技术资料

Cytoplasmic APOBEC3G has been reported to block wild-type human immunodeficiency virus type 1 (HIV-1) infection in some primary cells. It is not known whether cytoplasmic APOBEC3G has residual activity in activated T cells,even though virion-packaged APOBEC3G does restrict HIV-1 in activated T cells. Because we found that APOBEC3G expression is greater in activated CD4(+) T-helper type 1 (Th1) lymphocytes than in T-helper type 2 (Th2) lymphocytes,we hypothesized that residual target cell restriction of incoming Vif-positive virions that lack APOBEC3G,if present,would be greater in Th1 than Th2 lymphocytes. Infection of activated Th1 cells with APOBEC3-negative virions did result in decreased amounts of early and late reverse transcription products and integrated virus relative to infection of activated Th2 cells. Two-long terminal repeat (2-LTR) circles,which are formed in the nucleus when reverse transcripts do not integrate,were increased after APOBEC3-negative virus infection of activated Th1 cells relative to infection of activated Th2 cells. In contrast,2-LTR circle forms were decreased after infection of APOBEC3G-negative cells with APOBEC3G-containing virions relative to APOBEC3G-negative virions and with Th1 cell-produced virions relative to Th2 cell-produced virions. Increasing APOBEC3G in Th2 cells and decreasing APOBEC3G in Th1 cells modulated the target cell phenotypes,indicating causation by APOBEC3G. The comparison between activated Th1 and Th2 cells indicates that cytoplasmic APOBEC3G in activated Th1 cells partially restricts reverse transcription and integration of incoming Vif-positive,APOBEC3G-negative HIV-1. The differing effects of cytoplasmic and virion-packaged APOBEC3G on 2-LTR circle formation indicate a difference in their antiviral mechanisms. View Publication

The control of tyrosine phosphorylation depends on the fine balance between kinase and phosphatase activities. Protein tyrosine phosphatase 1B (PTP-1B) and T cell protein tyrosine phosphatase (TC-PTP) are 2 closely related phosphatases known to control cytokine signaling. We studied the functional redundancy of PTP-1B and TC-PTP by deleting 1 or both copies of these genes by interbreeding TC-PTP and PTP-1B parental lines. Our results indicate that the double mutant (tcptp(-/-)ptp1b(-/-)) is lethal at day E9.5-10.5 of embryonic development with constitutive phosphorylation of Stat1. Mice heterozygous for TC-PTP on a PTP-1B-deficient background (tcptp(+/-)ptp1b(-/-)) developed signs of inflammation. Macrophages from these animals were highly sensitive to IFN-gamma,as demonstrated by increased Stat1 phosphorylation and nitric oxide production. In addition,splenic T cells demonstrated increased IFN-gamma secretion capacity. Mice with deletions of single copies of TC-PTP and PTP-1B (tcptp(+/-)ptp1b(+/-)) exhibited normal development,confirming that these genes are not interchangeable. Together,these data indicate a nonredundant role for PTP-1B and TC-PTP in the regulation of IFN signaling. View Publication

OBJECTIVE: T lymphocytes play an important role in systemic sclerosis (SSc),a connective tissue disease characterized by inflammation,fibrosis,and vascular damage. While their precise role and antigen specificity are unclear,T cell-derived cytokines likely contribute to the induction of fibrosis. The aim of this study was to establish the role of cytokine dysregulation by T cells in the pathogenesis of SSc. METHODS: To identify relationships between a specific cytokine,T cell subset,and the disease course,we studied a large cohort of patients with diffuse cutaneous SSc (dcSSc) or limited cutaneous SSc (lcSSc). Using Luminex analysis and intracellular cytokine staining,we analyzed the intrinsic ability of CD4+ and CD8+ T cell subsets to produce cytokines following in vitro activation. RESULTS: High levels of the profibrotic type 2 cytokine interleukin-13 (IL-13) were produced following activation of peripheral blood effector CD8+ T cells from SSc patients as compared with normal controls or with patients with rheumatoid arthritis. In contrast,CD4+ T cells showed a lower and more variable level of IL-13 production. This abnormality correlated with the extent of fibrosis and was more pronounced in dcSSc patients than in lcSSc patients. CONCLUSION: Dysregulated IL-13 production by effector CD8+ T cells is important in the pathogenesis of SSc and is critical in the predisposition to more severe forms of cutaneous disease. Our study is the first to identify a specific T cell phenotype that correlates with disease severity in SSc and can be used as a marker of immune dysfunction in SSc and as a novel therapeutic target. View Publication

Retinoic acid (RA) produced by intestinal dendritic cells (DCs) imprints gut-homing specificity on lymphocytes and enhances Foxp3(+) regulatory T-cell differentiation. The expression of aldehyde dehydrogenase (ALDH) 1A in these DCs is essential for the RA production. However,it remains unclear how the steady-state ALDH1A expression is induced under specific pathogen-free (SPF) conditions. Here,we found that bone marrow-derived dendritic cells (BM-DCs) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) expressed Aldh1a2,an isoform of Aldh1a,but that fms-related tyrosine kinase 3 ligand-generated BM-DCs did not. DCs from mesenteric lymph nodes (MLN) and Peyer's patches (PP) of normal SPF mice expressed ALDH1A2,but not the other known RA-producing enzymes. Employing a flow cytometric method,we detected ALDH activities in 10-30% of PP-DCs and MLN-DCs. They were CD11c(high)CD4(-/low)CD8alpha(intermediate)CD11b(-/low) F4/80(low/intermediate)CD45RB(low)CD86(high)MHC class II(high)B220(-)CD103(+). Equivalent levels of aldehyde dehydrogenase activity (ALDHact) and ALDH1A2 expression were induced synergistically by GM-CSF and IL-4 in splenic DCs in vitro. In BM-DCs,however,additional signals via Toll-like receptors or RA receptors were required for inducing the equivalent levels. The generated ALDH1A2(+) DCs triggered T cells to express gut-homing receptors or Foxp3. GM-CSF receptor-deficient or vitamin A-deficient mice exhibited marked reductions in the ALDHact in intestinal DCs and the T cell number in the intestinal lamina propria,whereas IL-4 receptor-mediated signals were dispensable. GM-CSF(+)CD11c(-)F4/80(+) cells existed constitutively in the intestinal tissues. The results suggest that GM-CSF and RA itself are pivotal among multiple microenvironment factors that enable intestinal DCs to produce RA. View Publication

