Imprint of Initial Education and Loss of Ly49C/I in Activated Natural Killer Cells of TAP1-KO and C57BL/6 Wildtype Mice.

Natural killer (NK) cells are important effectors of the innate immune system and participate in the first line of defense against infections and tumors. Prior to being functional, these lymphocytes must be educated or licensed through interactions of their major histocompatibility complex class I molecules with self-specific inhibitory receptors that recognize them. In the absence of such contacts, caused by either the lack of expression of the inhibitory receptors or a very low level of major histocompatibility complex class I (MHC class I) proteins, NK cells are hypo-reactive at baseline (ex vivo). After stimulation (assessed through plate-bound antibodies against activating receptors or culture in the presence of cytokines such as interleukin (IL)-2 or IL-15) however, they can become cytotoxic and produce cytokines. This is particularly the case in transporter associated with antigen processing (TAP)-deficient mice, which we investigated in the present study. Transporter associated with antigen processing transports endogenous peptides from the cytosol to the endoplasmic reticulum, where they are loaded on nascent MHC class I molecules, which then become stable and expressed at the cell surface. Consequently, TAP-KO mice have very low levels of MHC class I expression. We present a study about phenotypic and functional aspects of NK cells in two mouse strains, C57BL/6 wildtype and TAP1-KO in spleen and lung. We observed that in both types of mice, on the same genetic background, the initial pattern of education, conferred to the cells via the inhibitory receptors Ly49C/I and NKG2A, was maintained even after a strong stimulation by the cytokines interleukin-2, interleukin-12, interleukin-15 and interleukin-18. Furthermore, the percentages of activated NK cells expressing Ly49C/I and Ly49I were strongly down-modulated under these conditions. We completed our investigations with phenotypic studies of NK cells from these mice.

Control of Viral Infection by Natural Killer Cell Inhibitory Receptors.

Major histocompatibility complex class I (MHC-I)-restricted immune responses are largely attributed to cytotoxic T lymphocytes (CTLs). However, natural killer (NK) cells, as predicted by the missing-self hypothesis, have opposing requirements for MHC-I, suggesting that they may also demonstrate MHC-I-restricted effects. In mice, the Ly49 inhibitory receptors prevent NK cell killing of missing-self targets in effector responses, and they have a proposed second function in licensing or educating NK cells via self-MHC-I in vivo. Here we show MHC-I-restricted control of murine cytomegalovirus (MCMV) infection in vivo that is NK cell dependent. Using mice lacking specific Ly49 receptors, we show that control of MCMV requires inhibitory Ly49 receptors and an inhibitory signaling motif and the capacity for MCMV to downregulate MHC-I. Taken together, these data provide definitive evidence that the inhibitory receptors are required for missing-self rejection and are relevant to MHC-I-restricted NK cell control of a viral infection in vivo.

Two Lineages of KLRA with Contrasting Transcription Patterns Have Been Conserved at a Single Locus during Ruminant Speciation.

Cattle possess the most diverse repertoire of NK cell receptor genes among all mammals studied to date. Killer cell receptor genes encoded within the NK complex and killer cell Ig-like receptor genes encoded within the leukocyte receptor complex have both been expanded and diversified. Our previous studies identified two divergent and polymorphic KLRA alleles within the NK complex in the Holstein-Friesian breed of dairy cattle. By examining a much larger cohort and other ruminant species, we demonstrate the emergence and fixation of two KLRA allele lineages (KLRA*01 and -*02) at a single locus during ruminant speciation. Subsequent recombination events between these allele lineages have increased the frequency of KLRA*02 extracellular domains. KLRA*01 and KLRA*02 transcription levels contrasted in response to cytokine stimulation, whereas homozygous animals consistently transcribed higher levels of KLRA, regardless of the allele lineage. KLRA*02 mRNA levels were also generally higher than KLRA*01 Collectively, these data point toward alternative functional roles governed by KLRA genotype and allele lineage. On a background of high genetic diversity of NK cell receptor genes, this KLRA allele fixation points to fundamental and potentially differential function roles.

Synergized regulation of NK cell education by NKG2A and specific Ly49 family members.

