Aberrant DAP12 signaling in the 129 strain of mice: implications for the analysis of gene-targeted mice

NK cells are implicated in antiviral responses, bone marrow transplantation and tumor immunosurveillance. Their function is controlled, in part, through the Ly49 family of class I binding receptors. Inhibitory Ly49s suppress signaling, while activating Ly49s (i.e., Ly49D) activate NK cells via the DAP12 signaling chain. Activating Ly49 signaling has been studied primarily in C57BL/6 mice, however, 129 substrains are commonly used in gene-targeting experiments. In this study, we show that in contrast to C57BL/6 NK cells, cross-linking of DAP12-coupled receptors in 129/J mice induces phosphorylation of DAP12 but not calcium mobilization or cytokine production. Consistent with poor-activating Ly49 function, 129/J mice reject bone marrow less efficiently than C57BL/6 mice. Sequence analysis of receptors and DAP12 suggests no structural basis for inactivity, and both the 129/J and C57BL/6 receptors demonstrate normal function in a reconstituted receptor system. Most importantly, reconstitution of Ly49D in 129/J NK cells demonstrated that the signaling deficit is within the NK cells themselves. These unexpected findings bring into question any NK analysis of 129/J, 129Sv, or gene-targeted mice derived from these strains before complete backcrossing, and provide a possible explanation for the differences observed in the immune response of 129 mice in a variety of models.     

MHC class I-Ly49 interactions shape the Ly49 repertoire on murine NK cells

This study aims to determine how the interaction of Ly49 receptors with MHC class I molecules shapes the development of the Ly49 repertoire. We have examined the percentage of NK cells that expressed Ly49A, Ly49G2, and Ly49D in single and double Ly49A/C-transgenic mice on four different MHC backgrounds, H-2(b), H-2(d), H-2(b/d), and beta(2)-microglobulin(-/-). The results show that the total numbers of NK cells were not different among the strains. The prior expression of a Ly49 receptor capable of binding to self MHC class I altered the percentage of NK cells expressing endogenous Ly49A, Ly49G2, and Ly49D even in mice in which no MHC ligand was present for the latter receptors. The NK cells in the Ly49-transgenic mice expressed the same level of endogenous Ly49 receptors as wild-type mice of a similar MHC background. In contrast, the number of NK T cells was reduced in mice in which the Ly49 transgene could bind to a MHC class I molecule. The onset of Ly49 receptor expression on NK cells during ontogeny was not altered in the presence of transgenic Ly49 receptors. These data support a sequential model and argue against a selection model for Ly49 repertoire development on NK cells.     

Independent control of Ly49g alleles: implications for NK cell repertoire selection and tumor cell killing

A novel murine NK cell-reactive mAb, AT8, was generated. AT8 recognizes Ly49G from 129/J, BALB/c, and related mouse strains, but does not bind to Ly49G(B6). Costaining with AT8 and a Ly49G(B6)-restricted Ab (Cwy-3) provides the first direct evidence that Ly49G protein is expressed from both alleles on a significant proportion of NK cells from four different types of F(1) hybrid mice. The observed level of biallelic Ly49G expression reproducibly followed the product rule in both freshly isolated and cultured NK cells. Surprisingly, the percentage of NK cells expressing both Ly49G alleles could be dramatically increased in vitro and in vivo through IL-2R- and IFN receptor-dependent signaling pathways, respectively. Unexpectedly, Ly49G(B6+) NK cells in an H-2(d), but not H-2(b), background were more likely to lyse D(d+) and Chinese hamster ovary tumor cells than Ly49G(BALB/129+) NK cells. Furthermore, Ly49G(B6+) NK cells also proliferated to a higher degree in response to poly(I:C) than NK cells expressing a non-Ly49G(B6) allele in an H-2(d), but not H-2(b), background. These results suggest that Ly49G(B6) has a lower affinity for H-2D(d) than Ly49G(BALB/129), and the genetic background calibrates the responsiveness of NK cells bearing self-specific Ly49. Other H-2D(d) receptors on the different Ly49G(+) NK cell subsets were unequally coexpressed, possibly explaining the disparate responses of Ly49G(B6+) NK cells in different hybrid mice. These data indicate that the stochastic mono- and biallelic expression of divergent Ly49G alleles increases the range of MHC affinities and the functional potential in the total NK cell population of heterozygous mice.     

