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Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are the major pathogens of human hand,foot,and mouth disease (HFMD). In our previous study,intramuscular immunization with the inactivated EV71 vaccine elicited effective immunity,while immunization with the inactivated CA16 vaccine did not. In this report,we focused on innate immune responses elicited by inactivated EV71 and CA16 antigens administered intradermally or intramuscularly. The distributions of the EV71 and CA16 antigens administered intradermally or intramuscularly were not obviously different,but the antigens were detected for a shorter period of time when administered intradermally. The expression levels of NF-kappaB pathway signaling molecules,which were identified as being capable of activating DCs,ILCs,and T cells,were higher in the intradermal group than in the intramuscular group. Antibodies for the EV71 and CA16 antigens colocalized with ILCs and DCs in skin and muscle tissues under fluorescence microscopy. Interestingly,ILC colocalization decreased over time,while DC colocalization increased over time. ELISpot analysis showed that coordination between DCs and ILCs contributed to successful adaptive immunity against vaccine antigens in the skin. EV71 and/or CA16 antigen immunization via the intradermal route was more capable of significantly increasing neutralizing antibody titers and activating specific T cell responses than immunization via the intramuscular route. Furthermore,neonatal mice born to mothers immunized with the EV71 and CA16 antigens were 100{\%} protected against wild-type EV71 or CA16 viral challenge. Together,our results provide new insights into the development of vaccines for HFMD. 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

Understanding how memory B cells are induced and relate to long-lived plasma cells is important for vaccine development. Immunity to oral vaccines has been considered short-lived because of a poor ability to develop IgA B-cell memory. Here we demonstrate that long-lived mucosal IgA memory is readily achieved by oral but not systemic immunization in mouse models with NP hapten conjugated with cholera toxin and transfer of B1-8(high)/GFP(+) NP-specific B cells. Unexpectedly,memory B cells are poorly related to long-lived plasma cells and less affinity-matured. They are α4β7-integrin(+)CD73(+)PD-L2(+)CD80(+) and at systemic sites mostly IgM(+),while 80% are IgA(+) in Peyer's patches. On reactivation,most memory B cells in Peyer's patches are GL7(-),but expand in germinal centres and acquire higher affinity and more mutations,demonstrating strong clonal selection. CCR9 expression is found only in Peyer's patches and appears critical for gut homing. Thus,gut mucosal memory possesses unique features not seen after systemic immunization. View Publication

Innate Defense Regulators (IDRs) are short synthetic peptides that target the host innate immune system via an intracellular adaptor protein which functions at key signaling nodes. In this work,further details of the mechanism of action of IDRs have been discovered. The studies reported here show that the lead clinical IDR,SGX94,has broad-spectrum activity against Gram-negative and Gram-positive bacterial infections caused by intracellular or extracellular bacteria and also complements the actions of standard of care antibiotics. Based on in vivo and primary cell culture studies,this activity is shown to result from the primary action of SGX94 on tissue-resident cells and subsequent secondary signaling to activate myeloid-derived cells,resulting in enhanced bacterial clearance and increased survival. Data from non-clinical and clinical studies also show that SGX94 treatment modulates pro-inflammatory and anti-inflammatory cytokine levels,thereby mitigating the deleterious inflammatory consequences of innate immune activation. Since they act through host pathways to provide both broad-spectrum anti-infective capability as well as control of inflammation,IDRs are unlikely to be impacted by resistance mechanisms and offer potential clinical advantages in the fight against emerging and antibiotic resistant bacterial infections. View Publication

The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed,in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature,dry ice,and liquid nitrogen) and in different preservation media (serum with cryoprotectant,commercial cryopreservation solution,and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion,flow cytometry,Cell Analysis System cell counting (CASY)),and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However,we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions,satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells. View Publication

HIV-infected slow progressors (SP) represent a heterogeneous group of subjects who spontaneously control HIV infection without treatment for several years while showing moderate signs of disease progression. Under conditions that remain poorly understood,a subgroup of these subjects experience failure of spontaneous immunological and virological control. Here we determined the frequency of SP subjects who showed loss of HIV control within our Canadian Cohort of HIV(+) Slow Progressors and identified the proinflammatory cytokine IL-32 as a robust biomarker for control failure. Plasmatic levels of the proinflammatory isoforms of IL-32 (mainly β and γ) at earlier clinic visits positively correlated with the decline of CD4 T-cell counts,increased viral load,lower CD4/CD8 ratio and levels of inflammatory markers (sCD14 and IL-6) at later clinic visits. We present here a proof-of-concept for the use of IL-32 as a predictive biomarker for disease progression in SP subjects and identify IL-32 as a potential therapeutic target. 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

Human mesenchymal stem cells (hMSCs) suppress T-cell and dendritic-cell function and represent a promising strategy for cell therapy of autoimmune diseases. Nevertheless,no information is currently available on the effects of hMSCs on B cells,which may have a large impact on the clinical use of these cells. hMSCs isolated from the bone marrow and B cells purified from the peripheral blood of healthy donors were cocultured with different B-cell tropic stimuli. B-cell proliferation was inhibited by hMSCs through an arrest in the G0/G1 phase of the cell cycle and not through the induction of apoptosis. A major mechanism of B-cell suppression was hMSC production of soluble factors,as indicated by transwell experiments. hMSCs inhibited B-cell differentiation because IgM,IgG,and IgA production was significantly impaired. CXCR4,CXCR5,and CCR7 B-cell expression,as well as chemotaxis to CXCL12,the CXCR4 ligand,and CXCL13,the CXCR5 ligand,were significantly down-regulated by hMSCs,suggesting that these cells affect chemotactic properties of B cells. B-cell costimulatory molecule expression and cytokine production were unaffected by hMSCs. These results further support the potential therapeutic use of hMSCs in immune-mediated disorders,including those in which B cells play a major role. View Publication

The ability of NK cells to directly recognize pathogens and be activated via Toll-like receptors (TLR) is increasingly recognized. Nevertheless,controversial results on the NK cell ability to be directly activated by lipopolysaccharide (LPS),the ligand of TLR4,have been recently reported. To start elucidating the reasons explaining the contrasting observations of the literature,we focused on the potential role of currently used NK cell purification procedures to condition putative NK cell responsiveness to LPS. To do so,human NK cells were isolated by negative selection,using three different commercial kits,to be comparatively evaluated for the production of IFNgamma in response to ultra-pure LPS and/or IL-2. Despite the lack of surface TLR4,we found that two out of the three NK cell-enriched populations released IFNgamma (and one of the two,IL-12p70 as well) in response to the LPS plus IL-2 combination,whereas the last one did not. However,the two LPS plus IL-2-responsive NK cell populations were found variably contaminated with 6-sulfo LacNAc(+) dendritic cells (slanDC),demonstrated responsible for triggering,via the production of IL-12p70 in response to LPS,the release of IFNgamma by IL-2-stimulated NK cells. Accordingly,slanDC depletion completely abrogated the capacity to produce both IL-12p70 and IFNgamma in response to LPS plus IL-2 by slanDC-containing NK cells. Taken together,our data uncover that two commercially available kits,specifically designed to isolate NK cells by negative selection,also co-purify variable amounts of slanDC. The latter cells may dramatically affect the outcome of experiments carried on to evaluate NK cell responsiveness to TLR agonists such as LPS. View Publication