Retinoic Acid Early Inducible-1 Research Articles

Kupffer Cells Regulate Natural Killer Cells Via the NK group 2, Member D (NKG2D)/Retinoic Acid Early Inducible-1 (RAE-1) Interaction and Cytokines in a Primary Biliary Cholangitis Mouse Model.

BackgroundKupffer cells and natural killer (NK) cells has been identified as contributing factors in the pathogenesis of hepatitis, but the detailed mechanism of these cell types in the pathogenesis of primary biliary cholangitis (PBC) is poorly understood.Material/MethodsIn this study, polyinosinic: polycytidylic acid (poly I: C), 2-octynoic acid-bovine serum albumin (2OA-BSA) and Freund’s adjuvant (FA) were injected to establish a murine PBC model, from which NK cells and Kupffer cells were extracted and isolated. The cells were then co-cultivated in a designed culture system, and then NK group 2, member D (NKG2D), retinoic acid early inducible-1 (RAE-1), F4/80, and cytokine expression levels were detected.ResultsThe results showed close crosstalk between Kupffer cells and NK cells. PBC mice showed increased surface RAE-1 protein expression and Kupffer cell cytokine secretion, which subsequently activated NK cell-mediated target cell killing via NKG2D/RAE-1 recognition, and increased inflammation. NK cell-derived interferon-γ (IFN-γ) and Kupffer cell-derived tumor necrosis factor α (TNF-α) were found to synergistically regulate inflammation. Moreover, interleukin (IL)-12 and IL-10 improved the crosstalk between NK cells and Kupffer cells.ConclusionsOur findings in mice are the first to suggest the involvement of the NKG2D/RAE-1 interaction and cytokines in the synergistic effects of NK and Kupffer cells in PBC.

Dendritic Epidermal T Cells in Allergic Contact Dermatitis

Allergic contact dermatitis (ACD) is a common inflammatory skin disease with a prevalence of approximately 20% in the European population. ACD is caused by contact allergens that are reactive chemicals able to modify non-immunogenic self-proteins to become immunogenic proteins. The most frequent contact allergens are metals, fragrances, and preservatives. ACD clinically manifests as pruritic eczematous lesions, erythema, local papules, and oedema. ACD is a T cell-mediated disease, involving both CD4+ and CD8+ T cells. In addition, γδ T cells appear to play an important role in the immune response to contact allergens. However, it is debated whether γδ T cells act in a pro- or anti-inflammatory manner. A special subset of γδ T cells, named dendritic epidermal T cells (DETC), is found in the epidermis of mice and it plays an important role in immunosurveillance of the skin. DETC are essential in sensing the contact allergen-induced stressed environment. Thus, allergen-induced activation of DETC is partly mediated by numerous allergen-induced stress proteins expressed on the keratinocytes (KC). Several stress proteins, like mouse UL-16-binding protein-like transcript 1 (Mult-1), histocompatibility 60 (H60) and retinoic acid early inducible-1 (Rae-1) α-ε family in mice and major histocompatibility complex (MHC) class I—chain-related A (MICA) in humans, are upregulated on allergen-exposed KC. Allergen-induced stress proteins expressed on the KC are consequently recognized by NKG2D receptor on DETC. This review focuses on the role of γδ T cells in ACD, with DETC in the spotlight, and on the role of stress proteins in contact allergen-induced activation of DETC.

