Effects on tumor development and metastatic dissemination by the NKG2D lymphocyte receptor expressed on cancer cells.

The stimulatory NKG2D lymphocyte receptor together with its tumor-associated ligands enable the immune system to recognize and destroy cancer cells. However, with dynamic changes unfolding, cancers exploit NKG2D and its ligands for immune evasion and suppression. Recent findings have added yet another functional dimension, wherein cancer cells themselves co-opt NKG2D for their own benefit to complement the presence of its ligands for self-stimulation of parameters of tumorigenesis. Those findings are here extended to in vivo tumorigenicity testing by employing orthotopic xenotransplant breast cancer models in mice. Using human cancer lines with ectopic NKG2D expression and RNA interference (RNAi)-mediated protein depletion among other controls, we show that NKG2D self-stimulation has tumor-promoting capacity. NKG2D signals had no notable effects on cancer cell proliferation and survival but acted at the level of angiogenesis, thus promoting tumor growth, tumor cell intravasation and dissemination. NKG2D-mediated effects on tumor initiation may represent another factor in the observed overall enhancement of tumor development. Altogether, these results may have an impact on immunotherapy approaches, which currently do not account for such NKG2D effects in cancer patients and thus could be misdirected as underlying assumptions are incomplete.

Immunobiology of human NKG2D and its ligands.

The NKG2D-DAP10 receptor complex activates natural killer (NK) cells and costimulates effector T cell subsets upon engagement of ligands that can be conditionally expressed under physiologically harmful conditions such as microbial infections and malignancies. These characteristics have given rise to the widely embraced concept of immunorecognition of "induced or damaged self," complementing the "missing self" paradigm that is represented by MHC class I allotypes and their interactions with inhibitory receptors on NK cells. However, this notion may only be partially sustainable, as various patterns of constitutive tissue distributions have become apparent among members of one NKG2D ligand family. This review summarizes the biological properties of NKG2D and its ligands and discusses the interactions and regulation of these molecules with emphasis of their significance in microbial infections, tumor immunology, and autoimmune disease.

NKG2D receptors induced by IL-15 costimulate CD28-negative effector CTL in the tissue microenvironment.

Unlike primary T cells in lymph nodes, effector CD8(+) CTL in tissues do not express the costimulatory receptor CD28. We report that NKG2D, the receptor for stress-induced MICA and MICB molecules expressed in the intestine, serves as a potent costimulatory receptor for CTL freshly isolated from the human intestinal epithelium. Expression and function of NKG2D are selectively up-regulated by the cytokine IL-15, which is released by the inflamed intestinal epithelium. These findings identify a novel CTL costimulatory pathway regulated by IL-15 and suggest that tissues can fine-tune the activation of effector T cells based on the presence or absence of stress and inflammation. Uncontrolled secretion of IL-15 could lead to excessive induction of NKG2D and thus contribute to the development of autoimmune disease by facilitating the activation of autoreactive T cells.

MICA engagement by human Vgamma2Vdelta2 T cells enhances their antigen-dependent effector function.

Vgamma2Vdelta2 T cells comprise 2%-5% of human peripheral blood T cells, recognize ubiquitous nonpeptide antigens, and expand up to 50-fold during microbial infection. It is not clear why these Vgamma2Vdelta2 T cells expand only after microbial infection. We show here that the stress-inducible molecule, MICA, is induced on the surface of dendritic and epithelial cells by infection with M. tuberculosis in vitro and in vivo. MICA engagement by the activating receptor, NKG2D, present on Vgamma2Vdelta2 T cells, resulted in a substantial enhancement of the TCR-dependent Vgamma2Vdelta2 T cell response to nonpeptide antigens and protein superantigens alike. Thus, a MICA-NKG2D interaction may be necessary for an effective innate immune response to microbe-associated antigens that also are constitutively present in vivo.

Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family.

