Inhibitory 2B4 contributes to NK cell education and immunological derangements in XLP1 patients

X‐linked lymphoproliferative disease 1 (XLP1) is an inherited immunodeficiency, caused by mutations in SH2D1A encoding Signaling Lymphocyte Activation Molecule (SLAM)‐associated protein (SAP). In XLP1, 2B4, upon engagement with CD48, has inhibitory instead of activating function. This causes a selective inability of cytotoxic effectors to kill EBV‐infected cells, with dramatic clinical sequelae. Here, we investigated the NK cell education in XLP1, upon characterization of killer Ig‐like receptor (KIR)/KIR‐L genotype and phenotypic repertoire of self‐HLA class I specific inhibitory NK receptors (self‐iNKRs). We also analyzed NK‐cell cytotoxicity against CD48⁺ or CD48^? KIR‐ligand matched or autologous hematopoietic cells in XLP1 patients and healthy controls. XLP1 NK cells may show a defective phenotypic repertoire with substantial proportion of cells lacking self‐iNKR. These NK cells are cytotoxic and the inhibitory 2B4/CD48 pathway plays a major role to prevent killing of CD48⁺ EBV‐transformed B cells and M1 macrophages. Importantly, self‐iNKR defective NK cells kill CD48^? targets, such as mature DCs. Self‐iNKR^? NK cells in XLP1 patients are functional even in resting conditions, suggesting a role of the inhibitory 2B4/CD48 pathway in the education process during NK‐cell maturation. Killing of autologous mature DC by self‐iNKR defective XLP1 NK cells may impair adaptive responses, further exacerbating the patients’ immune defect.     

WhatSAP — 2B4 sends mixed messages in the absence of SAP

2B4 (CD244), a member of the SLAM‐related receptor family, has important immuno‐regulatory functions including coactivating the cytotoxicity and cytokine secretion of NK cells. Immune modulation by 2B4 is dependent on the small intracellular signaling molecule SAP. In patients suffering from X‐linked lymphoproliferative disease (XLP1), SAP is nonfunctional, not only abolishing the activating function of 2B4, but rendering this receptor inhibitory. In this issue of European Journal of Immunology, Meazza et al. [Eur. J. Immunol. 2014. 44: 1526–1534.] demonstrate that 2B4‐mediated inhibition in NK cells from XLP1 patients is selective. While the activation of NK cells via ITAM‐based receptors is blocked by inhibitory 2B4, DNAM‐1, and NKG2D‐dependent NK‐cell activation is not affected by SAP deficiency. These findings provide an important insight into the different defective NK‐cell functions in XLP1 patients and demonstrate the differential integration of redundant receptor signaling pathways in NK cells.     

NKG2D performs two functions in invariant NKT cells: direct TCR-independent activation of NK-like cytolysis and co-stimulation of activation by CD1d

Invariant NKT cells are important in the activation and regulation of immune responses. They can also function as CD1d-restricted killer cells. However, the role of activating innate NK-cell receptors expressed on NKT cells in triggering cytolytic function is poorly characterized. Here, we initially confirmed that the cellular stress-ligand receptor NKG2D is expressed on CD4- NKT cells, whereas most CD4+ NKT cells lack this receptor. Interestingly, NKG2D+ NKT cells frequently expressed perforin, and both NKG2D and perforin localized at the site of contact with NKG2D ligand-expressing target cells. CD4- NKT cells degranulated in response to NKG2D engagement in a redirected activation assay independent of stimulation via their invariant TCR. NKT cells killed P815 cells coated with anti-NKG2D mAb and CD1d-negative K562 tumor target cells in an NKG2D-dependent manner. Furthermore, NKG2D engagement co-stimulated TCR-mediated NKT-cell activation in response to endogenous CD1d-presented ligands or suboptimal levels of anti-CD3 triggering. These data indicate that the CD4- subset of human NKT cells can mediate direct lysis of target cells via NKG2D engagement independent of CD1d, and that NKG2D also functions as a co-stimulatory receptor in these cells. NKG2D thus plays both a direct and a co-stimulatory role in the activation of NKT cells.     