Combining radiation therapy with immunotherapy: clinical translation

Ionizing radiation induces immunogenic cell death of tumors, an effect likely to contribute to the success associated with radiotherapy of cancer [1]. Recent discovery suggests that radiotherapy can be applied as a powerful adjuvant to immunotherapy and, in fact, can contribute to convert the irradiated tumor into an in situ vaccine, resulting in specific immunity against metastases [2]. Preclinical models of syngeneic tumors have reliably predicted clinical success, in distinct tumor settings and immunotherapy/radiation combinations [3-5]. As a first proof of principle trial, we translated the preclinical evidence of a successful combination with Flt3 ligand and RT [6] to a protocol of GM-CSF and RT, and demonstrated out of field objective responses in 27% of patients with multiple metastases of solid tumors, defined as an abscopal effect [7]. Parallel mechanistic studies in the lab in the syngeneic 4T1 mouse model of metastatic breast cancer demonstrated at intratravital microscopy that RT with anti-CTLA-4 increased the arrest of T cells in contact with tumor cells. The latter required interaction of NKG2D on CD8+ T cells with its ligand retinoic acid early inducible-1 (Rae-1) on the tumor cells, up-regulated by RT. Blocking NKG2D-Rae-1 interactions increased markedly the motility of anti-CTLA-4 treated T cells inhibiting their contact with irradiated tumor cells, and abrogated immune-mediated tumor rejection, suggesting a critical role of radiation-induced NKG2D ligands for the antitumor effects of anti-CTLA-4 [8]. In humans, a similar block of NKG2D is mediated by soluble MICA (sMICA), which is released by some tumors and reaches high levels in the serum [9]. Dranoff et al reported that in some patients sMICA levels dropped after initiation of Ipilimumab, due to the generation of anti-sMICA antibodies that led to its clearance [10]. Decreased levels of sMICA were associated with increased expression of NKG2D in T and NK cells, and corresponded to response to treatment. Anti-sMICA antibodies and sMICA levels can be measured in serum with ELISA by using recombinant MICA protein and anti-human MICA monoclonal antibodies [10]. Since RT is known to upregulate MICA on the surface of tumor cells [11] biopsies of tumors before and after radiotherapy and Ipilimumab could also be tested for expression of MICA.The preclinical success of the combination of anti-CTLA-4 antibody and RT was mirrored by abscopal responses in metastatic melanoma and NSCLC patients irradiated to one lesion, during Ipilimumab. This evidence inspired our current trial testing radiotherapy with CTLA-4 blockade in metastatic melanoma. In this study patients with newly diagnosed metastatic melanoma eligible to first line Ipilimumab are randomly assigned to Ipilimumab alone or Ipilimumab and radiotherapy to one metastatic lesion. Preliminary results in seven patients demonstrate feasibility of the combination, without additive toxicity. The novel role of radiotherapy as a powerful adjuvant to immunotherapy warrants more research to define the optimal immunotherapy/RT combinations: currently 35 trials of RT+immunotherapy are ongoing in USA.

Successful clinical translation of preclinical combinations of radiation and immunotherapy

Ionising radiation (IR) can induce immunogenic cell death in tumours, an effect likely to contribute to the success associated with radiotherapy (RT) for cancer. Recent studies suggest that radiotherapy can be applied as a powerful adjuvant to immunotherapy, and can even contribute to converting the irradiated tumour into an in situ vaccine, resulting in specific immunity against metastases [1]. Importantly, preclinical models of syngeneic tumours have reliably predicted clinical success in several distinct tumour settings and with several different immunotherapy/radiation combinations. As a proof-of-principle trial, we have translated the preclinical evidence of a successful combination with Flt3 ligand and RT to a protocol of granulocyte–monocyte colony-stimulating factor (GM-CSF) and IR, and demonstrated out-of-field objective responses in 27% of patients with multiple metastases of solid tumours, defined as an abscopal effect [2] In-parallel mechanistic studies in the laboratory were conducted in the syngeneic mouse models of metastatic breast cancer. Results showed that radiation was synergistic with anti-CTLA-4 treatment, a strategy to break immune tolerance to the tumor. Multiple mechanisms contributed to thus synergy, including the RT-induced upregulation of a chemokine that promoted homing of effector T cells to the tumor. Intravital microscopy demonstrated that while both IR and CTLA-4 blockade given as monotherapy enhanced the motility of activated CD8 T cells infiltrating 4T1 tumours, IR with anti-CTLA-4 increased the arrest of T cells in contact with tumour cells, promoting the formation of an immune synapse between cytotoxic T cells and their targets. The latter required interaction between NKG2D on CD8+ T cells and its ligand, retinoic acid early inducible-1 (Rae-1), on the tumour cells, which was up-regulated by IR. Blocking NKG2D–Rae-1 interactions markedly increased the motility of anti-CTLA-4 treated T cells within irradiated tumours, inhibiting their contact with tumour cells and also abrogated immune-mediated tumour rejection [4]. The preclinical success of the combination of anti-CTLA-4 antibody and IR was mirrored by abscopal responses seen in metastatic melanoma and non-small-cell lung cancer (NSCLC) patients irradiated in one lesion during ipilimumab therapy. These clinical observations strongly suggest that the effects of IR identified in experimental models are relevant to patients. IR can stimulate the anti-tumor immune response, increasing the proportion of patients who respond to checkpoint blockade treatment, an hypothesis currently being tested in clinical trials. The same pattern of abscopal responses has been observed in preclinical models was also demonstrated in clinical cases of lymphomas and breast cancers treated by combinations of RT and toll-like receptor agonists. Finally, blocking tumour growth factor beta (TGF-beta) during RT preclinically has demonstrated abscopal effects that have been confirmed in one patient accrued to a trial of anti-human TGFb antibody fresolimumab and RT. While promising, this evidence remains preliminary and warrants more research to define the optimal combinations of immunotherapy and RT, to best exploit this novel role of ionising radiation [3].

