Structure/function relationship of activating Ly-49D and inhibitory Ly-49G2 NK receptors.

Murine NK cells express Ly-49 family receptors capable of either inhibiting or activating lytic function. The overlapping patterns of expression of the various receptors have complicated their precise biochemical characterization. Here we describe the use of the Jurkat T cell line as the model for the study of Ly-49s. We demonstrate that Ly-49D is capable of delivering activation signals to Jurkat T cells even in the absence of the recently described Ly-49D-associated chain, DAP-12. Ly-49D signaling in Jurkat leads to tyrosine phosphorylation of TCRzeta and requires Syk/Zap70 family kinases and arginine 54 of Ly-49D, suggesting that Ly-49D signals via association with TCRzeta. Coexpression studies in 293-T cells confirmed the ability of Ly-49D to associate with TCRzeta. In addition, we have used this model to study the functional interactions between an inhibitory Ly-49 (Ly-49G2) and an activating Ly-49 (Ly-49D). Ly-49G2 blocks activation mediated by Ly-49D in an immunoreceptor tyrosine-based inhibitory motif (ITIM)-dependent manner. In contrast, Ly-49G2 was incapable of inhibiting activation by the TCR even though human killer cell inhibitory receptor (KIR) (KIR3DL2(GL183)) effectively inhibits TCR. Both the ability of Ly-49G2 to block Ly-49D activation and the failure of Ly-49G2 to inhibit TCR signaling were confirmed in primary murine NK cells and NK/T cells, respectively. These data demonstrate the dominant effects of the inhibitory receptors over those that activate and suggest an inability of the Ly-49 type II inhibitory receptors to efficiently inhibit type I transmembrane receptor signaling in T cells and NK cells.     

Activating Ly-49 receptors regulate LFA-1-mediated adhesion by NK cells

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors, and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions, yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore, we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1, and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors, leading to target cell death.     

Cutting edge: the mouse NK cell-associated antigen recognized by DX5 monoclonal antibody is CD49b (alpha 2 integrin, very late antigen-2)

DX5 mAb is a useful reagent because it stains NK cells from all mouse strains examined. We have identified the molecule recognized by DX5 mAb by using a retrovirus-mediated expression cloning system. A 5-kb cDNA encoding a protein that is reactive with the DX5 mAb was isolated from a NK cell cDNA library, and this molecule was identical with CD49b (very late Ag-2, alpha(2) integrin). The DX5 mAb reacted with transfectants expressing CD49b, and binding of DX5 to the NK cells and CD49b transfectants was blocked in the presence of other anti-CD49b mAbs. When NK1.1(+) NK cells were cultured with IL-2, they progressively lost reactivity with DX5 mAb as a consequence of cellular proliferation. Cytotoxicity mediated by the DX5(+) NK cells was dramatically higher as compared with DX5(-) NK cells. Therefore, DX5 mAb recognizes CD49b and can be used to define functionally distinct subsets of NK cells.     

Mouse CD94 participates in Qa-1-mediated self recognition by NK cells and delivers inhibitory signals independent of Ly-49

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94(bright) and CD94(dull) cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94(bright) but not CD94(dull) cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and "missing self" target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.     

Cutting edge: expression of functional CD94/NKG2A inhibitory receptors on fetal NK1.1+Ly-49- cells: a possible mechanism of tolerance during NK cell development.

Fetal liver- and thymus-derived NK1.1+ cells do not express known Ly-49 receptors. Despite the absence of Ly-49 inhibitory receptors, fetal and neonatal NK1.1+Ly-49- cells can distinguish between class Ihigh and class Ilow target cells, suggesting the existence of other class I-specific inhibitory receptors. We demonstrate that fetal NK1. 1+Ly-49- cell lysates contain CD94 protein and that a significant proportion of fetal NK cells are bound by Qa1b tetramers. Fetal and adult NK cells efficiently lyse lymphoblasts from Kb-/-Db-/- mice. Qa1b-specific peptides Qdm and HLA-CW4 leader peptide specifically inhibited the lysis of these blasts by adult and fetal NK cells. Qdm peptide also inhibited the lysis of Qa1b-transfected human 721.221 cells by fetal NK cells. Taken together, these results suggest that the CD94/NKG2A receptor complex is the major known inhibitory receptor for class I (Qa1b) molecules on developing fetal NK cells.     

