Human cytomegalovirus, MHC class I and inhibitory signalling receptors: more questions than answers

Summary: The human cytomegalovirus UL18 protein, an MHC class I homologue, has been shown to bind to leucocyte immunoglobulin-like receptor (LIR)-1, a member of a family of nine closely related immunoglobulin superfamily receptors expressed on leucocytes. The LIRs are related to the natural killer (NK)-cell immunoglobulin-like receptors and to several other immunoreceptors. Three groups of LIR molecules have been defined: those containing cytoplasmic domain inhibitory signalling motifs, those with short cytoplasmic domains and a charged residue within the trans-membrane domain, and a secreted molecule. LIR-1 and LIR-2 bind to a broad spectrum of cellular MHC class I antigens, including HLA-A, -B and -C alleles, LIR-2 is expressed by all monocytes and dendritic cells, whereas LIR-1 is additionally expressed by B cells and subsets of T and NK cells. Upon tyrosine phosphorylation, LIR- 1 and LIR-2 associate with the tyrosine phosphatase, SHP-1, and have been shown to inhibit FcγRI signalling when co-crosslinked in monocytes. Evidence for and against a role of UL18 as ail inhibitor of NK-cell function is discussed, as are possible functional outcomes of UL18-LIR-1 interactions in monocytic cells.     

Requirements for control of B‐cell lymphoma by NK cells

The role of NK cells in the control of endogenously arising tumors is still unclear. We monitored activation and effector functions of NK cells in a c‐myc‐transgenic mouse model of spontaneously arising lymphoma. At early stages, tumors demonstrated reduced MHC class I expression and increased expression of natural killer group 2D ligands (NKG2D‐L). NK cells in these tumors showed an activated phenotype that correlated with the loss of tumor MHC class I. With increasing tumor load however, NK‐cell effector functions became progressively paralyzed or exhausted. In later stages of disease, tumors re‐expressed MHC class I and lost NKG2D‐L, suggesting a role of these two signals for NK cell‐mediated tumor control. Testing a panel of lymphoma cell lines expressing various MHC class I and NKG2D‐L levels suggested that NK cell‐dependent tumor control required a priming and a triggering signal that were provided by MHC class I down‐regulation and by NKG2D‐L, respectively. Deleting either of the “two signals” resulted in tumor escape. At early disease stages, immune stimulation through TLR‐ligands in vivo efficiently delayed lymphoma growth in a strictly NK cell‐dependent manner. Thus, NK‐receptor coengagement is crucial for NK‐cell functions in vivo and especially for NK cell‐mediated tumor surveillance.     

Regulation of natural cytotoxicity receptors by heparan sulfate proteoglycans in ‐cis: A lesson from NKp44

NKp44 (NCR2) is a distinct member of natural cytotoxicity receptors (NCRs) family that can induce cytokine production and cytolytic activity in human NK cells. Heparan sulfate proteoglycans (HSPGs) are differentially expressed in various normal and cancerous tissues. HSPGs were reported to serve as ligands/co‐ligands for NKp44 and other NCRs. However, HSPG expression is not restricted to either group and can be found also in NK cells. Our current study reveals that NKp44 function can be modulated through interactions with HSPGs on NK cells themselves in ‐cis rather than on target cells in ‐trans. The intimate interaction of NKp44 and the NK cell‐associated HSPG syndecan‐4 (SDC4) in –cis can directly regulate membrane distribution of NKp44 and constitutively dampens the triggering of the receptor. We further demonstrate, that the disruption of NKp44 and SDC4 interaction releases the receptor to engage with its ligands in –trans and therefore enhances NKp44 activation potential and NK cell functional response.     

