Prevalent expression of MHC class I chain-related molecule A in human osteosarcoma

MHC class I chain-related molecule A (MICA) is one of the major ligands for activating immune-receptor NKG2D which is expressed on NK cells and cytotoxic T lymphocytes. The release and sustained expression of MICA protein can impair NKG2D-mediated cytotoxic activity by reducing NKG2D receptor on immune effector cells. The aim of the study was to investigate the expression and release of MICA in human osteosarcoma. RT-PCR, immunohistochemistry, western blotting and flow cytometry were used in analyzing the expression of MICA. Serum level of soluble MICA was quantitated by ELISA. Our data showed that MICA is prevalently expressed in osteosarcoma both in mRNA and protein level. Upregulation of MICA was found in osteosarcoma compared with benign tumors and normal bone tissues. Higher level of soluble MICA in serum can be detected in osteosarcoma patients. In conclusion, prevalent expression of MICA and higher serum level of soluble MICA may suggest a deficiency of MICA-NKG2D mediated immunosurveillance in osteosarcoma patients. Restoring the expression of NKG2D receptor on immune effector cells may contribute to a therapeutic strategy for human osteosarcoma.     

MHC class I chain-related protein A antibodies and shedding are associated with the progression of multiple myeloma

Monoclonal gammopathy of undetermined significance (MGUS) is a common disorder of aging and a precursor lesion to full-blown multiple myeloma (MM). The mechanisms underlying the progression from MGUS to MM are incompletely understood but include the suppression of innate and adaptive antitumor immunity. Here, we demonstrate that NKG2D, an activating receptor on natural killer (NK) cells, CD8(+) T lymphocytes, and MHC class I chain-related protein A (MICA), an NKG2D ligand induced in malignant plasma cells through DNA damage, contribute to the pathogenesis of MGUS and MM. MICA expression is increased on plasma cells from MGUS patients compared with normal donors, whereas MM patients display intermediate MICA levels and a high expression of ERp5, a protein disulfide isomerase linked to MICA shedding (sMICA). MM, but not MGUS, patients harbor circulating sMICA, which triggers the down-regulation of NKG2D and impaired lymphocyte cytotoxicity. In contrast, MGUS, but not MM, patients generate high-titer anti-MICA antibodies that antagonize the suppressive effects of sMICA and stimulate dendritic cell cross-presentation of malignant plasma cells. Bortezomib, a proteasome inhibitor with anti-MM clinical efficacy, activates the DNA damage response to augment MICA expression in some MM cells, thereby enhancing their opsonization by anti-MICA antibodies. Together, these findings reveal that the alterations in the NKG2D pathway are associated with the progression from MGUS to MM and raise the possibility that anti-MICA monoclonal antibodies might prove therapeutic for these disorders.     

Inhibition of selective signaling events in natural killer cells recognizing major histocompatibility complex class I.

Many studies have characterized the transmembrane signaling events initiated after T-cell antigen receptor recognition of major histocompatibility complex (MHC)-bound peptides. Yet, little is known about signal transduction from a set of MHC class I recognizing receptors on natural killer (NK) cells whose ligation dramatically inhibits NK cell-mediated killing. In this study we evaluated the influence of MHC recognition on the proximal signaling events in NK cells binding tumor targets. We utilized two experimental models where NK cell-mediated cytotoxicity was fully inhibited by the recognition of specific MHC class I molecules. NK cell binding to either class I-deficient or class I-transfected target cells initiated rapid protein tyrosine kinase activation. In contrast, whereas NK cell binding to class I-deficient targets led to inositol phosphate release and increased intracellular free calcium ([Ca2+]i), NK recognition of class I-bearing targets did not induce the activation of these phospholipase C-dependent signaling events. The recognition of class I by NK cells clearly had a negative regulatory effect since blocking this interaction using anti-class I F(ab')2 fragments increased inositol 1,4,5-trisphosphate release and [Ca2+]i and increased the lysis of the targets. These results suggest that one of the mechanisms by which NK cell recognition of specific MHC class I molecules can block the development of cell-mediated cytotoxicity is by inhibiting specific critical signaling events.     

