Increased bone marrow allograft rejection by depletion of NK cells expressing inhibitory Ly49 NK receptors for donor class I antigens

Natural killer (NK) cells are the major effectors of acute rejection of incompatible bone marrow cell (BMC) grafts in lethally irradiated mice. The immunogenetics of BMC rejection are largely controlled by the coexpression (or not) of inhibitory and stimulatory Ly49 receptors whose ligands are class I major histocompatibility complex (MHC) molecules. The majority of the BMC rejection studies involved low numbers of BMCs that were resisted by host NK cells. In the present study, larger numbers of BMCs were given in which rejection was not detected and the role of different Ly49 NK subsets not presumably involved in the rejection of a particular BMC haplotype was examined. Surprisingly, the data show that the removal of NK cell subsets expressing Ly49 inhibitory receptors for donor class I antigens, which would be predicted to have no effect on the BMC rejection capability, resulted in the marked rejection of BMCs where no resistance was normally seen. These results extend the "missing self" hypothesis to suggest that NK Ly49 inhibitory receptors can both inhibit activation and killing by those cells, but also can in some way influence the function of NK cells that do not express that inhibitory receptor in a cell-cell interaction. This suggests that caution must be exercised before removal of host NK cell subset is applied clinically because enhanced BMC rejection may result. Altering the balance of Ly49 NK subsets may also affect other in vivo activities of these cells.     

Synergistic effects of in vivo depletion of Ly-49A and Ly-49G2 natural killer cell subsets in the rejection of H2(b) bone marrow cell allografts

Subsets of murine natural killer (NK) cells exist that express the Ly-49 family of molecules that recognize different major histocompatibility complex (MHC) determinants. Bone marrow transplantation studies were performed to examine the in vivo functions of 2 of these subsets. Subsets of Ly-49A and Ly-49G2 NK share specificity for the same MHC class 1 ligand, D(d), binding of which results in an inhibitory signal to the NK cell but allows them to lyse H2(b) targets in vitro. We therefore examined the ability of these subsets to reject H2(b) bone marrow cell allografts in lethally irradiated mice. Surprisingly, depletion of Ly-49A(+) NK cells in BALB/c or B10.D2 mice (both H2(d)) had no effect on the rejection of H2(b) BMC. However, Ly-49A depletion did partially abrogate the ability of B10.BR (H2(k)) mice to reject H2(b) allografts. Although depletion of either Ly-49A(+) or Ly-49G2(+) NK cells alone had no effect on the ability of B10.D2 mice to reject H2(b) BMC, depletion of both subsets dramatically and synergistically abrogated rejection. Studies with various B10 congenic mice and their F(1) hybrids indicate that this synergy between Ly49A and Ly4G2 depletion occurs in every instance. Thus, Ly-49A(+) NK cells appear to play a role in the rejection H2(b) bone marrow allografts, but, in most strains of mice studied, Ly-49G2(+) NK cells must also be eliminated. The putative roles of these NK cell subsets in clinical transplantation remains to be elucidated. (Blood. 2000;95:3840-3844)     

The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells

Natural killer (NK) cells express inhibitory receptors for major histocompatibility complex (MHC) class I. If self-MHC is down-regulated or absent, lack of inhibition triggers "missing self" killing. NK cells developing in the absence of MHC class I are hypo-responsive, demonstrating that MHC class I molecules are required for NK-cell education. Here, we show that the number and the type of MHC class I alleles that are present during NK-cell education quantitatively determine the frequency of responding NK cells, the number of effector functions in individual NK cells, and the amount of interferon-gamma production in NK cells of specific Ly49 subsets. A relationship between the extent of inhibitory signals during education and functional responsiveness was corroborated by an enhanced probability of NK cells expressing more than one inhibitory receptor for a single host self-MHC class I allele to degranulate after activation. Our data suggest that the capacity of an individual NK cell to respond to stimulation is quantitatively controlled by the extent of inhibitory signals that are received from MHC class I molecules during NK-cell education.     

