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The Journal of Immunology, 1999, 163: 2628-2633.
Copyright © 1999 by The American Association of Immunologists

Levels of Ly-49 Receptor Expression Are Determined by the Frequency of Interactions with MHC Ligands: Evidence Against Receptor Calibration to a "Useful" Level1

Jennifer O. Manilay*,{ddagger}, Gerald L. Waneck{ddagger},{dagger} and Megan Sykes2,*,{ddagger}

* Bone Marrow Transplantation Section, {dagger} Laboratory of Molecular and Cellular Immunology, and {ddagger} Transplantation Biology Research Center, Surgical Service, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ly-49 receptor expression was studied in NK cells that developed in fully MHC-mismatched mixed bone marrow chimeras, in which host and donor MHC ligands were expressed solely on various proportions of hemopoietic cells or on both hemopoietic and nonhemopoietic cells. When hemopoietic cells were the only source of MHC ligand, a strong correlation between the level of down-regulation of Ly-49A, Ly-49C, and Ly-49G2 and the number of hemopoietic cells expressing their MHC ligands was observed on both donor and host NK cells. In some animals with low levels of donor hemopoietic chimerism, NK cells of donor origin expressed Ly-49 receptors at higher levels than was observed in normal mice of the same strain. This unexpected observation is inconsistent with the receptor calibration theory, which states that expression of Ly-49 inhibitory receptors is calibrated to an optimal level to maintain an NK cell repertoire that is sensitive to perturbations in normal class I ligand expression. Our data suggest a model in which Ly-49 receptors down-modulate in accordance with the frequency of their interactions with ligand-bearing cells, rather than a model in which these receptors calibrate to a specific "useful" level in response to ligands present in their environment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Ly-49 receptors on NK cells recognize MHC class I ligands (reviewed in Ref. 1). To date, nine members of the mouse Ly-49 receptor family have been identified and cloned (2, 3, 4, 5), and strain-specific allelic forms have been described (6). Although some Ly-49 members are activating receptors (7, 8), most of the known Ly-49 receptors transmit inhibitory signals that prevent NK cells from killing their targets. Ly-49-mediated inhibition is thought to be a major mechanism by which NK cells are prevented from killing normal self targets. The factors controlling the expression of Ly-49 receptors on the cell surface are not well understood.

Expression of Ly-49 genes, for the most part, has been shown to be monoallelic (9, 10, 11). However, studies of Ly-49A transgenic mice demonstrated that expression of the Ly-49A transgene in NK cells did not prevent expression of the endogenous Ly-49A gene. These studies also provided evidence for a transcriptional mechanism of regulation, because endogenous Ly-49A mRNA levels were lower in Dd+ NK cells expressing the Ly-49A transgene than in NK cells from nontransgenic controls (12).

NK cells developing in the presence of their MHC ligand express lower levels of the corresponding Ly-49 receptor than is observed among NK cells found in environments lacking the ligand. For example, Ly-49A, whose known inhibitory ligand is Dd, is expressed at lower levels in Dd+ mice than in C57BL/6 (B6, H-2b) mice (13). Ly-49G2, whose inhibitory ligands are Ld and Dd, is also expressed at lower levels in BALB/c (H-2d) mice than in B6 mice (14). Kb is one known inhibitory ligand for Ly-49C (15), and Ly-49C is expressed at lower levels in B6 mice than in BALB/c mice (16). These results have been interpreted to suggest that there is an optimal amount of Ly-49 receptor expression that allows NK cells to be sensitive to reductions in expression of self class I MHC, and hence makes the NK cells useful for destroying cells when they are, for example, virally infected (17). To prevent overinhibition, Ly-49 receptors should be expressed at the minimal useful level that allows for inhibition by normal cells, yet leaves them sensitive to reductions in MHC expression. NK cells that express unusually high levels of Ly-49-inhibitory receptors specific for self MHC would be functionally useless to their host (18).

