技术资料

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation,which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo,antibody blockade of TNFSF15 (TL1A),which is the ligand for TNFR25,inhibits lung inflammation and production of Th2 cytokines such as IL-13,even when administered days after airway antigen exposure. Similarly,blockade of TNFR25 by a dominant-negative (DN) transgene,DN TNFR25,confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant,NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells,but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung,and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics. 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

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

Clonal analysis is important for many areas of hematopoietic stem cell research,including in vitro cell expansion,gene therapy,and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle,they generally provide a low-resolution,biased,and incomplete assessment of clonality. To overcome those limitations,we labeled retroviral vectors with random sequence tags or barcodes." On integration� View Publication

Frequent hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) include aberrant NOTCH signaling and deletion of the CDKN2A locus,which contains 2 closely linked tumor suppressor genes (INK4A and ARF). When bone marrow cells or thymocytes transduced with a vector encoding the constitutively activated intracellular domain of Notch1 (ICN1) are expanded ex vivo under conditions that support T-cell development,cultured progenitors rapidly induce CD4+/CD8+ T-ALLs after infusion into healthy syngeneic mice. Under these conditions,enforced ICN1 expression also drives formation of T-ALLs in unconditioned CD-1 nude mice,bypassing any requirements for thymic maturation. Retention of Arf had relatively modest activity in suppressing the formation of T-ALLs arising from bone marrow-derived ICN1+ progenitors in which the locus is epigenetically silenced,and all resulting Arf (+/+) tumors failed to express the p19(Arf) protein. In striking contrast,retention of Arf in thymocyte-derived ICN1+ donor cells significantly delayed disease onset and suppressed the penetrance of T-ALL. Use of cultured thymocyte-derived donor cells expressing a functionally null Arf-GFP knock-in allele confirmed that ICN1 signaling can induce Arf expression in vivo. Arf activation by ICN1 in T cells thereby provides stage-specific tumor suppression but also a strong selective pressure for deletion of the locus in T-ALL. View Publication

HIV-1 infection is characterized by a progressive decline in CD4(+) T cells leading to a state of profound immunodeficiency. IL-2 therapy has been shown to improve CD4(+) counts beyond that observed with antiretroviral therapy. Recent phase III trials revealed that despite a sustained increase in CD4(+) counts,IL-2-treated patients did not experience a better clinical outcome [Abrams D,et al. (2009) N Engl J Med 361(16):1548-1559]. To explain these disappointing results,we have studied phenotypic,functional,and molecular characteristics of CD4(+) T cell populations in IL-2-treated patients. We found that the principal effect of long-term IL-2 therapy was the expansion of two distinct CD4(+)CD25(+) T cell populations (CD4(+)CD25(lo)CD127(lo)FOXP3(+) and CD4(+)CD25(hi)CD127(lo)FOXP3(hi)) that shared phenotypic markers of Treg but could be distinguished by the levels of CD25 and FOXP3 expression. IL-2-expanded CD4(+)CD25(+) T cells suppressed proliferation of effector cells in vitro and had gene expression profiles similar to those of natural regulatory CD4(+)CD25(hi)FOXP3(+) T cells (Treg) from healthy donors,an immunosuppressive T cell subset critically important for the maintenance of self-tolerance. We propose that the sustained increase of the peripheral Treg pool in IL-2-treated HIV patients may account for the unexpected clinical observation that patients with the greatest expansion of CD4(+) T cells had a higher relative risk of clinical progression to AIDS. View Publication

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5,were cross resistant to other small-molecule CCR5 antagonists,and were isolated from the patient's pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4(+) T cells. The V3 loop contained residues essential for viral resistance to APL,while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However,these mutations were context dependent,being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N' terminus of CCR5 in the presence of APL. In addition,the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However,recognition of drug-bound CCR5 was less efficient,resulting in a tropism shift toward effector memory cells upon infection of primary CD4(+) T cells in the presence of APL,with relative sparing of the central memory CD4(+) T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses,then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T(CM) subset of CD4(+) T cells and result in improved T cell homeostasis and immune function. View Publication

Accumulating evidence suggests that the regulation of gene expression by histone lysine methylation is crucial for several biological processes. The histone lysine methyltransferase G9a is responsible for the majority of dimethylation of histone H3 at lysine 9 (H3K9me2) and is required for the efficient repression of developmentally regulated genes during embryonic stem cell differentiation. However,whether G9a plays a similar role in adult cells is still unclear. We identify a critical role for G9a in CD4(+) T helper (Th) cell differentiation and function. G9a-deficient Th cells are specifically impaired in their induction of Th2 lineage-specific cytokines IL-4,IL-5,and IL-13 and fail to protect against infection with the intestinal helminth Trichuris muris. Furthermore,G9a-deficient Th cells are characterised by the increased expression of IL-17A,which is associated with a loss of H3K9me2 at the Il17a locus. Collectively,our results establish unpredicted and complex roles for G9a in regulating gene expression during lineage commitment in adult CD4(+) T cells. View Publication