Structural elucidation of the m157 mouse cytomegalovirus ligand for Ly49 natural killer cell receptors

Natural killer (NK) cells express activating and inhibitory receptors that, in concert, survey cells for proper expression of cell surface major histocompatibility complex (MHC) class I molecules. The mouse cytomegalovirus encodes an MHC-like protein, m157, which is the only known viral antigen to date capable of engaging both activating (Ly49H) and inhibitory (Ly49I) NK cell receptors. We have determined the 3D structure of m157 and studied its biochemical and cellular interactions with the Ly49H and Ly49I receptors. m157 has a characteristic MHC-fold, yet possesses several unique structural features not found in other MHC class I-like molecules. m157 does not bind peptides or other small ligands, nor does it associate with beta(2)-microglobulin. Instead, m157 engages in extensive intra- and intermolecular interactions within and between its domains to generate a compact minimal MHC-like molecule. m157's binding affinity for Ly49I (K(d) approximately 0.2 microM) is significantly higher than that of classical inhibitory Ly49-MHC interactions. Analysis of viral escape mutations on m157 that render it resistant to NK killing reveals that it is likely to be recognized by Ly49H in a binding mode that differs from Ly49/MHC-I. In addition, Ly49H+ NK cells can efficiently lyse RMA cells expressing m157, despite the presence of native MHC class I. Collectively, our results show that m157 represents a structurally divergent form of MHC class I-like proteins that directly engage Ly49 receptors with appreciable affinity in a noncanonical fashion.     