NKG2D is an activating receptor that is expressed on most natural killer (NK) cells, CD8 alphabeta T cells, and gammadelta T cells. Among its ligands is the distant major histocompatibility complex class I homolog MICA, which has no function in antigen presentation but is induced by cellular stress. To extend previous functional evidence, the NKG2D-MICA interaction was studied in isolation. NKG2D homodimers formed stable complexes with monomeric MICA in solution, demonstrating that no other components were required to facilitate this interaction. MICA glycosylation was not essential but enhanced complex formation. Soluble NKG2D also bound to cell surface MICB, which has structural and functional properties similar to those of MICA. Moreover, NKG2D stably interacted with surface molecules encoded by three newly identified cDNA sequences (N2DL-1, -2, and -3), which are identical to the human ULBP proteins and may represent homologs of the mouse retinoic acid-early inducible family of NKG2D ligands. Because of the substantial sequence divergence among these molecules, these results indicated promiscuous modes of receptor binding. Comparison of allelic variants of MICA revealed large differences in NKG2D binding that were associated with a single amino acid substitution at position 129 in the alpha2 domain. Varying affinities of MICA alleles for NKG2D may affect thresholds of NK-cell triggering and T-cell modulation.

Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA.

The major histocompatibility complex (MHC) class I homolog, MICA, is a stress-inducible ligand for NKG2D, a C-type lectin-like activating immunoreceptor. The crystal structure of this ligand-receptor complex that we report here reveals an NKG2D homodimer bound to a MICA monomer in an interaction that is analogous to that seen in T cell receptor-MHC class I protein complexes. Similar surfaces on each NKG2D monomer interact with different surfaces on either the alpha1 or alpha2 domains of MICA. The binding interactions are large in area and highly complementary. The central section of the alpha2-domain helix, disordered in the structure of MICA alone, is ordered in the complex and forms part of the NKG2D interface. The extensive flexibility of the interdomain linker of MICA is shown by its altered conformation when crystallized alone or in complex with NKG2D.

Blood pressure control in treated hypertensive patients: clinical performance of general practitioners.

BACKGROUND: The blood pressure of many treated hypertensive patients remains above recommended target levels. This discrepancy may be related to general practitioners' (GPs') actions. AIM: To assess clinical performance of GPs in blood pressure control in treated hypertensive patients and to explore the influence of patient and GP characteristics on clinical performance. DESIGN OF STUDY: Cross-sectional study conducted on 195 GPs with invitations to participate made via bulletins and by letter. SETTING: One hundred and thirty-two practices in the southern half of The Netherlands from November 1996 to April 1997. METHOD: Performance criteria were selected from Dutch national hypertension guidelines for general practice. GPs completed self-report forms immediately after follow-up visits of hypertensive patients treated with antihypertensive medication. RESULTS: The GPs recorded 3526 follow-up visits. In 63% of these consultations the diastolic blood pressure (DBP) was 90 mmHg or above. The median performance rates of the GPs were less than 51% for most of the recommended actions, even at a DBP of > or = 100 mmHg. Performance of non-pharmacological actions increased gradually with increasing DBP; prescribing an increase in antihypertensive medication and making a follow-up appointment scheduled within six weeks rose steeply at a DBP of > or = 100 mmHg. Patient and GP characteristics contributed little to clinical performance. Action performance rates varied considerably between GPs. CONCLUSION: GPs seem to target their actions at a DBP of below 100 mmHg, whereas guidelines recommend targeting at a DBP of below 90 mmHg.

Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells.

NKG2D is an activating receptor that stimulates innate immune responses by natural killer cells upon engagement by MIC ligands, which are induced by cellular stress. Because NKG2D is also present on most CD8alphabeta T cells, it may modulate antigen-specific T cell responses, depending on whether MIC molecules--distant homologs of major histocompatibility complex (MHC) class I with no function in antigen presentation--are induced on the surface of pathogen-infected cells. We found that infection by cytomegalovirus (CMV) resulted in substantial increases in MIC on cultured fibroblast and endothelial cells and was associated with induced MIC expression in interstitial pneumonia. MIC engagement of NKG2D potently augmented T cell antigen receptor (TCR)-dependent cytolytic and cytokine responses by CMV-specific CD28- CD8alphabeta T cells. This function overcame viral interference with MHC class I antigen presentation. Combined triggering of TCR-CD3 complexes and NKG2D induced interleukin 2 production and T cell proliferation. Thus NKG2D functioned as a costimulatory receptor that can substitute for CD28.