Mice lacking MHC class-I (MHC-I) display severe defects in natural killer (NK) cell functional maturation, a process designated as "education". Whether self-MHC-I specific Ly49 family receptors and NKG2A, which are closely linked within the NK gene complex (NKC) locus, are essential for NK cell education is still unclear. Here we show, using CRISPR/Cas9-mediated gene deletion, that mice lacking all members of the Ly49 family exhibit a moderate defect in NK cell activity, while mice lacking only two inhibitory Ly49 members, Ly49C and Ly49I, have comparable phenotypes. Furthermore, the deficiency of NKG2A, which recognizes non-classical MHC-Ib molecules, mildly impairs NK cell function. Notably, the combined deletion of NKG2A and the Ly49 family severely compromises the ability of NK cells to mediate "missing-self" and "induced-self" recognition. Therefore, our data provide genetic evidence supporting that NKG2A and the inhibitory members of Ly49 family receptors synergize to regulate NK cell education.

Chronic Ly49H Receptor Engagement in vivo Decreases NK Cell Response to Stimulation Through ITAM-Dependent and Independent Pathways Both in vitro and in vivo.

Natural killer (NK) cells play an important role in the innate immune response. The summation of activation and inhibitory signals delivered through cell surface membrane receptors determines NK cell function. However, the continuous engagement of an activating receptor on NK cells appears to render the cells hyporesponsive to stimulation through other unrelated activating receptors. The mechanism by which this takes place remains unclear. Herein we demonstrate that continuous in vivo engagement of the Ly49H receptor with its ligand, m157, results in Ly49H+ NK cells that are hyporesponsive to further stimulation by other ITAM-dependent and independent receptors, while Ly49H- NK cells remain unaffected. The hyporesponsiveness of the NK cell correlates with the degree of Ly49H receptor downmodulation on its cell surface. We observe defects in calcium flux in the hyporesponsive NK cells following stimulation through the NK1.1 receptor. In addition, we observe differences in signaling molecules that play a role in calcium flux, including spleen tyrosine kinase (Syk) at baseline and phosphorylated phospholipase C gamma 2 (p-PLCγ2) at both baseline and following stimulation through NK1.1. We also demonstrate that various ITAM associated activation receptors, including Ly49H, remain associated with their respective adaptor molecules. With regard to in vivo NK cell function, we did not find differences in the formation of metastatic lung lesions following IV injection of B16 melanoma cells. However, we did observe defects in rejection of missing-self targets in vivo. The data suggest that continuous engagement of the Ly49H activating receptor on NK cells results in hyporesponsiveness of the NK cells to all of the ITAM-dependent and independent receptors we analyzed due to altered signaling pathways downstream of the receptor and adaptor molecule.

Activation Receptor-Dependent IFN-γ Production by NK Cells Is Controlled by Transcription, Translation, and the Proteasome.

NK cells can recognize target cells such as virus-infected and tumor cells through integration of activation and inhibitory receptors. Recognition by NK cells can lead to direct lysis of the target cell and production of the signature cytokine IFN-γ. However, it is unclear whether stimulation through activation receptors alone is sufficient for IFN-γ production. In this study, we show that NK activation receptor engagement requires additional signals for optimal IFN-γ production, which could be provided by IFN-ß or IL-12. Stimulation of murine NK cells with soluble Abs directed against NK1.1, Ly49H, Ly49D, or NKp46 required additional stimulation with cytokines, indicating that a range of activation receptors with distinct adaptor molecules require additional stimulation for IFN-γ production. The requirement for multiple signals extends to stimulation with primary m157-transgenic target cells, which triggers the activation receptor Ly49H, suggesting that NK cells do require multiple signals for IFN-γ production in the context of target cell recognition. Using quantitative PCR and RNA flow cytometry, we found that cytokines, not activating ligands, act on NK cells to express Ifng transcripts. Ly49H engagement is required for IFN-γ translational initiation. Results using inhibitors suggest that the proteasome-ubiquitin-IKK-TPL2-MNK1 axis was required during activation receptor engagement. Thus, this study indicates that activation receptor-dependent IFN-γ production is regulated on the transcriptional and translational levels.

Modulation of NKG2D, NKp46, and Ly49C/I facilitates natural killer cell-mediated control of lung cancer.