Down-regulation of NKG2D and NKp80 ligands by Kaposi's sarcoma-associated herpesvirus K5 protects against NK cell cytotoxicity

Natural killer (NK) cells are important early mediators of host immunity to viral infections. The NK activatory receptors NKG2D and NKp80, both C-type lectin-like homodimeric receptors, stimulate NK cell cytotoxicity toward target cells. Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) down-regulates MHC class I molecules to avoid detection by cytotoxic T lymphocytes but renders cells susceptible to NK cell cytotoxicity. We now show that the KSHV immune evasion gene, K5, reduces cell surface expression of the NKG2D ligands MHC class I-related chain A (MICA), MICB, and the newly defined ligand for NKp80, activation-induced C-type lectin (AICL). Down-regulation of both MICA and AICL requires the ubiquitin E3 ligase activity of K5 to target substrate cytoplasmic tail lysine residues. The common MICA *008 allele has a frameshift mutation leading to a premature stop codon and is resistant to down-regulation because of the loss of lysine residues. K5-mediated ubiquitylation signals internalization but not degradation of MICA and causes a potent reduction in NK cell-mediated cytotoxicity. The down-regulation of ligands for both the NKG2D and NKp80 activation pathways provides KSHV with a powerful mechanism for evasion of NK cell antiviral functions.     

Altered natural killer cell repertoire in Tap-1 mutant mice.

We have analyzed the specificity and function of natural killer (NK) cells in mice with a homozygous deletion of the major histocompatibility complex (MHC)-encoded transporter gene associated with MHC class I-restricted antigen presentation (Tap-1). These mice express very low levels of class I molecules at the cell surface, and these molecules are either devoid of peptide or occupied only by TAP-independent peptides. NK cells in Tap-1 -/- mice, through normal in number, appeared tolerant toward autologous Tap-1 -/- Con A-activated blasts, Tap-1 -/- as well as allogeneic BALB/c bone marrow cells, and RMA-S tumor cell grafts. In contrast, they killed YAC-1 cells as efficiently as did NK cells from wild-type mice. Defective Tap-1 expression was sufficient to render nontransformed target cells sensitive to NK cell-mediated lysis. It is concluded that proper expression of TAP molecules is necessary for normal development of NK cells, as well as for rendering target cells resistant to NK cell-mediated lysis. These results support the hypothesis that class I molecules of the MHC influence the sensitivity of target cells to lysis by NK cells, as well as the development of the NK cell repertoire.     

Mechanisms of resistance to natural killer cell-mediated cytotoxicity in acute lymphoblastic leukemia

OBJECTIVE: Natural killer (NK) cell-mediated cytotoxicity contributes to the innate immune response against numerous malignancies, including leukemias. Acute lymphoblastic leukemias (ALL) often display a high degree of resistance, the mechanisms of which have not been elucidated. METHODS: We used the well-characterized NK cell line NK-92 as a model to investigate whether mechanisms commonly implicated in tumor escape from NK cell killing are relevant for ALL. RESULTS: We demonstrate selective resistance of B-precursor ALL to NK-92 cytotoxicity even in the absence of inhibitory killer cell immunoglobulin-like receptors (KIR), except for KIR2DL4. We also show that human leukocyte antigen-G, a ligand of KIR2DL4, expressed on a subset of ALL, does not mediate resistance of NK-cell mediated lysis. Similarly, intracellular adhesion molecule/lymphocyte function-associated antigen-1 interaction did not contribute significantly to resistance. In contrast the NK-sensitive T-ALL (MOLT-4) expressed moderate amounts of MHC class I chain-related gene AB (MICA/B) a ligand for the NK cell activating receptor NKG2D, while expression of MICA/B was absent in resistant B-ALL cell lines. CONCLUSIONS: The NK cell-resistance of B-lineage ALLs does not appear to involve inhibitory mechanisms, but suggests deficient NK cell activation. Thus, immunostrategies designed to enhance ALL sensitivity toward NK cell-mediated cytotoxicity should focus on mechanisms of NK cell activation.     

Recognition of beta 2-microglobulin-negative (beta 2m-) T-cell blasts by natural killer cells from normal but not from beta 2m- mice: nonresponsiveness controlled by beta 2m- bone marrow in chimeric mice.