Structural basis of mouse cytomegalovirus m152/gp40 interaction with RAE1γ reveals a paradigm for MHC/MHC interaction in immune evasion.

Natural killer (NK) cells are activated by engagement of the NKG2D receptor with ligands on target cells stressed by infection or tumorigenesis. Several human and rodent cytomegalovirus (CMV) immunoevasins down-regulate surface expression of NKG2D ligands. The mouse CMV MHC class I (MHC-I)-like m152/gp40 glycoprotein down-regulates retinoic acid early inducible-1 (RAE1) NKG2D ligands as well as host MHC-I. Here we describe the crystal structure of an m152/RAE1γ complex and confirm the intermolecular contacts by mutagenesis. m152 interacts in a pincer-like manner with two sites on the α1 and α2 helices of RAE1 reminiscent of the NKG2D interaction with RAE1. This structure of an MHC-I-like immunoevasin/MHC-I-like ligand complex explains the binding specificity of m152 for RAE1 and allows modeling of the interaction of m152 with classical MHC-I and of related viral immunoevasins.

Suppressing T cell motility induced by anti–CTLA-4 monotherapy improves antitumor effects

A promising strategy for cancer immunotherapy is to disrupt key pathways regulating immune tolerance, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4). However, the determinants of response to anti-CTLA-4 mAb treatment remain incompletely understood. In murine models, anti-CTLA-4 mAbs alone fail to induce effective immune responses to poorly immunogenic tumors but are successful when combined with additional interventions, including local ionizing radiation (IR) therapy. We employed an established model based on control of a mouse carcinoma cell line to study endogenous tumor-infiltrating CD8+ T lymphocytes (TILs) following treatment with the anti-CTLA-4 mAb 9H10. Alone, 9H10 monotherapy reversed the arrest of TILs with carcinoma cells in vivo. In contrast, the combination of 9H10 and IR restored MHC class I-dependent arrest. After implantation, the carcinoma cells had reduced expression of retinoic acid early inducible-1 (RAE-1), a ligand for natural killer cell group 2D (NKG2D) receptor. We found that RAE-1 expression was induced by IR in vivo and that anti-NKG2D mAb blocked the TIL arrest induced by IR/9H10 combination therapy. These results demonstrate that anti-CTLA-4 mAb therapy induces motility of TIL and that NKG2D ligation offsets this effect to enhance TILs arrest and antitumor activity.

Retinoic acids and hepatic stellate cells in liver disease

Quiescent hepatic stellate cells (HSCs) in healthy liver store 80% of total liver retinols and release them depending on the extracellular retinol status. However, HSCs activated by liver injury lose their retinols and produce a considerable amount of extracellular matrix, subsequently leading to liver fibrosis. Emerging evidence suggests that retinols and their metabolites such as retinoic acids (RAs) contribute to liver regeneration, fibrosis and tumor. However, it is not clear yet why HSCs lose retinol, which enzymes are involved in the retinol metabolism of HSCs and what function of retinol metabolites on HSCs upon liver injury. Recently, our group and collaborators have demonstrated that during activation, HSCs not only lose retinols but also metabolize them into RAs by alcohol dehydrogenases and retinaldehyde dehydrogenases. As transcriptional factors, metabolized RAs induce retinoic acid early inducible-1 and suppressor of cytokine signaling 1 in HSCs, which plays an important role in the interaction between HSCs and natural killer cells. In addition, RAs released from HSCs may induce hepatic cannabinoid receptor 1 expression in alcoholic liver steatosis or regulate immune responses upon liver inflammation. The present review summarizes the role of endogenous metabolized RAs on HSCs themselves and on other liver cells including hepatocytes and immune cells. Moreover, the effects of exogenous retinol and RA treatments on HSCs and liver disease are discussed.