Natural killer (NK) cells play a critical role in controlling malignancies. Susceptibility or resistance to lung cancer, for example, specifically depends on NK cell function. Nevertheless, intrinsic factors that control NK cell-mediated clearance of lung cancer are unknown. Here we report that NK cells exposed to exogenous major histocompatibility class I (MHCI) provide a significant immunologic barrier to the growth and progression of malignancy. Clearance of lung cancer is facilitated by up-regulation of NKG2D, NKp46, and other activating receptors upon exposure to environmental MHCI. Surface expression of the inhibitory receptor Ly49C/I, on the other hand, is down-regulated upon exposure to tumor-bearing tissue. We thus demonstrate that NK cells exhibit dynamic plasticity in surface expression of both activating and inhibitory receptors based on the environmental context. Our data suggest that altering the activation state of NK cells may contribute to immunologic control of lung and possibly other cancers.

Critical role for the Ly49 family of class I MHC receptors in adaptive natural killer cell responses.

Adaptive natural killer (NK) cell memory represents a new frontier in immunology. Work over the last decade has discovered and confirmed the existence of NK cells with antigen-specific memories, which had previously been considered a unique property of T and B cells. These findings have shown that antigen-specific NK cells gain their specificity without the use of RAG proteins, representing a novel mechanism for generating antigen specificity, but the details of this mechanism have remained a mystery. We have discovered that members of the Ly49 family of surface receptors are critically involved in both the sensitization and the challenge phases of an NK cell memory response, as is antigen presentation from their binding partner, the class I MHC. Moreover, we demonstrate that the Ly49-interacting component of a presented antigen dictates the specificity of the NK cell memory response, implicating Ly49 receptors themselves in antigen-specific recognition. Finally, we demonstrate that adaptive NK cell memories can protect against an otherwise lethal melanoma without T cell or B cell support. These findings offer insight into the mechanism behind NK cell antigen specificity and demonstrate the clinical potential of this adaptive immune cell.

Identification of MHC Class Ib Ligands for Stimulatory and Inhibitory Ly49 Receptors and Induction of Potent NK Cell Alloresponses in Rats.

Early studies indicate that rats may have a repertoire of MHC class Ib-reactive Ly49 stimulatory receptors capable of mounting memory-like NK cell alloresponses. In this article, we provide molecular and functional evidence for this assumption. Pairs of Ly49 receptors with sequence similarities in the lectin-like domains, but with opposing signaling functions, showed specificity for ligands with class Ia-like structural features encoded from the first telomeric MHC class Ib gene cluster, RT1-CE, which is syntenic with the H2-D/H2-L/H2-Q cluster in mice. The activating Ly49s4 receptor and its inhibitory counterparts, Ly49i4 and Ly49i3, reacted with all allelic variants of RT1-U, whereas Ly49s5 and Ly49i5 were specific for RT1-Eu NK cell cytolytic responses were predictably activated and inhibited, and potent in vivo NK alloresponses were induced by repeated MHC class Ib alloimmunizations. Additional Ly49-class Ib interactions, including RT1-Cl with the Ly49s4/Ly49i4/Ly49i3 group of receptors, were characterized using overexpressed receptor/ligand pairs, in vitro functional assays, and limited mutational analyses. Obvious, as well as subtle, Ly49-class Ib interactions led to ligand-induced receptor calibration and NK subset expansions in vivo. Together, these studies suggest that in vivo NK alloresponses are controlled by pleomorphic Ly49-class Ib interactions, some of which may not be easily detectable in vitro.

Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A.

Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In contrast, the movement of Ly49A was more constrained in educated NK cells compared to hyporesponsive NK cells. Either disrupting the actin cytoskeleton or adding cholesterol to the cells prohibited activating signaling, suggesting that the dynamics of receptor movements within the cell membrane are critical for the proper activation of NK cells. The faster and more dynamic movement of NKp46 in educated NK cells may facilitate a swifter response to interactions with target cells.

Natural killer cells attenuate cytomegalovirus-induced hearing loss in mice.

