocytes from indicated mice were stained with mAb 28-8-6 (anti-H2KbDb) and with mAb TU99 (anti-human β2m), respectively. In all histograms, the dotted line is the β2mKO mouse, the dashed line is the B6 mouse, and the solid line is the huβ2mTg μβ2mKO mouse. B, Ly49C MFI is shown. Relative Ly49C MFIs of Ly49C+ NK cells from indicated mice are compared, where Ly49C MFI of the β2mKO mouse is set as 100%. C, Ly49C-dependent licensing ratios are shown at a lower PK136 concentration (2 μg/ml). D, Ly49C-dependent licensing ratios are shown at a higher PK136 concentration (5 μg/ml) These results are shown as mean ± SD. Mice compared in this figure are listed in Table III.

Close modal

Finally, we compared the Ly49C-dependent licensing ratio in NK cells from β2m knockout (β2mKO), huβ2mTg muβ2mKO, and B6 mice. At a low concentration (2 μg/ml) of immobilized PK136 (anti-NK1.1), huβ2mTg muβ2mKO NK cells had a licensing ratio virtually identical to that of β2mKO cells, with both much lower than that of B6 cells (Fig. 6C). Similar results were obtained at a high concentration (5 μg/ml) of PK136 (Fig. 6D), indicating a role for species-specific β2m substitutions in Ly49C-dependent licensing.

We developed new transgenic mice to study the role of site 2 on the MHC molecule in Ly49-dependent NK cell licensing. For Ly49A, WT and site 2 mutant H2Dd molecules were well expressed as determined by FACS staining of splenocytes with two different mAbs specific for different extracellular domains of H2Dd. As expected, site 2 mutant H2Dd molecules on primary cells did not inhibit natural killing by Ly49A+ IL-2–activated NK cells in contrast to WT H2Dd. Moreover, site 2 mutant H2Dd did not bind Ly49A in cis as measured by Ly49A MFI, whereas WT Tg H2Dd showed an effect on Ly49A MFI even at levels only 50–60% of normal expression levels, consistent with previous reports (19). Most importantly, site 2 mutant H2Dd did not contribute to Ly49A-dependent NK cell licensing. These data were corroborated by investigation of licensing in human β2m Tg mice, which lack murine β2m. Despite otherwise normal MHC class I expression, both Ly49A- and Ly49-dependent licensing were perturbed in these animals, which is likely due to species-specific substitutions in β2m that influence site 2 interactions of MHC class I with Ly49 receptors in vitro (1317). Taken together, our studies indicate that site 2 in MHC class I molecules is critical for Ly49-dependent NK cell licensing as well as for inhibition of natural killing.

Our interpretations were dependent on appropriate transgenic expression of WT and mutant H2Dd molecules. Inasmuch as transgenic effects in any single transgenic mouse could be due to construct insertion and not necessarily to expression of the transgenic molecule, we studied two transgenic mutant H2Dd founder lines and analyzed mice on the KODO background to minimize effects of endogenous MHC class I. Our WT H2Dd transgenic mice produced licensing effects on Ly49A+ NK cells comparable with those of previously produced H2Dd transgenic mice (D8) (21), although the effects on licensing in D8 mice were slightly more robust. These effects could be related to a somewhat higher level of H2Dd in D8 versus our newly described WT H2Dd transgenic mice. Regardless, these data suggest that a relatively low level of H2Dd is sufficient to generate licensing effects, consistent with our previous observations with MHC heterozygous mice and with different MHC haplotypes (28). In contrast, we found no evidence for licensing of Ly49A+ NK cells in the two different R6A founder lines. The founder line R6A-1 expresses mutant H2Dd at levels comparable with those of the new WT H2Dd transgenic line, whereas the R6A-2 line expresses mutant H2Dd at levels much higher than those of WT H2Dd. However, neither R6A mutant demonstrated licensing effects on Ly49A+ NK cells. Moreover, the R6A-2 transgenic line expresses mutant H2Dd with a promoter construct identical to that used for our new WT H2Dd transgenic line, and R6A-2 still does not induce the licensing phenotype in Ly49A+ NK cells. These data with new mutant H2Dd Tg mice suggest that site 2 is required for Ly49A-dependent licensing.

Most importantly, these studies were corroborated by compound Tg and KO mice where we replaced mouse β2m with human β2m, which lacks appropriate murine β2m residues for Ly49A-dependent interactions. In addition, human β2m Tg mice on the H2b background allowed us to extend our findings to another receptor and MHC molecule, Ly49C and H2Kb, respectively. Finally, site 2 in both H2Dd and H2Kb is specifically involved in Ly49A- and Ly49C-dependent effector inhibition, respectively (1317). Therefore, these considerations strongly support the conclusion that site 2 in cognate self-MHC molecules is required for licensing of Ly49A+ and Ly49C+ NK cells as well as in effector inhibition.