Crystal structure of HLA-A2 bound to LIR-1, a host and viral major histocompatibility complex receptor

Leukocyte immunoglobulin-like receptor 1 (LIR-1), an inhibitory receptor expressed on monocytes, dendritic cells and lymphocytes, regulates cellular function by binding a broad range of classical and nonclassical major histocompatibility complex (MHC) class I molecules, and the human cytomegalovirus MHC class I homolog UL18. Here we describe the 3.4-A crystal structure of a complex between the LIR-1 D1D2 domains and the MHC class I molecule HLA-A2. LIR-1 contacts the mostly conserved beta(2)-microglobulin and alpha3 domains of HLA-A2. The LIR-1 binding site comprises residues at the interdomain hinge, and a patch at the D1 tip. The structure shows how LIR-1 recognizes UL18 and diverse MHC class I molecules, and indicates that a similar mode of MHC class I recognition is used by other LIR family members.     

Asymptomatic CMV infections in long‐term renal transplant recipients are associated with the loss of FcRγ from LIR‐1+ NK cells

While it is established that cytomegalovirus (CMV) disease affects NK‐cell profiles, the functional consequences of asymptomatic CMV replication are unclear. Here, we characterize NK cells in clinically stable renal transplant recipients (RTRs; n = 48) >2 years after transplantation. RTRs and age‐matched controls (n = 32) were stratified by their CMV serostatus and the presence of measurable CMV DNA. CMV antibody or CMV DNA influenced expression of NKG2C, LIR‐1, NKp30, NKp46, and FcRγ, a signaling adaptor molecule, on CD56^dim NK cells. Phenotypic changes ascribed to CMV were clearer in RTRs than in control subjects and affected NK‐cell function as assessed by TNF‐α and CD107a expression. The most active NK cells were FcRγ^–LIR‐1⁺NKG2C^– and displayed high antibody‐dependent cell cytotoxicity responses in the presence of immobilized CMV glycoprotein B reactive antibody. However, perforin levels in supernatants from RTRs with active CMV replication were low. Overall we demonstrate that CMV can be reactivated in symptom‐free renal transplant recipients, affecting the phenotypic, and functional profiles of NK cells. Continuous exposure to CMV may maintain and expand NK cells that lack FcRγ but express LIR‐1.     

Negative regulation of rat natural killer cell activity by major histocompatibility complex class I recognition

The cytolytic activity of human and mouse natural killer (NK) cells is negatively regulated by self major histocompatibility complex (MHC) class I molecules on potential target cells. In the rat, protection by RT1 class I gene products has so far not been formally shown although the complex effects of foreign and self RT1 genes on polyclonal NK cell activity suggest that MHC recognition can have both stimulatory and inhibitory effects. Here we report that the expression of self-MHC class I molecules on target cells strongly inhibits lysis by a long term NK cell line derived from LEW (RT1^l) rats and by LEW NK cells activated by short-term culture in the presence of interleukin-2. This was demonstrated with mouse-rat hybridoma target cells expressing different rat MHC alleles and with mouse tumor target cells transfected with classical (RT1.A^l) and nonclassical (RT1.C^l) rat MHC class I genes. With hybridoma target cells, the strongest reduction in lysis as compared to the parental mouse myeloma line was observed when “self” (LEW) MHC was expressed, while hybridomas expressing other MHC alleles showed less and variable reduction. Transfection of RT1.A^l protected both L-929 fibroblasts and P815 mastocytoma cells from lysis by the NK cell line, while RT1.C^l only protected P815 cells, indicating that additional target cell properties regulate rat NK cell activity.     

Expression of the UL16 glycoprotein of Human Cytomegalovirus protects the virus-infected cell from attack by natural killer cells

Background

Human Cytomegalovirus (HCMV) has acquired through evolution a number of genes to try to evade immune recognition of the virus-infected cell. Many of these mechanisms act to inhibit the MHC class I antigen presentation pathway, but any virus-infected cell which has down-regulated cell surface expression of MHC class I proteins, to avoid CTL attack, would be expected to become susceptible to lysis by Natural Killer cells. Surprisingly, however, HCMV infected fibroblasts were found to be resistant to NK cell mediated cytotoxicity. Expression of the UL16 glycoprotein could represent one mechanism to help the virus to escape from NK cell attack, as it has been shown to bind, in vitro, some of the ligands for NKG2D, the NK cell activating receptor. Here, we explored the role of UL16, in the context of a viral infection, by comparing the susceptibility to NK lysis of cells infected with HCMV and cells infected with a UL16 deletion mutant of this virus.

