技术资料

The interaction between the linker for activation of T cells (LAT) with PLC-gamma1 is important for TCR-mediated Ca(2+) signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells,implicating a breakdown of both central and peripheral tolerance. To bypass this developmental defect,we developed a conditional knock-in line in which only LATY136F is expressed in mature T cells after deletion of the wild type LAT allele. Analysis of LATY136F T cells indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling,proliferation,and IL-2 production. Furthermore,the deletion of LAT induced development of the lymphoproliferative syndrome in these mice. Although Foxp3(+) natural Treg cells were present in these mice after deletion,they were unable to suppress the proliferation of conventional T cells. Our data indicate that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4(+)CD25(+) regulatory T cells. View Publication

CD8(+) T lymphocytes play a key role in host defense,in particular against important persistent viruses,although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8(+) T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this,we examined CD8(+)CD161(+) T cells in healthy donors and those with hepatitis C virus and defined a population of CD8(+) T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation,including cytokines (e.g.,IL-17,IL-22),transcription factors (e.g.,retinoic acid-related orphan receptor gamma-t,P = 6 x 10(-9); RUNX2,P = 0.004),cytokine receptors (e.g.,IL-23R,P = 2 x 10(-7); IL-18 receptor,P = 4 x 10(-6)),and chemokine receptors (e.g.,CCR6,P = 3 x 10(-8); CXCR6,P = 3 x 10(-7); CCR2,P = 4 x 10(-7)). CD161(+)CD8(+) T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-gamma and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8(+) T cells in normal humans and a substantial fraction of tissue-infiltrating CD8(+) T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man. View Publication

The activating receptor NKG2D,expressed by natural killer (NK) cells and CD8(+) T cells,has a role in the specific killing of transformed cells. We examined NKG2D expression in patients with glioblastoma multiforme and found that NKG2D was downregulated on NK cells and CD8(+) T cells. Expression of NKG2D on lymphocytes significantly increased following tumor resection and correlated with an increased ability to kill NKG2D ligand-positive tumor targets. Despite the presence of soluble NKG2D ligands in the sera of glioblastoma patients,NKG2D downregulation was primarily caused by tumor-derived tumor growth factor-beta,suggesting that blocking of this cytokine may have therapeutic benefit. View Publication

Recently,it has been shown that certain combinations of TLR ligands act in synergy to induce the release of IL-12 by DCs. In this study,we sought to define the critical parameters underlying TLR synergy. Our data show that TLR ligands act synergistically if MyD88- and TRIF-dependent ligands are combined. TLR4 uses both of these adaptor molecules,thus activation via TLR4 proved to be a synergistic event on its own. TLR synergy did not affect all aspects of DC activation but enhanced primarily the release of certain cytokines,particularly IL-12,whereas the expression of costimulatory molecules remained unchanged. Consequently,synergistic activation of DC did not affect their ability to induce T cell proliferation but resulted in T(H)1-biased responses in vitro and in vivo. Furthermore,we examined the impact of TLR ligand combinations on primary DC in vitro but observed only modest effects with a combination of CpG + Poly (I:C). However,noticeable synergy in terms of IL-12 production by DCs was detectable in vivo after systemic administration of CpG + Poly (I:C). Finally,we show that synergy is partially dependent on IFNAR signaling but does not require the release of IFNs to the enviroment,suggesting an autocrine action of type I IFNs. View Publication

Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented,their ability to directly recognize pathogens is less well defined. We have recently reported FimH,a bacterial fimbrial protein,as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here,we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha] and cytotoxicity. Importantly,NK cells directly recognize FimH-expressing pathogens as FimH(+),but not FimH(-),bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling,as NK cells from both TLR4(-/-) and MyD88(-/-) mice as well as human NK-92 cells,which lack TLR4,were all unresponsive to FimH. In addition,TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4-MyD88 pathways to aid innate protection against cancer or microbial infections. View Publication

