NKG2D Receptor Expression Research Articles

A Functional Variation In the NKG2D Gene Regulates NKG2D Receptor Expression and Is Associated with Better Transplant Outcomes After Fully-HLA-Matched Unrelated Bone Marrow Transplantation

AbstractAbstract 221A functional variation in the NKG2D gene regulates NKG2D receptor expression and is associated with better transplant outcomes after fully-HLA-matched unrelated bone marrow transplantationNKG2D, an activating receptor expressed by NK cells and some T cells, plays important roles in both immunity to microbial infections and cancer immune surveillance. A single nucleotide polymorphism (SNP rs1049174) in the 3′-untranslated region (3′UTR) of the NKG2D gene determines two genotypes, HNK1 genotype with high NK cytotoxicity, and the LNK1 genotype with low NK cytotoxicity. Individuals with the HNK1 genotype have reduced risk of cancer (Cancer Res 2006; 66(1):63-70). In the present study, we analyzed the impact of SNP rs1049174 on transplant outcomes after HLA-matched unrelated bone marrow transplantation. The NKG2D genotypes were retrospectively analyzed in a cohort of 360 pairs of patients and their unrelated donors transplanted through the Japan Marrow Donor Program. The donor HNK1 genotype was associated with lower transplant related mortality (TRM) in patients with standard risk (hazard ratio [HR], 0.58; 95% confidence interval [CI], 0.35–0.97; P=0.04) (Fig.1). The donor HNK1 genotype was also associated with a trend toward better OS (HR, 0.69; 95% CI, 0.46–1.04; P=0.08). The host NKG2D genotypes did not significantly influence the transplant outcomes. NK cells from HNK1 donors expressed higher levels of NKG2D on the cell surface, and also expressed higher levels of NKG2D mRNA. Luciferase reporter gene constructs transfected into NK cells showed that the LNK1 construct more effectively attenuated gene expression compared with the HNK1 construct (60% vs. 35%). Bioinformatics tools predicted an interaction between the rs1049174 SNP motif with the micro-RNA-1245 (miR1245). Next, in an mRNA/miRNA binding assay, the LNK allele showed higher affinity to the miR1245 than the HNK1 allele. Moreover transfection of NK cells possessing the LNK1 allele with a miR1245 mimic resulted in 25 fold decrease in mRNA NKG2D level versus only 2 fold decrease in mRNA NKG2D expression in HNK1 allele derived NK cells, thus suggesting that the 3′UTR SNP may regulate the expression of NKG2D through its binding to the putative repressor miR1245. These findings provide evidence that the NKG2D 3′UTR polymorphism is functional, and that the NK cells from donors with the HNK1 genotype exert higher cytotoxicity through their increased expression of NKG2D, thereby resulting in an improved outcome for bone marrow transplantation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

FK506 causes cellular and functional defects in human natural killer cells

The role of NK cells in allogeneic HCT has been increasingly appreciated, particularly in the GVL effect. Although FK506 has been used widely to prevent GVHD, its action was considered to be primarily through activated T cells. In this study, we provide direct evidence for the first time that human NK cells are immediate targets of FK506. Our in vivo data from patients undergoing peripheral blood stem cell transplantation or BMT showed a reduced number of NK cells with down-regulated CD25 expression in their peripheral blood compartment. Likewise, FK506 caused profound inhibition of NK cell proliferation in vitro and suppressed NK cytotoxicity and cytokine secretion in response to IL-2. These defects were accompanied by impaired cell clustering and selective down-regulation of adhesion molecules, ICAM-1, CD2, CD49d, and CD58. Furthermore, FK506 specifically inhibited expression of NKG2D, CD48, and DNAM1 receptors without affecting that of 2B4, NKp30, NKp44, and NKp46. As a result, natural cytotoxicity against K562 tumor targets was impaired, while leaving redirected ADCC via 2B4 intact. Finally, FK506-treated NK cells showed impaired IL-2R signaling and inhibition of STAT3. Collectively, these signaling impairments and selective down-regulation of NK receptors by FK506 may underlie the proliferative and functional defects of NK cells. Thus, our data provide a new insight into the mechanism of immunosuppression by FK506, which should be considered to interpret the outcome of graft transplantation.