2B4: an NK cell activating receptor with unique specificity and signal transduction mechanism

2B4 is a cell surface glycoprotein of the Ig-superfamily structurally related to CD2-like molecules such as CD2, CD48, CD58, CD84, Ly-9, and SLAM. Engagement of 2B4 on NK cells with specific antibodies or with its ligand CD48 enhances NK cell-mediated cytotoxicity. 2B4 is also expressed on both CD8+ T cells and myeloid cells, but its function in these cells remains unknown. Signal transduction through 2B4 involves recruitment of the SH2-containing adapter molecule SAP to cytoplasmic tyrosines. SAP is deficient in patients affected by X-linked lymphoproliferative disorder (XLP), which is triggered following EBV infection. Thus, an interruption of signaling through 2B4 and related molecules may impair NK cell recognition of virally infected cells and contribute to XLP.     

DNAX accessory molecule‐1 (CD226) promotes human hepatocellular carcinoma cell lysis by Vγ9Vδ2 T cells

Human Vγ9Vδ2 T lymphocytes can be activated by nonpeptidic antigens such as the mevalonate pathway‐derived isopentenyl pyrophosphate or synthetic phosphoantigen such as bromohydrin pyrophosphate. They display a strong cytotoxic activity against several tumor types, including hepatocellular carcinoma (HCC). Little is known about the mechanisms underlying Vγ9Vδ2 T‐cell recognition of tumor cells, but there is strong evidence that activating NK receptors play a role in γδ T‐cell cytotoxicity. In this study, we showed that the two NK receptors DNAX accessory molecule‐1 (DNAM‐1) and CD96 were expressed by Vγ9Vδ2 T cells. The ligands Nectin‐like‐5 specific of both DNAM‐1 and CD96, and also Nectin‐2, an additional ligand of DNAM‐1, were present on all HCC cell lines analyzed. Furthermore, we demonstrated by mAb‐mediated masking experiments that cytotoxicity against HCC cells as well as IFN‐γ production in γδ T cells were dependent on DNAM‐1. Our experiments indicated that Nectin‐like‐5 but not Nectin‐2 was involved in DNAM‐1‐dependent γδ T‐cell functions. We did not reveal a role for CD96 in the killing of HCC cells. Finally, we showed by combined mAb‐mediated blockade that DNAM‐1 and NKG2D could cooperate in the cell lysis of HCC.     

Potential pathways for regulation of NK and T cell responses: differential X-linked lymphoproliferative syndrome gene product SAP interactions with SLAM and 2B4

SAP, the gene that is altered or absent in the X-linked lymphoproliferative syndrome (XLP), encodes a small protein that comprises a single SH2 domain and binds to the cell-surface protein SLAM which is present on activated or memory T and B cells. Because defective NK cell activity also has been reported in XLP patients, we studied the SAP gene in NK cells. SAP was induced upon viral infection of SCID mice and shown to be expressed in NK cells by in vitro culturing in the presence of IL-2. Moreover, SAP was expressed in the NK cell lines YT and RNK 16. Because SLAM, the cell-surface protein with which SAP interacts, and 2B4, a membrane protein having sequence homologies with SLAM, also were found to be expressed on the surfaces of activated NK and T cell populations, they may access SAP functions in these populations. Whereas we found that 2B4 also binds SAP, 2B4-SAP interactions occurred only upon tyrosine phosphorylation of 2B4. By contrast, SLAM-SAP interactions were independent of phosphorylation of Y281 and Y327 on SLAM. As CD48, the ligand for 2B4, is expressed on the surface of Epstein-Barr virus (EBV)-infected B cells, it is likely that SAP regulates signal transduction through this pair of cell-surface molecules. These data support the hypothesis that XLP is a result of both defective NK and T lymphocyte responses to EBV. The altered responses may be due to aberrant control of the signaling cascades which are initiated by the SLAM-SLAM and 2B4-CD48 interactions.     