Critical residues at the Ly49 natural killer receptor's homodimer interface determine functional recognition of m157, a mouse cytomegalovirus MHC class I-like protein

NK cell function is regulated by Ly49 receptors in mice and killer cell Ig-like receptors in humans. Although inhibitory Ly49 and killer cell Ig-like receptors predominantly ligate classical MHC class I molecules, recent studies suggest that their activating counterparts recognize infection. The quintessential example is resistance to the mouse CMV in C57BL/6 mice, which depends on the functional recognition of m157, a mouse CMV-encoded MHC class I-like molecule, by Ly49H, an activating NK cell receptor. We have taken advantage of the natural variation in closely related members of the Ly49C-like receptors and the availability of Ly49 crystal structures to understand the molecular determinants of the Ly49H-m157 interaction and to identify amino acid residues discriminating between m157 binding and nonbinding receptors. Using a site-directed mutagenesis approach, we have targeted residues conserved in receptors binding to m157 (Ly49H and Ly49I(129)) but different from receptors lacking m157 recognition (Ly49C, Ly49I(B6), and Ly49U). Wild-type and mutant receptors were transfected into reporter cells, and physical binding as well as functional activation by m157 was studied. Our findings suggested that the Ly49 MHC class I contact "site 2," I226, may not be involved in m157 binding. In contrast, residue Y146 and G151, mapping at the receptor homodimer interface, are likely critical for functional recognition of the m157 glycoprotein. Our combined functional and three-dimensional modeling approach suggested that the architecture of the Ly49H dimer is crucial to accessing m157, but not MHC class I. These results link Ly49 homodimerization variability to the direct recognition of pathogen products.     

Functional consequences of natural sequence variation of murine cytomegalovirus m157 for Ly49 receptor specificity and NK cell activation

The Ly49H activating receptor on C57BL/6 (B6) NK cells plays a key role in early resistance to murine cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. The m157 molecule is also recognized by the Ly49I inhibitory receptor from the 129/J mouse strain. The m157 gene is highly sequence variable among MCMV isolates, with many m157 variants unable to bind Ly49H(B6). In this study, we have sought to define if m157 variability leads to a wider spectrum of interactions with other Ly49 molecules and if this modifies host susceptibility to MCMV. We have identified novel m157-Ly49 receptor interactions, involving Ly49C inhibitory receptors from B6, BALB/c, and NZB mice, as well as the Ly49H(NZB) activation receptor. Using an MCMV recombinant virus in which m157(K181) was replaced with m157(G1F), which interacts with both Ly49H(B6) and Ly49C(B6), we show that the m157(G1F)-Ly49C interactions cause no apparent attenuating effect on viral clearance in B6 mice. Hence, when m157 can bind both inhibitory and activation NK cell receptors, the outcome is still activation. Thus, these data indicate that whereas m157 variants predominately interact with inhibitory Ly49 receptors, these interactions do not profoundly interfere with early NK cell responses.     

A "chimeric" C57l-derived Ly49 inhibitory receptor resembling the Ly49D activation receptor.

Ly49D is a natural killer (NK) cell activation receptor that is responsible for differential mouse inbred strain-determined lysis of Chinese hamster ovary (CHO) cells. Whereas C57BL/6 NK cells kill CHO, BALB/c-derived NK cells cannot kill because they lack expression of Ly49D. Furthermore, the expression of Ly49D, as detected by monoclonal antibody 4E4, correlates well with CHO lysis by NK cells from different inbred strains. However, one discordant mouse strain was identified; C57L NK cells express the mAb 4E4 epitope but fail to lyse CHO cells. Herein we describe a Ly49 molecule isolated from C57L mice that is recognized by mAb 4E4 (anti-Ly49D). Interestingly, this molecule shares extensive similarity to Ly49D(B6) in its extracellular domain, but its cytoplasmic and transmembrane domains are identical to the inhibitory receptor Ly49A(B6), including a cytoplasmic ITIM. This molecule bears substantial overall homology to the previously cloned Ly49O molecule from 129 mice the serologic reactivity and function of which were undefined. Cytotoxicity experiments revealed that 4E4(+) LAK cells from C57L mice failed to lyse CHO cells and inhibited NK cell function in redirected inhibition assays. MHC class I tetramer staining revealed that the Ly49O(C57L)-bound H-2D(d) and lysis by 4E4(+) C57L LAK cells is inhibited by target H-2D(d). The structural basis for ligand binding was also examined in the context of the recent crystallization of a Ly49A-H-2D(d) complex. Therefore, this apparently "chimeric" Ly49 molecule serologically resembles an NK cell activation receptor but functions as an inhibitory receptor.     