Kv1.3 potassium channels maintain the membrane potential of effector memory (T(EM)) T cells that are important mediators of multiple sclerosis,type 1 diabetes mellitus,and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5),containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin,is a potent and selective blocker of these channels. However,a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability,ShK-192,contains a nonhydrolyzable phosphotyrosine surrogate,a methionine isostere,and a C-terminal amide. ShK-192 shows the same overall fold as ShK,and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys(411) of the channel. ShK-192 blocks Kv1.3 with an IC(50) of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 microg/kg,approximately 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24,48,and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T(EM) cells and suppresses delayed type hypersensitivity when administered at 10 or 100 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells. View Publication

The CD31(+) subset of human naive CD4(+) T cells is thought to contain the population of cells that have recently emigrated from the thymus,while their CD31(-) counterparts have been proposed to originate from CD31(+) cells after homeostatic cell division. Naive T-cell maintenance is known to involve homeostatic cytokines such as interleukin-7 (IL-7). It remains to be investigated what role this cytokine has in the homeostasis of naive CD4(+) T-cell subsets defined by CD31 expression. We provide evidence that IL-7 exerts a preferential proliferative effect on CD31(+) naive CD4(+) T cells from adult peripheral blood compared with the CD31(-) subset. IL-7-driven proliferation did not result in loss of CD31 expression,suggesting that CD31(+) naive CD4(+) T cells can undergo cytokine-driven homeostatic proliferation while preserving CD31. Furthermore,IL-7 sustained or increased CD31 expression even in nonproliferating cells. Both proliferation and CD31 maintenance were dependent on the activation of phosphoinositide 3-kinase (PI3K) signaling. Taken together,our data suggest that during adulthood CD31(+) naive CD4(+) T cells are maintained by IL-7 and that IL-7-based therapies may exert a preferential effect on this population. View Publication

During persistent murine cytomegalovirus (MCMV) infection,the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells,which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation,MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead,we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported,at least in part,by memory T cells primed early in infection,as well as by recruitment of naive T cells at late times. Thus,these data show that memory inflation is maintained by a continuous replacement of short-lived,functional cells during chronic MCMV infection. View Publication

We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct,allowing its regulated expression (and,at the same time,deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes,miRNA-based RNAi (including two shRNA constructs at once),and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture,efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny,and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo. View Publication

The mutagenic enzyme activation-induced cytidine deaminase (AID) is required for immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in germinal center (GC) B cells. Deregulated expression of AID is associated with various B-cell malignancies and,currently,it remains unclear how AID activity is extinguished to avoid illegitimate mutations. AID has also been shown to be alternatively spliced in malignant B cells,and there is limited evidence that this also occurs in normal blood B cells. The functional significance of these splice variants remains unknown. Here we show that normal GC human B cells and blood memory B cells similarly express AID splice variants and show for the first time that AID splicing variants are singly expressed in individual normal B cells as well as malignant B cells from chronic lymphocytic leukemia patients. We further demonstrate that the alternative AID splice variants display different activities ranging from inactivation of CSR to inactivation or heightened SHM activity. Our data therefore suggest that CSR and SHM are differentially switched off by varying the expression of splicing products of AID at the individual cell level. Most importantly,our findings suggest a novel tumor suppression mechanism by which unnecessary AID mutagenic activities are promptly contained for GC B cells. View Publication

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation,we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells,cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid,B-lymphoid,and erythroid lineages,but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization,which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice. View Publication

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However,it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous,endogenously HIV-1-infected CD4+ T cells. Here,we stimulate primary CD4+ T cells,purified ex vivo from HIV-1-infected viremic patients,with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that,subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules,HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells,while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However,the degree of NK cell cytolytic activity against autologous,endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively,our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells. View Publication

The dynamic interplay between dendritic cells (DCs) and human immunodeficiency virus type-1 (HIV-1) is thought to result in viral dissemination and evasion of antiviral immunity. Although initial observations suggested that the C-type lectin receptor (CLR) DC-SIGN was responsible for the trans-infection function of the virus,subsequent studies demonstrated that trans-infection of CD4(+) T cells with HIV-1 can also occur through DC-SIGN-independent mechanisms. We demonstrate that a cell surface molecule designated DCIR (for DC immunoreceptor),a member of a recently described family of DC-expressing CLRs,can participate in the capture of HIV-1 and promote infection in trans and in cis of autologous CD4(+) T cells from human immature monocyte-derived DCs. The contribution of DCIR to these processes was revealed using DCIR-specific siRNAs and a polyclonal antibody specific for the carbohydrate recognition domain of DCIR. Data from transfection experiments indicated that DCIR acts as a ligand for HIV-1 and is involved in events leading to productive virus infection. Finally,we show that the neck domain of DCIR is important for the DCIR-mediated effect on virus binding and infection. These results point to a possible role for DCIR in HIV-1 pathogenesis by supporting the productive infection of DCs and promoting virus propagation. View Publication