Natural killer cell licensing in mice with inducible expression of MHC class I

Mouse natural killer (NK) cells acquire effector function by an education process termed “licensing” mediated by inhibitory Ly49 receptors which recognize self-MHC class I. Ly49 receptors can bind to MHC class I on targets (in trans) and also to MHC class I on the NK-cell surface (in cis). Which of these interactions regulates NK-cell licensing is not yet clear. Moreover, there are no clear phenotypic differences between licensed and unlicensed NK cells, perhaps because of the previously limited ability to study NK cells with synchronized licensing. Here, we produced MHC class I-deficient mice with inducible MHC class I consisting of a single-chain trimer (SCT), ovalbumin peptide-β2 microgloblin-H2K^b (SCT-K^b). Only NK cells with a Ly49 receptor with specificity for SCT-K^b were licensed after MHC class I induction. NK cells were localized consistently in red pulp of the spleen during induced NK-cell licensing, and there were no differences in maturation or activation markers on recently licensed NK cells. Although MHC class I-deficient NK cells were licensed in hosts following SCT-K^b induction, NK cells were not licensed after induced SCT-K^b expression on NK cells themselves in MHC class I-deficient hosts. Furthermore, hematopoietic cells with induced SCT-K^b licensed NK cells more efficiently than stromal cells. These data indicate that trans interaction with MHC class I on hematopoietic cells regulates NK-cell licensing, which is not associated with other obvious phenotypic changes.The ability of natural killer (NK) cells to be activated by their targets is controlled by their activation and inhibitory receptors (1, 2). Activation receptors can recognize ligands expressed on their targets, triggering NK cells if their inhibitory receptors are not engaged. In mice, most Ly49 molecules are inhibitory receptors that recognize MHC class I molecules on target cells (3). Engagement of Ly49 receptors by MHC class I leads to signals that block NK-cell triggering during effector responses. These receptors explain the “missing-self” hypothesis, which postulates that NK cells survey tissues for normal levels of the ubiquitously expressed MHC class I molecule (4). In the absence of MHC class I, inhibition is released, and NK cells then can be activated through their activation receptors to kill and produce cytokines. Thus, the NK-cell inhibitory receptors control NK-cell responses at the effector level.NK cells in MHC class I-deficient mice, such as β₂-microglobulin (β2m)^−/− and TAP^−/− mice, do not receive inhibitory signals and should be overactive (5–7). Instead, however, NK cells from these animals are hyporesponsive to activation receptor cross-linking. Moreover, NK cells apparently lacking known MHC-specific receptors also are hyporesponsive (8). Previously, we reported that the NK-cell inhibitory receptors have a second function that can explain these paradoxical findings (7). Through recognition of self-MHC class I via their inhibitory MHC class I-specific receptors, NK cells become licensed (or educated), resulting in the functional competence of their activation receptors. For example, in C57BL/6 (H2^b) mice, Ly49C⁺ NK cells are licensed by recognition of self-H2K^b and secrete more IFN-γ after NK1.1 stimulation than do Ly49C⁻ NK cells. In contrast, IFN-γ production is comparable in Ly49A⁺ and Ly49A⁻ NK cells, indicating that Ly49A⁺ NK cells are not licensed in C57BL/6 mice because Ly49A does not recognize an H2^b class I allele. On the other hand, Ly49A⁺ NK cells are specifically licensed in mice expressing its MHC class I ligand, H2D^d, as self-MHC. Thus, the recognition of self-MHC class I leads to enhanced function of NK-cell activation receptors, and this enhancement is influenced by self-MHC–specific Ly49 receptors in mice.Other studies suggest that a similar process occurs for human NK cells that preferentially express killer Ig-like receptors (KIRs) for HLA class I recognition and effector inhibition. Although the mouse lectin-like Ly49 receptors and human KIRs are structurally distinct, they share many other features, supporting the concept that each species evolved a different genetic solution for the MHC class I-specific inhibitory receptors; that is, Ly49 receptors and KIRs now are considered to be outstanding examples of convergent evolution (9, 10). As with NK cells bearing self-MHC–specific Ly49 receptors in mice, human NK cells with self-HLA–specific KIRs display enhanced function (11–13). In contrast, NK cells from the same donor but without expression of self-HLA–specific KIRs show decreased function. Moreover, NK cells bearing the same KIR but without its cognate ligand in other donors do not display enhanced function. Thus, licensing (i.e., the education effects of self-MHC class I recognition by relevant NK-cell receptors) is now accepted as being operational in mice and humans (13).Other than expression of self-MHC–specific Ly49 receptors or KIRs and enhanced capacity to signal through their activation receptors, licensed mouse and human NK cells are not clearly distinguishable from unlicensed NK cells (7, 11, 12). However, this apparent similarity could result from a continuous process in which small numbers of NK cells are educated and then join “older” NK cells, and major phenotypic changes during licensing may be transient. Therefore previous phenotypic studies of licensed NK cells may have been limited by the inability to investigate NK cells in which licensing is synchronized.Crystallographic studies revealed two potential interaction sites on MHC class I at which interactions with Ly49 receptors might take place: Site 1 is on the left side of the peptide-binding cleft (when viewed from above with the α1 helix shown on top); site 2 is below the peptide-binding cleft (14). Mutational studies demonstrated that the Ly49 receptors engage site 2 in trans to inhibit effector functions, because point mutations at site 2 abrogated the capacity of transfected MHC class I molecules to inhibit NK cells in an Ly49-dependent manner (15–17). Furthermore, we recently showed that these same residues are involved in conferring Ly49-dependent licensing effects because transgenic (Tg) expression of site 2-mutant MHC class I molecules failed to induce licensed NK cells, whereas wild-type MHC class I alleles allowed licensing (18). Thus, Ly49 receptors interact with the same site on their MHC class I ligands for effector inhibition and licensing, providing another link of these two processes.Because NK cells also express MHC class I molecules, prior studies suggested that Ly49 receptors also can interact with their ligands on the same NK-cell surface, i.e., in cis as well as in trans with their target cell (19). Site 2 on MHC class I molecules is involved in both cis and trans interactions of Ly49 receptors with their MHC class I ligands. Studies of Tg mice expressing a mutated Ly49A receptor that cannot engage in in cis interactions but retains the ability to engage in in trans interactions showed that NK cells expressing mutated Ly49A were not licensed, suggesting that NK cells are licensed via cis interactions (20). However, the possibility that the mutated Ly49A disrupted other unknown signals or interactions required for NK-cell licensing cannot be ruled out completely. Moreover, we and others recently identified the importance of trans interactions in NK-cell licensing because unlicensed splenic β2m^−/− NK cells acquired the licensed phenotype when adoptively transferred to wild-type hosts (21, 22). On the other hand, non–H2D^d-expressing Ly49A⁺ NK cells can acquire H2D^d through contact with H2D^d-expressing cells in an Ly49A-dependent manner in vitro and in vivo (23, 24), suggesting that the transferred NK cells could have acquired host MHC class I, allowing potential interactions with MHC class I in cis as well as in trans. Thus, it remains unclear whether cis or trans interactions are required for NK-cell licensing.Previously, we used mice in which an MHC class I single-chain trimer (SCT) consisting of ovalbumin (OVA) peptide (SIINFEKL), β2m, and H2K^b heavy chain (SCT-K^b) is expressed in an MHC class I-deficient environment (7). The SCT-K^b molecule is specifically and solely recognized by Ly49C when stably expressed as a transgene in C57BL/6 mice, conferring the licensed phenotype to Ly49C⁺ NK cells. Here we were able to monitor Ly49C⁺ NK cells after acute induction of SCT-K^b and found that unlicensed Ly49C⁺ NK cells became licensed without changes in NK-cell maturation or activation markers or localization in spleen. Furthermore, our data showed trans interaction with hematopoietic cells is required for NK-cell licensing, but cis interaction is not.     