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.     

The MHC class I molecule H-2Dp inhibits murine NK cells via the inhibitory receptor Ly49A.

MHC class I molecules strongly influence the phenotype and function of mouse NK cells. NK cell-mediated lysis is prevented through the interaction of Ly49 receptors on the effector cell with appropriate MHC class I ligands on the target cell. In addition, host MHC class I molecules have been shown to modulate the in vivo expression of Ly49 receptors. We have previously reported that H-2Dd and H-2Dp MHC class I molecules are able to protect (at the target cell level) from NK cell-mediated lysis and alter the NK cell specificity (at the host level) in a similar manner, although the mechanism behind this was not clear. In this study, we demonstrate that the expression of both H-2Dd and H-2Dp class I molecules in target cells leads to inhibition of B6 (H-2b)-derived Ly49A+ NK cells. This inhibition could in both cases be reversed by anti-Ly49A Abs. Cellular conjugate assays showed that Ly49A-expressing cells indeed bind to cells expressing H-2Dp. The expression of Ly49A and Ly49G2 receptors on NK cells was down-regulated in H-2Dp-transgenic (B6DP) mice compared with nontransgenic B6 mice. However, B6DP mice expressed significantly higher levels of Ly49A compared with H-2Dd-transgenic (D8) mice. We propose that both H-2Dd and H-2Dp MHC class I molecules can act as ligands for Ly49A.     

Structural and functional aspects of the Ly49 natural killer cell receptors

Natural killer cells are part of the first line of innate immune defence against virus-infected cells and cancer cells in the vertebrate immune system. They are called 'natural' killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. The Ly49 NK receptors are type II transmembrane glycoproteins, structurally characterized as disulphide-linked homodimers. They share extensive homology with C-type lectins, and they are encoded by a multigene family that in mice maps on chromosome 6. A fine balance between inhibitory and activating signals regulates the function of NK cells. Inhibitory Ly49 molecules bind primarily MHC class I ligands, whereas the ligands for activating Ly49 molecules may include MHC class I, but also interestingly MHC class I-like molecules expressed by viruses, as is the case for Ly49H, which binds the m157 gene product of murine cytomegalovirus. In this study, we review the function and X-ray crystal structure of the Ly49 NK cell receptors hitherto determined (Ly49A, Ly49C and Ly49I), and the structural features of the Ly49/MHC class I interaction as revealed by the X-ray crystal structures of Ly49A/H-2Dd and the recently determined Ly49C/H-2Kb.     

Ly49C-Dependent Control of MCMV Infection by NK Cells Is Cis-Regulated by MHC Class I Molecules

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. We previously identified a mutant strain (MCMV^G1F) whose variant m157 also binds the inhibitory Ly49C receptor. Here we show that simultaneous binding of m157 to the two receptors hampers Ly49H-dependent NK cell activation as Ly49C-mediated inhibition destabilizes NK cell conjugation with their targets and prevents the cytoskeleton reorganization that precedes killing. In B6 mice, as most Ly49H⁺ NK cells do not co-express Ly49C, the overall NK cell response remains able to control MCMVm157^G1F infection. However, in B6 Ly49C transgenic mice where all NK cells express the inhibitory receptor, MCMV infection results in altered NK cell activation associated with increased viral replication. Ly49C-mediated inhibition also regulates Ly49H-independent NK cell activation. Most interestingly, MHC class I regulates Ly49C function through cis-interactions that mask the receptor and restricts m157 binding. B6 Ly49C Tg, β2m ko mice, whose Ly49C receptors are unmasked due to MHC class I deficient expression, are highly susceptible to MCMVm157^G1F and are unable to control a low-dose infection. Our study provides novel insights into the mechanisms that regulate NK cell activation during viral infection.     