DAP10 and DAP12 form distinct, but functionally cooperative, receptor complexes in natural killer cells.

Many of the activating receptors on natural killer (NK) cells are multisubunit complexes composed of ligand-binding receptors that are noncovalently associated with membrane-bound signaling adaptor proteins, including CD3zeta, FcstraightepsilonRIgamma, DAP12, and DAP10. Because the DAP10 and DAP12 genes are closely linked, expressed in NK cells, and have remarkably similar transmembrane segments, it was of interest to determine the specificity of their interactions with ligand-binding receptors and to examine their signaling properties. Despite their similarities, DAP10, DAP12, FcstraightepsilonRIgamma, and CD3zeta form specific receptor complexes with their ligand-binding partners in NK cells and transfectants. The transmembrane regions of DAP10 and DAP12 are sufficient to confer specific association with their partners. Although cross-linking of either DAP10- or DAP12-associated receptors has been shown to be sufficient to trigger NK cell-mediated cytotoxicity against Fc receptor-bearing cells, substantial synergy was observed in the induction of cytokine production when both receptors were engaged. Activation of the Syk/ZAP70 tyrosine kinases by the immunoreceptor tyrosine-based activation motif-containing DAP12 adaptor and of the phosphatidylinositol 3-kinase pathway by the YxNM-containing DAP10 adaptor may play an important role in the stimulation of NK cells and T cells.

Impaired assembly yet normal trafficking of MHC class I molecules in Tapasin mutant mice.

Loading of peptides onto major histocompatibility complex class I molecules involves a multifactorial complex that includes tapasin (TPN), a membrane protein that tethers empty class I glycoproteins to the transporter associated with antigen processing. To evaluate the in vivo role of TPN, we have generated Tpn mutant mice. In these animals, most class I molecules exit the endoplasmic reticulum (ER) in the absence of stably bound peptides. Consequently, mutant animals have defects in class I cell surface expression, antigen presentation, CD8+ T cell development, and immune responses. These findings reveal a critical role of TPN for ER retention of empty class I molecules. Tpn mutant animals should prove useful for studies on alternative antigen-processing pathways that involve post-ER peptide loading.

Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA.

Stress-inducible MICA, a distant homolog of major histocompatibility complex (MHC) class I, functions as an antigen for gammadelta T cells and is frequently expressed in epithelial tumors. A receptor for MICA was detected on most gammadelta T cells, CD8+ alphabeta T cells, and natural killer (NK) cells and was identified as NKG2D. Effector cells from all these subsets could be stimulated by ligation of NKG2D. Engagement of NKG2D activated cytolytic responses of gammadelta T cells and NK cells against transfectants and epithelial tumor cells expressing MICA. These results define an activating immunoreceptor-MHC ligand interaction that may promote antitumor NK and T cell responses.

An activating immunoreceptor complex formed by NKG2D and DAP10.

Many immune receptors are composed of separate ligand-binding and signal-transducing subunits. In natural killer (NK) and T cells, DAP10 was identified as a cell surface adaptor protein in an activating receptor complex with NKG2D, a receptor for the stress-inducible and tumor-associated major histocompatibility complex molecule MICA. Within the DAP10 cytoplasmic domain, an Src homology 2 (SH2) domain-binding site was capable of recruiting the p85 subunit of the phosphatidylinositol 3-kinase (PI 3-kinase), providing for NKG2D-dependent signal transduction. Thus, NKG2D-DAP10 receptor complexes may activate NK and T cell responses against MICA-bearing tumors.

Analysis of the acute cytotoxicity of the Erlanger silver catheter.

The Erlanger silver catheter consists of a new form of polyurethane, which contains finely dispersed metallic silver. The aim of this study was to establish the biocompatibility of this intravenous catheter by investigating the acute cytotoxicity of extracts from the Erlanger silver catheter on human fibroblasts and lymphocytes. Extracts of the Erlanger silver catheter were not cytotoxic for MRC-5 human fibroblasts nor for sensitized phytohemagglutinin (PHA)-stimulated human lymphocytes. The addition of silver powder of up to 2% by weight to the basic catheter polyurethane Tecothane led to no increase in acute cytotoxicity in comparison with untreated Tecothane. The Erlanger silver catheter is a new intravenous catheter with good biocompatibility.