It has also become clear that the presence of MHC ligand on cells other than the NK cell itself can influence the expression of Ly-49 receptors. Our previous studies showed that Ly-49A expression on Dd-negative NK cells was influenced by Dd expression on both hemopoietic and radioresistant (nonhemopoietic) host elements in fully allogeneic bone marrow chimeras (19). Consistent with these results, in mice with mosaic expression of a Dd transgene (Tg),3 the surface levels of Ly-49A were reduced on both Tg+ and Tg- NK cells, compared with wild-type animals (20). Thus, the presence of Dd in the environment, and not necessarily on the same cell, is sufficient to induce Ly-49A down-regulation.

In the present study, we have utilized fully MHC-mismatched, mixed allogeneic bone marrow chimeras prepared with a nonmyeloablative conditioning regimen (21) to examine the relationship between levels of expression of several Ly-49 molecules and the presence of their MHC ligands on hemopoietic and nonhemopoietic cells. A quantitative correlation was observed between the number of cells expressing MHC ligand and the amount of down-regulation of Ly-49A, Ly-49C, and Ly-49G2 on NK cells in these chimeras. Surprisingly, in animals with relatively low levels of hemopoietic chimerism, some Ly-49 receptors were expressed at higher levels than that observed in normal mice. This observation was surprising because these Ly-49-overexpressing NK cells should theoretically not be useful (18). Our data demonstrate that, in mixed chimeras, Ly-49 receptor expression is regulated by the number of MHC ligand-expressing cells in the environment, but is not calibrated to a specific useful level.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Six- to ten-week-old C57BL/6 (H-2b) and BALB/c (H-2d) mice were purchased from the Frederick Cancer Research Facility (FCRF, Frederick, MD). Animals were housed in sterile microisolator cages and received autoclaved feed and autoclaved, acidified drinking water.

Preparation of mixed chimeras

BALB/c->B6 and B6->BALB/c mixed chimeras were prepared using a previously described nonmyeloablative conditioning regimen, followed by bone marrow transplantation (BMT) (21). Briefly, recipients were treated with depleting doses of anti-CD4 (GK1.5) and anti-CD8 (2.43) mAbs on day -5 before BMT, and received 7 Gy thymic irradiation and 3 Gy whole body irradiation on day 0. Either 5 x 106, 10 x 106, or 20 x 106 donor bone marrow cells were administered on day 0. The different bone marrow dosages were given to obtain animals with varying levels of donor chimerism. Animals receiving such treatment demonstrate long-term mixed chimerism in all hemopoietic lineages (21). B6 and BALB/c mice receiving the conditioning regimen without BMT served as nontransplanted, conditioned controls. Animals were sacrificed 5–7 wk post-BMT. Chimerism in the bone marrow and spleen was assessed by flow cytometry. Percentage of donor chimerism in the bone marrow was calculated with the following formula, where percentage of donor chimerism = 100% x (%34-2-12+) - background]/[[(%34-2-12-) - background] + [(%34-2-12+) - background]].

Percentage of donor NK chimerism in the spleen was calculated with a similar formula, except that values for (% DX5+ 34-2-12+) and (% DX5+ 34-2-12-) cells were used.

Flow cytometry

Preparation of tissues. Peripheral blood was collected into heparinized tubes, and white blood cells were prepared by hypotonic lysis of RBC. On the day of sacrifice, spleens were harvested and gently crushed in ACK lysing buffer (Biowhittaker, Walkersville, MD) to lyse red cells, and were resuspended in RPMI 1640 (Mediatech, Herndon, VA) containing 2% FBS (Sigma, St. Louis, MO) and 0.04 M gentamicin sulfate (Life Technologies, Grand Island, NY). Bone marrow cells were harvested from the femurs and tibiae by flushing the bones with FACS medium consisting of 1x HBSS, 0.1% sodium azide, and 0.1% BSA.