Slow receptor acquisition by NK cells regenerated in vivo from transplanted fetal liver or adult bone marrow stem cells

Adult mouse natural killer (NK) cells express two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2, whereas fetal and neonatal NK cells express only CD94/NKG2. After birth, Ly49(+) NK cells slowly increase and CD94/NKG2(+) NK cells decrease. The aim of this study was to determine whether murine NK cells develop differently from transplants of fetal liver and adult marrow stem cells and whether the adult marrow microenvironment is critical for NK receptor maturation. Enriched populations of stem cells were transplanted into adult mice, and the kinetics of NK receptor acquisition was examined. NK cells from osteopetrotic Csf1(op)/Csf1(op) mice, in which hematopoiesis within the marrow is severely limited, were also analyzed.NK cells regenerated from both fetal and adult stem cells initially resembled neonatal NK cells in their slow acquisition of Ly49 over several weeks, although the adult stem cell-derived NK cells matured approximately 10 days sooner. NK cells from adult Csf1(op)/Csf1(op) mice expressed normal levels of Ly49. Maturation of the NK receptor repertoire is a slow process regardless of their stem cell origin or reduced marrow space caused by osteopetrosis.     

Augmentation of antitumor effects by NK cell inhibitory receptor blockade in vitro and in vivo

Subsets of natural killer (NK) cells are characterized by the expression of inhibitory and/or stimulatory receptors specific for major histocompatibility complex (MHC) class I determinants. In mice, these include the Ly49 family of molecules. One mechanism by which tumor cells may evade NK cell killing is by expressing the appropriate MHC class I and binding inhibitory Ly49 receptors. Therefore, the question of whether blocking the interaction between the Ly49 inhibitory receptors on NK and MHC class I cells on tumor cells augments antitumor activity was investigated. Blockade of Ly49C and I inhibitory receptors using F(ab')(2) fragments of the 5E6 monoclonal antibody (mAb) resulted in increased cytotoxicity against syngeneic tumors and decreased tumor cell growth in vitro. The effect of 5E6 F(ab')(2) was specific for the MHC of the tumor, as the use of F(ab')(2) of the mAb against Ly49G2 failed to increase NK activity. Treatment of leukemia-bearing mice with 5E6 F(ab')(2) fragments or adoptive transfer of NK cells treated ex vivo with the F(ab')(2) resulted in significant increases in survival. These results demonstrate that blockade of NK inhibitory receptors enhances antitumor activity both in vitro and in vivo, suggesting that NK inhibitory receptors can be responsible for diminishing antitumor responses. Therefore, strategies to block inhibitory receptors may be of potential use in increasing the efficacy of immunotherapy. (Blood. 2001;97:3132-3137)     

Impaired natural killer cell self-education and "missing-self" responses in Ly49-deficient mice

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.     

Eri1 regulates microRNA homeostasis and mouse lymphocyte development and antiviral function

Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here, we show that mice deficient in Eri1, a conserved 3'-to-5' exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK-cell development and maturation. Eri1(-/-) NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow (BM) and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Eri1 was required for immune-mediated control of mouse CMV (MCMV) infection. Ly49H(+) NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1(-/-) T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1(-/-) T cells. Thus, mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK-cell development and antiviral immunity.     