Congenital cytomegalovirus (CMV) infection is the most common non-hereditary cause of sensorineural hearing loss (SNHL) yet the mechanisms of hearing loss remain obscure. Natural Killer (NK) cells play a critical role in regulating murine CMV infection via NK cell recognition of the Ly49H cell surface receptor of the viral-encoded m157 ligand expressed at the infected cell surface. This Ly49H NK receptor/m157 ligand interaction has been found to mediate host resistance to CMV in the spleen, and lung, but is much less effective in the liver, so it is not known if this interaction is important in the context of SNHL. Using a murine model for CMV-induced labyrinthitis, we have demonstrated that the Ly49H/m157 interaction mediates host resistance in the temporal bone. BALB/c mice, which lack functional Ly49H, inoculated with mCMV at post-natal day 3 developed profound hearing loss and significant outer hair cell loss by 28 days of life. In contrast, C57BL/6 mice, competent for the Ly49H/m157 interaction, had minimal hearing loss and attenuated outer hair cell loss with the same mCMV dose. Administration of Ly49H blocking antibody or inoculation with a mCMV viral strain deleted for the m157 gene rendered the previously resistant C57BL/6 mouse strain susceptible to hearing loss to a similar extent as the BALB/c mouse strain indicating a direct role of the Ly49H/m157 interaction in mCMV-dependent hearing loss. Additionally, NK cell recruitment to sites of infection was evident in the temporal bone of inoculated susceptible mouse strains. These results demonstrate participation of NK cells in protection from CMV-induced labyrinthitis and SNHL in mice.

Kinetic and thermodynamic studies of the interaction between activating and inhibitory Ly49 natural killer receptors and MHC class I molecules.

Natural killer (NK) cells are lymphocytes of the innate immune system that eliminate virally infected or malignantly transformed cells. NK cell function is regulated by diverse surface receptors that are both activating and inhibitory. Among them, the homodimeric Ly49 receptors control NK cell cytotoxicity by sensing major histocompatibility complex class I molecules (MHC-I) on target cells. Although crystal structures have been reported for Ly49/MHC-I complexes, the underlying binding mechanism has not been elucidated. Accordingly, we carried out thermodynamic and kinetic experiments on the interaction of four NK Ly49 receptors (Ly49G, Ly49H, Ly49I and Ly49P) with two MHC-I ligands (H-2Dd and H-2Dk). These Ly49s embrace the structural and functional diversity of the highly polymorphic Ly49 family. Combining surface plasmon resonance, fluorescence anisotropy and far-UV circular dichroism (CD), we determined that the best model to describe both inhibitory and activating Ly49/MHC-I interactions is one in which the two MHC-I binding sites of the Ly49 homodimer present similar binding constants for the two sites (∼106 M-1) with a slightly positive co-operativity in some cases, and without far-UV CD observable conformational changes. Furthermore, Ly49/MHC-I interactions are diffusion-controlled and enthalpy-driven. These features stand in marked contrast with the activation-controlled and entropy-driven interaction of Ly49s with the viral immunoevasin m157, which is characterized by strong positive co-operativity and conformational selection. These differences are explained by the distinct structures of Ly49/MHC-I and Ly49/m157 complexes. Moreover, they reflect the opposing roles of NK cells to rapidly scan for virally infected cells and of viruses to escape detection using immunoevasins such as m157.

Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C.

Murine natural killer (NK) cells are regulated by the interaction of Ly49 receptors with major histocompatibility complex class I molecules (MHC-I). Although the ligands for inhibitory Ly49 were considered to be restricted to classical MHC (MHC-Ia), we have shown that the non-classical MHC molecule (MHC-Ib) H2-M3 was a ligand for the inhibitory Ly49A. Here we establish that another MHC-Ib, H2-Q10, is a bona fide ligand for the inhibitory Ly49C receptor. H2-Q10 bound to Ly49C with a marginally lower affinity (∼5 µm) than that observed between Ly49C and MHC-Ia (H-2K(b)/H-2D(d), both ∼1 µm), and this recognition could be prevented by cis interactions with H-2K in situ To understand the molecular details underpinning Ly49·MHC-Ib recognition, we determined the crystal structures of H2-Q10 and Ly49C bound H2-Q10. Unliganded H2-Q10 adopted a classical MHC-I fold and possessed a peptide-binding groove that exhibited features similar to those found in MHC-Ia, explaining the diverse peptide binding repertoire of H2-Q10. Ly49C bound to H2-Q10 underneath the peptide binding platform to a region that encompassed residues from the α1, α2, and α3 domains, as well as the associated ß2-microglobulin subunit. This docking mode was conserved with that previously observed for Ly49C·H-2K(b) Indeed, structure-guided mutation of Ly49C indicated that Ly49C·H2-Q10 and Ly49C·H-2K(b) possess similar energetic footprints focused around residues located within the Ly49C ß4-stand and L5 loop, which contact the underside of the peptide-binding platform floor. Our data provide a structural basis for Ly49·MHC-Ib recognition and demonstrate that MHC-Ib represent an extended family of ligands for Ly49 molecules.