Interactions of Ly49 family receptors with MHC class I ligands in trans and cis

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.     

Cloning and characterization of a novel activating Ly49 closely related to Ly49A.

The majority of the known Ly49 family members have been isolated from either C57BL/6 (B6) or BALB/c mice. Interestingly, the anti-Ly49 Ab reactivities observed in 129/J mice are different from those of B6 mice. Furthermore, immunoprecipitation of 129/J NK cell lysates with YE1/32 and YE1/48, Abs specific for the inhibitory Ly49A in B6, resulted in detection of the activation-associated DAP12 molecule. These results indicated a need for a more detailed study of this strain. Therefore, a cloning strategy was devised to isolate Ly49 cDNAs from 129/J mice. An immunoreceptor tyrosine-based inhibitory motif-containing, Ly49D-related clone was discovered that we have named Ly49O, and one immunoreceptor tyrosine-based inhibitory motif-lacking, Ly49A-related clone was discovered that we have named Ly49P. No anti-Ly49 mAb reacted with Ly49O, whereas the molecule encoded by the Ly49P cDNA was found to react with YE1/32 and YE1/48. Ly49P was found to associate with mouse DAP12, and Ab-mediated cross-linking of Ly49P resulted in mouse DAP12 phosphorylation and Ca2+ mobilization, indicating that Ly49P is a competent activation receptor. Ly49P, therefore, represents a novel member of the Ly49 activating receptor subfamily.     

Regulation of NKT cells by Ly49: analysis of primary NKT cells and generation of NKT cell line

TCRalphabeta(+)NK1.1(+) (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to alpha-galactosylceramide (alpha-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with alpha-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to alpha-GalCer. Moreover, alpha-GalCer presented by normal dendritic cells stimulated purified Ly49(-), but not Ly49(+), splenic NKT cells, whereas MHC class I-deficient dendritic cells presented alpha-GalCer to both Ly49(+) and Ly49(-) NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an alpha-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49(-)NK1.1(+)CD4(+) thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.     

Ly-49P activates NK-mediated lysis by recognizing H-2Dd

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd alpha1/alpha2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.     

Probing Natural Killer Cell Education by Ly49 Receptor Expression Analysis and Computational Modelling in Single MHC Class I Mice

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for “missing self” recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an “educating impact” on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.     

Ly49G2 receptor blockade reduces tumor burden in a leukemia model but not in a solid tumor model

Background NK cell activity is regulated in part by inhibitory receptors that bind to MHC class I molecules. It is possible to enhance NK cell cytotoxicity against tumor cells by preventing the interaction of these inhibitory receptors with their MHC class I ligands. Results In this study, we determined that Ly49G2 is an inhibitory receptor in AKR mice for self-MHC class I, and AKR Ly49G2 has an identical sequence to BALB/c Ly49G2. Blockade of Ly49G2 receptors in vivo resulted in decreased growth of BW-Sp3 lymphoma cells when the tumor cells were given i.v. but not when the tumor cells were inoculated into the flank forming a solid tumor. However, NK cells were involved in inhibiting the growth of BW-Sp3 tumor cells in the flank. Conclusion These data demonstrate that the effectiveness of inhibitory receptor blockade depends upon the tissue location of the tumor cells.     

B6 strain Ly49I inhibitory gene expression on T cells in FVB.Ly49IB6 transgenic mice fails to prevent normal T cell functions