Ly49 and CD94/NKG2 receptor acquisition by NK cells does not require lymphotoxin-beta receptor expression

A crucial step in murine natural killer (NK) cell development, mediated by bone marrow stromal cells, is the induction of Ly49 and CD94/NKG2 receptor expression. The signals that regulate Ly49 receptor expression are still largely undetermined. It has been shown that interaction between lymphotoxin alpha1beta2 (LTalpha1beta2) and LTbeta receptor (LTbetaR), expressed on lymphoid progenitor cells and nonlymphoid bone marrow stromal cells, respectively, is important for both quantitative and functional NK cell development. Therefore, we have investigated the role of LT-LTbetaR-mediated signaling in Ly49 and CD94/NKG2 receptor acquisition. We show that the NK receptor repertoire of LTbetaR-/- mice can only be partially analyzed because of the residual 129/Ola mouse genetic background, due to a physical linkage of the LTbetaR locus and the loci encoding the Ly49 and CD94/NKG2 receptors. Therefore, we transferred wild-type B6 lymphoid-committed progenitor cells into LTbetaR-/- mice, which differentiated into NK cells with a normal NK cell receptor repertoire. Also, administration of LTbetaR-immunoglobulin (Ig), which acts as a soluble receptor for LTalpha1beta2, resulted in reduced NK cell percentages but did not influence the Ly49 and CD94/NKG2 receptor acquisition on remaining NK cells. These results indicate that LTbetaR-mediated signals are not required for Ly49 and CD94/NKG2 receptor acquisition.     

Implications of CD94 deficiency and monoallelic NKG2A expression for natural killer cell development and repertoire formation

Natural killer (NK) cells are believed to achieve self-tolerance through the expression of self-MHC-specific inhibitory receptors, such as members of the Ly49 and CD94/NKG2 families. Individual Ly49 genes are stochastically expressed by NK subsets and are expressed in a monoallelic fashion, but little is known about the mechanisms underlying CD94/NKG2A expression. We show here that, like Ly49 genes, mouse Nkg2a is stochastically and monoallelically expressed. Thus, a single general mechanism controls expression of all known MHC-specific receptors by mouse NK cells. In addition, we find that DBA/2J mice are naturally CD94-deficient and do not express cell-surface CD94/NKG2A receptors, even on neonatal NK cells. Thus, self-tolerance of neonatal NK cells cannot be attributed to CD94/NKG2A expression. Taken together, the results lead to a reconsideration of current models of NK cell development and self-tolerance.     

NK-cell purging of leukemia: superior antitumor effects of NK cells H2 allogeneic to the tumor and augmentation with inhibitory receptor blockade

Natural killer (NK) cells are composed of subsets characterized by the expression of inhibitory or activating receptors, or both, specific for different major histocompatibility complex (MHC) class I determinants. We have previously shown that inhibitory receptor blockade of syngeneic NK cells was an effective means of ex vivo purging of leukemia-contaminated bone marrow and that the transplantation of mice with the purged bone marrow cells (BMCs) resulted in long-term, relapse-free survival. We have extended the investigation to assess the antitumor effects mediated by NK cells H2-allogeneic to tumor cells. We demonstrate that various tumor cell lines are more susceptible to lysis by H2-allogeneic NK cells than by syngeneic NK cells in vitro even though comparable percentages of Ly49 NK cells were present. Using allogeneic NK cells to purge leukemia-contaminating BMCs before transplantation resulted in a higher proportion of mice with long-term survival than using syngeneic NK cells. Allogeneic NK cells did not suppress hematopoietic reconstitution as measured by granulocyte/monocyte-colony-forming unit (CFU-GM), complete blood count (CBC), and donor chimerism at various days after transplantation. Inhibitory receptor blockade of allogeneic NK cells also significantly increased these antitumor effects at lower NK/tumor ratios compared with those of syngeneic NK cells. These results demonstrate that H2-allogeneic NK cells mediate more potent antitumor effects than syngeneic NK cells without adverse hematologic effects and thus may be useful in cancer therapy.     