Results

Cells infected with the UL16 knockout virus were killed at substantially higher levels than cells infected with the wild-type virus. This increased killing could be correlated with a UL16-dependent reduction in surface expression of ligands for the NK cell activating receptor NKG2D.

Conclusions

Expression of the UL16 glycoprotein was associated with protection of HCMV-infected cells from NK cell attack. This observation could be correlated with the downregulation of cell surface expression of NKG2D ligands. These data represent a first step towards understanding the mechanism(s) of action of the UL16 protein.

     

Slp‐76 is a critical determinant of NK‐cell mediated recognition of missing‐self targets

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying “missing‐self” recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl‐N‐nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of “missing‐self” targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp‐76—a critical adapter molecule downstream of ITAM‐containing surface receptors. The Slp‐76 Ace mutation behaved as a hypomorphic allele—while no major defects were observed in conventional T‐cell development/function, a marked defect in NK cell mediated elimination of β2‐microglobulin (β2M) deficient target cells was observed. Further studies revealed Slp‐76 to control NK‐cell receptor expression and maturation; however, activation of Slp‐76^ace/ace NK cells through ITAM‐containing NK‐cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK‐β2M^?/? target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp‐76^ace/ace NK cells. Overall these studies establish Slp‐76 as a critical determinant of NK‐cell development and NK cell mediated elimination of missing‐self target cells in mice.     

ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor

The human cytomegalovirus glycoprotein, UL16, binds to two members of a novel family of molecules, the ULBPs, and to the MHC class I homolog, MICB. The ULBPs are GPI-linked glycoproteins belonging to the extended MHC class I family but are only distantly related to MICB. The ULBP and MICB molecules are ligands for the activating receptor, NKG2D/DAP10, and this interaction is blocked by a soluble form of UL16. The ULBPs stimulate cytokine and chemokine production from NK cells, and expression of ULBPs in NK cell-resistant target cells confers susceptibility to NK cell cytotoxicity. Masking of NK cell recognition of ULBP or MIC antigens by UL16 provides a potential mechanism by which human cytomegalovirus-infected cells might evade attack by the immune system.     

Herpes Simplex Virus Infection Downmodulates NKG2D Ligand Expression

Herpes simplex virus (HSV) type 1 infection may cause orofacial infections in humans. The virus resides in a latent form in neural ganglia and occasionally reactivates and infects epithelial cells. Natural killer (NK) cells have been implicated in immune control of herpes virus infections, possibly by downmodulating major histocompatibility complex (MHC) class I and by other, as yet unidentified, mechanisms. Upon HSV‐1 infection of cell lines, surface levels of NKG2D ligands MHC class I related proteins (MIC) A and UL16 binding protein 2 were downmodulated due to late viral gene product(s). As also MHC class I levels were reduced by HSV‐1, NK cell recognition of HeLa cells was not affected by infection. Total cellular MICA contents remained unchanged, suggesting masking, internalization or intracellular retention of MICA as possible mechanisms of viral downregualtion of MICA surface levels. Furthermore, NK cells from patients with active HSV‐1 infection had a tendency towards increased expression level of the activating receptor NKG2D. These data support a role for NKG2D–MICA interactions in immune responses to HSV‐1 reactivation.     