Toll-like receptor 4 engagement contributes to expression of NKG2D ligands by renal tubular epithelial cells

Engagement of Toll-like receptor (TLR) 4 on intrinsic kidney cells is critical for the full development of renal ischaemia-reperfusion injury (IRI). Effects of TLR signalling in renal parenchymal cells include the production of cytokines, chemokines and other soluble mediators which contribute to local inflammation and leucocyte accumulation. Whether engagement of TLR4 on kidney cells results in additional pro-inflammatory modifications of the renal microenvironment remains to be determined. Renal IRI was induced by clamping of the renal pedicles, and expression of NKG2D ligands in mice deficient in TLR4 or its adaptor molecule MyD88, or else pretreated with blocking antibodies against the endogenous TLR4 ligand HMGB1, was compared to that in wild-type mice. Cultures of isolated renal tubular epithelial cells (TECs) from WT, TLR4(-/-) and MyD88(-/-) mice were stimulated with the TLR4 ligand lipopolysaccharide (LPS), or mineral oil occlusion was used to simulate IRI in vitro, prior to determination of NKG2D ligand expression. Chimeric mice lacking TLR4 in either the bone marrow derived or the parenchymal compartment were also subjected to IRI. In this study, we demonstrate a substantial increase in the expression of the NKG2D ligands retinoic acid early inducible-1 (RAE-1), murine ULBP-like transcript 1 (MULT-1) and histocompatibility-60 (H-60) in mouse kidneys during renal IRI. Expression of NKG2D ligands was attenuated in mice deficient in either TLR4 or the adaptor molecule MyD88. Antibody blockade of HMGB1 reduced NKG2D ligand expression by a comparable extent to TLR4 deficiency and did not result in further reduction of NKG2D ligand expression in TLR4(-/-) mice. Isolated TECs from normal mice but not those with defects in the TLR4-MyD88 signalling pathway expressed RAE-1 and MULT-1 upon exposure to LPS and after being subjected to in vitro conditions resembling ischaemia-reperfusion. TLR4 competence in the parenchymal but not the bone marrow-derived compartment was required for RAE-1 up-regulation in mouse kidneys after ischaemia, while TLR4 signalling in both compartments contributed to the intrarenal expression of MULT-1 during IRI. Expression of the NKG2D ligands RAE-1 and MULT-1 on kidney cells in response to TLR4 engagement by HMGB1 represents another mechanism by which TLR4 signalling may participate in the pathogenesis of renal IRI.

NKG2D–retinoic acid early inducible‐1 recognition between natural killer cells and kupffer cells in a novel murine natural killer cell–dependent fulminant hepatitis†

Increasing evidence suggests the contribution of natural killer (NK) cells to pathogenesis of human hepatitis, but the detailed mechanisms have yet to be clearly elucidated. In this study, injection of polyinosinic:polycytidylic acid (poly I:C) and D-galactosamine (D-GalN) was used to establish a novel murine fulminant hepatitis model: results showed that predepletion of either NK cells or Kupffer cells could completely abolish the liver injury. Injection of poly I:C/D-GalN into mice could promote tumor necrosis factor-alpha production and surface retinoic acid early inducible-1 (Rae1) protein expression by Kupffer cells, which then activated NK cells to produce interferon-gamma via NKG2D-Rae1 recognition. NK cell-derived interferon-gamma and Kupffer cell-derived tumor necrosis factor-alpha synergistically mediated the severe liver injury. Moreover, Kupffer cell-derived interleukin-12 and interleukin-18 were also found to improve cross talk between NK cells and Kupffer cells. These results provide the first in vivo evidence that NKG2D/ligand interaction is involved in the synergic effects of NK cells and Kupffer cells on acute liver injury.

NKG2D‐Rea1 recognition between NK cells and Kupffer cells in a novel NK cell‐dependent fulminant hepatitis

Increasing evidences suggest the contribution of NK cells in the pathogenesis of human hepatitis, but the mechanisms remain to be further investigated. In this study, we demonstrate that poly I:C can induce severe liver injury in D‐galactosamine (D‐GalN) sensitized mice. Further experiments show that the liver injury induced by poly I:C/D‐GalN depends on the cooperation between NK cells and Kupffer cells. poly I:C/GalN induce Kupffer cells to produce TNF‐¦Á and to express the retinoic acid early inducible‐1 (RAE 1) proteins, which then stimulates NK cells to produce IFN‐¦Ã via NKG2D‐RAE 1 interaction. The NK‐derived IFN‐¦Ã and Kupffer cells‐derived TNF‐¦Á induce severe liver injury via synergic effects. These results suggest that NK cells and Kupffer cells have a synergic effect on liver injury and provide the first in vivo evidence that NKG2D/ligand interaction are involved in the cooperation between NK cells and macrophages. Our results also provide an NK cells‐mediated hepatitis model.