Recognition of a virus-encoded ligand by a natural killer cell activation receptor

Natural killer (NK) cells express inhibitory and activation receptors that recognize MHC class I-like molecules on target cells. These receptors may be involved in the critical role of NK cells in controlling initial phases of certain viral infections. Indeed, the Ly49H NK cell activation receptor confers in vivo genetic resistance to murine cytomegalovirus (MCMV) infections, but its ligand was previously unknown. Herein, we use heterologous reporter cells to demonstrate that Ly49H recognizes MCMV-infected cells and a ligand encoded by MCMV itself. Exploiting a bioinformatics approach to the MCMV genome, we find at least 11 ORFs for molecules with previously unrecognized features of predicted MHC-like folds and limited MHC sequence homology. We identify one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-mediated cytotoxicity, and cytokine and chemokine production by freshly isolated NK cells. We hypothesize that the other ORFs with predicted MHC-like folds may be involved in immune evasion or interactions with other NK cell receptors.     

2B4 (CD244)-CD48 interactions provide a novel MHC class I-independent system for NK-cell self-tolerance in mice

Natural killer (NK) cells must be able to eliminate infected and transformed cells while remaining tolerant of normal cells. NK-cell self-tolerance is thought to be maintained by self-major histocompatibility complex (MHC) class I recognition; however, there are examples where NK cells are not regulated by MHC class I and yet remain self-tolerant. Here, we show that 2B4 (CD244) and CD48 represent a second system for murine NK-cell self-recognition. 2B4 and MHC class I receptors act nonredundantly to inhibit NK lysis of syngeneic tumor cells. NK cells from beta2 microglobulin (beta2m)-deficient mice and NK cells that lack expression of self-MHC-binding inhibitory receptors are inhibited by 2B4. Moreover, we provide the first in vivo evidence for MHC-independent NK self-recognition in a bone marrow rejection assay. These data suggest that NK-cell self-tolerance can be mediated by molecules other than MHC.     

Genetic and antibody-mediated reprogramming of natural killer cell missing-self recognition in vivo

Natural killer (NK) cells are lymphocytes of the innate immune system able to recognize and kill tumors lacking self-MHC class I molecules. This “missing-self” recognition is mediated by the lack of engagement of MHC class I-specific inhibitory NK cell receptors that include the killer cell Ig-like receptors (KIR) in humans and Ly49 molecules in mice. A promising immunotherapeutic strategy against MHC class I⁺ cancer cells is to block NK cell inhibitory receptors using monoclonal antibodies (mAb). However, interactions between MHC class I molecules and their inhibitory receptors are also required for the acquisition of NK cell functional competence, a process referred as to “education.” In addition, inhibitory receptors are involved in self-tolerance on educated NK cells. Here, we developed a preclinical mouse model in which all NK cells are educated by a single transgenic inhibitory receptor, human KIR2DL3, through the engagement with its HLA-Cw3 ligand. This approach revealed that NK cells could be reprogrammed to control the development of mouse syngenic tumors in vivo. Moreover, in vivo anti-KIR mAb treatment induced the killing of HLA⁺ target cells without breaking self-tolerance. Finally, the long-term infusion of anti-KIR mAb neither abolished NK cell education nor tumor cell recognition. Therefore, these results strongly support the use of inhibitory receptor blockade in cancer patients.     

Stable masking by H-2Dd cis ligand limits Ly49A relocalization to the site of NK cell/target cell contact

Ly49A is an inhibitory receptor, which counteracts natural killer (NK) cell activation on the engagement with H-2D(d) (D(d)) MHC class I molecules (MHC-I) on target cells. In addition to binding D(d) on apposed membranes, Ly49A interacts with D(d) ligand expressed in the plane of the NK cells' membrane. Indeed, multivalent, soluble MHC-I ligand binds inefficiently to Ly49A unless the NK cells' D(d) complexes are destroyed. However, it is not known whether masked Ly49A remains constitutively associated with cis D(d) also during target cell interaction. Alternatively, it is possible that Ly49A has to be unmasked to significantly interact with its ligand on target cells. These two scenarios suggest distinct roles of Ly49A/D(d) cis interaction for NK cell function. Here, we show that Ly49A contributes to target cell adhesion and efficiently accumulates at synapses with D(d)-expressing target cells when NK cells themselves lack D(d). When NK cells express D(d), Ly49A no longer contributes to adhesion, and ligand-driven recruitment to the cellular contact site is strongly reduced. The destruction of D(d) complexes on NK cells, which unmasks Ly49A, is necessary and sufficient to restore Ly49A adhesive function and recruitment to the synapse. Thus, cis D(d) continuously sequesters a considerable fraction of Ly49A receptors, preventing efficient Ly49A recruitment to the synapse with D(d)+ target cells. The reduced number of Ly49A receptors that can functionally interact with D(d) on target cells explains the modest inhibitory capacity of Ly49A in D(d) NK cells. This property renders Ly49A NK cells more sensitive to react to diseased host cells.     

