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Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells and are key cells of the immune system,acquiring different phenotypes in accordance with their localization during the immune response. A subset of inflammatory DCs is derived from circulating monocytes (Mo) and has a key role in inflammation and infection. The pathways controlling Mo-DC differentiation are not fully understood. Our objective was to investigate the possible role of nicotinamide adenine dinucleotide phosphate reduced form oxidases (NOXs) in Mo-DC differentiation. In this study,we revealed that Mo-DC differentiation was inhibited by NOX inhibitors and reactive oxygen species scavengers. We show that the Mo-DC differentiation was dependent on p22phox,and not on gp91phox/NOX2,as shown by the reduced Mo-DC differentiation observed in chronic granulomatous disease patients lacking p22phox. Moreover,we revealed that NOX5 expression was strongly increased during Mo-DC differentiation,but not during Mo-macrophage differentiation. NOX5 was expressed in circulating myeloid DC,and at a lower level in plasmacytoid DC. Interestingly,NOX5 was localized at the outer membrane of the mitochondria and interacted with p22phox in Mo-DC. Selective inhibitors and small interfering RNAs for NOX5 indicated that NOX5 controlled Mo-DC differentiation by regulating the JAK/STAT/MAPK and NFκB pathways. These data demonstrate that the NOX5-p22phox complex drives Mo-DC differentiation,and thus could be critical for immunity and inflammation. View Publication

Targeting of myeloid-dendritic cell receptor DC-SIGN by numerous chronic infectious agents,including Porphyromonas gingivalis,is shown to drive-differentiation of monocytes into dysfunctional mDCs. These mDCs exhibit alterations of their fine-tuned homeostatic function and contribute to dysregulated immune-responses. Here,we utilize P. gingivalis mutant strains to show that pathogen-differentiated mDCs from primary human-monocytes display anti-apoptotic profile,exhibited by elevated phosphorylated-Foxo1,phosphorylated-Akt1,and decreased Bim-expression. This results in an overall inhibition of DC-apoptosis. Direct stimulation of complex component CD40 on DCs leads to activation of Akt1,suggesting CD40 involvement in anti-apoptotic effects observed. Further,these DCs drove dampened CD8(+) T-cell and Th1/Th17 effector-responses while inducing CD25(+)Foxp3(+)CD127(-) Tregs. In vitro Treg induction was mediated by DC expression of indoleamine 2,3-dioxygenase,and was confirmed in IDO-KO mouse model. Pathogen-infected &CMFDA-labeled MoDCs long-lasting survival was confirmed in a huMoDC reconstituted humanized mice. In conclusion,our data implicate PDDCs as an important target for resolution of chronic infection. View Publication

Autologous chondrocyte implantation is the current gold standard cell therapy for cartilage lesions. However,in some instances,the heavily compromised health of the patient can either impair or limit the recovery of the autologous chondrocytes and a satisfactory outcome of the implant. Allogeneic human articular chondrocytes (hAC) could be a good alternative,but the possible immunological incompatibility between recipient and hAC donor should be considered. Herein,we report that allogeneic hAC inhibited T lymphocyte response to antigen-dependent and -independent proliferative stimuli. This effect was maximal when T cells and hAC were in contact and it was not relieved by the addition of exogenous lymphocyte growth factor interleukin (IL)-2. More important,hAC impaired the differentiation of peripheral blood monocytes induced with granulocyte monocyte colony-stimulating factor and IL-4 (Mo) to professional antigen-presenting cells,such as dendritic cells (DC). Indeed,a marked inhibition of the onset of the CD1a expression and an ineffective downregulation of CD14 antigens was observed in Mo-hAC co-cultures. Furthermore,compared to immature or mature DC,Mo from Mo-hAC co-cultures did not trigger an efficacious allo-response. The prostaglandin (PG) E2 present in the Mo-hAC co-culture conditioned media is a putative candidate of the hAC-mediated inhibition of Mo maturation. Altogether,these findings indicate that allogeneic hAC inhibit,rather than trigger,immune response and strongly suggest that an efficient chondrocyte implantation could be possible also in an allogeneic setting. View Publication

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses,host defense mechanisms,and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system,DCs are very rare in blood,accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore,alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity,affordability,high purity,and high yield of cells is imperative to consider. In the current study,two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability,proliferation,and phenotype were assessed using viability dyes,MTT assay,and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method,the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded textgreater 70% CD11c+ MDDCs. Therefore,our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs. View Publication