Decrease in Phosphorylation of ERK following Decreased Expression of NK Cell-Activating Receptors in Human NK Cell Line Exposed to Asbestos

YT-CB5, which had been continuously cultured with chrysotile B (CB)asbestos, showed impaired cytotoxicity with decreased expression of NKG2D and 2B4 NK cell-activating receptors. In the present study, the phosphorylation of extracellular signal-regulated kinase (ERK), which is known to induce degranulation downstream of many NK cell-activating receptors, was examined in YT-CB5 by flow cytometry and compared with the control line YT-Org. YT-CB5 exhibited impaired phosphorylation of ERK1/2 induced by the recognition of K562 cells, downstream of a process mediated by Src family kinase and phosphoinositide 3-kinase. YT-CB5 also exhibited impaired phosphorylation of ERK1/2 following incubation with K562 cells in the presence of anti-2B4 antibodies, where co-stimulation by 2B4 augmented the phosphorylation of ERK1/2 in YT-CB5 to a similar degree as in YT-Org. The phosphorylation of ERK1/2 induced by an inhibitor against phosphatase (PP) 1 and PP2A was also lower in YT-CB5 compared with YT-Org. Moreover, bead-bound antibodies to NKG2D, which contribute to cytotoxicity against K562 cells, induced negligible phosphorylation of ERK1/2 in YT-CB5, although antibodies to 2B4 induced a comparatively greater level of phosphorylation. Additionally, peripheral blood (PB-) NK cells with low expression of NKG2D showed lower phosphorylation of ERK1/2 mediated by anti-NKG2D antibodies compared with PB-NK cells with high expression of NKG2D. These results indicate that signal transduction events leading to the phosphorylation of ERK is impaired in YT-CB5 due to decreased expression of NKG2D. Further studies are required to clarify whether this suppressive effect of asbestos exposure on NK cells might promote lung cancer and mesothelioma in people who have inhaled asbestos.

The Traffic of the NKG2D/Dap10 Receptor Complex during Natural Killer (NK) Cell Activation

NKG2D is an important activating receptor for triggering the NK cell cytotoxic activity, although chronic engagement of specific ligands by NKG2D is also known to provoke decreased cell surface expression of the receptor and compromised NK cell function. We have studied the dynamics of surface NKG2D expression and how exposure to the specific ligand major histocompatibility complex class I chain-related molecule B (MICB) affects receptor traffic and fate. While in the NKL cell line and "resting" NK cells NKG2D was found principally at the cell surface, in activated primary NK cells an intracellular pool of receptor could also be found recycling to the plasma membrane. Exposure of NK cells to targets expressing MICB resulted in degradation of approximately 50% of total NKG2D protein and lysosomal degradation of the DAP10 adaptor molecule. Consistent with these observations, confocal microscopy experiments demonstrated that DAP10 trafficked to secretory lysosomes in both transfected NKL cells and in activated primary NK cells upon interaction with MICB-expressing target cells. Interestingly, polarization to the synapse of secretory lysosomes containing DAP10 was also observed. The implications of the intracellular traffic of the NKG2D/DAP10 receptor complex for NK cell activation are discussed. We propose that the rapid degradation of NKG2D/DAP10 observed coincident with recruitment of the receptor to the cytotoxic immune synapse may explain the loss of NKG2D receptor expression after chronic exposure to NKG2D ligands.

Insights into the Mechanism Responsible for Spontaneous Acceptance of Liver Allografts (141.41)

Abstract Transplantation of fully histoincompatable livers between strains of rodents often results in spontaneous acceptance of the graft. Lewis rats reject DA livers in 14 days while DA recipients of Lewis livers have long-term graft survival. We sought to determine the mechanism of this spontaneous graft survival. NK cells and subsets of T cells express NKG2D. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We examined NKG2D receptor and ligand expression post-transplant. Groups of Lewis rats received livers from DA (rejection combination) or Lewis donors (syngeneic) and DA rats received livers from Lewis (non-rejection combination) or DA donors (syngeneic). NKG2D expression was low in syngeneic grafts but increased 7-fold in Lewis→DA grafts and 17-fold in DA→Lewis grafts by day 7 post-transplant. Ligands of NKG2D were not expressed in normal liver but were up regulated in all liver grafts by day 1 post-transplant, suggesting that ischemia/reperfusion injury and trauma induce expression of these ligands. RAE1L and RRLT were significantly up regulated specifically in DA→Lewis liver grafts by day 7 post-transplant. In marked contrast, the expression of RAE1L and RRLT in Lewis→ DA liver grafts was similar to the levels detected in syngeneic liver grafts suggesting that signals that lead to an increase in NKG2D ligand expression are absent in this strain combination. In conclusion, the absence of NKG2D ligand expression in the liver graft may play a role in preventing rejection, and decreasing the expression of these proteins may be a useful strategy to promote graft non-responsiveness. Supported by AI044095