Molecular and cellular pathogenesis of X-linked lymphoproliferative disease

Summary: X‐linked lymphoproliferative disease (XLP) is an inherited immune defect caused by mutations in the Src homology 2 domain‐containing gene 1A, which encodes the adapter protein, signaling lymphocytic activation molecule (SLAM)‐associated protein (SAP). SAP is expressed in T cells, natural killer (NK) cells, and NKT cells, where it binds to the cytoplasmic domain of the surface receptor SLAM (CD150) and the related receptors, 2B4 (CD244), CD84, Ly9 (CD229), NK‐T‐B‐antigen, and CD2‐like receptor‐activating cytotoxic T cells. SAP also binds to the Src family tyrosine kinase Fyn and recruits it to SLAM, which leads to the generation of downstream phosphotyrosine signals. While the roles of the SLAM family receptors are only beginning to be understood, experiments suggest that these molecules regulate important aspects of lymphocyte function, such as proliferation, cytokine secretion, cytotoxicity, and antibody production. Thus, in XLP patients who lack functional SAP, the SLAM family receptors may not signal properly. This property likely contributes to the phenotypes of XLP, including fulminant infectious mononucleosis, lymphoma, and hypogammaglobulinemia. Further studies of SAP and the SLAM family receptors will provide insights into XLP and elucidate the signaling events regulating lymphocyte ontogeny and function.     

NKG2D engagement of colorectal cancer-specific T cells strengthens TCR-mediated antigen stimulation and elicits TCR independent anti-tumor activity

The NKG2D receptor is expressed by human NK, gammadelta T and alpha/beta T lymphocytes and its engagement results in the stimulation of effector cells. We evaluated the role of NKG2D receptor in anti-colorectal cancer (CRC) immune response. The cell surface expression of stress-inducible NKG2D ligands MICA/B (MHC class I-related chain molecules A/B) and ULBP (UL16 binding protein) by a panel of CRC lines was evaluated by flow cytometry. MICA and ULBP2/3 were widely expressed by the analyzed lines, with a minority of them being also ULBP-1+, whereas MICB was undetectable. CD8+ and CD4+ HLA-restricted anti-tumor T cell clones of a CRC patient were used to evaluate whether NKG2D engagement could mediate tumor recognition. Three out of four CD8+ T cell clones recognized the autologous tumor with a marginal NKG2D engagement, a finding that was correlated with the weak expression of NKG2D ligands by the autologous tumor. On the contrary, NKG2D triggering of these CD8+ T cell clones induced recognition of allogeneic CRC lines showing high expression of MICA and ULBP. A costimulatory role of NKG2D was observed with one CD4+/NKG2D+ T cell clone when stimulated by tumors sharing the HLA class II alleles and expressing NKG2D ligands. Taken together these data indicate that the engagement of NKG2D, depending on the expression of its ligands by target cells, can influence the pattern of anti-tumor reactivity by T lymphocytes.     

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA‐I) molecules can regulate survival of NK cells and their anti‐tumor killing activity. Herein, we have analysed whether interaction of sHLA‐I with CD8 and/or different isoforms of killer Ig‐like receptors (KIR) induced secretion of transforming growth factor (TGF)‐β1. CD8⁺KIR^? NK cell clones secreted TGF‐β1 upon the interaction of sHLA‐I with CD8 molecule. sHLA‐Cw4 or sHLA‐Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF‐β1 production elicited through CD8. On the other hand, sHLA‐Cw4 or sHLA‐Cw3 alleles induced secretion of TGF‐β1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF‐β1 strongly reduced NK cell‐mediated tumor cell lysis and production of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Also, TGF‐β1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL‐2‐dependent surface upregulation of some of these receptors was prevented by TGF‐β1. Furthermore, TGF‐β1 hampered IL‐2‐induced NK cell proliferation but not IL‐2‐mediated rescue from apoptosis of NK cells. Depletion of TGF‐β1 restored all the NK cell‐mediated functional activities analysed. Taken together these findings suggest that sHLA‐I antigens may downregulate the NK cell‐mediated innate response by inducing TGF‐β1 release.     