Inhibitory Ly49 receptors expressed on NK cells provide a mechanism for tolerance to normal self tissues. The immunoregulatory tyrosine-based inhibitory motifs present in some Ly49s are able to transmit an inhibitory signal upon ligation by MHC class I ligands. In our system, as well as others, mice transgenic for inhibitory Ly49 receptors express these receptors on both NK and T cells. FVB (H2(q)) mice transgenic for the B6 strain Ly49I (Ly49I(B6)) express the inhibitory Ly49 receptor on the surface of both T and NK cells. Although Ly49I functions to prevent NK-mediated rejection of H2(b) donor bone marrow cells in this transgenic mouse strain, the T cells do not appear to be affected by the expression of the Ly49I transgene. FVB.Ly49I T cells have normal proliferative capabilities both in vitro and in vivo in response to the Ly49I ligand, H2(b). In vivo functional T cell assays were also done, showing that transgenic T cells were not functionally affected. T cells in these mice also appear to undergo normal T cell development and activation. Only upon stimulation with suboptimal doses of anti-CD3 in the presence of anti-Ly49I is T cell proliferation inhibited. These data are in contrast with findings in Ly49A, and Ly49G2 receptor transgenic models. Perhaps Ly49I-H2(b) interactions are weaker or of lower avidity than Ly49A-H-2D(d) interactions, especially in T cells.     

Natural Killer Cell Tolerance Persists Despite Significant Reduction of Self MHC Class I on Normal Target Cells in Mice

Background

A major group of murine inhibitory receptors on Natural Killer (NK) cells belong to the Ly49 receptor family and recognize MHC class I molecules. Infected or transformed target cells frequently downmodulate MHC class I molecules and can thus avoid CD8⁺ T cell attack, but may at the same time develop NK cell sensitivity, due to failure to express inhibitory ligands for Ly49 receptors. The extent of MHC class I downregulation needed on normal cells to trigger NK cell effector functions is not known.

Methodology/Principal Findings

In this study, we show that cells expressing MHC class I to levels well below half of the host level are tolerated in an in vivo assay in mice. Hemizygous expression (expression from only one allele) of MHC class I was sufficient to induce Ly49 receptor downmodulation on NK cells to a similar degree as homozygous expression, despite a strongly reduced cell surface level of MHC class I. Co-expression of weaker MHC class I ligands in the host did not have any further effect on the degree of Ly49 downmodulation. Furthermore, a single MHC class I allele could downmodulate up to three Ly49 receptors on individual NK cells. Only when NK cells simultaneously expressed several Ly49 receptors and hemizygous MHC class I levels, a putative threshold for Ly49 downmodulation was reached.

Conclusion

Collectively, our findings suggest that in interactions between NK cells and normal untransformed cells, MHC class I molecules are in most cases expressed in excess compared to what is functionally needed to ensure self tolerance and to induce maximal Ly49 downmodulation. We speculate that the reason for this is to maintain a safety margin for otherwise normal, autologous cells over a range of MHC class I expression levels, in order to ensure robustness in NK cell tolerance.     

Altered donor and recipient Ly49+ NK cell subsets in allogeneic H-2d --> H-2b and H-2b --> H-2d bone marrow chimeras

NK cells reject non-self hematopoietic bone marrow (BM) grafts via Ly49 receptor-mediated MHC class I-specific recognition and calibration of receptor expression levels. In this paper we investigated how Ly49+ subset frequencies were regulated dependent on MHC class I expression. The development of donor and host Ly49A+ (recognizes H-2Dd and H-2Dk ligands) and Ly49C/I+ (Ly49CBALB/c recognizes H-2Kb, H-2Kd, and H-2Dd, and Ly49CB6 recognizes only H-2Kb) NK cell frequencies were monitored for 120 days in murine-mixed allogeneic BM chimeras. C57BL/6 (H-2b) BM was transplanted into BALB/c (H-2d) mice and vice versa. Peripheral NK cell populations were examined every 5 days. Chimerism was found to be stable with 80-90% donor NK cells. In contrast to syngeneic controls reexpressing pretransplant patterns, donor and host NK cells revealed new and mainly reduced subset frequencies 55 days after allogeneic transplantation. Recipient NK cells acquired these later than donor NK cells. In H-2d --> H-2b chimeras Ly49A+, Ly49C/I+, and Ly49A+/Ly49C/I+ proportions were mainly diminished upon interaction with cognate ligands. Also in H-2b --> H-2d chimeras, Ly49A+ and Ly49A+/Ly49C/I+ subsets were reduced, but there was a transient normalization of Ly49C/I+ proportions in the noncognate host. After 120 days all subsets were reduced. Therefore, down-regulation of developing Ly49A+ and Ly49C/I+ chimeric NK cell frequencies by cognate ligands within 7-8 wk after BM transplantation may be important for successful engraftment.