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Human embryonic stem cell-derived dendritic cells (hESC-DCs) may potentially provide a platform to generate off-the-shelf" therapeutic cancer vaccines. To apply hESC-DCs for cancer immunotherapy in a semiallogeneic setting� View Publication

HIV elite controllers (EC) are a rare group of HIV-infected patients who are able to maintain undetectable viral loads during a long period of time in the absence of antiretroviral treatment. Adaptive immunity and host genetic factors,although implicated,do not entirely explain this phenomenon. On the other hand,plasmacytoid dendritic cells (pDCs) are the principal type I interferon (IFN) producers in response to viral infection,and it is unknown whether pDCs are involved in the control of HIV infection in EC. In our study,we analyzed peripheral pDC levels and IFN-α production by peripheral blood mononuclear cells (PBMCs) in EC compared to other groups of HIV-infected patients,the ability of pDCs to reduce HIV production in vitro,and the mechanisms potentially involved. We showed preserved pDC counts and IFN-α production in EC. We also observed a higher capacity of pDCs from EC to reduce HIV production and to induce T cell apoptosis,whereas pDCs from viremic patients barely responded without previous Toll-like receptor 9 (TLR-9) stimulus. The preserved functionality of pDCs from EC to reduce viral production may be one of the mechanisms involved in the control of HIV viremia in these subjects. These results demonstrate the importance of innate immunity in HIV pathogenesis,and an understanding of pDC mechanisms would be helpful for the design of new therapies. View Publication

Symptomatic dengue virus infection ranges in disease severity from an influenza-like illness to life-threatening shock. One model of the mechanism underlying severe disease proposes that weakly neutralizing,dengue serotype cross-reactive antibodies induced during a primary infection facilitate virus entry into Fc receptor-bearing cells during a subsequent secondary infection,increasing viral replication and the release of cytokines and vasoactive mediators,culminating in shock. This process has been termed antibody-dependent enhancement of infection and has significantly hindered vaccine development. Much of our understanding of this process has come from studies using mouse monoclonal antibodies (MAbs); however,antibody responses in mice typically exhibit less complexity than those in humans. A better understanding of the humoral immune response to natural dengue virus infection in humans is sorely needed. Using a high-efficiency human hybridoma technology,we isolated 37 hybridomas secreting human MAbs to dengue viruses from 12 subjects years or even decades following primary or secondary infection. The majority of the human antibodies recovered were broadly cross-reactive,directed against either envelope or premembrane proteins,and capable of enhancement of infection in vitro; few exhibited serotype-specific binding or potent neutralizing activity. Memory B cells encoding enhancing antibodies predominated in the circulation,even two or more decades following infection. Mapping the epitopes and activity of naturally occurring dengue antibodies should prove valuable in determining whether the enhancing and neutralizing activity of antibodies can be separated. Such principles could be used in the rational design of vaccines that enhance the induction of neutralizing antibodies,while lowering the risk of dengue shock syndrome. View Publication

Chylomicron remnants (CMRs) contribute directly to human monocyte activation in vitro,by increasing reactive oxygen species (ROS) production and cell migration. In this study,the effects of the oxidative state of CMR on the degree of monocyte activation was investigated. CMR-like particles (CRLPs) were prepared in three different oxidative states,normal (CRLPs),protected from oxidation by incorporation of the antioxidant,probucol (pCRLPs),or oxidised with CuSO(4) (oxCRLPs). Lipid accumulation and ROS production were significantly increased in primary human monocytes incubated with CRLPs,whilst secretion on monocyte chemoattractant protein-1 was reduced,but oxCRLPs had no additional effect. In contrast,pCRLPs were taken up by monocytes to a lesser extent and had no significant effect on ROS or MCP-1 secretion. These studies suggest that the oxidative state of CMRs modulates their stimulation of the activation of peripheral blood human monocytes and that dietary antioxidants may provide some protection against these atherogenic effects. View Publication

Molluscum contagiosum poxvirus (MCV) type 1 and type 2 encode two chemokine-like proteins MC148R1 and MC148R2. It is believed that MC148R proteins function by blocking the inflammatory response. However,the mechanism of the proposed biological activities of MC148R proteins and the role of the additional C-terminal cysteines that do not exist in other chemokines are not understood. Here,we demonstrated in two different assay systems that His-tagged MC148R1 displaces the interaction between CXCL12α and CXCR4. The N-terminal cysteines but not the additional C-terminal cysteines modulate this displacement. His-tagged MC148R1 blocked both CXCL12α-mediated and MIP-1α-mediated chemotaxis. In contrast,MC148R2 blocked MIP-1α-mediated but not CXCL12α-mediated chemotaxis. Immunoprecipitation by antibodies to MC148R1 or CXCL12α followed by immunoblotting and detection by antibodies to the other protein demonstrated physical interaction of His-tagged CXCL12α and His-tagged MC148R1. Interaction with chemokines might mask the receptor interaction site resulting in decreased binding and impairment of the biological activities. View Publication