NK cells from malignant pleural effusions are not anergic but produce cytokines and display strong antitumor activity on short‐term IL‐2 activation

NK cells are a major component of innate immunity and exert a potent antitumor effect both in vitro and in vivo. However, NK cells infiltrating solid tumors have been shown to display severely impaired functional capabilities. In this study, we analyzed NK cells present in pleural effusions (PEs) of patients with primary or metastatic tumors of different origin, including mesothelioma and lung, breast, colon, gastric, bladder, and uterus carcinoma. In all instances, freshly isolated PE‐NK cells displayed a CD56^bright phenotype and expressed normal levels of both activating receptors and HLA class I‐specific inhibitory receptors. In addition, they rapidly released large amounts of IFN‐γ and TNF‐α after stimulation. Upon culture in IL‐2, they acquired a potent cytolytic activity against both allogeneic and autologous tumor cells. Tumor cell lysis was primarily mediated by NKG2D and NKp30 and partially by NKp46 and DNAM‐1, in agreement with the expression of the corresponding ligands on tumor cells. The finding that PE‐NK cells are not functionally impaired and that they can efficiently kill tumor cells upon short‐term IL‐2 activation may offer important clues for the development of novel approaches in tumor immunotherapy.     

Balance between activating NKG2D,DNAM‐1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast‐like synoviocytes (FLS) are central components of the aggressive, tumour‐like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA‐FLS and NK cells. We used cultured RA‐FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA‐FLS/NK cell cross‐talk. We show that RA‐FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM‐1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA‐FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA‐E expressed on RA‐FLS further enhanced Nishi cell degranulation in co‐culture with RA‐FLS. Using cultured RA‐FLS and the human NK cell line Nishi as an in vitro model system of RA‐FLS/NK cell cross‐talk, our results suggest that cell‐mediated cytotoxicity of RA‐FLS may be one mechanism by which NK cells influence local joint inflammation in RA.     

Critical role of the NKG2D receptor for NK cell‐mediated control and immune escape of B‐cell lymphoma

Little is known on the control of lymphomas by NK cells. Here, we study the role of the NK group 2D (NKG2D) receptor for the immunosurveillance of lymphoma. By using transplantable tumors as well as a λ‐myc‐transgenic model of endogenously arising lymphoma and NKG2D‐deficient mice, we show that NK cells eliminate tumor cells in vivo after receiving two signals. One step involved the activation of NK cells giving rise to IFN‐γ expression, which was effected by MHCI^low tumor cells or DCs. However, this was necessary but not sufficient to mediate cytotoxicity. Triggering cytotoxicity additionally required a second step, which could be mediated by engagement of the NKG2D receptor. Thus, NKG2D‐deficient NK cells could become activated in vivo, but they were not able to reject transplanted lymphomas or to degranulate in animals bearing autochthonous lymphomas. Tumor growth in NKG2D‐deficient λ‐myc‐transgenic mice was significantly accelerated compared to NKG2D‐competent animals. Whereas the latter developed tumors that lost expression of NKG2D ligands (NKG2D‐L) in late disease stages, this did not occur in NKG2D‐deficient mice. This indicates that NK cells and the NKG2D receptor play a role for control of lymphomas and that selection for NKG2D‐L loss mutants provides a mechanism of tumor escape.     

CD59 is physically and functionally associated with natural cytotoxicity receptors and activates human NK cell-mediated cytotoxicity

Triggering of cytotoxicity in human NK cells is induced by the combined engagement of several triggering receptors. These include primary receptors such as NKG2D and the natural cytotoxicity receptors (NCR) NKp30, NKp46 and NKp44, while other molecules, including 2B4, NTB-A and NKp80, function as co-receptors. As reported in the present study, during an attempt to identify novel NK receptors or co-receptors, we found that CD59 functions as a co-receptor in human NK cell activation; engagement of CD59 by specific mAb delivers triggering signals to human NK cells, resulting in enhancement of cytotoxicity. Similar to other NK co-receptors, the triggering function of CD59, a glycosylphosphatidylinositol (GPI)-linked protein, depends on the simultaneous engagement of primary receptors such as NCR. Accordingly, CD59-dependent triggering was virtually restricted to NK cells expressing high surface densities of NKp46, and mAb-mediated modulation of NKp46 resulted in markedly decreased responses to anti-CD59 mAb. Biochemical analysis revealed that CD59 is physically associated with NKp46 and NKp30. Moreover, engagement of CD59 resulted in tyrosine phosphorylation of CD3zeta chains associated with these NCR, but not those associated with CD16. Thus, CD59-mediated costimulation of NK cells requires direct physical interaction of this GPI-linked protein with primary triggering NK receptors.     