Blockade of NKG2D on NKT cells prevents hepatitis and the acute immune response to hepatitis B virus

Hepatitis B virus (HBV) is a hepadnavirus that is a major cause of acute and chronic hepatitis in humans. Hepatitis B viral infection itself is noncytopathic, and it is the immune response to the viral antigens that is thought to be responsible for hepatic pathology. Previously, we developed a transgenic mouse model of primary HBV infection and demonstrated that the acute liver injury is mediated by nonclassical natural killer (NK)T cells, which are CD1d-restricted, but nonreactive to alpha-GalCer. We now demonstrate a role for NKG2D and its ligands in this nonclassical NKT cell-mediated immune response to hepatitis B virus and in the subsequent acute hepatitis that ensues. Surface expression of NKG2D and one of its ligands (retinoic acid early inducible-1 or RAE-1) are modulated in an HBV-dependent manner. Furthermore, blockade of an NKG2D-ligand interaction completely prevents the HBV- and CD1d-dependent, nonclassical NKT cell-mediated acute hepatitis and liver injury. This study has major implications for understanding activation of NKT cells and identifies a potential therapeutic target in treating hepatitis B viral infection.

NKG2D recognition mediates Toll-like receptor 3 signaling-induced breakdown of epithelial homeostasis in the small intestines of mice.

Toll-like receptors (TLRs) and NK receptors are the two most important receptor families in innate immunity. Although it has been observed that TLR signaling can induce or up-regulate the expression of the ligands for stimulatory NK receptors on monocytes or muscle cells, there is not yet a report indicating whether TLR signaling can break down self-tolerance through NK receptors. The present work reports that TLR3 signaling by polyinosinic-polycytidylic acid stimulation induces intestinal epithelial cells (IECs) to express retinoic acid early inducible-1 (a ligand for NKG2D) and to induce NKG2D expression on CD8alphaalpha intestinal intraepithelial lymphocytes by IL-15 derived from TLR3-activated IECs. The blockade of interaction between NKG2D and Rae1 inhibits the cytotoxicity of intraepithelial lymphocytes against IECs in a cell-cell contact-dependent manner and therefore alleviates polyinosinic-polycytidylic acid-induced epithelial destruction and acute mucosal injury of small intestine. These results demonstrate that TLR signaling induces tissue injury through the NKG2D pathway, suggesting that TLR signaling may break down self-tolerance through induction of abnormal expression of ligands for stimulatory NK receptors.

Genomic analysis of RAE-1 genes (35.30)

Abstract NKG2D is an activating receptor on NK cells and activated CD8 T cells that has been implicated in tumor immunity, viral immunity and autoimmunity. The ligands of NKG2D are tightly regulated stress-inducible molecules, whose expression is triggered by transformation or infection. In mice, these NKG2D ligands include the retinoic acid early inducible-1 (RAE-1) proteins. However, the mechanisms of transcriptional regulation in RAE-1 genes are poorly understood. We examined what transcription factors regulate RAE-1 expression. We found that RAE-1 delta has six exons and RAE-1 epsilon has seven exons. We isolated genomic DNAs of upstream from exon one which are thought of RAE-1 promoters in RAE-1 delta or RAE-1 epsilon. We found that consensus sequences of several transcription factors are present in RAE promoters. Therefore, these transcription factors may regulate RAE-1 expressions. This work is supported by Grants-in Aid for Science Research from the Ministries of Education, Culture, Sports, Science, and Technology of Japan, Grants-in Aid for Science Research from the Ministriesof Health, Labor, and Welfare of Japan, Human Frontier Science Program CDA, The Mitsubishi Foundation, The Sumitomo Foundation, Takeda Science Foundation and Uehara Memorial Foundation.