Although structural analysis suggested that there are potentially two sites on H2Dd for Ly49A binding (12), our data also extend previous studies indicating that site 2 is necessary for both trans and cis functional interactions with Ly49A (13, 14, 19). In the case of trans interactions, Ly49A on an NK cell interacts with H2Dd on a tumor target and confers inhibitory effects. Previous site-directed mutagenesis of both site 1 and site 2 residues established that site 2 is the interaction site for trans recognition of a tumor transfectant (13, 14). We also showed that site 2 on H2Dd on primary cells is critical for the Ly49A-dependent inhibitory effect.

In terms of cis interactions, Held and colleagues (18, 19) demonstrated that H2Dd on the NK cell itself can interact in cis with Ly49A on the same cell. Prior site-directed mutagenesis has established that site 2 is involved in these cis interactions and that Ly49A reactivity with anti-Ly49A mAbs provide an indirect measure of such interactions. In this study, we provide additional evidence of the importance of site 2 for cis interactions of Ly49A with H2Dd because site 2 mutant H2Dd and H2Dd with human β2m do not alter anti-Ly49A reactivity compared with that of H2b mice.

Notably, however, site 2 substitutions via species-specific differences in β2m affected Ly49C-dependent licensing but had less effect on Ly49C reactivity with mAb 4LO. These effects could be due to a higher number of residues in contact with β2m between Ly49A and Ly49C or the specific residues contacted (12, 13, 16)(17). Regardless, although mAb 4LO reactivity is only an indirect marker of cis interactions, these data nonetheless suggest that cis interactions may be less important for licensing interactions between Ly49C and H2Kb. Although this interpretation will require additional studies, it may highlight the subtle differences between Ly49A and Ly49C with respect to functional interaction with their MHC ligands, such as the previously noted effect of MHC-bound peptides on Ly49C but not Ly49A interactions (2931). Despite these differences, both receptors interact with site 2 in their cognate MHC ligand for licensing and effector inhibition.

Our conclusions come with two caveats. 1) Our current interpretation is partially based on the assumption that the R6A mutation specifically disrupts a critical interaction at site 2. It is theoretically possible that this mutation causes a more profound conformational change in the H2Dd structure that affects site 1. However, we think this is unlikely to be the case because Ab binding is preserved and the mutant MHC class I molecule can still be functionally recognized in allo-MLR. Moreover, we provide corroborating data with the species-specific β2m studies. 2) It should be noted that we have not formally excluded the possibility that site 1 may be involved in licensing because we did not test any site 1 mutant MHC class I molecules in this study. Our expectation is that site 1 mutant MHC class I molecules will still permit licensing to occur, although interpretations of such experiments may be problematic because there are no data, other than the crystallographic results, showing that disruption of putative site 1 residues perturbs interaction between Ly49 receptors and MHC class I. Such experiments may need to simultaneously mutate multiple residues putatively involved in site 1 recognition, but then more profound conformational changes may occur that disturb site 2. Nonetheless, at the current time, the available data strongly support a role for site 2 in NK cell licensing by Ly49 receptors.

Therefore, the most important conclusions from this study are that both licensing and inhibition of effector function involve the same interaction site on the cognate MHC ligand for Ly49A and Ly49C. Both functions for both Ly49s are related to tolerance. That is, a licensed Ly49A+ or Ly49C+ NK cell should be inhibited by the same site on the same MHC allele that conferred licensing in the first place. Thus, the current study provides additional evidence that licensing and inhibition of effector function are related properties of NK cells and their receptors.

We thank Mike White, James Mohan, Kate Render, Melissa Berrien, and Darryl Higuchi for help in the development of transgenic animals. We thank Chella David and Ted Hansen for KO and Tg mice. We also thank Megan Cooper, Julie Elliott, and Helena Jonsson for critical review of the manuscript.

This work was supported by the Howard Hughes Medical Institute and by grants from the National Institutes of Health (to W.M.Y.). The Transgenic Knockout Microinjection Core and Speed Congenics Core Facilities were supported in part by the Rheumatic Diseases Core Center grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

The online version of this article contains supplemental material.

Abbreviations used in this article:

B6

C57BL/6

huβ2m

human β2m

huβ2mTg μβ2mKO

human β2m transgenic, murine β2m knockout

KODO

H2Kb−/− Db−/−

β2m

β2-microglobulin

MFI

mean fluorescence intensity

β2mKO

β2-microglobulin knockout

mu

murine

TKO

triple knockout

WT

wild-type.

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The authors have no financial conflicts of interest.