The role of the NKG2D immunoreceptor in immune cell activation and natural killing

Little is known concerning the stimulatory receptors responsible for tumor cell lysis by NK cells. We generated a monoclonal antibody specific for murine NKG2D in order to investigate its function. Blocking of NKG2D inhibited natural cytotoxicity of all tumor cells tested that express ligands for the receptor. Staining analysis showed that NKG2D is also expressed by activated CD8(+) T cells and macrophages, and subsets of TCRgammadelta(+) and NK1.1(+) T cells. Contradicting reports that NKG2D is solely a costimulatory receptor, we observed that cross-linking of NKG2D directly stimulates NK cells and activated macrophages. In contrast, NKG2D costimulates activated CD8(+) T cells. Thus, NKG2D engagement directly stimulates NK cells and macrophages, costimulates CD8(+) T cells, and plays a substantial role in natural killing.     

Suppression of human anti-porcine natural killer cell xenogeneic responses by combinations of monoclonal antibodies specific to CD2 and NKG2D and extracellular signal-regulated kinase kinase inhibitor

Natural killer (NK) cells can destroy xenogeneic tissues by antibody-dependent cell cytotoxicity (ADCC) and direct lysis. Unlike ADCC, activating interactions between human NK receptors and their cognate ligands in pigs are not fully elucidated. We set up this study to identify human NK activating receptors recognizing porcine cells isolated from distinct organs, e.g., aorta, cornea and liver, and to provide a molecular basis for effective immunosuppressive regimens. Among the array of NK receptors tested, NKp46, 2B4, CD49d, CD48, CD2 and NKG2D, only CD2 and NKG2D were shown to be involved in both cytotoxicity and cytokine (interferon-γ and tumour necrosis factor-α) production against porcine targets. Simultaneous blocking of CD2 and NKG2D by combining its monoclonal antibodies further suppressed xenogeneic NK responses. Moreover, addition of a suboptimal dose of PD98059, an extracellular signal-regulated kinase (ERK) kinase inhibitor, to those cells maximally reduced NK cytotoxicity, suggesting that ERK plays an important role in NK-mediated xenoreactivity. These impairments in NK cells were tightly associated with defective intracellular calcium mobilization and the subsequent degranulation process. Therefore, our data demonstrate a distinct role of CD2 and NKG2D on human NK cells in recognizing porcine grafts and further provide a potentially efficacious combinational regimen using anti-CD2 and anti-NKG2D monoclonal antibodies with PD98059 in a pig-to-human transplantation model.     

Distinct interactions of the X-linked lymphoproliferative syndrome gene product SAP with cytoplasmic domains of members of the CD2 receptor family.

X-linked lymphoproliferative syndrome (XLP; Duncan's disease) is a primary immunodeficiency disease that manifests as an inability to regulate the immune response to Epstein-Barr virus (EBV) infection. Here we examine the ability of the product of the gene defective in XLP, SAP (DSHP/SH2D1A), to associate with the cytoplasmic domains of several members of the CD2 subfamily of cell surface receptors, including SLAM, 2B4, and CD84. While recruitment of SAP to SLAM occurred in a phosphorylation-independent manner, SAP was found to bind preferentially to tyrosine-phosphorylated cytoplasmic domains within 2B4 and CD84. Missense or nonsense mutations in the SAP open reading frame were identified in five of seven clinically diagnosed XLP patients from different kindreds. Four of these variants retained the ability to bind to the cytoplasmic tails of SLAM and CD84. While ectopic expression of wild-type SAP was observed to block the binding of SHP-2 to SLAM, mutant SAP derivatives that retained the ability to bind SLAM did not inhibit recruitment of SHP-2 to SLAM. In contrast, SAP binding to CD84 had no effect on the ability of CD84 to recruit SHP-2, but instead displaced SHP-1 from the cytoplasmic tail of CD84. These results suggest that mutations in the gene encoding the XLP protein SAP lead to functional defects in the protein that include receptor binding and SHP-1 and SHP-2 displacement and that SAP utilizes different mechanisms to regulate signaling through the CD2 family of receptors.     