West Nile virus (WNV) infection is a mosquito-borne zoonosis with increasing prevalence in the United States. WNV infection begins in the skin,and the virus replicates initially in keratinocytes and dendritic cells (DCs). In the skin and cutaneous lymph nodes,infected DCs are likely to interact with invariant natural killer T cells (iNKTs). Bidirectional interactions between DCs and iNKTs amplify the innate immune response to viral infections,thus controlling viral load and regulating adaptive immunity. iNKTs are stimulated by CD1d-bound lipid antigens or activated indirectly by inflammatory cytokines. We exposed human monocyte-derived DCs to WNV Kunjin and determined their ability to activate isolated blood iNKTs. DCs became infected as judged by synthesis of viral mRNA and Envelope and NS-1 proteins,but did not undergo significant apoptosis. Infected DCs up-regulated the co-stimulatory molecules CD86 and CD40,but showed decreased expression of CD1d. WNV infection induced DC secretion of type I interferon (IFN),but no or minimal interleukin (IL)-12,IL-23,IL-18 or IL-10. Unexpectedly,we found that the WNV-infected DCs stimulated human iNKTs to up-regulate CD69 and produce low amounts of IL-10,but not proinflammatory cytokines such as IFN-γ or tumour necrosis factor (TNF)-α. Both CD1d and IFNAR blockade partially abrogated this iNKT response,suggesting involvement of a T cell receptor (TCR)-CD1d interaction and type I interferon receptor (IFNAR) signalling. Thus,WNV infection interferes with DC-iNKT interactions by preventing the production of proinflammatory cytokines. iNKTs may be a source of IL-10 observed in human flavivirus infections and initiate an anti-inflammatory innate response that limits adaptive immunity and immune pathology upon WNV infection. View Publication

Viral infection triggers induction of antiviral cytokines and effectors,which are critical mediators of innate antiviral immune response. It has been shown that the processing body-associated protein LSm14A is involved in the induction of antiviral cytokines in cell lines but in vivo evidence is lacking. By generating LSm14A-deficient mice,in this study,we show that LSm14A plays a critical and specific role in the induction of antiviral cytokines in dendritic cells (DCs) but not in macrophages and fibroblasts. Induction of antiviral cytokines triggered by the DNA viruses HSV-1 and murid herpesvirus 68 and the RNA virus vesicular stomatitis virus but not Sendai virus was impaired in Lsm14a(-/-) DCs,which is correlated to the functions of the adaptor protein MITA/STING in the antiviral signaling pathways. LSm14A deficiency specifically downregulated MITA/STING level in DCs by impairing its nuclear mRNA precursor processing and subsequently impaired antiviral innate and adaptive immune responses. Our findings reveal a nuclear mRNA precursor processing and cell-specific regulatory mechanism of antiviral immune responses. 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

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

PURPOSE We conducted an open-label,single-arm Phase I/II clinical trial in metastatic CRPC (mCRPC) patients eligible for docetaxel combined with treatment with autologous mature dendritic cells (DCs) pulsed with killed LNCaP prostate cancer cells (DCVAC/PCa). The primary and secondary endpoints were safety and immune responses,respectively. Overall survival (OS),followed as a part of the safety evaluation,was compared to the predicted OS according to the Halabi and MSKCC nomograms. EXPERIMENTAL DESIGN Twenty-five patients with progressive mCRPC were enrolled. Treatment comprised of initial 7 days administration of metronomic cyclophosphamide 50 mg p.o. DCVAC/PCa treatment consisted of a median twelve doses of 1 × 107 dendritic cells per dose injected s.c. (Aldara creme was applied at the site of injection) during a one-year period. The initial 2 doses of DCVAC/PCa were administered at a 2-week interval,followed by the administration of docetaxel (75 mg/m2) and prednisone (5 mg twice daily) given every 3 weeks until toxicity or intolerance was observed. The DCVAC/PCa was then injected every 6 weeks up to the maximum number of doses manufactured from one leukapheresis. RESULTS No serious DCVAC/PCa-related adverse events have been reported. The median OS was 19 months,whereas the predicted median OS was 11.8 months with the Halabi nomogram and 13 months with the MSKCC nomogram. Kaplan-Meier analyses showed that patients had a lower risk of death compared with both MSKCC (Hazard Ratio 0.26,95% CI: 0.13-0.51) and Halabi (Hazard Ratio 0.33,95% CI: 0.17-0.63) predictions. We observed a significant decrease in Tregs in the peripheral blood. The long-term administration of DCVAC/PCa led to the induction and maintenance of PSA specific T cells. We did not identify any immunological parameter that significantly correlated with better OS. CONCLUSIONS In patients with mCRPC,the combined chemoimmunotherapy with DCVAC/PCa and docetaxel was safe and resulted in longer than expected survival. Concomitant chemotherapy did not preclude the induction of specific anti-tumor cytotoxic T cells. View Publication