A major mechanism of hypercoagulability in the antiphospholipid syndrome (APS) is antiphospholipid Ab-mediated upregulation of tissue factor (TF) on monocytes via activation of TLRs,p38 MAPK,and NF-kappaB pathways. We examined whether monocyte signaling pathways are differentially activated by IgG from patients with vascular thrombosis (VT) alone compared with IgG from patients with pregnancy morbidity (PM) alone. We purified IgG from 49 subjects. A human monocyte cell line and ex vivo healthy monocytes were treated with 100 microg/ml IgG for 6 h,and cell extracts were examined by immunoblot using Abs to p38 MAPK and NF-kappaB. To further investigate intracellular signaling pathways induced by these IgGs,specific inhibitors of p38 MAPK,NF-kappaB,TLR4,and TLR2 were used to determine their effect on TF activity. Only IgG from patients with VT but no PM (VT+/PM-) caused phosphorylation of NF-kappaBand p38 MAPK and upregulation of TF activity in monocytes. These effects were not seen with IgG from patients with PM alone (VT-/PM+),anti-phospholipid Ab-positive patients without APS,or healthy controls. TF upregulation caused by the VT+/PM- samples was reduced by inhibitors of p38 MAPK,NF-kappaB,and TLR4. The effects of VT+/PM- IgG on signaling and TF upregulation were concentrated in the fraction that bound beta-2-glycoprotein I. Our findings demonstrate that IgGs from patients with diverse clinical manifestations of APS have differential effects upon phosphorylation of NF-kappaB and p38 MAPK and TF activity that may be mediated by differential activation of TLR4. View Publication

In humans,recent clinical and experimental data from hematopoietic stem cell transplantation revealed that donor-derived alloreactive NK cells exert a beneficial graft versus leukemia effect. The existence of donor-derived alloreactive NK cells can be predicted on the basis of donor killer cell Ig-like receptor (KIR) gene profile and HLA class I typing of both donor and recipient. Moreover,the size of the alloreactive NK cell population can be directly assessed by the combined use of anti-KIR-specific mAb. In this study,in an attempt to improve the definition of alloreactive NK cell subsets,we assessed the KIR genotype and phenotype in a cohort of 44 donors. This approach allowed the identification of two different KIR2DL3 alleles (KIR2DL3*005 and the novel allele KIR2DL3*015) that did not react with the anti-KIR2DL3-specific ECM41 mAb. In contrast,both alleles were recognized at the cell surface by several mAb reacting with KIR2DL2/L3/S2. Notably,KIR2DL3*005 was also stained by the anti-KIR2DL1/S1-specific EB6B and 11PB6 mAb. Functional analysis revealed that,despite its particular mAb reactivity,the specificity of KIR2DL3*005 for HLA-C molecules did not differ from that of other KIR2DL2/L3 alleles. Finally,site-directed mutagenesis demonstrated that glutamine at position 35 is required for ECM41 staining,whereas glutamic acid 35 and arginine 50 are relevant for staining with EB6B or 11PB6 mAb. Our present data represent a substantial progress in the characterization of the NK cell repertoire and an improved phenotypic/functional definition of given KIR(+) subsets. View Publication

Monomeric HIV envelope vaccines fail to elicit broadly neutralizing antibodies or to protect against infection. Neutralizing antibodies against HIV bind to native functionally active Env trimers on the virion surface. Gag-Env pseudovirions recapitulate the native trimer and could serve as an effective epitope presentation platform for study of the neutralizing antibody response in HIV-infected individuals. To address if pseudovirions can recapitulate native HIV virion epitope structures,we carefully characterized these particles,concentrating on the antigenic structure of the coreceptor binding site. By blue native gel shift assays,Gag-Env pseudovirions were shown to contain native trimers that were competent for binding to neutralizing monoclonal antibodies. In enzyme-linked immunosorbent assay,pseudovirions exhibited increased binding of known CD4-induced antibodies after addition of CD4. Using flow cytometric analysis,fluorescently labeled pseudovirions specifically identified a subset of antigen-specific B cells in HIV-infected subjects. Interestingly,the sequence of one of these novel human antibodies,identified during cloning of single HIV-specific B cells and designated 2C6,exhibited homology to mAb 47e,a known anti-CD4-induced coreceptor binding site antibody. The secreted monoclonal antibody 2C6 did not bind monomeric gp120,but specifically bound envelope on pseudovirions. A recombinant form of the antibody 2C6 acted as a CD4-induced epitope-specific antibody in neutralization assays,yet did not bind monomeric gp120. These findings imply specificity against a quaternary epitope presented on the pseudovirion envelope spike. These data demonstrate that Gag-Env pseudovirions recapitulate CD4 and coreceptor binding pocket antigenic structures and can facilitate identification of B-cell clones that secrete neutralizing antibodies. View Publication