Abs. The following mAbs were used: RM4-4 (anti-CD4) FITC, 53-5.8 (anti-CD8ß) FITC, 5E6 (anti-Ly-49C/I) FITC, purified DX5 (rat IgM anti-mouse pan-NK cell marker), DX5 FITC, mouse anti-rat (MAR) IgM PE, rat IgG2a PE, 34-2-12 (anti-Dd) biotin, KH95 (anti Db) biotin, 1D3 (anti-CD19) biotin, and 2C11 (anti-CD3{epsilon}) biotin, all purchased from PharMingen (San Diego, CA). MAC-1 (anti-CD11b) FITC was purchased from Caltag (South San Francisco, CA). FITC-conjugated and biotinylated HOPC, a nonreactive mouse IgG2a mAb, was used as a negative staining control. The YE1/48 (rat IgG2b anti-mouse Ly-49A)-producing hybridoma was kindly provided by Dr. John Ortaldo (National Cancer Institute, Frederick, MD). The 4D11-producing hybridoma cell line was obtained from the American Type Culture Collection (Manassas, VA). YE1/48 and 4D11 mAbs were purified from bioreactor supernatants and were FITC conjugated using the Quick Tag kit (Boehringer Mannheim, Indianapolis, IN). mAb 2.4G2 (rat anti-mouse Fc{gamma}R) was added to all tubes before addition of other mAbs. Biotinylated mAbs were developed with either phycoerythrin-streptavidin or CyChrome-streptavidin (PharMingen), in two-color or three-color staining protocols, respectively.

For mAb staining, cells were resuspended in FACS media. For multiple color staining, cells were incubated with mAbs for 30 min at 4°C, and washed once before the addition of the next mAb/fluorochrome. Cells were incubated with phycoerythrin-streptavidin and CyChrome-streptavidin for 10 min at 4°C, then washed, and analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). For two-color stains, propidium iodide was used to exclude dead cells during analysis. For three-color staining, dead cells, RBC, macrophages, and granulocytes were excluded on the basis of forward angle and 90° light scatter characteristics, and then specific cell populations were collected, as described in Results. A total of 1000–5000 gated events was collected for analysis. Data analysis was performed using either CELLQuest (Becton Dickinson) or WinList (Verity Software House, Topsham, ME) software.

Data analysis. The relative median fluorescence intensity (MFI) was calculated by dividing the observed MFI of the peak of cells staining with anti-Ly-49A, anti-Ly-49C/I, and anti-Ly-49G2 among gated B6 or BALB/c NK cells from mixed chimeras by the observed MFI of the same cell population from normal B6 or BALB/c mice, respectively, and then multiplying the quotient by 100%. Plotting of scattergrams, simple regression analysis, calculation of Pearson correlation coefficients (r), and drawing of the best fit lines were performed using Microsoft Excel software. Statistical significance of the best fit line was tested using the t-distribution, where t = 0.5 x [log((1 + r)/(1 - r))]/[1/(n - 3)]1/2, in which r = the Pearson correlation coefficient and n = the number of samples. Statistical significance was achieved if p < 0.05. Correlation coefficients were interpreted as follows: 0.9 < ||r|| < 1 = strong correlation, 0.5 < ||r|| < 0.9 = moderate correlation, and 0.0 < ||r|| < 0.5 = weak correlation. An r2 value >= 0.8 was interpreted as a strongly linear correlation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of mixed chimeras and Ly-49 receptor expression

Using three-color flow cytometry, we examined the expression of Ly-49A, Ly-49C, and Ly-49G2 on freshly isolated splenic NK cells developing in BALB/c->B6 and B6->BALB/c mixed chimeras. B6 NK cells were examined by gating on DX5+CD3-Dd- cells. BALB/c NK cells were examined by gating on DX5+CD3-Db- spleen cells. Bone marrow engraftment and the presence of chimerism were confirmed in each individual animal by flow cytometry analysis of the bone marrow and spleen for the presence of donor MHC class I Ags at the time of sacrifice (data not shown). A strong linear correlation between the amount of bone marrow chimerism and the amount of donor NK cell chimerism in the spleen was observed (BALB/c->B6 mixed chimeras, r = 0.89426, r2 = 0.7997, n = 31; B6->BALB/c mixed chimeras, r = 0.93496, r2 = 0.8742, n = 10).