2B4 (CD244), NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells

The recently described family of SLAM-related receptors plays an important role in the modulation of lymphocyte activity. The members of this family expressed on human NK cells are 2B4 (CD244), NTB-A and CRACC (CS1). The ligands of these surface receptors are also present on all human NK cells, suggesting that 2B4, NTB-A and CRACC are engaged during the contact of neighboring NK cells. Here we investigate the functional consequence of this interaction. We show that blocking the engagement of 2B4, NTB-A and CRACC has no effect on the proliferation or the development of the cytotoxic potential of human NK cells. However, triggering of 2B4, NTB-A or CRACC by their physiological ligands on MHC class I-negative target cells induces potent NK cell cytotoxicity. This suggests that the engagement of inhibitory receptors by MHC class I on neighboring NK cells blocks 2B4-, NTB-A- and CRACC-induced NK cell cytotoxicity, thereby ensuring that NK cells do not kill each other. In support of this, limiting inhibitory receptor engagement by antibodies leads to the autologous killing of NK cells in a 2B4-, NTB-A- and CRACC-dependent manner.     

NKG2D engagement on human NK cells leads to DNAM-1 hypo-responsiveness through different converging mechanisms

Natural killer (NK) cell activation is regulated by activating and inhibitory receptors that facilitate diseased cell recognition. Among activating receptors, NKG2D and DNAM-1 play a pivotal role in anticancer immune responses since they bind ligands upregulated on transformed cells. During tumor progression, however, these receptors are frequently downmodulated and rendered functionally inactive. Of note, NKG2D internalization has been associated with the acquisition of a dysfunctional phenotype characterized by the cross-tolerization of unrelated activating receptors. However, our knowledge of the consequences of NKG2D engagement is still incomplete. Here, by cytotoxicity assays combined with confocal microscopy, we demonstrate that NKG2D engagement on human NK cells impairs DNAM-1-mediated killing through two different converging mechanisms: by the upregulation of the checkpoint inhibitory receptor TIGIT, that in turn suppresses DNAM-1-mediated cytotoxic function, and by direct inhibition of DNAM-1-promoted signaling. Our results highlight a novel interplay between NKG2D and DNAM-1/TIGIT receptors that may facilitate neoplastic cell evasion from NK cell-mediated clearance.     

Role of NKG2D in tumor cell lysis mediated by human NK cells: cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin

NKG2D is a recently described activating receptor expressed by both NK cells and CTL. In this study we investigated the role of NKG2D in the natural cytolysis mediated by NK cell clones. The role of NKG2D varied depending on the type of target cells analyzed. Lysis of various tumors appeared to be exclusively natural cytotoxicity receptors (NCR) dependent. In contrast, killing of another group of target cells, including not only the epithelial cell lines HELA and IGROV-1, but also the FO-1 melanoma, the JA3 leukemia, the Daudi Burkitt lymphoma and even normal PHA-induced lymphoblasts, involved both NCR and NKG2D. Notably, NK cell clones expressing low surface densities of NCR (NCR(dull)) could lyse these tumors in an exclusively NKG2D-dependent fashion. Remarkably, not all of these targets expressed MICA/B, thus implying the existence of additional ligands recognized by NKG2D, possibly represented by GPI-linked molecules. Finally, we show that the engagement of different HLA class I-specific inhibitory receptors by either specific antibodies or the appropriate HLA class I ligand led to inhibition of NKG2D-mediated NK cell triggering.     

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.