Cellular Expression, Trafficking, and Function of Two Isoforms of Human ULBP5/RAET1G

BackgroundThe activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised.Methodology/Principal FindingsWe pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy.Conclusions/SignificanceWe demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality.

Rapid NKG2D Reconstitution on NK Cells Possibly Plays a Major Role in the Development of Graft-Versus-Leukemia Effect in the Early Period after Unrelated Cord Blood Transplantation.

Background: Cord blood transplantation (CBT) is being increasingly used for treatment of hematologic malignancies due to its potent anti-leukemic effects and the low incidence of graft-versus-host disease (GVHD). Recent study has shown that recovery of natural killer (NK) cells is prompt in the blood of CBT recipients both in numbers and functions during the first two months, suggesting that NK cells play an important role in the GVL effect in CBT. NKG2D is an activating receptor expressed by NK cells, which recognizes human MHC class I chain related A and B (MICA/B) and UL16-binding protein 1–4 (ULBP1-4) on target cells, and regulates NK cell activity. However, NKG2D reconstitution following CBT is presently unclear. In this study, we investigated the dynamic expression of NKG2D and other receptors on CD3−CD56+ NK cells in the early period after CBT.Material and methods: The study panel comprised 21 leukemia patients receiving allogeneic hematopoietic cell transplants. Of these, 10 received unrelated CBT and 11 received allogeneic transplants with peripheral blood (PB) combined with bone marrow (BM). To examine the recovery of NKG2D, and other receptors expressed by NK cells, flow cytometry was used to evaluate samples obtained from the recipients on day of clinical engraftment, 30, 60, and 90, respectively following transplantation.Results: Recovery of NK cells was prompt in both transplantation groups (CBT and PB combining with BM), which is consistent with the previous reports. Interestingly, there are significant differences in NKG2D reconstitution between CBT and PB combining with BM transplantation in early stage after transplantation. CBT recipients showed significantly increased expression of NKG2D compared to PB combining with BM transplantation recipients on day of clinical engraftment (79.58±8.71 VS 36.7±14.33 P<0.03 ) and day 30 after transplantation ( 80.03±13.49 VS 56.8±18.21 p<0.035 ). However there was no differences for NKG2D expression between the CBT and PB combined with BM transplantation groups on day 60(82.55±9.10 VS 82.3±28.64 p<0.01 )and on day 90 (85.82±18.21 VS 90.3±27.89 p<0.01), respectively. The inhibitory NK receptor NKG2A reconstitution occurred at the early period, and the expression was not significantly changed during the first three months in CBT, although the level was lower than the other transplantation group. The reconstitution of other inhibitory NK receptors (CD94, CD158a and CD158b) and activating receptor (NKp46) was not found statistically significant differences between the two transplantation groups. T and B cells reconstitution started gradually, and remained at low level at the end of investigation period in CBT recipients.Conclusion: NK cells contribute to GVL effects after transplantation maybe due to rapid NKG2D reconstitution. Rapid NKG2D reconstitution on NK cells in CBT recipients than blood combined bone marrow transplantation suggests that CBT appears superior to blood combined bone marrow transplantation in the treatment of leukemia patients.