Levels of Ly-49 receptor expression are determined by the frequency of interactions with MHC ligands: evidence against receptor calibration to a "useful" level.

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.     

Receptor glycosylation regulates Ly-49 binding to MHC class I

Murine NK cells express the Ly-49 family of class I MHC-binding receptors that control their ability to lyse tumor or virally infected host target cells. X-ray crystallography studies have identified two predominant contact sites (sites 1 and 2) that are involved in the binding of the inhibitory receptor, Ly-49A, to H-2D(d). Ly-49G2 (inhibitory) and Ly-49D (activating) are highly homologous to Ly-49A and also recognize H-2D(d). However, the binding of Ly-49D and G(2) to H-2D(d) is of lower affinity than Ly-49A. All Ly-49s contain N-glycosylation motifs; however, the importance of receptor glycosylation in Ly-49-class I interactions has not been determined. Ly-49D and G(2) contain a glycosylation motif (NTT (221-223)), absent in Ly-49A, adjacent to one of the proposed binding sites for H-2D(d) (site 2). The presence of a complex carbohydrate group at this critical site could interfere with class I binding. In this study, we are able to demonstrate for the first time that Ly-49D binds H-2D(d) in the presence of mouse beta(2)-microglobulin. We also demonstrate that glycosylation of the NTT (221-23) motif of Ly-49D inteferes with recognition of H-2D(d). Alteration of the Ly-49D-NTT (221-23) motif to abolish glycosylation at this site resulted in enhanced H-2D(d) binding and receptor activation. Furthermore, glycosylation of Ly-49G2 at NTT (221-23) also reduces receptor binding to H-2D(d) tetramers. Therefore, the addition of complex carbohydrates to the Ly-49 family of receptors may represent a mechanism by which NK cells regulate affinity for host class I ligands.     

A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from beta 2-microglobulin-deficient mice

Mouse NK cells express inhibitory NK receptors that recognize target cell MHC class I molecules and activation receptors that are less well defined. The Ly-49D activation receptor on C57BL/6 NK cells recognizes Chinese hamster ovary cells and triggers natural killing. In this study, we demonstrate that a Chinese hamster classical MHC class I molecule is the ligand for Ly-49D in a reporter gene assay system as well as in NK cell killing assays. Ly-49D recognizes the Chinese hamster class I molecule better when it is expressed with Chinese hamster beta(2)-microglobulin (beta(2)m) than murine beta(2)m. However, it is still controversial that Ly-49D recognizes H-2D(d), as we were unable to demonstrate the specificity previously reported. Using this one ligand-one receptor recognition system, function of an NK activation receptor was, for the first time, investigated in NK cells that are tolerized in beta(2)m-deficient mice. Surprisingly, Ly-49D-killing activity against ligand-expressing targets was observed with beta(2)m-deficient mouse NK cells, albeit reduced, even though "tolerized" function of Ly-49D was expected. These results indicate that Ly-49D specifically recognizes the Chinese hamster MHC class I molecule associated with Chinese hamster beta(2)m, and indicate that the Ly-49D NK cell activation receptor is not tolerized in beta(2)m deficiency.     

Interaction of Ly-49D+ NK cells with H-2Dd target cells leads to Dap-12 phosphorylation and IFN-gamma secretion

Murine Ly-49D augments NK cell function upon recognition of target cells expressing H-2Dd. Ly-49D activation is mediated by the immunoreceptor tyrosine-based activation motif-containing signaling moiety Dap-12. In this report we demonstrate that Ly-49D receptor ligation can lead to the rapid and potent secretion of IFN-gamma. Cytokine secretion can be induced from Ly-49D+ NK cells after receptor ligation with Ab or after interaction with target cells expressing their H-2Dd ligand. Consistent with the dominant inhibitory function of Ly-49G, NK cells coexpressing Ly-49D and Ly-49G show a profound reduction in IFN-gamma secretion after interaction with targets expressing their common ligand, H-2Dd. Importantly, we are able to demonstrate for the first time that effector/target cell interactions using Ly-49D+ NK cells and H-2Dd targets result in the rapid phosphorylation of Dap-12. However, Dap-12 is not phosphorylated when Ly-49D+ NK cells coexpress the inhibitory receptor, Ly-49G. These studies are novel in describing Ly-49 activation vs inhibition, where two Ly-49 receptors recognize the same class I ligand, with the dominant inhibitory receptor down-regulating phosphorylation of Dap-12, cytokine secretion, and cytotoxicity in NK cells.     