Quantitative relationship between Ly-49 receptor expression and hemopoietic cell expression of MHC ligand

The known inhibitory ligands of Ly-49G2 are Dd and Ld (4). We examined the expression of Ly-49G2 on B6- and BALB/c-derived NK cells from BALB/c->B6 mixed chimeras, in which Dd and Ld are expressed on hemopoietic (BALB/c) cells only. The data are presented in Fig. 1Go. A statistically significant, negative correlation was observed between the level of Ly-49G2 expression on both B6- and BALB/c-derived NK cells and the percentage of BALB/c hemopoietic cells (Fig. 1Go, left panels). As the level of BALB/c hemopoietic chimerism reached a maximum of 80%, the levels of Ly-49G2 on B6 NK cells decreased to a minimum ~70% of that in NK cells of B6 naive controls (Fig. 1Go, top left panel), and the levels on BALB/c NK cells reached a minimum of 125% of that on NK cells of naive BALB/c controls (Fig. 1Go, bottom left panel). Examples of the FACS profiles from individual animals are shown in Fig. 2Go. A similar correlation was observed when the amount of Ly-49G2 expression was plotted against the percentage of donor chimerism in the bone marrow (data not shown). In B6->BALB/c mixed chimeras, in which Dd and Ld were expressed on both nonhemopoietic and hemopoietic cells, Ly-49G2 expression on both B6- and BALB/c-derived cells did not show a significant correlation with the level of chimerism (Fig. 1Go, right panels).



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  		FIGURE 1. The relationship between Ly-49G2 surface expression and percentage of donor chimerism in mixed chimeras. NK cells from the spleens of BALB/c->B6 (left panels) and B6 ->BALB/c (right panels) mixed chimeras were analyzed by flow cytometry, as described in Materials and Methods. Ly-49G2 expression on B6-derived NK cells is shown in the top panels, while Ly-49G2 expression on BALB/c-derived NK cells is shown in the bottom panels. The left panels show a moderate correlation between the level of Ly-49G2 surface expression and the percentage of donor (BALB/c) NK cell chimerism in the spleen, and that maximal down-modulation occurred at the highest levels of chimerism. The right panels show no correlation, and that maximal down-modulation occurred when Ly-49G2’s inhibitory ligands, Ld and Dd, were expressed on both nonhemopoietic and hemopoietic cells. r = Pearson correlation coefficient. p < 0.05 indicates that simple linear regression analysis of the data was statistically significant.

 


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  		FIGURE 2. Increased down-modulation of Ly-49G2 in animals with increased proportions of Ld and Dd ligand in the environment. Examples of FACS profile staining of Ly-49G2 expression on BALB/c NK cells in individual BALB/c->B6 mixed chimeras with differing levels of chimerism are shown. The level of donor chimerism is indicated at the right of each histogram. MFI = median fluorescence intensity of staining in the marked region. The data show that Ly-49G2 is expressed at higher levels in animals with lower levels of BALB/c chimerism, and in animals with lowest levels of chimerism, Ly-49G2 is expressed at higher levels than in normal BALB/c mice.

 
A similar negative correlation was also observed between the relative intensity of Ly-49A expression and the level of donor hemopoiesis in BALB/c->B6 mixed chimeras (Fig. 3Go, left panels). Similar to results for Ly-49G2, reduced levels of Ly-49A expression on both B6- and BALB/c-derived NK cells were observed when Dd was expressed increasing percentages of hemopoietic cells. Maximal down-regulation was also observed at the highest levels of chimerism (Fig. 3Go, left panels). Also similar to results for Ly-49G2, no significant relationship between chimerism and Ly-49A expression was observed for B6 NK cells when Dd ligand was expressed on both nonhemopoietic and hemopoietic cells (B6->BALB/c mixed chimeras) (Fig. 3Go, upper right panel). However, for BALB/c NK cells in B6->BALB/c mixed chimeras, the intensity of Ly-49A staining tended to increase with increasing amounts of B6 chimerism (r = 0.7478, Fig. 3Go, lower right panel).