Expression of NK cells activation receptors after occupational exposure to toxics: A preliminary study

The expression of NK cells activation receptors was assessed by comparative study of two groups of women workers at a chemical reagents factory, located in Zapopan, Jalisco, Mexico. Twenty of them were exposed to environmental toxics identified and quantified by gas chromatography, and 20 women unexposed to toxic substances. The expression of the surface markers CD56+ and CD3+, and of the activation receptors and co-receptors on NK cells was quantified by flow cytometry. To assess the cellular damage produced by chronic exposure to the toxics, the thiobarbituric acid reacting substances (TBARS) generated and the total plasma antioxidizing capacity (TPAC) were quantified in both groups. The exposed women had been exposed at least to 12 volatile toxic compounds, benzene, benz(a)pyrene, ethylbenzene, dimethylbenz(a)anthracene, xylene, toluene, styrene, chloroform, formaldehyde, iodine, chlorine and fluorine. Significant difference between the two groups was in the proportion of CD3 lymphocytes, 72.7 ± 10.3% in the unexposed women versus 66.8 ± 7.9% in the exposed group ( p < 0.05). The density of expression of NKG2D and NKp30 receptors was significantly higher in the unexposed women compared to the exposed group: NKG2D were 31.3 ± 6.3 and NKp30 were 9.5 ± 5.2 in the unexposed women and 5.14 ± 2.9 ( p < 0.01) and 4.6 ± 1.9 ( p < 0.05), respectively in the exposed women. No statistically significant differences were found in the expression of NKp80, NKp46 and 2B4 receptors. The concentration of TBARS was lower in women from the unexposed group than the corresponding data from women of the exposed group. However, no significant difference was observed in TPAC between the two groups studied. The results of this preliminary study suggest that from the five activation receptors and co-receptors of NK cells evaluated (NKp30, NKp46, NKp80, NKG2D and 2B4), only NKp30 and NKG2D receptor expression was diminished in women exposed to toxics when compared with data from unexposed women. These results suggest that the occupational exposure to mixture of toxics is one of the important factors in the diminution of the NK cell receptor expression.

Peripheral blood gamma-delta T cells in advanced-stage cancer patients

Gamma-delta T (gammadelta T) cells form a subgroup which has been reported to play a role in both natural and acquired immunity. Their levels have been found to increase in some tumour tissues. The aim of this study was to investigate the ratio of gammadelta T cells to all T cells in the peripheral blood of advanced-stage cancer patients; the level of gammadelta T cells expressing the Vdelta2-T-cell receptor (TCR) chain; NKG2D receptor expression; and apoptotic (Annexin-V) gammadelta T-cell levels. Twenty patients with advanced-stage cancer and 13 healthy controls were included. No statistical differences were found between control and patient groups in terms of the gammadelta T/total T-cell ratio (P=0.53), the Vgamma2-TCR expressing gammadelta T-cell ratio (P=0.19) or the Annexin-V ratio (P=0.48). However, NKG2D expression in gammadelta T cells was significantly different between the control and patient groups (P=0.014). In summary it was shown that the levels of NKG2D receptors, which are responsible for the cytolytic effect of gammadelta T cells, were lower in cancer patients than in healthy adults. However, no significant differences were observed in the other parameters studied between groups.

Relations between different coping strategies for social stress, tumor development and neuroendocrine and immune activity in male mice

This study analyzes the effects of acute social stress and different coping strategies employed in response to it on the development of B16F10 melanoma pulmonary metastases, the activation of the HPA axis and the NKG2D receptor expression. To this end, male OF1 mice were subjected to 24 h of social stress using the sensorial contact model. This model includes three 5-min sessions of direct social interaction with resident cagemates selected for consistent levels of aggression. Subjects’ behavior was videotaped and assessed. Six days after the first social interaction (1st social stress), the animals were inoculated with tumor cells or vehicle, and six days later, both tumor-bearing and non tumor-bearing mice were subjected to a second 24 h sensorial contact social stress session (2nd social stress). One hour after the 2nd social interaction, corticosterone levels and NKG2D receptor expression were determined. Lung metastatic foci numbers were determined 21 days after inoculation (15 days post-stress). Social stress increased the number of pulmonary metastases and the serum corticosterone level. A combination of cluster and discriminant analyses established the existence of two types of coping strategies: (1) a passive–reactive strategy characterized by subjects dedicating a greater percentage of time to submission, flee and avoidance behaviors; and (2) an active–proactive strategy, characterized by subjects dedicating a greater percentage of time to attack and non social exploration behaviors. Subjects belonging to the passive–reactive group were found to have a higher number of tumor foci, a higher level of corticosterone and a lower NKG2D receptor expression than subjects in the active–proactive group. These data indicate the relationship between different coping strategies for social stress and tumor development.

