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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

BACKGROUND Human rhinoviruses (HRVs) are a major trigger of asthma exacerbations,with the bronchial epithelium being the major site of HRV infection and replication. Mast cells (MCs) play a key role in asthma where their numbers are increased in the bronchial epithelium with increasing disease severity. OBJECTIVE In view of the emerging role of MCs in innate immunity and increased localization to the asthmatic bronchial epithelium,we investigated whether HRV infection of MCs generated innate immune responses which were protective against infection. METHODS The LAD2 MC line or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV at increasing multiplicities of infection (MOI) without or with IFN-β or IFN-λ. After 24 h,innate immune responses were assessed by RT-qPCR and IFN protein release by ELISA. Viral replication was determined by RT-qPCR and virion release by TCID50 assay. RESULTS HRV infection of LAD2 MCs induced expression of IFN-β,IFN-λ and IFN-stimulated genes. However,LAD2 MCs were permissive for HRV replication and release of infectious HRV particles. Similar findings were observed with CBMCs. Neutralization of the type I IFN receptor had minimal effects on viral shedding,suggesting that endogenous type I IFN signalling offered limited protection against HRV. However,augmentation of these responses by exogenous IFN-β,but not IFN-λ,protected MCs against HRV infection. CONCLUSION AND CLINICAL RELEVANCE MCs are permissive for the replication and release of HRV,which is prevented by exogenous IFN-β treatment. Taken together,these findings suggest a novel mechanism whereby MCs may contribute to HRV-induced asthma exacerbations. View Publication

SAMHD1 restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid cells and CD4(+) T cells,while Vpx can mediate SAMHD1 degradation to promote HIV-1 replication. Although the restriction mechanisms of SAMHD1 have been well-described,SAMHD1 expression and Vpx-mediated SAMHD1 degradation during chronic HIV-1 infection were poorly understood. Flow cytometric analysis was used to directly visualize ex vivo,and after in vitro SIV-Vpx treatment,SAMHD1 expression in CD4(+) T cells and monocytes. Here we report activated CD4(+) T cells without SAMHD1 expression were severely reduced,and SAMHD1 in CD4(+) T cells became susceptible to SIV-Vpx mediated degradation during chronic HIV-1 infection,which was absent from uninfected donors. These alterations were irreversible,even after long-term fully suppressive antiretroviral treatment. Although SAMHD1 expression in CD4(+) T cells and monocytes was not found to correlate with plasma viral load,Vpx-mediated SAMHD1 degradation was associated with indicators of immune activation. In vitro assays further revealed that T-cell activation and an upregulated IFN-I pathway contributed to these altered SAMHD1 properties. These findings provide insight into how immune activation during HIV-1 infection leads to irreparable aberrations in restriction factors and in subsequent viral evasion from host antiviral defenses. View Publication

The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study,we demonstrate that IFN regulatory factor 2 (IRF2),an ISG and a negative regulator of IFN signaling,influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2(-/-) mice after peripheral inoculation. Irf2(-/-) mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2(-/-) mouse brains despite the presence of peripheral neutralizing antibodies,suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell-deficient μMT mice are significantly more susceptible to viral infection,yet WT B cells and serum are unable to rescue the Irf2(-/-) mice. Collectively,our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections. View Publication

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis-associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex,SHARPIN,HOIP and HOIL-1,to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production,independently of tumor necrosis factor. Consistent with this,Traf1(-/-) mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling,providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP. View Publication

Human NK cell deficiencies are rare yet result in severe and often fatal disease,particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells,and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8,which encodes an interferon regulatory factor,as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells,and this impairment in terminal maturation was also observed in Irf8-/-,but not Irf8+/-,mice. We then determined that impaired maturation was NK cell intrinsic,and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together,these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease,thereby emphasizing a critical role for NK cells in human antiviral defense. View Publication

Autoantigen-specific regulatory immunity emerges in the spleen of mice recovering from experimental autoimmune uveitis (EAU),a murine model for human autoimmune uveoretinitis. This regulatory immunity provides induced tolerance to ocular autoantigen,and requires melanocortin 5 receptor (MC5r) expression on antigen presenting cells with adenosine 2 A receptor (A2Ar) expression on T cells. During EAU it is not well understood what roles MC5r and A2Ar have on promoting regulatory immunity. Cytokine profile analysis during EAU revealed MC5r and A2Ar each mediate distinct T cell responses,and are responsible for a functional regulatory immune response in the spleen. A2Ar stimulation at EAU onset did not augment this regulatory response,nor bypass the MC5r requirement to induce regulatory immunity. The importance of this pathway in human autoimmune uveitis was assayed. PBMC from uveitis patients were assayed for MC5r expression on monocytes and A2Ar on T cells,and comparison between uveitis patients and healthy controls had no significant difference. The importance for MC5r and A2Ar expression in EAU to promote the induction of protective regulatory immunity,and the expression of MC5r and A2Ar on human immune cells,suggests that it may be possible to utilize the melanocortin-adenosinergic pathways to induce protective immunity in uveitic patients. View Publication

Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study,to our knowledge,we have,for the first time,used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands,CD80 and CD86,and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays,using PBMCs from type 1 diabetes patients,had significant improvements in CD80,but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265,abatacept,and belatacept,depending on which type of APC was used,with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2 In a cynomolgus model of T cell-dependent Ab response,the CTLA4-Ig variant MEDI5265 could be formulated at textgreater100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2 The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent,s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies. View Publication

The regulatory properties of B cells have been studied in autoimmune diseases; however,their role in allergic diseases is poorly understood. We demonstrate that Semaphorin 4C (Sema4C),an axonal guidance molecule,plays a crucial role in B cell regulatory function. Mice deficient in Sema4C exhibited increased airway inflammation after allergen exposure,with massive eosinophilic lung infiltrates and increased Th2 cytokines. This phenotype was reproduced by mixed bone marrow chimeric mice with Sema4C deficient only in B cells,indicating that B lymphocytes were the key cells affected by the absence of Sema4C expression in allergic inflammation. We determined that Sema4C-deficient CD19(+)CD138(+) cells exhibited decreased IL-10 and increased IL-4 expression in vivo and in vitro. Adoptive transfer of Sema4c(-/-) CD19(+)CD138(+) cells induced marked pulmonary inflammation,eosinophilia,and increased bronchoalveolar lavage fluid IL-4 and IL-5,whereas adoptive transfer of wild-type CD19(+)CD138(+)IL-10(+) cells dramatically decreased allergic airway inflammation in wild-type and Sema4c(-/-) mice. This study identifies a novel pathway by which Th2-mediated immune responses are regulated. It highlights the importance of plasma cells as regulatory cells in allergic inflammation and suggests that CD138(+) B cells contribute to cytokine balance and are important for maintenance of immune homeostasis in allergic airways disease. Furthermore,we demonstrate that Sema4C is critical for optimal regulatory cytokine production in CD138(+) B cells. View Publication

The aryl hydrocarbon receptor (AhR),a transcription factor known for mediating xenobiotic toxicity,is expressed in B cells,which are known targets for environmental pollutants. However,it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up-regulated upon B-cell receptor (BCR) engagement and IL-4 treatment. Addition of a natural ligand of AhR,FICZ,induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1,showing that the AhR pathway is functional in B cells. AhR-deficient (Ahr(-/-)) B cells proliferate less than AhR-sufficient (Ahr(+/+)) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr(-/-) B cells are outcompeted by Ahr(+/+) cells. Transcriptome comparison of AhR-deficient and AhR-sufficient B cells identified cyclin O (Ccno),a direct target of AhR,as a top candidate affected by AhR deficiency. View Publication

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection,but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal β-glucans stimulate IFN-β production by dendritic cells (DCs) following detection by the Dectin-1 receptor,but the effects of β-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal β-glucan particle-stimulated DCs. We demonstrate that β-glucan-stimulated DCs induce CD8 T cell proliferation,activation marker (CD44 and CD69) expression,and production of IFN-γ,IL-2,and granzyme B. Moreover,we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by β-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically,type I IFNs promote Ag presentation on MHC I molecules,CD86 and CD40 expression,and the production of IL-12 p70,IL-2,IL-6,and TNF-α by β-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation,although,in the context of LPS stimulation,this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection,which may be useful in the rational design of vaccines directed against pathogens and tumors. View Publication

Increasing amounts of pathogen replication usually lead to a proportionate increase in size and effector differentiation of the CD8(+) T cell response,which is attributed to increased Ag and inflammation. Using a murine CMV that is highly sensitive to the antiviral drug famciclovir to modulate virus replication,we found that increased virus replication drove increased effector CD8(+) T cell differentiation,as expected. Paradoxically,however,increased virus replication dramatically decreased the size of the CD8(+) T cell response to two immunodominant epitopes. The decreased response was due to type I IFN-dependent depletion of conventional dendritic cells and could be reproduced by specific depletion of dendritic cells from day 2 postinfection or by sterile induction of type I IFN. Increased virus replication and type I IFN specifically inhibited the response to two immunodominant epitopes that are known to be dependent on Ag cross-presented by DCs,but they did not inhibit the response to inflationary" epitopes whose responses can be sustained by infected nonhematopoietic cells. Our results show that type I IFN can suppress CD8(+) T cell responses to cross-presented Ag by depleting cross-presenting conventional dendritic cells." View Publication