Spontaneous mutations in the human CMV HLA class I homologue UL18 affect its binding to the inhibitory receptor LIR-1/ILT2/CD85j

Human cytomegalovirus (HCMV) down-regulates cell surface expression of HLA class I molecules (HLA-I). UL18, an HCMV-encoded HLA-I homologue, has been proposed to protect virus-infected cells against NK cell recognition by engaging the inhibitory receptor leukocyte Ig-like receptor (LIR)-1, which also binds a broad spectrum of HLA-I alleles, including HLA-G1. Because genetic and biological differences exist among HCMV strains, we characterized laboratory (AD169) and clinical (4636, 13B, 109B) strain-derived UL18 proteins. Compared to the known AD169-derived UL18, mutations were found in clinical strain-derived UL18. They were clustered in the alpha3 domain (13B), previously shown to be critical for LIR-1 binding, or in the alpha1 domain (4636). Iotan cytotoxicity assays, pretreatment of LIR-1+ NKL with soluble 4636-UL18 completely abolished LIR-1-dependent protection from NK lysis, conferred by the expression of HLA-G1 on target cells (721.221-HLA-G1+). Similarly, flow cytometry, Biacore and ELISA experiments showed 4636-UL18 and 13B-UL18 to have the strongest binding capacity to LIR-1. Our results suggest the importance of two independent UL18 regions for LIR-1 binding, one localized on the tip of the alpha3 domain, and another composed of two loops that emerge from the alpha1 domain. Strain variations in these domains may result in different UL18-mediated effects on LIR-1+ cells during the course of HCMV infection.     

Peptide selectivity discriminates NK cells from KIR2DL2‐ and KIR2DL3‐positive individuals

Natural killer cells are controlled by peptide selective inhibitory receptors for MHC class I, including the killer cell immunoglobulin‐like receptors (KIRs). Despite having similar ligands, KIR2DL2 and KIR2DL3 confer different levels of protection to infectious disease. To investigate how changes in peptide repertoire may differentially affect NK cell reactivity, NK cells from KIR2DL2 and KIR2DL3 homozygous donors were tested for activity against different combinations of strong inhibitory (VAPWNSFAL), weak inhibitory (VAPWNSRAL), and antagonist peptide (VAPWNSDAL). KIR2DL3‐positive NK cells were more sensitive to changes in the peptide content of MHC class I than KIR2DL2‐positive NK cells. These differences were observed for the weakly inhibitory peptide VAPWNSRAL in single peptide and double peptide experiments (p < 0.01 and p < 0.03, respectively). More significant differences were observed in experiments using all three peptides (p < 0.0001). Mathematical modeling of the experimental data demonstrated that VAPWNSRAL was dominant over VAPWNSFAL in distinguishing KIR2DL3‐ from KIR2DL2‐positive donors. Donors with different KIR genotypes have different responses to changes in the peptide bound by MHC class I. Differences in the response to the peptide content of MHC class I may be one mechanism underlying the protective effects of different KIR genes against infectious disease.     

Association of NKG2A with treatment for chronic hepatitis C virus infection

Natural killer (NK) cells are critical to the immune response to viral infections. Their functions are controlled by receptors for major histocompatibility complex (MHC) class I, including NKG2A and killer‐cell immunoglobulin‐like receptors (KIR). In order to evaluate the role of MHC class I receptors in the immune response to hepatitis C virus infection we have studied patients with chronic HCV infection by multi‐parameter flow cytometry directly ex vivo. This has permitted evaluation of combinatorial expression of activating and inhibitory receptors on single NK cells. Individuals with chronic HCV infection had fewer CD56^dim NK cells than healthy controls (4·9 ± 3·4% versus 9·0 ± 5·9%, P < 0·05). Expression levels of the inhibitory receptor NKG2A was up‐regulated on NK cells from individuals with chronic hepatitis C virus (HCV) (NKG2A mean fluorescence intensity 5692 ± 2032 versus 4525 ± 1646, P < 0·05). Twelve individuals were treated with pegylated interferon and ribavirin. This resulted in a down‐regulation of NKG2A expression on CD56^dim NK cells. Individuals with a sustained virological response (SVR) had greater numbers of NKG2A‐positive, KIR‐negative NK cells than those without SVR (27·6 ± 9·6% NK cells versus 17·6 ± 5·7, P < 0·02). Our data show that NKG2A expression is dysregulated in chronic HCV infection and that NKG2A‐positive NK cells are associated with a beneficial response to pegylated interferon and ribavirin therapy.     

The ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognize HLA-G1 and HLA-E molecules co-expressed on target cells

Previous studies on NK recognition of HLA-G1 employed as targets 721.221 transfectants (.221-G1) that unknowingly co-expressed the HLA-E molecule, subsequently found to be a major ligand for the CD94/NKG2 receptors. In the present study we re-evaluated the relative role played by CD94/NKG2 and ILT2(LIR1) molecules in recognition of HLA-G1 by NK clones. We employed as targets .221-G1 cells and a surface HLA-E-negative transfectant, .221-G1(E_neg), generated by site-directed mutagenesis of the HLA-G1 leader sequence. The antagonistic effects of receptor- (i.e. CD94/NKG2A, ILT2) and ligand-specific mAb (i.e. HLA-G, HLA-E) were assessed. In addition, binding of an ILT2-Ig fusion protein to the .221-AEH, expressing only HLA-E, and the .221-G1(E_neg) transfectants was analyzed. Our data demonstrate that NK recognition of cells expressing HLA-G1 involves at least two non-overlapping receptor-ligand systems: the CD94/NKG2 interaction with HLA-E, and the engagement of the ILT2(LIR1) receptor by HLA-G1 molecules.     

Manipulating the Properties of Poly(1,4‐Cyclohexylenedimethylene Terephthalate) (PCT) Just by Tuning Steric Configuration of 1.4‐Cyclohexanedimethanol (CHDM)

The properties of poly(1,4‐cyclohexylenedimethylene terephthalate) (PCT) are manipulated only by tuning the steric configuration of 1,4‐cyclohexanedimethanol (CHDM). The chemical structures and compositions of PCTs are confirmed by nuclear magnetic resonance (¹H NMR and ¹³C NMR) before their thermal and mechanical properties are investigated by differential scanning calorimeter, thermogravimetric analyzer, and dynamic mechanical analysis. Results show that the ratio of cis‐ to trans‐CHDM in PCTs has significant influences on their properties. The melting temperature of PCTs is varied from 251 to 313 °C, and the glass transition temperature is increased from 73 to 92 °C when the content of trans‐CHDM in PCTs is increased from 10% to 96%. In addition, the crystallizability of PCT is also increased with the content of trans‐CHDM. However, its thermal stability is decreased seriously when the melting temperature of PCT is higher than 280 °C. It is concluded that when the content of trans‐CHDM is lower than 50% (PCT‐10, PCT‐28, and PCT‐46), the balance of melting temperature, glass transition temperature, and thermal stability for PCT can be achieved. Just by varying the cis/trans ratio of CHDM, rather than the addition of other components, the properties of PCT can be manipulated.     

Poly(p‐phenylene vinylene) Derivatives with Different Contents of cis‐Olefins and their Effect on the Optical Properties

Soluble poly[(2,5‐dihexyloxy‐1,4‐phenylene vinylene)‐alt‐(2,5‐diphenyl‐1,4‐phenylene vinylene)] (DPO‐PPV) with a majority of cis‐vinylenes (>87%) has been synthesized by the Wittig reaction. A series of DPO‐PPV derivatives with different contents of cis‐olefins from approximately 0.05% to 87% was successfully prepared using the photoisomerization technique. On the basis of ¹H NMR, Fourier transform IR (FTIR), ultraviolet (UV), and photoluminescence (PL) spectroscopy, as well as fluorescence quantum efficiency measurements, the effect of cis‐olefins on the optical properties of the polymer in solution and in film was systemically studied. The results indicate that with a cis‐configuration above 75%, the luminescence of DPO‐PPV is decayed due to cis‐trans isomerization with rotational motions of the double bonds; however, the luminescence is dramatically improved on increasing of the trans‐configuration.