Expression of membrane/soluble MHC class I chain-related molecule A and NKG2D receptor in osteosarcoma and its clinical significance

To study the expression of membrane MICA (mMICA), soluble MICA (sMICA) and NKG2D receptor in cases of osteosarcoma and to analyze its clinical significance. Expression of mMICA in osteosarcoma tissue of 43 cases was detected with immunohistochemistry. Expression of NKG2D in peripheral blood lymphocytes of 16 cases was analyzed by flow cytometry. Serum level of soluble MICA (sMICA) was measured by ELISA. mMICA was widely expressed in osteosarcoma tissue (37/43). Expression of NKG2D in peripheral blood lymphocytes was significantly decreased. High levels of mMICA and NKG2D expression were associated with better differentiation and earlier tumor stage of osteosarcoma (P < 0.05). A significant increase in serum level of sMICA was demonstrated in patients with metastasis and advanced tumor. The mMICA expression in tumor tissue, NKG2D expression in peripheral lymphocytes and serum sMICA level correlate with the differentiation and stage of osteosarcoma. These parameters may thus represent potential diagnostic and prognostic markers in patients with osteosarcoma. Manipulation of the MICA-NKG2D pathway may become a target of immunotherapy for osteosarcoma.

Suppressive Effect of a Standardized Mistletoe Extract on the Expression of Activatory NK Receptors and Function of Human NK Cells

Despite long-term use of mistletoe extracts for cancer treatment, their mode of action remains elusive. In this study, it was studied in vitro if mistletoe extract is able to modulate the expression of natural cytotoxic receptors (NCRs) and NKG2D receptor, which stimulate natural killer cell-mediated cytotoxicity. Unexpectedly, a mistletoe extract, ABNOBA viscum Fraxini, inhibited the expression level of NKp46 and NKG2D receptors in dose- and time-dependent manners. The levels of NKp30 and NKG2D receptors were remarkably induced and NKp44 was slightly induced after 48 h treatment with IL-2 and IL-15 in both mRNA and surface expression. The activatory NK receptors were not induced significantly after treatment with IL-12, IL-18, and IL-21 for 48 h. Induction of activatory NK receptors by IL-2 and IL-15 was suppressed almost to the untreated levels by treatment with mistletoe extract, which appeared to induce apoptosis of NK cells in a dose-dependent manner. However, the treatment with IL-2 and IL-15 did not prevent the mistletoe-induced NK-cell death. Mistletoe extract inhibited significantly the cytotoxic activity of resting and IL-2- or IL-15-stimulated NK cells. These results suggest that inhibition of survival and function of NK cells by mistletoe extract may curtail in part the therapeutic effects of mistletoe.

Pretherapy predictive immunomodulation of NK cell activity and expression of activating and inhibitory receptors in stage IV melanoma patients

12514 Background: As melanoma (MM) is an immunogenic tumor with poor response to chemotherapy immunomodulating agents are applied in order to potentiate cytotoxic effect of chemotherapy and enhance antitumor immune response. Beside therapeutic benefit of IFN-α and IL-2, 13-cis retinoic acid (RA), as an antiproliferative, differentiating and immunomodulating agent is also investigated. The effect of these agents on NK cells, as main innate immune system effectors, is being investigated. Methods: 35 patients with MM in stage IV prior to therapy and 20 controls were investigated. We evaluated NK activity and the expression of activating (NKG2D and CD161) and inhibitory (CD158a and CD158b) receptors on freshly isolated PBL. Predicitive immunomodulation was performed in 18 h in vitro treated PBL with rh IL-2 (200U/ml), IFN (250U/ml), RA (10−6M), and their combination. Results: Native NK cell cytotoxic activity and expression of NKG2D and CD161 activating receptors on fresh NK cells in MM patients is significantly decreased compared to controls. Predictive treatments with IL-2, IFN, IFN and RA, unlike RA alone, gave a significant increase in NK cell activity of MM patients. Singly, IFN also induced a significant increase in CD161 expression on NK cells in patients. The treatments gave no change in the expression of CD158b, while RA, alone, induced significant decrease in the expression of the inhibitory CD158a antigen. Evaluation of mRNA of transcription molecule IRF-1 shows that it is promptly up-regulated by IFNα, more by IFNα and RA, while single RA has no effect on mRNA induction. Conclusions: Considering that the mechanism of applied immunomodulating agents is continually investigated in order to optimize the dose and schedule of their administration and, also, considering controversial clinical results of RA application, alone, or with IFN, in this study we give novel results that show no significant effect of RA, whereas, IFN-induced increase in NK cell activity of MM patients is for the first time associated with the increase in the expression of NKG2D and CD161 receptors on CD16+NK cells, and not with the decrease in some inhibitory receptors, as the activity of NK cells is regulated by the balance of these two types of signals. No significant financial relationships to disclose.

