技术资料

Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription,nuclear import,and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell-based gene therapy trials performed to date,cells have been activated via their cognate antigen receptor. To couple activation with gene transfer,we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However,transduction of highly purified resting T cells with these anti-CD3-displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here,we describe interleukin-7 (IL-7)-displaying HIV-1-derived vectors. Like recombinant IL-7,these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore,a single exposure to the IL-7-displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells,preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether,these results suggest that IL-7-displaying vectors could constitute interesting tools for T-cell-targeted gene therapy. 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

IL-7 plays a major role in T lymphocyte homeostasis and has been proposed as an immune adjuvant for lymphopenic patients. This prospect is based,at least in part,on the short-term expansion of peripheral T cells in rIL7-treated mice and primates. Nevertheless,in vivo,following initial increases in T cell proliferation and numbers,lymphocytes return to a quiescent state. As the bases for this cell cycle exit have not yet been elucidated,it is important to assess the long-term biological effects of IL-7 on quiescent human T lymphocyte subsets. In this study,we find that IL-7-stimulated CD4+ naive lymphocytes enter into cell cycle with significantly delayed kinetics as compared with the memory population. Importantly though,these lymphocytes exit from the cell cycle despite the continuous replenishment of rIL-7. This response is distinct in memory and naive CD4+ lymphocytes with memory cells starting to exit from cycle by day 10 vs day 18 for naive cells. Return to quiescence is associated with a cessation in IL-7R signaling as demonstrated by an abrogation of STAT-5 phosphorylation,despite an up-regulation of surface IL-7Ralpha. Indeed,an initial 10-fold decrease in IL-7Ralpha mRNA levels is followed by increased IL-7Ralpha expression in naive as well as memory T cells,with kinetics paralleling cell cycle exit. Altogether,our data demonstrate that IL-7 promotes the extended survival of both naive and memory CD4+ T cells,whereas cycling of these two subsets is distinct and transient. Thus,IL-7 therapy should be designed to allow optimal responsiveness of naive and memory T cell subsets. View Publication

Dysregulation of airway inflammation contributes to lung disease in cystic fibrosis (CF). Inflammation is mediated by inflammatory cytokines,including IL-8,which illustrates an increase in biological half-life and proinflammatory activity when bound to glycosaminoglycans (GAGs). The aim of this project was to compare IL-8 and IL-18 for their relative stability,activity,and interaction with GAGs,including chondroitin sulfate,hyaluronic acid,and heparan sulfate,present in high quantities in the lungs of patients with CF. Bronchoalveolar lavage fluid was collected from patients with CF (n = 28),non-CF controls (n = 14),and patients with chronic obstructive pulmonary disease (n = 12). Increased levels of IL-8 and reduced concentrations of IL-18 were detected in bronchial samples obtained from CF individuals. The low level of IL-18 was not a defect in IL-18 production,as the pro- and mature forms of the molecule were expressed and produced by CF epithelial cells and monocytes. There was,however,a marked competition between IL-8 and IL-18 for binding to GAGs. A pronounced loss of IL-18 binding capacity occurred in the presence of IL-8,which displaced IL-18 from these anionic-matrices,rendering the cytokine susceptible to proteolytic degradation by neutrophil elastase. As a biological consequence of IL-18 degradation,reduced levels of IL-2 were secreted by Jurkat T lymphocytes. In conclusion,a novel mechanism has been identified highlighting the potential of IL-8 to determine the fate of other inflammatory molecules,such as IL-18,within the inflammatory milieu of the CF lung. 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

Rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles that code a five-amino acid sequence motif in positions 70-74 of the HLA-DRbeta-chain,called the shared epitope (SE). The mechanistic basis of SE-RA association is unknown. We recently found that the SE functions as an allele-specific signal-transducing ligand that activates an NO-mediated pathway in other cells. To better understand the role of the SE in the immune system,we examined its effect on T cell polarization in mice. In CD11c(+)CD8(+) dendritic cells (DCs),the SE inhibited the enzymatic activity of indoleamine 2,3 dioxygenase,a key enzyme in immune tolerance and T cell regulation,whereas in CD11c(+)CD8(-) DCs,the ligand activated robust production of IL-6. When SE-activated DCs were cocultured with CD4(+) T cells,the differentiation of Foxp3(+) T regulatory cells was suppressed,whereas Th17 cells were expanded. The polarizing effects could be seen with SE(+) synthetic peptides,but even more so when the SE was in its natural tridimensional conformation as part of HLA-DR tetrameric proteins. In vivo administration of the SE ligand resulted in a greater abundance of Th17 cells in the draining lymph nodes and increased IL-17 production by splenocytes. Thus,we conclude that the SE acts as a potent immune-stimulatory ligand that can polarize T cell differentiation toward Th17 cells,a T cell subset that was recently implicated in the pathogenesis of autoimmune diseases,including RA. View Publication

Interleukin-10 (IL-10) has potent immunoregulatory effects on the maturation and the antigen-presenting cell (APC) function of dendritic cells (DCs). The molecular basis underlying these effects in DCs,however,is ill defined. It is well established that the transcription factor NF-kappaB is a key regulator of DC development,maturation,and APC function. This study was initiated to determine the effects of IL-10 on the NF-kappaB signaling pathway in immature DCs. IL-10 pretreatment of myeloid DCs cultured from bone marrow resulted in reduced DNA binding and nuclear translocation of NF-kappaB after anti-CD40 antibody or lipopolysaccharide (LPS) stimulation. Furthermore,inhibited NF-kappaB activation was characterized by reduced degradation,phosphorylation,or both of IkappaBalpha and IkappaBepsilon but not IkappaBbeta and by reduced phosphorylation of Ser536,located in the trans-activation domain of p65. Notably,IL-10-mediated inhibition of NF-kappaB coincided with suppressed IkappaB kinase (IKK) activity in vitro. Furthermore,IL-10 blocked inducible Akt phosphorylation,and inhibitors of phosphatidylinositol 3-kinase (PI3K) effectively suppressed the activation of Akt,IKK,and NF-kappaB. These findings demonstrate that IL-10 targets IKK activation in immature DCs and that suppressing the PI3K pathway in part mediates blockade of the pathway. View Publication

Redirecting Ag specificity by transfer of TCR genes into PBLs is an attractive method to generate large numbers of cytotoxic T cells for immunotherapy of cancer and viral diseases. However,transferred TCR chains can pair with endogenous TCR chains,resulting in the formation of mispaired TCR dimers and decreased or unspecific reactivity. TCR gene transfer into hematopoietic stem cells (HSCs) is an alternative to create T cells with desired Ag specificity,because in this case expression of endogenous TCR chains is then less likely owing to allelic exclusion. We generated TCR-transduced T cells from peripheral T cells using the lymphocytic choriomeningitis virus-specific P14 TCR. After transfer of the P14 TCR genes into HSCs and subsequent reconstitution of irradiated mice,TCR-engineered HSC-derived T cells were produced. We then compared the Ag-specific T cell populations with P14 TCR-transgenic T cells for their therapeutic efficiency in three in vivo models. In this study,we demonstrate that TCR-transduced T cells and TCR-engineered HSC-derived T cells are comparable in controlling lymphocytic choriomeningitis virus infection in mice and suppress growth of B16 tumor cells expressing the cognate Ag in a comparable manner. View Publication

We investigated the contribution of host immune cells to bacterial killing in a whole-blood bactericidal activity (WBA) assay,an ex vivo model used to test efficacy of drugs against mycobacterium tuberculosis (Mtb). We performed WBA assays with immuno-magnetic depletion of specific cell types,in the presence or absence of rifampicin. Innate immune cells decreased Mtb growth in absence of drug,but appeared to diminish the cidal activity of rifampicin,possibly attributable to intracellular bacterial sequestration. Adaptive immune cells had no effect with or without drug. The WBA assay may have potential for testing adjunctive host-directed therapies acting on phagocytic cells. View Publication