Recognition of class I MHC by NK receptor Ly-49C: identification of critical residues.

The Ly-49 family of inhibitory receptors plays a major role in regulating mouse NK cell cytotoxicity. Two of its members, Ly-49C and I, are recognized by the mAb 5E6, which also defines a subset of NK cells involved in the hybrid resistance phenomenon. Previous studies have shown that Ly-49C binds to a broad spectrum of class I MHC molecules, while Ly-49I apparently does not bind to any class I MHC molecules tested. In the present investigation we have defined the amino acid residues of Ly-49C that are critical for determining its ligand specificities. First, using quantitative COS cell adhesion assays, we demonstrated that Ly-49CB6 bound to Dd, Db, Kb, or Kk as well as to murine leukemic cell lines GM979 (H-2s) and IC-21 (H-2b). In contrast, COS cells expressing Ly-49IB6 did not significantly bind to any of the class I MHC tested. To determine which amino acid residues of Ly-49C are critical for their specific binding to class I MHC, a series of chimeric and mutant Ly-49C and I were generated and tested. Exchanging the critical residues between Ly-49C and I significantly affected their binding specificities. Finally, we identified the epitopes on Ly-49C recognized by mAbs 5E6 and 4LO3311 that functionally inhibit Ly-49C recognition of its ligands. These results further define the class I specificities of Ly-49C and provide insight into the structural basis for how class I MHC is recognized by the Ly-49 family of NK receptors.     

Ly-49W, an activating receptor of nonobese diabetic mice with close homology to the inhibitory receptor Ly-49G, recognizes H-2D(k) and H-2D(d)

The diversity and ligand specificity of activating Ly-49 receptors expressed by murine NK cells are largely unknown. We cloned a new Ly-49-activating receptor, expressed by NK cells of the nonobese diabetic mouse strain, which we have designated Ly-49W. Ly-49W is highly related to the known inhibitory receptor Ly-49G in its carbohydrate recognition domain, exhibiting 97.6% amino acid identity in this region. We demonstrate that the 4D11 and Cwy-3 Abs, thought to be Ly-49G specific, also recognize Ly-49W. Rat RNK-16 cells transfected with Ly-49W mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49W can activate NK-mediated lysis. We further show that Ly-49W is allo-MHC specific: Ly-49W transfectants of RNK-16 only lysed Con A blasts expressing H-2(k) or H-2(d) haplotypes, and Ab-blocking experiments indicated that H-2D(k) and D(d) are ligands for Ly-49W. Ly-49W is the first activating Ly-49 receptor demonstrated to recognize an H-2(k) class I product. Ly-49G and Ly-49W represent a new pair of NK receptors with very similar ligand-binding domains, but opposite signaling functions.     

Immature NK cells suppress dendritic cell functions during the development of leukemia in a mouse model

To analyze the mechanisms by which cancer cells escape from hosts' immune surveillance, we investigated the changes in immune status during the progression of leukemia induced by injecting mice with WEHI-3B cells. In the bone marrow (BM) of leukemic mice, only DX5(+)CD3(-) cells were continuously increased, despite the progression of leukemia. In addition, DX5(+)CD3(-) cells were rapidly increased in peripheral blood (PB) 20 days after inoculation. We also found that myeloid dendritic cells (DCs) expressing low levels of I-A(d) and having low allo-T cell stimulatory activity were markedly increased in PB and spleen. The increase in DX5(+) cells in BM was thought to be induced by soluble factors from leukemic cells. DX5(+) cells from leukemic mice were CD3(-), B220(-), Gr-1(-), CD14(-), CD94(-), Ly-49C/F(-), asialo GM1(+), CD25(+), CD122(+), Thy-1(bright), and c-kit(dim) and showed low killing activity against YAC-1 cells, suggesting that those DX5(+) cells were immature NK cells. NK cells from leukemic PB down-regulated the expression of I-A(d) on DCs, an effect mediated by TGF-beta. Moreover, these NK cells significantly suppressed the allo-T cell stimulatory activity of DCs, an effect requiring cell-to-cell contact between NK cells and DCs and thought to involve CD25. Importantly, NK cells from leukemic PB inhibited generation of autotumor-specific CTL induced by DCs in primary MLR or by DC immunization. In conclusion, we identified circulating immature NK cells with immunosuppressive activities. These cells may be important for understanding the involvement of the host immune system during the development of leukemia.     