NKp46 and NKG2D receptor expression in NK cells with CD56dim and CD56bright phenotype: regulation by histamine and reactive oxygen species

The cytotoxicity of natural killer (NK) cells is dependent on the interaction between target cell ligands and a series of stimulatory receptors on NK cells. Two of these triggering receptors, the NKp46 natural cytotoxicity receptor (NKp46) and the major histocompatibility complex (MHC) class I-interactive NKG2D receptor, are deficiently expressed by NK cells recovered from patients with acute myeloid leukaemia (AML), but little is known regarding the regulation of NKp46 and NKG2D expression. Here we report that mononuclear and polymorphonuclear phagocytes downregulate the cell surface density of NKp46 and NKG2D on NK cells with CD56(dim) phenotype in vitro by a mechanism that is dependent on the availability of phagocyte-derived reactive oxygen species (ROS). Histamine maintained NKp46 and NKG2D expression despite the presence of inhibitory phagocytes by targeting an H2 receptor on phagocytes. By contrast, NKp46 and NKG2D expression by the CD56(bright) subset of NK cells was resistant to inhibition by phagocytes. Our findings are suggestive of a novel mechanism of relevance to the regulation of NKp46/NKG2D receptor expression. Moreover, our findings suggest that the previously reported action of histamine on NK cell-mediated killing of leukaemic cells may be related to the preservation of activatory NK-cell receptors.

TNFα Stimulates NKG2D-Mediated Lytic Activity of Acute Myeloid Leukemic Cells.

The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFα, they display cytolytic activity towards various cellular targets including CFU-GM. This mechanism is dependent on stimulation of Granzyme B/Perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patient with acute myeloid leukemia (AML) (FAB M0 to M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by pool >15% of leukemic cells. Futhermore, CD34+ hematopoietic progenitors undergoing granulo-monocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which upon stimulation by TNFα leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFα accumulation, such as AML or myelodisplasic syndromes.

TNFα stimulates NKG2D-mediated lytic activity of acute myeloid leukemic cells

The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show here that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFalpha, they display cytolytic activity toward various cellular targets including CFU-GM. This mechanism is dependent on stimulation of the granzyme B/perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP 10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patients with acute myeloid leukemia (AML) (FAB M0-M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by a pool >15% of leukemic cells. Furthermore, CD34+ hematopoietic progenitors undergoing granulomonocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which, upon stimulation by TNFalpha, leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFalpha accumulation, such as AML or myelodisplasic syndromes.

CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor–β–dependent manner

Tumor growth promotes the expansion of CD4+CD25+ regulatory T (T reg) cells that counteract T cell–mediated immune responses. An inverse correlation between natural killer (NK) cell activation and T reg cell expansion in tumor-bearing patients, shown here, prompted us to address the role of T reg cells in controlling innate antitumor immunity. Our experiments indicate that human T reg cells expressed membrane-bound transforming growth factor (TGF)–β, which directly inhibited NK cell effector functions and down-regulated NKG2D receptors on the NK cell surface. Adoptive transfer of wild-type T reg cells but not TGF-β −/− T reg cells into nude mice suppressed NK cell–mediated cytotoxicity, reduced NKG2D receptor expression, and accelerated the growth of tumors that are normally controlled by NK cells. Conversely, the depletion of mouse T reg cells exacerbated NK cell proliferation and cytotoxicity in vivo. Human NK cell–mediated tumor recognition could also be restored by depletion of T reg cells from tumor-infiltrating lymphocytes. These findings support a role for T reg cells in blunting the NK cell arm of the innate immune system.