Crystal structure of the MHC class I homolog MIC-A, a gammadelta T cell ligand.

The major histocompatibility complex (MHC) class I homolog MIC-A functions as a stress-inducible antigen that is recognized by a subset of gammadelta T cells independent of beta2-microglobulin and bound peptides. Its crystal structure reveals a dramatically altered MHC class I fold, both in detail and overall domain organization. The only remnant of a peptide-binding groove is a small cavity formed as the result of disordering a large section of one of the groove-defining helices. Loss of beta2-microglobulin binding is due to a restructuring of the interaction interfaces. Structural mapping of sequence variation suggests potential receptor binding sites on the underside of the platform on the side opposite of the surface recognized by alphabeta T cell receptors on MHC class I-peptide complexes.

Population study of allelic diversity in the human MHC class I-related MIC-A gene.

The polymorphism of major histocompatibility complex (MHC) class I HLA-A, -B, and -C molecules may have evolved through pathogen-driven selection of alleles with diverse peptide-binding specificities. Two MHC-encoded molecules that are distantly related to class I, MIC-A and MIC-B, do not function in the presentation of pathogen-derived peptides to T cells with alphabeta T-cell receptors (TCRs), but are broadly recognized by intraepithelial T cells with gammadelta TCRs. However, both MIC-A and MIC-B are polymorphic, displaying an unusual distribution of a number of variant amino acids in their extracellular alpha1, alpha2, and alpha3 domains. In order to further define the polymorphism of MIC-A, we examined its alleles among 275 individuals with common and rare HLA genotypes. Of 16 previously defined alleles, 12 were confirmed and 5 new alleles were identified. A two-by-two analysis of MIC-A and HLA-B alleles uncovered a number of statistically significant associations. These results confirm and extend previous knowledge on the polymorphism of MIC-A. The strong positive linkage of certain MIC-A and HLA-B alleles may have implications for studies related to MHC-associated diseases and transplantation.

Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB.

Human MHC class I-related molecules, MICA and MICB, are stress-induced antigens that are recognized by a subset of gamma delta T cells expressing the variable region Vdelta1. This functional association has been found to be limited to intestinal epithelium, where these T cells are prevalent and where MICA and, presumably, MICB are mainly expressed. However, increased frequencies of Vdelta1 gamma delta T cells have been observed in various epithelial tumors; moreover, MICA/B are expressed on diverse cultured epithelial tumor cells. With freshly isolated tumor specimens, expression of MICA/B was documented in many, but not all, carcinomas of the lung, breast, kidney, ovary, prostate, and colon. In tumors that were positive for MICA/B, the frequencies of Vdelta1 gamma delta T cells were significantly higher than in those that were negative. Vdelta1 gamma delta T cell lines and clones derived from different tumors recognized MICA/B on autologous and heterologous tumor cells. In accord with previous evidence, no constraints were observed in these interactions, such as those imposed by specific peptide ligands. Thus, MICA/B are tumor-associated antigens that can be recognized, in an apparently unconditional manner, by a subset of tumor-infiltrating gamma delta T cells. These results raise the possibility that an induced expression of MICA/B, by conditions that may be related to tumor homeostasis and growth, could play a role in immune responses against tumors.

Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells.

Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation.

Expression, purification, crystallization and crystallographic characterization of the human MHC class I related protein MICA.

Crystals of the human MHC-encoded molecule MICA, a homologue of MHC class I proteins, have been grown in hanging-drop vapor-diffusion trials using ammonium sulfate as a precipitating agent with recombinant protein expressed in a baculovirus-based system. Cryo-preserved crystals of MICA belong to the cubic space group F4132 with lattice constants a = b = c = 260.7 A and diffract to a resolution limit of 3.0 A when cryo-preserved. These crystals do not diffract when handled conventionally.

Diversification, expression, and gamma delta T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules.

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial gamma delta T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative alpha-helical regions of the alpha1 alpha2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their alpha1 alpha2 domains, they were recognized by diverse human intestinal epithelial gamma delta T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of gamma delta T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.