The B cell-activating factor of the TNF family receptor (BAFF-R),encoded by the TNFRSF13C gene,is critically important for transitional B cell survival to maturity. Thus,ligation of BAFF-R by BAFF delivers a potent survival signal. Reports implicating the BAFF/BAFF-R signaling axis in the pathogenesis of autoimmune human diseases and B lineage malignancies have largely prompted studies focusing on BAFF expression; however,there is an equally critical need to better understand BAFF-R expression. Initial BAFF-R expression,although characterized in murine B cells,has not yet been reported in human B lymphopoiesis. In this study,we first demonstrate that BAFF-R expression is absent from early precursors and is acquired by bone marrow B cells newly expressing the BCR. We next focused on identifying the specific genomic region that controls BAFF-R expression in mature B cells (i.e.,the TNFRSF13C promoter). To accomplish this,we used in silico tools examining interspecies genomic conservation in conjunction with reporter constructs transfected into malignant B and plasma cell lines. DNase protection assays using nuclear extracts from BAFF-R-expressing cells suggested potential regulatory sites,which allowed the generation of EMSA probes that bound NFs specific to BAFF-R-expressing cells. With a more stringent analysis of interspecies homology,these assays identified a site at which a single nucleotide substitution could distinctly impact promoter activity. Finally,chromatin immunoprecipitation assays revealed the in vivo binding of the specific transcription factor c-Rel to the most proximal genomic region,and c-Rel small interfering RNA transfections in BAFF-R-expressing lines demonstrated a coincident knockdown of both c-Rel and BAFF-R mRNA. View Publication

RATIONALE: Chitin is a ubiquitous polysaccharide in fungi,insects,allergens,and parasites that is released at sites of infection. Its role in the generation of tissue inflammation,however,is not fully understood. OBJECTIVES: We hypothesized that chitin is an important adjuvant for adaptive immunity. METHODS: Mice were injected with a solution of ovalbumin and chitin. MEASUREMENTS AND MAIN RESULTS: We used in vivo and ex vivo/in vitro approaches to characterize the ability of chitin fragments to foster adaptive immune responses against ovalbumin and compared these responses to those induced by aluminum hydroxide (alum). In vivo,ovalbumin challenge caused an eosinophil-rich pulmonary inflammatory response,Th2 cytokine elaboration,IgE induction,and mucus metaplasia in mice that had been sensitized with ovalbumin plus chitin or ovalbumin plus alum. Toll-like receptor-2,MyD88,and IL-17A played critical roles in the chitin-induced responses,and MyD88 and IL-17A played critical roles in the alum-induced responses. In vitro,CD4(+) T cells from mice sensitized with ovalbumin plus chitin were incubated with ovalbumin-stimulated bone marrow-derived dendritic cells. In these experiments,CD4(+) T-cell proliferation,IL-5,IL-13,IFN-γ,and IL-17A production were appreciated. Toll-like receptor-2,MyD88,and IL-17A played critical roles in these in vitro adjuvant properties of chitin. TLR-2 was required for cell proliferation,whereas IL-17 and TLR-2 were required for cytokine elaboration. IL-17A also inhibited the generation of adaptive Th1 responses. CONCLUSIONS: These studies demonstrate that chitin is a potent multifaceted adjuvant that induces adaptive Th2,Th1,and Th17 immune responses. They also demonstrate that the adjuvant properties of chitin are mediated by a pathway(s) that involves and is regulated by TLR-2,MyD88,and IL-17A. View Publication

BACKGROUND: Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects,including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments,with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However,current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover,FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific,whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations,we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs,and maintained the capacity to suppress conventional T cell responses directed against tyrosinase,as well as bystander T cell responses. Using this methodology in a model tumor system,murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging. CONCLUSIONS/SIGNIFICANCE: These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy. View Publication

Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair,but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization,but show altered recruitment/retention kinetics in the diabetic environment. Moreover,diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative,chemotaxis,adhesion,and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response,enhanced neovascularization,and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing. View Publication