Differential roles for IL-15R alpha-chain in NK cell development and Ly-49 induction

IL-15Ralpha-deficient (IL-15Ralpha(-/-)) mice lack NK cells. However, when bone marrow (BM) progenitors from IL-15Ralpha(-/-) mice were cultured with IL-7, stem cell factor and flt3 ligand, followed by IL-15, they were able to differentiate into functional NK cells, indicating that IL-15Ralpha is not critical for NK cell development. Whereas NK cells generated in vitro from IL-15Ralpha(-/-) BM progenitors expressed CD94/NKG2, they failed to express Ly-49 receptors. In keeping with this, when IL-15Ralpha(-/-) BM cells were transferred into wild type recipients, they gave rise to NK cells in vivo, but with greatly reduced expression of Ly-49 receptors. Furthermore, the small numbers of NK cells found in IL-15(-/-) as well as IL-15Ralpha(-/-) but not flt3 ligand(-/-) mice expressed much lower levels of Ly-49 receptors than those from wild type mice. These results indicate a novel role for IL-15Ralpha-chain in Ly-49 induction on developing NK cells.     

Natural killing of xenogeneic cells mediated by the mouse Ly-49D receptor.

NK lymphocytes lyse certain xenogeneic cells without prior sensitization. The receptors by which NK cells recognize xenogeneic targets are largely uncharacterized but have been postulated to possess broad specificity against ubiquitous target ligands. However, previous studies suggest that mouse NK cells recognize xenogeneic targets in a strain-specific manner, implicating finely tuned, complex receptor systems in NK xenorecognition. We speculated that mouse Ly-49D, an activating NK receptor for the MHC I ligand, H2-Dd, might display public specificities for xenogeneic target structures. To test this hypothesis, we examined the lysis of xenogeneic targets by mouse Ly-49D transfectants of the rat NK cell line RNK-16 (RNK. Ly-49D). Of the xenogeneic tumor targets tested, RNK.Ly-49D, but not untransfected RNK-16, preferentially lysed tumor cells derived from Chinese hamsters and lymphoblast targets from rats. Ly-49D-dependent recognition of Chinese hamster cells was independent of target N-linked glycosylation. Mouse Ly-49D also specifically stimulated the natural killing of lymphoblast targets derived from wild-type and MHC-congenic rats of the RT1lv1 and RT1l haplotypes, but not of the RT1c, RT1u, RT1av1, or RT1n haplotypes. These studies demonstrate that Ly-49D can specifically mediate cytotoxicity against xenogeneic cells, and they suggest that Ly-49D may recognize xenogeneic MHC-encoded ligands.     

Autocrine IL-15 mediates intestinal epithelial cell death via the activation of neighboring intraepithelial NK cells

Intestinal intraepithelial lymphocytes (IELs), which reside between the basolateral faces of intestinal epithelial cells (IECs), provide a first-line defense against pathogens via their cytotoxic activity. Although IEC-derived IL-7 and IL-15 are key regulatory cytokines for the development and activation of IELs, we report here that IL-15 but not IL-7 mediates the reciprocal interaction between IELs and IECs, an important interaction for the regulation of appropriate mucosal immunohomeostasis. IL-15-treated IELs induced cell death in IECs via the cytotoxic activity in vitro. Among the different subsets of IL-15-treated IELs, CD4(-)CD8(-)TCR(-) IELs, which express NK marker (DX5 or NK1.1), showed the most potent syngenic IEC killing activity. These intraepithelial NK cells expressed Ly-49 molecules, NKG2 receptors, and perforin. These results suggest the possibility that the cell death program of IECs could be regulated by self-produced IL-15 through the activation of intraepithelial NK cells.     

Ly-49s3 is a promiscuous activating rat NK cell receptor for nonclassical MHC class I-encoded target ligands