The role of major histocompatibility complex (MHC) class I expression in control of the sensitivity of normal cells to natural killer (NK) cells was studied by the use of mutant mice made deficient for expression of beta 2-microglobulin (beta 2m) through homologous recombination in embryonal stem cells. T-cell blasts from beta 2m-deficient (beta 2m -/-) mice were killed by NK cells from normal mice in vitro, while beta 2m +/- blasts were resistant. The beta 2m defect also affected the NK effector cell repertoire: NK cells from beta 2m -/- mice failed to kill beta 2m -/- blasts, while they retained the ability to kill the prototype NK cell target lymphoma YAC-1, although at reduced levels. The inability to recognize beta 2m -/- blasts could be transferred with beta 2m -/- bone marrow to irradiated beta 2m-expressing mice. In contrast, the development of CD8+ T cells (deficient in beta 2m -/- mice) was restored in such chimera. These results indicate that loss of MHC class I/beta 2m expression is sufficient to render normal cells sensitive to NK cells, and that the same defect in the hemopoietic system of a mouse renders its NK cells tolerant to beta 2m-deficient but otherwise normal cells. In the beta 2m -/- mice, NK cells may be selected or educated by other bone marrow cells to tolerate the MHC class I deficiency. Alternatively, the specificity may be controlled directly by the class I molecules on the NK cells themselves.     

Therapy-induced antibodies to MHC class I chain-related protein A antagonize immune suppression and stimulate antitumor cytotoxicity

The activation of NKG2D on innate and adaptive cytotoxic lymphocytes contributes to immune-mediated tumor destruction. Nonetheless, tumor cell shedding of NKG2D ligands, such as MHC class I chain-related protein A (MICA), results in immune suppression through down-regulation of NKG2D surface expression. Here we show that some patients who respond to antibody-blockade of cytotoxic T lymphocyte-associated antigen 4 or vaccination with lethally irradiated, autologous tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor generate high titer antibodies against MICA. These humoral reactions are associated with a reduction of circulating soluble MICA (sMICA) and an augmentation of natural killer (NK) cell and CD8(+) T lymphocyte cytotoxicity. The immunotherapy-induced anti-MICA antibodies efficiently opsonize cancer cells for dendritic cell cross-presentation, which is correlated with a diversification of tumor antigen recognition. The anti-MICA antibodies also accomplish tumor cell lysis through complement fixation. Together, these findings establish a key role for the NKG2D pathway in the clinical activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade and granulocyte-macrophage colony-stimulating factor secreting tumor cell vaccines. Moreover, these results highlight the therapeutic potential of anti-MICA antibodies to overcome immune suppression and effectuate tumor destruction in patients.     

A basolateral sorting motif in the MICA cytoplasmic tail

The MHC class I chain-related MICA molecule is a stress-induced, highly polymorphic, epithelia-specific, membrane-bound glycoprotein interacting with the activating NK cell receptor NKG2D and/or gut-enriched Vdelta1-bearing gammadelta T cells. We have previously reported the presence of a MICA transmembrane-encoded short-tandem repeat harboring a peculiar allele, A5.1, characterized by a frame shift mutation leading to a premature intradomain stop codon, thus denying the molecule of its 42-aa cytoplasmic tail. Given that this is the most common population-wide MICA allele found, we set out to analyze the functional consequences of cytoplasmic tail deletion. Here, we show native expression of MICA at the basolateral surface of human intestinal epithelium, the site of putative interaction with intraepithelial T and NK lymphocytes. We then demonstrate, in polarized epithelial cells, that although the full-length MICA protein is sorted to the basolateral membrane, the cytoplasmic tail-deleted construct as well as the naturally occurring A5.1 allele are aberrantly transported to the apical surface. Site-directed mutagenesis identified the cytoplasmic tail-encoded leucine-valine dihydrophobic tandem as the basolateral sorting signal. Hence, the physiological location of MICA within epithelial cells is governed by its cytoplasmic tail, implying impairment in A5.1 homozygous individuals, perhaps relevant to the immunological surveillance exerted by NK and T lymphocytes on epithelial malignancies.     

Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity.