Ly49Q defines 2 pDC subsets in mice

Plasmacytoid dendritic cells (pDCs) play an important primary role for antiviral innate immunity by rapidly producing large amounts of type 1 interferon (IFN) upon viral infection. To study pDC biology, we generated a monoclonal antibody, termed 2E6, that recognizes pDCs. Molecular cloning of a cDNA encoding the 2E6 antigen revealed that it is a type II C-type lectin, Ly49Q, that consists of 247 amino acids with high homology to the natural killer (NK) receptor family Ly49, with an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic domain. Ly49Q is expressed on pDCs but not on NK cells or myeloid dendritic cells. B220+, CD11c+, CD11b- pDCs in bone marrow were divided into Ly49Q+ and Ly49Q- subsets. While both subsets produced IFN-alpha upon cytosine-phosphate-guanosine (CpG) and herpes simplex virus stimulation, Ly49Q- pDCs responded poorly to influenza virus. In addition, Ly49Q- pDCs produced inflammatory cytokines such as interleukin 6 (IL-6), IL-12, and tumor necrosis factor alpha (TNF-alpha) upon stimulation at lower levels than those produced by Ly49Q+ pDCs. In contrast to bone marrow, Ly49Q+ pDCs were only found in peripheral blood, lymph nodes, and spleen. These results indicate that Ly49Q is a specific marker for peripheral pDCs and that expression of Ly49Q defines 2 subsets of pDCs in bone marrow.     

Reduction of GVHD and enhanced antitumor effects after adoptive infusion of alloreactive Ly49-mismatched NK cells from MHC-matched donors

We investigated if an infusion of alloreactive natural killer (NK) cells would reduce GVHD and mediate antitumor effects in mice undergoing MHC-matched allogeneic stem cell transplantation (SCT). Balb/c mice bearing RENCA tumors underwent an allogeneic SCT from MHC-matched B10.d2 donors and were given a single infusion of either Ly49 ligand-matched, ligand-mismatched, or no donor NK cells. Recipients of Ly49 ligand-mismatched NK cells had a reduced incidence of graft-versus-host disease (GVHD; 39% vs 100%; P < .01), and prolonged survival (median 84 days vs 39 days; P < .01) compared with SCT recipients not receiving NK cells. Recipients of Ly49 ligand-matched NK cells had the same incidence of GVHD and similar survival compared with controls not receiving NK cells. Pulmonary tumor burden was significantly (P < .01) lower in recipients that received Ly49-mismatched or Ly49-matched NK cells compared with recipients not receiving NK cells. These data provide in vivo evidence that a single infusion of alloreactive donor NK cells reduces GVHD and mediates antitumor effects following MHC-matched allogeneic transplantation.     

Ly49E-dependent inhibition of natural killer cells by urokinase plasminogen activator

The Ly49 natural killer (NK)-cell receptor family comprises both activating and inhibitory members, which recognize major histocompatibility complex (MHC) class I or MHC class I-related molecules and are involved in target recognition. As previously shown, the Ly49E receptor fails to bind to a variety of soluble or cell-bound MHC class I molecules, indicating that its ligand is not an MHC class I molecule. Using BWZ.36 reporter cells, we demonstrate triggering of Ly49E by the completely distinct, non-MHC-related protein urokinase plasminogen activator (uPA). uPA is known to be secreted by a variety of cells, including epithelial and hematopoietic cells, and levels are up-regulated during tissue remodeling, infections, and tumorigenesis. Here we show that addition of uPA to Ly49E-positive adult and fetal NK cells inhibits interferon-gamma secretion and reduces their cytotoxic potential, respectively. These uPA-mediated effects are Ly49E-dependent, as they are reversed by addition of anti-Ly49E monoclonal antibody and by down-regulation of Ly49E expression using RNA interference. Our results suggest that uPA, besides its established role in fibrinolysis, tissue remodeling, and tumor metastasis, could be involved in NK cell-mediated immune surveillance and tumor escape.