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  		FIGURE 3. The relationship between Ly-49A surface expression and percentage of donor chimerism in mixed chimeras. NK cells were analyzed as described in Fig. 1Go. Similar to Ly-49G2 expression, a moderate negative correlation was observed between the levels of Ly-49A expression and the percentage of donor (BALB/c) chimerism in BALB/c->B6 mixed chimeras (left panels). Maximum down-regulation was also observed at the highest levels of chimerism (left panels) and when Ly-49A’s inhibitory ligand, Dd, was expressed on both hemopoietic and nonhemopoietic cells in B6->BALB/c mixed chimeras (right panels). Down-regulation of Ly-49AB6 to a level that was 18% of that of normal B6 mice was observed in one animal with only 8.50% donor (BALB/c) NK cell chimerism in the spleen (data not shown). r = Pearson correlation coefficient.

 
Ly-49CBALB/c recognizes both Kb and H-2d class I ligands (15), both of which are present in B6->BALB/c and BALB/c->B6 mixed chimeras. Ly-49C/IB6 is known to recognize Kb (15). For B6 NK cells, no correlation was observed between levels of chimerism and Ly-49C/I expression in either type of chimera (Fig. 4Go, upper panels). In contrast, a strong negative correlation was observed between the proportion of hemopoietic cells expressing Kb and the level of expression of Ly-49C on BALB/c NK cells in B6->BALB/c mixed chimeras (Fig. 4Go, lower right panel). In the reciprocal BALB/c->B6 chimeras, Ly-49CBALB/c was expressed at a level that was about 40% of the level observed in normal BALB/c mice regardless of the level of chimerism (Fig. 4Go, lower left panel).



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  		FIGURE 4. The relationship between Ly-49C surface expression and percentage of donor chimerism in mixed chimeras. Ly-49C/IB6 is inhibited by Kb, whereas Ly-49CBALB/c is inhibited by both Kb and H-2d Ags. Therefore, Ly-49CBALB/c ligands are expressed on both hemopoietic and nonhemopoietic cells in BALB/c->B6 and B6->BALB/c mixed chimeras. A strong inverse correlation between the expression of Ly-49C and the percentage of donor (B6) NK chimerism in the spleen was observed (lower right panel), demonstrating the strong influence of Kb ligand on hemopoietic cells on Ly-49CBALB/c expression. Maximum down-modulation of Ly-49CBALB/c was observed when Kb was expressed in both the nonhemopoietic and the hemopoietic environments (lower left panel). r = Pearson correlation coefficient.

 
Examination of the graphs for Ly-49G2, Ly-49A, and Ly-49C expression (Figs. 1Go, 2Go, and 3Go) shows that Ly-49 receptors on NK cells of a given strain can be expressed at higher levels in mixed chimeras than is found in normal mice of the same strain (represented by data points greater than 100% relative MFI in each graph). For example, when compared with normal BALB/c NK cells, BALB/c NK cells in mixed chimeras expressed higher levels of Ly-49G2 when relatively small numbers of BALB/c cells were present in the environment (Fig. 1Go, lower left panel). The highest levels of Ly-49G2 were found on NK cells developing in animals with the lowest levels of chimerism. For example, the first point on the graph in Fig. 1Go, lower left panel, represents a BALB/c->B6 chimera with 22% BALB/c NK chimerism and Ly-49G2 expression on BALB/c NK cells that is 198% of that observed in normal BALB/c mice. The histogram illustrating this increased expression is presented in Fig. 2Go (chimera 1). The graphs for Ly-49A expression on BALB/c-derived cells (Fig. 3Go, lower left panel) illustrate a similar phenomenon. For the same animal described above (with 22% donor NK chimerism), Ly-49A expression on BALB/c NK cells is 140% of that observed on NK cells in normal BALB/c mice.