In addition to their role in peptide antigen presentation, class I MHC proteins also play a critical role in inhibiting natural killer (NK) cytotoxicity through interaction with NK inhibitory receptors. Thus, NK cells are cytotoxic to virus-infected and tumor cells that have lost class I MHC protein expression. However, the nature of the receptors involved in the triggering of lysis of target cells is poorly understood. CD16 (Fcgamma receptor III) has been described as a receptor expressed on NK cells that facilitates antibody-dependent cellular cytotoxicity (ADCC) by binding to the Fc portion of various antibodies. However, we show here that CD16 has a broader function and is directly involved in the lysis of some virus-infected cells and tumor cells, independent of antibody binding. The presence of a putative CD16 ligand on appropriate target cells has also been demonstrated by the use of a CD16-Ig fusion protein.     

The natural killer cell receptor Ly-49A recognizes a peptide-induced conformational determinant on its major histocompatibility complex class I ligand.

Natural killer (NK) cells are inhibited from killing cellular targets by major histocompatibility complex (MHC) class I molecules. In the mouse, this can be mediated by the Ly-49A NK cell receptor that specifically binds the H-2Dd MHC class I molecule, then inhibits NK cell activity. Previous experiments have indicated that Ly-49A recognizes the alpha 1/alpha 2 domains of MHC class I and that no specific MHC-bound peptide appeared to be involved. We demonstrate here that alanine-substituted peptides, having only the minimal anchor motifs, stabilized H-2Dd expression and provided resistance to H-2Dd-transfected, transporter associated with processing (TAP)-deficient cells from lysis by Ly-49A+ NK cells. Peptide-induced resistance was blocked only by an mAb that binds a conformational determinant on H-2Dd. Moreover, stabilization of "empty" H-2Dd heavy chains by exogenous beta 2-microglobulin did not confer resistance. In contrast to data for MHC class I-restricted T cells that are specific for peptides displayed MHC molecules, these data indicate that NK cells are specific for a peptide-induced conformational determinant, independent of specific peptide. This fundamental distinction between NK cells and T cells further implies that NK cells are sensitive only to global changes in MHC class I conformation or expression, rather than to specific pathogen-encoded peptides. This is consistent with the "missing self" hypothesis, which postulates that NK cells survey tissues for normal expression of MHC class I.     

Genes encoding putative natural killer cell C-type lectin receptors in teleostean fishes

Mammalian natural killer (NK) cells are cytotoxic lymphocytes that express receptors specific for MHC class I molecules. The NK cell receptors belong to two structurally unrelated families, the killer cell Ig-like receptors and the killer cell C-type lectin receptors. We describe a cDNA clone derived from the bony (cichlid) fish Paralabidochromis chilotes and show that it encodes a protein related to the CD94/NK cell group 2 (NKG2) subfamily of the killer cell C-type lectin receptors. The gene encoding this receptor in a related species, Oreochromis niloticus, has a similar structure to the human CD94/NKG2 genes and is a member of a multigene cluster that resembles the mammalian NK cell gene complex. Thus, the CD94/NKG2 subfamily of NK cell receptors must have arisen before the divergence of fish and tetrapods and may have retained its function (possibly monitoring the expression of MHC class I molecules) for >400 million years.     

Transfer of NKG2D and MICB at the cytotoxic NK cell immune synapse correlates with a reduction in NK cell cytotoxic function

Although transfer of membrane proteins has been shown to occur during immune cell interactions, the functional significance of this process is not well understood. Here we describe the intercellular transfer of NKG2D and MHC class I chain-related molecule (MIC) B proteins at the cytotoxic natural killer cell immune synapse (cNK-IS). MICB expressed on the 721.221 cell line induced clustering of NKG2D at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing F-actin. Moreover, natural killer (NK) cell membrane-connective structures formed during cytotoxic interactions contained F-actin, perforin, and NKG2D. NKG2D transfer depended on binding to MICB and was specific because transfer of other molecules not involved in NK-IS formation was not observed. Transfer of MICB to NK cells also was noted, suggesting a bidirectional exchange of receptor/ligand pairs at cNK-IS. Experiments designed to test the functional significance of these observations revealed that brief interactions between NK cells and MICB expressing target cells led to a reduction in NKG2D-dependent NK cytotoxicity. These data demonstrate interchange of an activating receptor and its ligand at the cNK-IS and document a correlation between synapse organization, intercellular protein transfer, and compromised NK cell function after interaction with a susceptible target cell.     