Expression of the inhibitory Ly49E receptor is not critically involved in the immune response against cutaneous, pulmonary or liver tumours.

Natural killer (NK) lymphocytes are part of the innate immune system and are important in immune protection against tumourigenesis. NK cells display a broad repertoire of activating and inhibitory cell surface receptors that regulate NK cell activity. The Ly49 family of NK receptors is composed of several members that recognize major histocompatibility complex class I (MHC-I) or MHC-I-related molecules. Ly49E is a unique inhibitory member, being triggered by the non-MHC-I-related protein urokinase plasminogen activator (uPA) in contrast to the known MHC-I-triggering of the other inhibitory Ly49 receptors. Ly49E also has an uncommon expression pattern on NK cells, including high expression on liver DX5(-) NK cells. Furthermore, Ly49E is the only Ly49 member expressed by epidermal γδ T cells. As γδ T cells and/or NK cells have been shown to be involved in the regulation of cutaneous, pulmonary and liver malignancies, and as uPA is involved in tumourigenesis, we investigated the role of the inhibitory Ly49E receptor in the anti-tumour immune response. We demonstrate that, although Ly49E is highly expressed on epidermal γδ T cells and liver NK cells, this receptor does not play a major role in the control of skin tumour formation or in lung and liver tumour development.

Ly49 receptors: evolution, genetic diversity, and impact on immunity.

Natural killer (NK) cells express cell surface receptors that recognize class I major histocompatibility complex (MHC-I) molecules to distinguish between healthy and unhealthy cells. The multigenic and polymorphic nature of the MHC-I genes has influenced the convergent evolution of similarly polymorphic and diversified NK cell receptor families: the C-type lectin-like Ly49 receptors in mice, and the killer cell immunoglobulin-like receptors (KIRs) in humans. Although structurally distinct, both receptor families have similar functions in terms of MHC-I recognition and downstream signal transduction, and they regulate multiple aspects of NK cell biology during development and after maturation as fully differentiated and functionally competent cells. The Ly49 gene locus has undergone rapid, lineage-specific expansions and contractions resulting in multiple distinct haplotypes of variable gene number, allelic diversity, and MHC-I ligand specificity. This in turn has influenced the type and degree of Ly49 receptor expression on NK cells, and their contribution to immunity in different mouse strains. In this review, we have attempted to describe the evolutionary processes that have shaped strain-specific Ly49 receptor repertoires, and their impact on NK cell functions during health and disease.

Selection, tuning, and adaptation in mouse NK cell education.

Natural killer (NK) cells recognize transformed cells with an array of germline-encoded inhibitory and activating receptors. Inhibitory Ly49 receptors bind major histocompatibility complex class I (MHC-I) molecules, providing a mechanism by which NK cells maintain self-tolerance yet eliminate cells expressing reduced levels of MHC-I. Additionally, MHC-I molecules are required for NK cell education, a process in which NK cells acquire responsiveness. In this review, we discuss three facets of MHC class I-dependent education of mouse NK cells: skewing of the inhibitory receptor repertoire, induction of functional responsiveness, and tuning in response to changes in MHC-I expression. We discuss prevailing models for education such as licensing and disarming and propose a model for positive selection of 'useful' NK cell subsets. Furthermore, we argue that both repertoire skewing and functional NK cell education may be altered in mature NK cells subject to changes in MHC-I input and suggest that this process may provide increased dynamics to the NK cell system.