     

Fine tuning of natural killer cell specificity and maintenance of self tolerance in MHC class I-deficient mice

TAP1 −/−, β2-microglobulin (β2m) −/− and TAP1/β2m −/− mice all express low but quantitatively different levels of MHC class I molecules. Using these mice, we have addressed questions relating to the fine tuning of natural killer (NK) cell specificity and maintenance of self tolerance in the NK cell system. NK cells from B6 wild-type mice killed target cells from TAP1 −/−, β2m −/− and TAP1/β2m −/− mice in vivo and rejected bone marrow grafts from the same mice in vivo at equivalent levels. NK cells from TAP1 −/−, β2m −/− mice did not kill target cells or reject bone marrow grafts from TAP1/β2m −/− mice. NK cells in all MHC class I-deficient mice were tolerant to autologous MHC class I-deficient cells, as revealed by in vitro cytotoxicity assays using NK cell effectors activated with the interferon-inducing agent Tilorone, or by in vivo bone marrow graft experiments. However, the self-tolerant state of MHC class I-deficient NK cells was broken by in vitro stimulation with IL-2 for 4 days. Under these conditions, NK cells from the MHC class I-deficient mice killed autologous MHC class I-deficient cells while MHC class I-positive targets were spared. The C-type lectin inhibitory receptor Ly49C has a specificity for H-2K^b and is expressed on a subset of NK1.1⁺ cells in B6 mice. Wild-type and all MHC class I-deficient mice had similar numbers of Ly49C-positive NK1.1⁺ cells. However, Ly49C expression was markedly down-regulated on NK1.1⁺ cells from B6 mice, as compared to TAP1 −/−, β2m −/− and TAP1/β2m −/− mice. In vitro stimulation of NK cells with IL-2 for 4 days did not significantly change this pattern. The present results are discussed in relation to the role of MHC class I molecules and Ly49 receptors in shaping the NK cell repertoire and raise new questions about maintenance of self tolerance in the NK cell system.     

Biology of T memory type 1 cells

Summary: Engagement of inhibitory natural killer (NK) cell receptors for MHC class I molecules (NKR) can impair NK‐cell activation programs. Inhibitory NKR thus confer to NK cells the capacity to discriminate between MHC class I⁺ and MHC class I^? target cells, and are therefore involved in the control of NK‐cell tolerance to self, as well as in the elimination of MHC class I^? distressed cells by NK cells. In human and mouse, a subset of αβ T cells also express inhibitory NKR at their surface, but the biological function of inhibitory NKR on T cells remains to be precisely elucidated. We refer to these cells as T memory type 1 (Tm1) cells, and review here the phenotypic and functional features of this subset of memory‐phenotype CD8⁺αβ T cells. In vitro studies suggest that inhibitory NKR are involved in the peripheral control of T‐cell self‐tolerance. In vitro and in vivo analysis have revealed a novel biological function for inhibitory NKR when expressed on T cells. Indeed, engagement of inhibitory NKR on T cells provides them with survival signals against activation‐induced cell death. Thus, sensing of self‐MHC class I molecules by inhibitory NKR displayed on αβ T cells leads to the in vivo accumulation of Tm1 cells. The authors thank C. Arpin, J. Marvel (INSERM 503, CERVI, Lyon), C. Melief, R. Toes (Leiden University Medical Center, The Netherlands), D. Raulet (Berkeley, USA) and N. Glaichenhaus (IPMC, Nice) for helpful discussions and help, as well as F. Romagné (Immunotech) for the generous gift of MHC class I tetramers. This work was supported by institutional grants from INSERM, CNRS, Ministère de l’Enseignement Supérieur et de la Recherche, and specific grants from Ligue Nationale contre le Cancer (E. V., Equipe labellisée La Ligue; S. U.), from Association pour la Recherche contre le Cancer (E.V.), and from SIDACTION (S.U.).