Previous studies of the rapid rejection of MHC-disparate lymphocytes in rats, named allogeneic lymphocyte cytotoxicity, have indicated that rat NK cells express activating receptors for nonclassical MHC class I allodeterminants from the RT1-C/E/M region. Using an expression cloning system that identifies activating receptors associated with the transmembrane adapter molecule DAP12, we have cloned a novel rat Ly-49 receptor that we have termed Ly-49 stimulatory receptor 3 (Ly-49s3). A newly generated anti-Ly-49s3 Ab, mAb DAR13, identified subpopulations of resting and IL-2-activated NK cells, but not T or B lymphocytes. Depletion of Ly-49s3-expressing NK cells drastically reduced alloreactivity in vitro, indicating that this subpopulation is responsible for a major part of the observed NK alloreactivity. DAR13-mediated blockade of Ly-49s3 inhibited killing of MHC-congenic target cells from the av1, n, lv1, and c haplotypes, but not from the u or b haplotypes. A putative ligand was mapped to the nonclassical MHC class I region (RT1-C/E/M) using intra-MHC recombinant strains. Relative numbers of Ly-49s3(+) NK cells were reduced, and surface levels of Ly-49s3 were lower, in MHC congenic strains expressing the putative Ly-49s3 ligand(s). In conclusion, we have identified a novel Ly-49 receptor that triggers rat NK cell-mediated responses.     

Reciprocal transfer of class I MHC allele specificity between activating Ly-49P and Ly-49W receptors by exchange of beta 4-beta 5 loop residues

Receptors of the Ly-49 multigene family regulate rodent NK cell functions. Ly-49Rs are highly polymorphic and exist in either activating or inhibitory forms. Examples of both Ly-49 receptor types have been shown to recognize class I MHC ligands. Ly-49Rs can distinguish between class I alleles, but the molecular basis of this discrimination is unknown. Two activating receptors, Ly-49P and Ly-49W, differ in class I recognition, recognizing H-2D(d), or H-2D(d) and D(k), respectively. In this report, we demonstrate that specificity for H-2D(k) can be transferred from Ly-49W to Ly-49P by substituting 3 aa predicted to reside in the beta4-beta5 loop of Ly-49W into Ly-49P. Replacement of these same residues of Ly-49W with corresponding residues in Ly-49P eliminates H-2D(k) recognition while still preserving H-2D(d) recognition. Further mutagenesis indicates that all 3 aa facilitate optimal class I specificity exchange. These results provide the first evidence for a specific site on Ly-49Rs, the beta4-beta5 loop, in determining class I MHC allele specificity.     

Ly-6G+CCR2- myeloid cells rather than Ly-6ChighCCR2+ monocytes are required for the control of bacterial infection in the central nervous system

Myeloid cell recruitment is a characteristic feature of bacterial meningitis. However, the cellular mechanisms important for the control of Streptococcus pneumoniae infection remain largely undefined. Previous pharmacological or genetic studies broadly depleted many myeloid cell types within the meninges, which did not allow defining the function of specific myeloid subsets. Herein we show that besides CD11b(+)Ly-6G(+)CCR2(-) granulocytes, also CD11b(+)Ly-6C(high)CCR2(+) but not Ly-6C(low)CCR2(-) monocytes were recruited in high numbers to the brain as early as 12 h after bacterial challenge. Surprisingly, CD11b(+)Ly-6C(high)CCR2(+) inflammatory monocytes modulated local CXCL2 and IL-1beta production within the meninges but did not provide protection against bacterial infection. Consistent with these results, CCR2 deficiency strongly impaired monocyte recruitment to the infected brains but was redundant for disease pathogenesis. In contrast, specific depletion of polymorphonuclear granulocytes caused elevated local bacterial titer within the brains, led to an aggravated clinical course, and enhanced mortality. These findings demonstrate that Ly-6C(high)CCR2(+) inflammatory monocytes play a redundant role for the host defense during bacterial meningitis and that predominantly CD11b(+)Ly-6G(+)CCR2(-) myeloid cells are involved in the restriction of the extracellular bacteria.     

Transgenic Ly-49A inhibits antigen-driven T cell activation and delays diabetes

Activation of islet-specific T cells plays a significant role in the development of type 1 diabetes. In an effort to control T cell activation, we expressed the inhibitory receptor, Ly-49A, on islet-specific mouse CD4 cells. Ag-mediated activation of Ly-49A T cells was inhibited in vitro when the Ly-49A ligand, H-2D(d), was present on APCs. Ag-driven T cell proliferation, cytokine production, and changes in surface receptor expression were significantly reduced. Inhibition was also evident during secondary antigenic challenge. Addition of exogenous IL-2 did not rescue cells from inhibition, suggesting that Ly-49A engagement does not lead to T cell anergy. Importantly, in an adoptive transfer model, Ly-49A significantly delays the onset of diabetes. Together these results demonstrate that the inhibitory receptor Ly-49A effectively limits Ag-specific CD4 cell responses even in the presence of sustained autoantigen expression in vivo.