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1,also known as CD71),which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fcγ receptors and the complement component C1q,suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition,in 2 disseminated multiple myeloma xenograft mouse models,we show that a single dose of ch128.1Av results in significant antitumor activity,including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally,we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches,including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies. View Publication

How HIV controllers (HICs) maintain undetectable viremia without therapy is unknown. The strong CD8(+) T-cell HIV suppressive capacity found in many,but not all,HICs may contribute to long-lasting viral control. However,other earlier defense mechanisms may be involved. Here,we examined intrinsic HIC cell resistance to HIV-1 infection. After in vitro challenge,monocyte-derived macrophages and anti-CD3-activated CD4(+) T cells from HICs showed low HIV-1 susceptibility. CD4 T-cell resistance was independent of HIV-1 coreceptors and affected also SIVmac infection. CD4(+) T cells from HICs expressed ex vivo higher levels of p21(Waf1/Cip1),which has been involved in the control of HIV-1 replication,than cells from control subjects. However,HIV restriction in anti-CD3-activated CD4(+) T cells and macrophages was not associated with p21 expression. Restriction inhibited accumulation of reverse transcripts,leading to reduction of HIV-1 integrated proviruses. The block could be overcome by high viral inocula,suggesting the action of a saturable mechanism. Importantly,cell-associated HIV-1 DNA load was extremely low in HICs and correlated with CD4(+) T-cell permissiveness to infection. These results point to a contribution of intrinsic cell resistance to the control of infection and the containment of viral reservoir in HICs. View Publication

How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice,N-glycan branching by the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity,cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis,spontaneous inflammatory demyelination and neurodegeneration,the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3),including genetic variants in interleukin-7 receptor-α (IL7RA*C),interleukin-2 receptor-α (IL2RA*T),MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3),optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway,namely N-glycosylation. View Publication

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected,we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs,where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection,as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability,expansion,and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro,it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether,these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression. View Publication

The lipocalin mouse 24p3 has been implicated in diverse physiological processes,including apoptosis,iron trafficking,development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However,a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3,we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid,myeloid,and erythroid cells,which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead,apoptotic defects were unique to many mature hematopoietic cell types,including neutrophils,cytokine-dependent mast cells,thymocytes,and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells,thus explaining their resistance to the ensuing cell death. The results of these studies,in conjunction with those of previous studies,reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly,these functions are limited to relatively mature blood cell compartments. View Publication

Mast cells (MCs) play an important role in the regulation of protective adaptive immune responses against pathogens. However,it is still unclear whether MCs promote such host defense responses via direct effects on T cells or rather by modifying the functions of antigen-presenting cells. To identify the underlying mechanisms of the immunoregulatory capacity of MCs,we investigated the impact of MCs on dendritic cell (DC) maturation and function. We found that murine peritoneal MCs underwent direct crosstalk with immature DCs that induced DC maturation as evidenced by enhanced expression of costimulatory molecules. Furthermore,the MC/DC interaction resulted in the release of the T-cell modulating cytokines IFN-γ,IL-2,IL-6 and TGF-β into coculture supernatants and increased the IL-12p70,IFN-γ,IL-6 and TGF-β secretion of LPS-matured DCs. Such MC-primed" DCs subsequently induced efficient CD4+ T-cell proliferation. Surprisingly� View Publication

Regulatory T cells (Tregs) contribute significantly to the tolerogenic nature of the liver. The mechanisms,however,underlying liver-associated Treg induction are still elusive. We recently identified the vitamin A metabolite,retinoic acid (RA),as a key controller that promotes TGF-β-dependent Foxp3(+) Treg induction but inhibits TGF-β-driven Th17 differentiation. To investigate whether the RA producing hepatic stellate cells (HSC) are part of the liver tolerance mechanism,we investigated the ability of HSC to function as regulatory APC. Different from previous reports,we found that highly purified HSC did not express costimulatory molecules and only upregulated MHC class II after in vitro culture in the presence of exogenous IFN-γ. Consistent with an insufficient APC function,HSC failed to stimulate naive OT-II TCR transgenic CD4(+) T cells and only moderately stimulated α-galactosylceramide-primed invariant NKT cells. In contrast,HSC functioned as regulatory bystanders and promoted enhanced Foxp3 induction by OT-II TCR transgenic T cells primed by spleen dendritic cells,whereas they greatly inhibited the Th17 differentiation. Furthermore,the regulatory bystander capacity of the HSC was completely dependent on their ability to produce RA. Our data thus suggest that HSC can function as regulatory bystanders,and therefore,by promoting Tregs and suppressing Th17 differentiation,they might represent key players in the mechanism that drives liver-induced tolerance. View Publication