Immunogenicity and Engraftment of Mouse Embryonic Stem Cells in Allogeneic Recipients

Embryonic stem cells (ESCs) are pluripotent and therefore able to differentiate both in vitro and in vivo into specialized tissues under appropriate conditions, a property that could be exploited for cellular therapies. However, the immunological nature of these cells in vivo has not been well understood. In vitro, mouse-derived ESCs fail to stimulate T cells, but they abrogate ongoing alloresponses by a process that requires cell-cell contact. We further show that despite a high expression of the NKG2D ligand retinoic acid early inducible-1 by mouse ESCs, they remain resistant to natural killer cell lysis. In vivo, allogeneic mouse ESCs populate the thymus, spleen, and liver of sublethally irradiated allogeneic host mice, inducing apoptosis to T cells and establishing multilineage mixed chimerism that significantly inhibits alloresponses to donor major histocompatibility complex antigens. Immunohistochemical imaging revealed a significant percentage of ESC-derived cells in the splenic marginal zones, but not in the follicles. Taken together, the data presented here reveal that nondifferentiated mouse embryonic stem cells are non-immunogenic and appear to populate lymphoid tissues in vivo, leading to T-cell deletion by apoptosis.

Cutting edge: Toll-like receptor signaling in macrophages induces ligands for the NKG2D receptor.

Macrophages recognize the presence of infection by using the Toll-like receptor (TLR) family of proteins that detect ligands on bacterial, viral, and fungal pathogens. We show that murine macrophages stimulated with pathogen products known to signal through TLRs express ligands for the NKG2D receptor, found on NK cells, activated CD8(+) T cells and activated macrophages. TLR signaling, through the MyD88 adaptor, up-regulates transcription of the retinoic acid early inducible-1 (RAE-1) family of NKG2D ligands, but not H-60 or murine UL16-binding protein-like transcript-1. RAE-1 proteins are found on the surface of activated, but not resting, macrophages and can be detected by NKG2D on NK cells resulting in down-regulation of this receptor both in vitro and in vivo. RAE-1-NKG2D interactions provide a mechanism by which NK cells and infected macrophages communicate directly during an innate immune response to infection.

NKG2D-mediated natural killer cell protection against cytomegalovirus is impaired by viral gp40 modulation of retinoic acid early inducible 1 gene molecules.

Natural killer (NK) cells play a critical role in the innate immune response against cytomegalovirus (CMV) infections. Although CMV encodes several gene products committed to evasion of adaptive immunity, viral modulation of NK cell activity is only beginning to be appreciated. A previous study demonstrated that the mouse CMV m152-encoded gp40 glycoprotein diminished expression of ligands for the activating NK cell receptor NKG2D on the surface of virus-infected cells. Here we have defined the precise ligands that are affected and have directly implicated NKG2D in immune responses to CMV infection in vitro and in vivo. Murine CMV (MCMV) infection potently induced transcription of all five known retinoic acid early inducible 1 (RAE-1) genes (RAE-1α, RAE-1β, RAE-1δ, RAE-1ɛ, and RAE-1γ), but not H-60. gp40 specifically down-regulated the cell surface expression of all RAE-1 proteins, but not H-60, and diminished NK cell interferon γ production against CMV-infected cells. Consistent with previous findings, a m152 deletion mutant virus (Δm152) was less virulent in vivo than the wild-type Smith strain of MCMV. Treatment of BALB/c mice with a neutralizing anti-NKG2D antibody before infection increased titers of Δm152 virus in the spleen and liver to levels seen with wild-type virus. These experiments demonstrate that gp40 impairs NK cell recognition of virus-infected cells through disrupting the RAE-1–NKG2D interaction.

Cutting edge: tumor rejection mediated by NKG2D receptor-ligand interaction is dependent upon perforin.

We have investigated the primary immunity generated in vivo by MHC class I-deficient and -competent tumor cell lines that expressed the NKG2D ligand retinoic acid early inducible-1 (Rae-1) beta. Rae-1beta expression on class I-deficient RMA-S lymphoma cells enhanced primary NK cell-mediated tumor rejection in vivo, whereas RMA-Rae-1beta tumor cells were rejected by a combination of NK cells and CD8(+) T cells. Rae-1beta expression stimulated NK cell cytotoxicity and IFN-gamma secretion in vitro, but not proliferation. Surprisingly, only NK cell perforin-mediated cytotoxicity, but not production of IFN-gamma, was critical for the rejection of Rae-1beta-expressing tumor cells in vivo. This distinct requirement for perforin activity contrasts with the NK cell-mediated rejection of MHC class I-deficient RMA-S tumor cells expressing other activating ligands such as CD70 and CD80. Thus, these results indicated that NKG2D acted as a natural cytotoxicity receptor to stimulate perforin-mediated elimination of ligand-expressing tumor cells.

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, Kärre 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.