Evidence for high affinity interactions between Ly-49AB6 and Dd and between Ly-49CBALB/c and Kb

In the presence of relatively low numbers of Dd+ cells, Ly-49AB6 in BALB/c->B6 mixed chimeras was down-regulated to levels 25–30% of the level observed in normal B6 mice (Fig. 3Go, upper left panel). This high level of sensitivity of Ly-49AB6 to the presence of relatively low numbers of cells expressing Dd suggests that a very high affinity interaction may occur between these molecules.

Ly-49CBALB/c expression in B6->BALB/c mixed chimeras also showed a high level of sensitivity to increasing numbers of Kb ligand-expressing cells (Fig. 4Go, lower right panel), as reflected by the steep slope of the line (-1.7761). Thus, despite the fact that the H-2d haplotype also contains a ligand for Ly-49CBALB/c (15), expression of this receptor on BALB/c NK cells was highly sensitive to the presence of H-2b-bearing cells, suggesting that Kb may have a higher affinity than the H-2d ligand for Ly-49C. This possibility is consistent with the observation that Ly-49C is expressed at reduced levels in B6 (H-2b) mice compared with BALB/c (H-2d) mice (16).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several lines of evidence have indicated that the level of cell surface expression of Ly-49 receptors is regulated by the presence of MHC ligands. Ly-49A expression levels are lower on NK cells from mice in which its ligand, Dd, is expressed than on those from ligand-negative mice (13, 19, 22, 23). Previous studies from our laboratory demonstrated that Dd expression on either hemopoietic or nonhemopoietic cells could induce this down-modulation of Ly-49A expression, and that the degree of down-regulation was proportional to the level of ligand expression in the environment (19). Studies of mosaic MHC Tg mice showed an inverse linear correlation between the level of Ly-49A expression on both MHC Tg-positive and Tg-negative NK cells and the percentage of MHC Tg-positive cells in the mice (24). However, the use of mosaic mice did not allow specific assessment of the effect of MHC ligand expression on hemopoietic vs nonhemopoietic tissues, as is made possible by the mixed chimera model. We demonstrate in this study, for three different Ly-49 receptors (Ly-49A, Ly-49C, and Ly-49G2), that the amount of down-regulation of Ly-49 receptor expression is inversely proportional to the number of hemopoietic cells expressing the major MHC ligand for that receptor. This relationship is less obvious when MHC ligand is expressed on both nonhemopoietic and hemopoietic cells, perhaps because the total MHC ligand load provided by nonhemopoietic cells exceeds that provided in the hemopoietic compartment.

Previous studies from our laboratory (19), involving lethally irradiated B10->(B10 x B10.A)F1 chimeras, demonstrated only partial Ly-49A down-regulation on B10 NK cells compared with that observed in (B10 + F1)->B10 mixed chimeras. Thus, expression of the Dd ligand on F1 hemopoietic cells had a more powerful ability to down-regulate Ly-49A expression than did ligand expression on nonhemopoietic cells. In those studies, however, the level of ligand expression on radioresistant F1 cells, which expressed only heterozygous levels of Dd, was submaximal and caused less marked Ly-49A down-regulation on B10 NK cells than was observed in the presence of homozygous levels of Dd on either hemopoietic or nonhemopoietic cells. Therefore, the amount of Dd ligand per cell present on the host cells determines the expression level of Ly-49A (19). Our current study using mixed allogeneic chimeras extends these observations by demonstrating that, in addition to the amount of MHC ligand per cell, the total number of MHC-expressing cells present in the environment influences the level of Ly-49 receptor expression. Taken together, our previous study (19) and our present results suggest that the amount of MHC ligand expressed per cell and the number of MHC ligand-bearing cells are independent determinants of Ly-49 receptor levels on NK cells.