Interaction of ICOS with its ligand is essential for germinal center formation,T cell immune responses,and development of autoimmune diseases. Human ICOS deficiency has been identified worldwide in nine patients with identical ICOS mutations. In vitro studies of the patients to date have shown only mild T cell defect. In this study,we report an in-depth analysis of T cell function in two siblings with novel ICOS deficiency. The brother displayed mild skin infections and impaired Ig class switching,whereas the sister had more severe symptoms,including immunodeficiency,rheumatoid arthritis,inflammatory bowel disease,interstitial pneumonitis,and psoriasis. Despite normal CD3/CD28-induced proliferation and IL-2 production in vitro,peripheral blood T cells in both patients showed a decreased percentage of CD4 central and effector memory T cells and impaired production of Th1,Th2,and Th17 cytokines upon CD3/CD28 costimulation or PMA/ionophore stimulation. The defective polarization into effector cells was associated with impaired induction of T-bet,GATA3,MAF,and retinoic acid-related orphan nuclear hormone receptor (RORC). Reduced CTLA-4(+)CD45RO(+)FoxP3(+) regulatory T cells and diminished induction of inhibitory cell surface molecules,including CTLA-4,were also observed in the patients. T cell defect was not restricted to CD4 T cells because reduced memory T cells and impaired IFN-gamma production were also noted in CD8 T cells. Further analysis of the patients demonstrated increased induction of receptor activator of NF-kappaB ligand (RANKL),lack of IFN-gamma response,and loss of Itch expression upon activation in the female patient,who had autoimmunity. Our study suggests that extensive T cell dysfunction,decreased memory T cell compartment,and imbalance between effector and regulatory cells in ICOS-deficient patients may underlie their immunodeficiency and/or autoimmunity. View Publication

RATIONALE: Tolerogenic dendritic cells and natural regulatory T cells have been implicated in the process of infectious tolerance in human allergic asthma. However,the significance of the influence of natural regulatory T cells on tolerogenic dendritic cells in the disease has not been investigated. OBJECTIVES: We aimed to characterize the mechanism of induction of the tolerogenic phenotype in circulating blood dendritic cells by allergic asthmatic natural regulatory T cells. METHODS: The study was performed in a cohort of 21 subjects with allergic asthma,21 healthy control subjects,and 21 subjects with nonallergic asthma. We cultured blood dendritic cells with natural regulatory T cells to study the induction of tolerogenic dendritic cells. Flow cytometry and proliferation assays were employed to analyze phenotype and function of dendritic cells as well as IL-10 production from natural regulatory T cells. MEASUREMENTS AND MAIN RESULTS: Dendritic cells cultured with natural regulatory T cells up-regulated IL-10,down-regulated costimulatory molecules,and stimulated the proliferation of CD4(+)CD25(-) effector T cells less potently. Allergic asthmatic natural regulatory T cells were significantly less efficient in inducing this tolerogenic phenotype of dendritic cells compared with healthy control and nonallergic asthmatic counterparts. Furthermore,this defective function of natural regulatory T cells was associated with their decreased IL-10 expression,disease severity,and could be reversed by oral corticosteroid therapy. CONCLUSIONS: These results provided the first evidences of impaired induction of tolerogenic dendritic cells mediated by natural regulatory T cells in human allergic asthma. View Publication

Immune dysfunction develops in patients with many cancer types and may contribute to tumor progression and failure of immunotherapy. Mechanisms underlying cancer-associated immune dysfunction are not fully understood. Efficient IFN signaling is critical to lymphocyte function; animals rendered deficient in IFN signaling develop cancer at higher rates. We hypothesized that altered IFN signaling may be a key mechanism of immune dysfunction common to cancer. To address this,we assessed the functional responses to IFN in peripheral blood lymphocytes from patients with 3 major cancers: breast cancer,melanoma,and gastrointestinal cancer. Type-I IFN (IFN-alpha)-induced signaling was reduced in T cells and B cells from all 3 cancer-patient groups compared to healthy controls. Type-II IFN (IFN-gamma)-induced signaling was reduced in B cells from all 3 cancer patient groups,but not in T cells or natural killer cells. Impaired-IFN signaling was equally evident in stage II,III,and IV breast cancer patients,and downstream functional defects in T cell activation were identified. Taken together,these findings indicate that defects in lymphocyte IFN signaling arise in patients with breast cancer,melanoma,and gastrointestinal cancer,and these defects may represent a common cancer-associated mechanism of immune dysfunction. View Publication