Natural Killer andNK-LikeT-Cell Activation in Colorectal Carcinoma Patients Treated with Autologous Tumor-Derived Heat Shock Protein 96

Heat shock proteins (HSPs) are involved in the activation of both adaptive and innate immune systems. Here, we report that vaccination with autologous tumor-derived HSP96 of colorectal cancer patients, radically resected for liver metastases, induced a significant boost of natural killer (NK) activity detected as cytokine secretion and cytotoxicity in the presence of NK-sensitive targets. Increased NK activity was associated with a raise in CD3-CD56+ NK and/or CD3+CD56+ NK-like T cells, displaying enhanced expression of NKG2D and/or NKp46 receptors. Up-regulated expression of CD83 and CD40 and increased interleukin-12 release on stimulation were observed in CD14+ cells from post-HSP96 peripheral blood mononuclear cells, suggesting an indirect pathway of NK stimulation by HSP96-activated monocytes. Additionally, CD3-CD56+ and CD3+CD56+ lymphocytes were found to undergo functional and phenotypic activation on in vitro exposure to HSP96 even in the absence of monocytes, supporting a potential direct activity of HSP96 on these cell subsets. This evidence was confirmed by the specific binding of FITC-conjugated HSP96 to a subset of both CD3-CD56+ and CD3+CD56+ cells in peripheral blood mononuclear cells from colorectal cancer patients. Altogether, these findings identify the activation of the NK compartment as an additional immunologic effect of autologous tumor-derived HSP96 administration in cancer patients.

Opposing effect of IFNγ and IFNα on expression of NKG2 receptors: Negative regulation of IFNγ on NK cells

The effector functions of natural killer (NK) cells are regulated by integrated signals across an array of stimulatory and inhibitory receptors interacting with target cell surface ligands. The regulatory effect of interferon-α (IFNα) and interferon-γ (IFNγ) on expression of the family of NKG2 receptors, stimulatory NKG2D receptor and inhibitory NKG2A receptor, and cytolysis of the target tumor cells (MICA + and HLA-E +) were studied. Results show that IFNγ and IFNα influence NK cell function differently. Interferon-α stimulates expression of stimulatory NKG2D receptors and inhibits the expression of inhibitory NKG2A receptors on NK cells. Contrary to the stimulatory effect of IFNα, IFNγ inhibits cytolysis by NK cells of tumor cells expressing MICA or HLA-E cell surface proteins. Blocking NKG2D or NKG2A receptor activity with monoclonal antibodies partly attenuates the inhibitory effect of IFNγ while promoting the effects of IFNα on NK cytolysis. These results show for the first time that IFNγ negatively regulates NK cells through NKG2 receptors, and that the balance between stimulatory and inhibitory signals through the NKG2 family of receptors may be controlled by two opposing interferons. Modulating the balance between stimulatory and inhibitory signals through cell surface receptors on NK cells may open a new approach to NK cell-based biotherapy for cancer and infectious diseases.

Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells.

The surface density of the triggering receptors responsible for the natural killer (NK)-mediated cytotoxicity is crucial for the ability of NK cells to kill susceptible target cells. In this study, we show that transforming growth factor beta1 (TGFbeta1) down-regulates the surface expression of NKp30 and in part of NKG2D but not that of other triggering receptors such as NKp46. The TGFbeta1-mediated inhibition of NKp30 surface expression reflects gene regulation at the transcriptional level. NKp30 has been shown to represent the major receptor involved in the NK-mediated killing of dendritic cells. Accordingly, the TGFbeta1-dependent down-regulation of NKp30 expression profoundly inhibited the NK-mediated killing of dendritic cells. On the contrary, killing of different NK-susceptible tumor cell lines was variably affected, reflecting the differential usage of NKp30 and/or NKG2D in the lysis of such tumors. Our present data suggest a possible mechanism by which TGFbeta1-producing dendritic cells may acquire resistance to the NK-mediated attack.