SHIP1 intrinsically regulates NK cell signaling and education, resulting in tolerance of an MHC class I-mismatched bone marrow graft in mice.

NK cells are an important component of host immune defense against malignancy and infection. NK cells are educated by MHC class I ligands to ensure self-tolerance while also promoting lytic competency against altered self and damaged self targets. However, the intracellular molecular events that culminate in tolerance and functional competency of educated NK cells remain undefined. Mice with germline deficiency in SHIP1 were shown to have a defective NK cell compartment. However, SHIP1 is expressed in all hematopoietic lineages, and consequently several hematolymphoid phenotypes have already been identified in certain cell types that are the result of SHIP1 deficiency in cells in separate and distinct lineages, that is, cell-extrinsic phenotypes. Thus, it was previously impossible to determine the NK cell-intrinsic role of SHIP1. In the present study, through the creation of an NK cell-specific deletion mouse model of SHIP1, we show that SHIP1 plays a profound NK lineage-intrinsic role in NK cell homeostasis, development, education, and cytokine production. Moreover, we show SHIP1 expression by NK cells is required for in vivo-mismatched bone marrow allograft rejection as well as for NK memory responses to hapten.

Murine cytomegalovirus strains co-replicate at multiple tissue sites and establish co-persistence in salivary glands in the absence of Ly49H-mediated competition.

Infection with multiple genetically distinct strains of pathogen is common and can lead to positive (complementation) or negative (competitive) within-host interactions. These interactions can alter aspects of the disease process and help shape pathogen evolution. Infection of the host with multiple strains of cytomegalovirus (CMV) occurs frequently in humans and mice. Profound, NK-cell-mediated (apparent) competition has been identified in C57BL/6 mice, and prevented the replication and shedding of certain co-infecting CMV strains. However, the frequency of such strong competition has not been established. Other within-host interactions such as complementation or alternative forms of competition remain possible. Moreover, high rates of recombination in both human CMV and murine CMV (MCMV) suggest prolonged periods of viral co-replication, rather than strong competitive suppression. An established model was employed to investigate the different possible outcomes of multi-strain infection in other mouse strains. In this study, co-replication of up to four strains of MCMV in the spleen, liver and salivary glands was observed in both MCMV-susceptible and MCMV-resistant mice. In the absence of apparent competition, no other forms of competition were unmasked. In addition, no evidence of complementation between viral strains was observed. Importantly, co-replication of MCMV strains was apparent for up to 90 days in the salivary glands. These data indicated that competition was not the default outcome of multi-strain CMV infection. Prolonged, essentially neutral, co-replication may be the norm, allowing for multi-strain transmission and prolonged opportunities for recombination.

Acquisition of activation receptor ligand by trogocytosis renders NK cells hyporesponsive.

Because NK cells secrete cytotoxic granules and cytokines that can destroy surrounding cells and help shape the subsequent immune response, they must be kept under tight control. Several mechanisms, at different levels, are in place to control NK cell function. In this study, we describe a novel mechanism regulating NK cell function in which NK cells acquire ligands for activating receptors from target cells by trogocytosis, rendering the NK cells hyporesponsive. In this model, murine NK cells acquire m157, the murine CMV-encoded ligand for the Ly49H-activating receptor, from target cells both in vitro and in vivo. Although acquisition of m157 requires cell-to-cell contact, it does not require the expression of the Ly49H receptor by the NK cell. Acquired m157 protein is expressed on the NK cell surface with a glycosylphosphatidylinisotol linkage and interacts with the Ly49H receptor expressed on the NK cell. This interaction results in blocking the Ly49H receptor that prevents the NK cells from recognizing m157-expressing targets and continuous engagement of the Ly49H-activating receptor, which results in the hyporesponsiveness of the Ly49H(+) NK cell to stimulation through other activating receptors. Thus, NK cell acquisition of a ligand for an activation receptor by trogocytosis renders them hyporesponsive. This mechanism, by which mature NK cell function can be altered, has important implications in regard to how NK cells respond to tumors in specific microenvironments as well as the use of expanded NK cells in treating various malignancies.