Takayasu arteritis: insights into immunopathology

Takayasu arteritis is an acute and sometimes chronic form of vasculitis involving the aorta, its main branches and pulmonary arteries. Although its etiology is still unknown, immunopathologic analyses revealed that the infiltrating cells mainly consisted of gammadelta T-cells as well as alphabeta T-cells and NK cells. The infiltrating gammadelta T-cells, cytotoxic T-lymphocytes (CTLs), and natural killer (NK) cells directly injured the vascular cells by releasing a cytolytic factor, perforin. Expression of heat-shock protein (HSP)-65 as well as human leukocyte antigen (HLA) class I and II was enhanced in Takayasu arteritis lesions, supporting the pathogenic role of gammadelta T-cells and alphabeta T-cells. T-cell receptor (TCR) alphabeta gene usage by the infiltrating cells was restricted, strongly suggesting that a specific antigen was targeted. TCR gammadelta gene usage by the infiltrating cells was also restricted. Furthermore, it has been reported that a strong association with a specific haplotype of major histocompatibility complex (MHC) class I chain-related (MIC), MICA gene with Takayasu arteritis, suggesting that the HLA-linked gene susceptible to the disease is mapped near the MICA gene. This also supports a pathogenic role of gammadelta T-cells in Takayasu arteritis because gammadelta T-cells were shown to recognize MICA molecule, which can be stress-induced. These findings suggest that unknown stress, such as infection, may trigger the autoimmune process of inflammation involved in Takayasu arteritis.     

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.     

A sustained response to low dose interferon-alpha in a case of refractory pure red cell aplasia

Acquired pure red cell aplasia (PRCA) may be the result of a cellular or humoral autoimmune process. One proposed mechanism is the destruction of erythroid progenitors by self-reactive, cytotoxic T cells or natural killer (NK) cells. These cells normally express MHC class I receptors (KIR) which inhibit cytotoxicity when the target cell expresses the HLA class I antigen(s) they bind. Therefore, loss of these antigens on maturing erythroid progenitors may render them susceptible to destruction by the pathogenic cells. Interferon-alpha (INF-alpha) increases HLA class I expression on hematopoietic precursor cells. Therefore, we initiated a trial of INF-alpha in a patient with refractory PRCA. Following treatment, he developed transfusion independence, and a sustained normal hematocrit. Analysis of bone marrow erythroid cells revealed an increase in expression of HLA class I molecules. INF-alpha should be used in a controlled trial in patients with PRCA to determine its activity and mechanism of action.     

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.     

Ectopic expression of retinoic acid early inducible-1 gene (RAE-1) permits natural killer cell-mediated rejection of a MHC class I-bearing tumor in vivo

In 1986, K?rre and colleagues reported that natural killer (NK) cells rejected an MHC class I-deficient tumor cell line (RMA-S) but they did not reject the same cell line if it expressed MHC class I (RMA). Based on this observation, they proposed the concept that NK cells provide immune surveillance for "missing self," e.g., they eliminate cells that have lost class I MHC antigens. This seminal observation predicted the existence of inhibitory NK cell receptors for MHC class I. Here, we present evidence that NK cells are able to reject tumors expressing MHC class I if the tumor expresses a ligand for NKG2D. Mock-transfected RMA cells resulted in tumor formation. In contrast, when RMA cells were transfected with the retinoic acid early inducible gene-1 gamma or delta (RAE-1), ligands for the activating receptor NKG2D, the tumors were rejected. The tumor rejection was mediated by NK cells, and not by CD1-restricted NK1.1(+) T cells. No T cell-mediated immunological memory against the parental tumor was generated in the animals that had rejected the RAE-1 transfected tumors, which succumbed to rechallenge with the parental RMA tumor. Therefore, NK cells are able to reject a tumor expressing RAE-1 molecules, despite expression of self MHC class I on the tumor, demonstrating the potential for NK cells to participate in immunity against class I-bearing malignancies.