HIV-1 infection leads to numerous B cell abnormalities,including hypergammaglobulinemia,nonspecific B cell activation,nonspecific class switching,increased cell turnover,breakage of tolerance,increased immature/transitional B cells,B cell malignancies,as well as a loss of capacity to generate and maintain memory,all of which contribute to a global impairment of the immune humoral compartment. Several cytokines and soluble factors,which are increased in sera of HIV-1-infected individuals,have been suggested to directly or indirectly contribute to these B cell dysfunctions,and one of these is the B cell-activating factor (BAFF). We report in this study that HIV-1 (X4- and R5-tropic) upregulates BAFF expression and secretion by human monocytes. Moreover,we show that the virus-mediated production of BAFF by monocytes relies on a type I IFN response by a small percentage of plasmacytoid dendritic cells (pDCs) present in the monocyte cultures. HIV-1-induced type I IFN by pDCs triggers BAFF production in both classical and intermediate monocytes,but not in nonclassical monocytes,which nonetheless display a very strong basal BAFF production. We report also that basal BAFF secretion was higher in monocytes obtained from females compared with those from male donors. This study provides a novel mechanistic explanation for the increased BAFF levels observed during HIV-1 infection and highlights the importance of pDC/monocyte crosstalk to drive BAFF secretion. View Publication

The ontogeny of human Langerhans cells (LCs) remains poorly characterized,in particular the nature of LC precursors and the factors that may drive LC differentiation. Here we report that thymic stromal lymphopoietin (TSLP),a keratinocyte-derived cytokine involved in epithelial inflammation,cooperates with transforming growth factor (TGF)-β for the generation of LCs. We show that primary human blood BDCA-1(+),but not BDCA-3(+),dendritic cells (DCs) stimulated with TSLP and TGF-β harbor a typical CD1a(+)Langerin(+) LC phenotype. Electron microscopy established the presence of Birbeck granules,an intracellular organelle specific to LCs. LC differentiation was not observed from tonsil BDCA-1(+) and BDCA-3(+) subsets. TSLP + TGF-β LCs had a mature phenotype with high surface levels of CD80,CD86,and CD40. They induced a potent CD4(+) T-helper (Th) cell expansion and differentiation into Th2 cells with increased production of tumor necrosis factor-α and interleukin-6 compared with CD34-derived LCs. Our findings establish a novel LC differentiation pathway from BDCA-1(+) blood DCs with potential implications in epithelial inflammation. Therapeutic targeting of TSLP may interfere with tissue LC repopulation from circulating precursors. View Publication

Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity,and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs,but no receptors for high-mannose have so far been reported on human pDCs. Here we show that upon activation with HIV-1 or by a synthetic compound triggering the same receptor in human pDCs as single-stranded RNA,human pDCs upregulate the mannose receptor (MR,CD206). To examine the functional outcome of this upregulation,inactivated intact or viable HIV-1 particles with various degrees of mannosylation were cultured with pDCs. Activation of pDCs was determined by assaying secretion of IFN-alpha,viability,and upregulation of several pDC-activation markers: CD40,CD86,HLA-DR,CCR7,and PD-L1. The level of activation negatively correlated with degree of mannosylation,however,subsequent reduction in the original mannosylation level had no effect on the pDC phenotype. Furthermore,two of the infectious HIV-1 strains induced profound necrosis in pDCs,also in a mannose-independent manner. We therefore conclude that natural mannosylation of HIV-1 is not involved in HIV-1-mediated immune suppression of pDCs. View Publication