Mature CD4(+)Vbeta5(+) T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or TCR revision. In Vbeta5 transgenic mice,this latter tolerance pathway results in the appearance of CD4(+)Vbeta5(-)TCRbeta(+) T cells,coinciding with Rag1,Rag2,and TdT expression and the accumulation of V(beta)-DJ(beta) recombination intermediates in peripheral CD4(+) T cells. Because postthymic RAG-dependent TCR rearrangement has remained controversial,we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We show in this study that Rag deletion in post-positive selection T cells in Vbeta5 transgenic mice blocks TCR revision in vivo and that mature peripheral T cells sorted to remove cells bearing endogenous TCRbeta-chains can express newly generated TCRbeta molecules in adoptive hosts. These findings unambiguously demonstrate postthymic,RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4(+) T cells. View Publication

The viral infection of higher vertebrates elicits potent innate and adaptive host immunity. However,an excessive or inappropriate immune response also may lead to host pathology that often is more severe than the direct effects of viral replication. Therefore,several mechanisms exist that regulate the magnitude and class of the immune response. Here,we have examined the potential involvement of regulatory T (Treg) cells in limiting pathology induced by influenza A virus (IAV) infection. Using lymphocyte-deficient mice as hosts,we showed that Treg cell reconstitution resulted in a significant delay in weight loss and prolonged survival following infection. The adoptively transferred Treg cells did not affect the high rate of IAV replication in the lungs of lymphocyte-deficient hosts,and therefore their disease-ameliorating effect was mediated through the suppression of innate immune pathology. Mechanistically,Treg cells reduced the accumulation and altered the distribution of monocytes/macrophages in the lungs of IAV-infected hosts. This reduction in lung monocytosis was associated with a specific delay in monocyte chemotactic protein-2 (MCP-2) induction in the infected lungs. Nevertheless,Treg cells failed to prevent the eventual development of severe disease in lymphocyte-deficient hosts,which likely was caused by the ongoing IAV replication. Indeed,using T-cell-deficient mice,which mounted a T-cell-independent B cell response to IAV,we further showed that the combination of virus-neutralizing antibodies and transferred Treg cells led to the complete prevention of clinical disease following IAV infection. Taken together,these results suggested that innate immune pathology and virus-induced pathology are the two main contributors to pathogenesis during IAV infection. View Publication

The contribution of mammary epithelial cells (MEC) to human immunodeficiency virus type 1 (HIV-1) in breast milk remains largely unknown. While breast milk contains CD4(+) cells throughout the breast-feeding period,it is not known whether MEC directly support HIV-1 infection or facilitate infection of CD4(+) cells in the breast compartment. This study evaluated primary human MEC for direct infection with HIV-1 and for indirect transfer of infection to CD4(+) target cells. Primary human MEC were isolated and assessed for expression of HIV-1 receptors. MEC were exposed to CCR5-,CXCR4- and dual-tropic strains of HIV-1 and evaluated for viral reverse transcription and integration and productive viral infection. MEC were also tested for the ability to transfer HIV to CD4(+) target cells and to activate resting CD4(+) T cells. Our results demonstrate that MEC express HIV-1 receptor proteins CD4,CCR5,CXCR4,and galactosyl ceramide (GalCer). While no evidence for direct infection of MEC was found,HIV-1 virions were observed in MEC endosomal compartments. Coculture of HIV-exposed MEC resulted in productive infection of activated CD4(+) T cells. In addition,MEC secretions increased HIV-1 replication and proliferation of infected target cells. Overall,our results indicate that MEC are capable of endosomal uptake of HIV-1 and can facilitate virus infection and replication in CD4(+) target cells. These findings suggest that MEC may serve as a viral reservoir for HIV-1 and may enhance infection of CD4(+) T lymphocytes in vivo. View Publication

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular bulk degradation pathway that plays critical roles in eliminating intracellular pathogens,presenting endogenous Ags,and regulating T lymphocyte survival and proliferation. In this study,we have investigated the role of autophagy in regulating the endoplasmic reticulum (ER) compartment in T lymphocytes. We found that ER content is expanded in mature autophagy-related protein (Atg) 7-deficient T lymphocytes. Atg7-deficient T cells stimulated through the TCR display impaired influx,but not efflux,of calcium,and ER calcium stores are increased in Atg7-deficient T cells. Treatment with the ER sarco/ER Ca(2+)-ATPase pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T lymphocytes,suggesting that this impairment is caused by an intrinsic defect in ER. Furthermore,we found that the stimulation-induced redistribution of stromal interaction molecule-1,a critical event for the store-operated Ca(2+) release-activated Ca(2+) channel opening,is impaired in Atg7-deficient T cells. Together,these findings indicate that the expanded ER compartment in Atg7-deficient T cells contains increased calcium stores,and the inability of these stores to be depleted causes defective calcium influx in these cells. Our results demonstrate that autophagy plays an important role in maintaining ER and calcium homeostasis in T lymphocytes. View Publication