The receptor calibration theory, as postulated by Sentman et al. (18), hypothesizes that in order for an NK cell to be tolerant to self, it must express at least one Ly-49 receptor that recognizes a self MHC ligand. According to this theory, the strength of the inhibitory signal is dependent on the overall avidity of interaction between the Ly-49-inhibitory receptors and their MHC ligands; thus, the affinity of specific Ly-49 receptors for their ligands, the number of receptors expressed by the NK cell, and the number of MHC ligand molecules expressed by the target contribute to the inhibitory signal. Because NK cell function is thought to be controlled by a balance between activating and inhibitory signals (25), if the inhibitory signal generated upon Ly-49 recognition of an MHC ligand is below a certain threshold, the signals transmitted to the NK cell would favor activation, and target lysis would proceed. To avoid overinhibition and to achieve sensitivity to perturbations in class I MHC expression, the theory states that self MHC-specific Ly-49 receptors are calibrated to a minimal, specific useful level, dictated by the level of class I ligand per cell in the environment. This theory is consistent with the observation of lower levels of Ly-49 receptor expression in mice that express the MHC ligand, and with the increased Ly-49 receptor levels observed on NK cells of class I-deficient mice (23, 26). It is also consistent with the observation that expression of specific Ly-49 receptors is higher in environments in which the MHC ligand is absent than in those in which it is present (13, 14, 16, 19, 23, 26).

The quantitative relationship between Ly-49 receptor expression and the number of cells expressing the relevant ligand suggests a model wherein interactions with MHC ligands on surrounding cells in the environment dynamically modulate the expression of their receptors on NK cells. In BALB/c->B6 chimeras with low levels of BALB/c chimerism, BALB/c NK cells expressed Ly-49G2 at levels twice as high as those detected in normal BALB/c mice. Ly-49A expression was also higher than in normal BALB/c mice in these chimeras. These observations are inconsistent with the above concept that Ly-49 receptor levels are calibrated in a manner that makes them sensitive to perturbations in the level of self class I MHC expression on normal self cells in the environment. In fully MHC-mismatched, mixed allogeneic chimeras, the expression of Dd and Ld ligand per BALB/c cell is similar in animals with low levels or high levels of donor chimerism. As such, the receptor calibration theory predicts that Ly-49 receptors specific for MHC ligand should be calibrated to the same level of expression in animals with low levels or high levels of chimerism. This prediction is inconsistent with our observation of higher than normal Ly-49 receptor expression, despite the presence of a significant, but not predominant, population of hemopoietic cells expressing normal levels of the class I ligand. We therefore prefer to use the term modulation rather than calibration to describe the regulation of Ly-49 receptor levels, because the latter implies a specific level to which Ly-49 receptor expression is regulated. Modulation, on the other hand, implies that Ly-49 receptors are simply down-modulated as a consequence of ligation. The frequency of receptor ligation will be determined not only by the level of class I ligand expressed on the cells in the environment, but also by the proportion of such cells.

The receptor calibration model would also predict that BALB/c NK cells expressing Ly-49G2 above the level normally observed in BALB/c mice would not be useful, because their excessively high levels of Ly-49G2 expression would inhibit their ability to kill self targets expressing abnormally low levels of Dd and Ld (17) (illustrated in Fig. 5Go). Further studies are needed to test whether or not the cells that overexpress Ly-49 receptors in mixed chimeras lose their ability to lyse targets that have lower than normal surface levels of class I MHC. However, it has been demonstrated that Ly-49G2+ NK cells from B6 mice lyse Dd low tumor targets more efficiently than Dd high targets (4). Similarly, killing of Dd low lymphoblast targets was conducted more efficiently by Ly-49Alow NK cells than by Ly-49Ahigh NK cells (27). These data support our hypothesis, and the part of the original receptor calibration theory that states that expression of high levels of Ly-49 receptor makes NK cells less sensitive to low MHC levels.



