技术资料

HCMV is a highly sophisticated virus that has developed various mechanisms for immune evasion and viral dissemination throughout the body (partially mediated by neutrophils). NK cells play an important role in elimination of HCMV-infected cells. Both neutrophils and NK cells utilize similar sets of chemokine receptors to traffic,to and from,various organs. However,the mechanisms by which HCMV attracts neutrophils and not NK cells are largely unknown. Here,we show a unique viral protein,vCXCL1,which targets three chemokine receptors: CXCR1 and CXCR2 expressed on neutrophils and CXCR1 and CX3CR1 expressed on NK cells. Although vCXCL1 attracted both cell types,neutrophils migrated faster and more efficiently than NK cells through the binding of CXCR2. Therefore,we propose that HCMV has developed vCXCL1 to orchestrate its rapid systemic dissemination through preferential attraction of neutrophils and uses alternative mechanisms to counteract the later attraction of NK cells. View Publication

Group 2 innate lymphoid cells (ILC2 cells) are important for type 2 immune responses and are activated by the epithelial cytokines interleukin 33 (IL-33),IL-25 and thymic stromal lymphopoietin (TSLP). Here we demonstrated that IL-1β was a critical activator of ILC2 cells,inducing proliferation and cytokine production and regulating the expression of epithelial cytokine receptors. IL-1β also governed ILC2 plasticity by inducing low expression of the transcription factor T-bet and the cytokine receptor chain IL-12Rβ2,which enabled the conversion of these cells into an ILC1 phenotype in response to IL-12. This transition was marked by an atypical chromatin landscape characterized by the simultaneous transcriptional accessibility of the locus encoding interferon-γ (IFN-γ) and the loci encoding IL-5 and IL-13. Finally,IL-1β potentiated ILC2 activation and plasticity in vivo,and IL-12 acted as the switch that determined an ILC2-versus-ILC1 response. Thus,we have identified a previously unknown role for IL-1β in facilitating ILC2 maturation and plasticity. View Publication

Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here,we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7),a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2,a receptor that recognizes NAP-2,abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration. View Publication

Natural killer (NK) cells play a key role in immunity,but how HLA class I (HLA-I) and killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1) polymorphism impacts disease outcome remains unclear. KIR3DL1 (*001/*005/*015) tetramers were screened for reactivity against a panel of HLA-I molecules. This revealed different and distinct hierarchies of specificity for each KIR3DL1 allotype,with KIR3DL1*005 recognizing the widest array of HLA-I ligands. These differences were further reflected in functional studies using NK clones expressing these specific KIR3DL1 allotypes. Unexpectedly,the Ile/Thr80 dimorphism in the Bw4-motif did not categorically define strong/weak KIR3DL1 recognition. Although the KIR3DL1*001,*005,and *015 polymorphisms are remote from the KIR3DL1-HLA-I interface,the structures of these three KIR3DL1-HLA-I complexes showed that the broader HLA-I specificity of KIR3DL1*005 correlated with an altered KIR3DL1*005 interdomain positioning and increased mobility within its ligand-binding site. Collectively,we provide a generic framework for understanding the impact of KIR3DL1 polymorphism on the recognition of HLA-I allomorphs. View Publication

Acute myeloid leukemia (AML) is the most common type of acute leukemia affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (Gemtuzumab ozogamicin) have been shown to improve overall survival,validating CD33 as a target for antibody-based therapy of AML. Here we report the in vitro efficacy of BI 836858,a fully human,Fc-engineered,anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine,two hypomethylating agents that show efficacy in older patients,did not compromise BI 836858-induced NK cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a ten-day course of DAC (pre-DAC,days 4,11 and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared to pre-DAC treatment. Analysis of ligands (L) to activating receptors (NKG2D showed significantly increased NKG2DL expression in day 28 post-DAC samples compared to pre-DAC samples; when NKG2DL receptor was blocked using antibodies,BI 836858-mediated ADCC was significantly decreased,suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML. View Publication

Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover,Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands,such as HIV and CpG respectively,turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions,and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection,but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here,we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α,TNF-α,IFN-γ and IL-12,and CCR5-interacting chemokines (MIP-1α and MIP-1β) in NK-pDCs co-cultures. At high HIV-1BaL concentrations,the addition of NK cells did not promote the release of these mediators,suggesting that once efficiently triggered by the virus,pDCs could not integrate new activating signals delivered by NK cells. However,high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly,we identified the alarmin HMGB1,released at pDC-NK cell synapse,as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover,HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1,HMGB1-specific antibodies,sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether,these findings identify HMGB1 as a trigger for IFN-α-mediated TRAIL expression at the surface of pDCs and NK cells,and they suggest a novel mechanism of innate control of HIV-1 infection. View Publication

NLRC5 is a transcriptional regulator of MHC class I (MHCI),which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK-T-cell crosstalk,raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly,NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice,we show that NK cells surprisingly break tolerance even towards 'self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore,during chronic LCMV infection,the total CD8(+) T-cell population is severely decreased in these mice,a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets,having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions. View Publication

The hepatitis C virus (HCV) infects ∼200 million people worldwide. The majority of infected individuals develop persistent infection,resulting in chronic inflammation and liver disease,including cirrhosis and hepatocellular carcinoma. The ability of HCV to establish persistent infection is partly due to its ability to evade the immune response through multiple mechanisms,including suppression of NK cells. NK cells control HCV replication during the early phase of infection and regulate the progression to chronic disease. In particular,IFN-$\gamma$ produced by NK cells limits viral replication in hepatocytes and is important for the initiation of adaptive immune responses. However,NK cell function is significantly impaired in chronic HCV patients. The cellular and molecular mechanisms responsible for impaired NK cell function in HCV infection are not well defined. In this study,we analyzed the interaction of human NK cells with CD33(+) PBMCs that were exposed to HCV. We found that NK cells cocultured with HCV-conditioned CD33(+) PBMCs produced lower amounts of IFN-$\gamma$,with no effect on granzyme B production or cell viability. Importantly,this suppression of NK cell-derived IFN-$\gamma$ production was mediated by CD33(+)CD11b(lo)HLA-DR(lo) myeloid-derived suppressor cells (MDSCs) via an arginase-1-dependent inhibition of mammalian target of rapamycin activation. Suppression of IFN-$\gamma$ production was reversed by l-arginine supplementation,consistent with increased MDSC arginase-1 activity. These novel results identify the induction of MDSCs in HCV infection as a potent immune evasion strategy that suppresses antiviral NK cell responses,further indicating that blockade of MDSCs may be a potential therapeutic approach to ameliorate chronic viral infections in the liver. View Publication

Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT),underscoring the need to improve the graft-versus-leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest,but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs,but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement,while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally,adoptive transfers of activated-pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL. View Publication

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells,including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM,we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients,we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However,we discovered,next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC,a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients),as well as CDC (56 patients). Similarly,experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly,all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients. View Publication

Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood,the more mature CD56(dim) NK cell efficiently kills malignant targets at rest,whereas the less mature CD56(bright) NK cells cannot. In this study,we show that resting CD56(bright) NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56(dim) NK cells. Consistent with this,forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity,and loss of PTEN in CD56(bright) NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell-activating and inhibitory receptor expression yet,as in humans,did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell's ability to organize immunological synapse components including decreases in actin accumulation,polarization of the microtubule organizing center,and the convergence of cytolytic granules. In summary,our data suggest that PTEN normally works to limit the NK cell's PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56(bright) NK cell to the cytolytic CD56(dim) NK cells. View Publication

NK cells are innate immune cells known for their cytolytic activities toward tumors and infections. They are capable of expressing diverse killer Ig-like receptors (KIRs),and KIRs are implicated in susceptibility to Crohn's disease (CD),a chronic intestinal inflammatory disease. However,the cellular mechanism of this genetic contribution is unknown. In this study,we show that the licensing" of NK cells View Publication