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  		FIGURE 5. The functional implications of overexpression of self-specific Ly-49 receptors on NK cells in mixed chimeras. A, A representation of an NK cell from a normal mouse, expressing normal levels of Ly-49G2 (left), and a representation of an NK cell from a mixed chimera expressing higher than normal levels of Ly-49G2 (right) are shown. When either of these NK cells interacts with a target that expresses normal levels of the MHC ligand (center), both NK cells are prevented from lysing the target, because the inhibitory signal transmitted to both NK cells is adequate to prevent activation of the NK cell. Thus, expression of self MHC-specific Ly-49 receptors at higher than normal levels should not affect NK cell tolerance to self. B, However, a problem may arise when the same NK cells interact with targets that express reduced levels of MHC ligand (center). The balance of activating and inhibitory signals transmitted to the normal NK cell (left) will favor activation, because the avidity of interaction between Ly-49 receptors and MHC ligands on the target with reduced levels of MHC is lower than the avidity of interaction between Ly-49 receptors and MHC ligands on the target with normal levels of MHC (as shown in A). That is, the inhibitory signal is weaker than normal, allowing NK lysis to proceed. However, the NK cell from the mixed chimera, which expresses higher than normal levels of Ly-49G2, will still be inhibited, because the avidity of interaction between Ly-49G2 and MHC ligand in this case is equal to the avidity of interaction between Ly-49G2 on normal NK cells and MHC ligand on normal targets (A); i.e., the inhibitory signal is still adequate to overcome activating signals. Thus, the NK cell from the mixed chimera theoretically would be insensitive to perturbations in MHC levels.

 
Our data lead us to favor a model in which Ly-49 receptors are down-modulated in response to the frequency of contact between NK cells and other MHC ligand-bearing cells in the environment. In BALB/c->B6 mixed chimeras, for example, the frequency of such contacts with H-2d-bearing cells depends on the proportion of BALB/c cells in the hemopoietic system, and is invariably lower than the frequency that would occur in a normal BALB/c mouse, in which both hemopoietic and nonhemopoietic cells express H-2d. Our observations of high expression of Ly-49G2BALB/c and Ly-49ABALB/c in BALB/c->B6 mixed chimeras (Figs. 1Go and 2Go, lower left panels) are consistent with this hypothesis. Future studies will address whether or not this regulation of Ly-49 receptor levels is determined during NK cell development and fixed for the life of the cell, or whether it is a dynamic process that is plastic throughout the life span of a given NK cell. Experiments using mosaic MHC class I transgenic mice showed that Ly-49A was down-regulated on H-2d-negative NK cells after adoptive transfer to H-2d recipients (24), and that NK cells lacking MHC ligand up-regulate Ly-49 receptor expression upon activation in vitro (24, 28), suggesting that Ly-49 expression by mature NK cells is indeed plastic.

In conclusion, we have shown that the level of Ly-49 receptor expression on NK cells is dependent on the number of MHC ligand-bearing cells in the environment. The ability of NK cells expressing an Ly-49 molecule and its MHC ligand to up-regulate Ly-49 receptor levels well above that in normal mice of that strain shows that the level of Ly-49 receptor expression is not calibrated to a specific useful level for receiving inhibitory signals from ligand-bearing cells. Rather, the level of Ly-49 receptor expression is determined largely by the frequency of interactions with ligand-bearing cells in the environment. In mixed chimeras, two entirely different MHC haplotypes are expressed, and Ly-49 receptor expression is down-regulated in proportion to the frequency with which MHC ligands are encountered. The dominant down-regulation of Ly-49 receptors induced by the presence of MHC ligand on either donor or host cells is likely to have functional consequences for NK cell recognition in mixed chimeras, and could limit the extent to which tolerance to nonself may be induced.


   Acknowledgments
 
We thank Dr. Henry Winn and Dr. Yong Zhao for helpful review of the manuscript; Guiling Zhao, Denise Pearson, Kirsten Swenson, and Juanita Shaffer for expert technical assistance; Vantran Tru for excellent animal husbandry; Dr. David Schoenfeld for assistance with the statistical analysis; and Diane Plemenos for expert assistance with the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant RO1 HL49915. J.O.M. was supported by National Research Service Award 5 F31 HL09733-02. Back

2 Address correspondence and reprint requests to Dr. Megan Sykes, Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital, MGH East, Building 149-5102, 13th Street, Boston, MA 02129. E-mail address: Back

3 Abbreviations used in this paper: Tg, transgene; BMT, bone marrow transplantation; MFI, median fluorescence intensity. Back

Received for publication March 15, 1999. Accepted for publication June 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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