技术资料

The cytidine deaminase AID (encoded by Aicda in mice and AICDA in humans) is critical for immunoglobulin class-switch recombination (CSR) and somatic hypermutation (SHM). Here we show that AID expression was induced by the HoxC4 homeodomain transcription factor,which bound to a highly conserved HoxC4-Oct site in the Aicda or AICDA promoter. This site functioned in synergy with a conserved binding site for the transcription factors Sp1,Sp3 and NF-kappaB. HoxC4 was 'preferentially' expressed in germinal center B cells and was upregulated by engagement of CD40 by CD154,as well as by lipopolysaccharide and interleukin 4. HoxC4 deficiency resulted in impaired CSR and SHM because of lower AID expression and not some other putative HoxC4-dependent activity. Enforced expression of AID in Hoxc4(-/-) B cells fully restored CSR. Thus,HoxC4 directly activates the Aicda promoter,thereby inducing AID expression,CSR and SHM. View Publication

Imatinib is currently the first line therapy for newly diagnosed patients with chronic myeloid leukemia. However,20-25% of patients do not achieve durable complete cytogenetic responses. The mechanism underlying this primary resistance is unknown,but variations in BCR-ABL1 kinase activity may play a role and can be investigated by measuring the autophosphorylation levels of BCR-ABL1 or of a surrogate target such as Crkl. In this study we used flow cytometry to investigate the in vitro inhibition of Crkl phosphorylation by imatinib in CD34(+) cells in diagnostic samples from two groups of patients distinguished by their cytogenetic response. No difference in inhibition of Crkl phosphorylation was observed in the two groups. The observation that increasing the dose of imatinib in vivo did not increase the level of cytogenetic response in some non-responders suggests that in at least a proportion of patients imatinib resistance may be due to activation of BCR-ABL1-independent pathway. 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

Threats of bioterrorism have renewed efforts to better understand poxvirus pathogenesis and to develop a safer vaccine against smallpox. Individuals with atopic dermatitis are excluded from smallpox vaccination because of their propensity to develop eczema vaccinatum,a disseminated vaccinia virus (VACV) infection. To study the underlying mechanism of the vulnerability of atopic dermatitis patients to VACV infection,we developed a mouse model of eczema vaccinatum. Virus infection of eczematous skin induced severe primary erosive skin lesions,but not in the skin of healthy mice. Eczematous mice exhibited lower natural killer (NK) cell activity but similar cytotoxic T lymphocyte activity and humoral immune responses. The role of NK cells in controlling VACV-induced skin lesions was demonstrated by experiments depleting or transferring NK cells. The proinflammatory cytokine interleukin (IL)-17 reduced NK cell activity in mice with preexisting dermatitis. Given low NK cell activities and increased IL-17 expression in atopic dermatitis patients,these results can explain the increased susceptibility of atopic dermatitis patients to eczema vaccinatum. View Publication

The rising incidence of autoimmune diseases such as multiple sclerosis (MS) in developed countries might be due to a more hygienic environment,particularly during early life. To investigate this concept,we developed a model of neonatal exposure to a common pathogen-associated molecular pattern,LPS,and determined its impact on experimental autoimmune encephalomyelitis (EAE). Mice exposed to LPS at 2 wk of age showed a delayed onset and diminished severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE,induced at 12 wk,compared with vehicle-exposed animals. Spinal cord transcript levels of CD3epsilon and F4/80 were lower in LPS- compared with PBS-exposed EAE animals with increased IL-10 levels in the LPS-exposed group. Splenic CD11c(+) cells from LPS-exposed animals exhibited reduced MHC class II and CD83 expression but increased levels of CD80 and CD86 both before and during EAE. MOG-treated APC from LPS-exposed animals stimulated less T lymphocyte proliferation but increased expansion of CD4(+)FoxP3(+) T cells compared with APC from PBS-exposed animals. Neuropathological studies disclosed reduced myelin and axonal loss in spinal cords from LPS-exposed compared with PBS-exposed animals with EAE,and this neuroprotective effect was associated with an increased number of CD3(+)FoxP3(+) immunoreactive cells. Analyses of human brain tissue revealed that FoxP3 expression was detected in lymphocytes,albeit reduced in MS compared with non-MS patients' brains. These findings support the concept of early-life microbial exposure influencing the generation of neuroprotective regulatory T cells and may provide insights into new immunotherapeutic strategies for MS. View Publication

Persistent viral infections are a major health concern worldwide. During persistent infection,overwhelming viral replication and the rapid loss of antiviral T-cell function can prevent immune-mediated clearance of the infection,and therapies to reanimate the immune response and purge persistent viruses have been largely unsuccessful. Adoptive immunotherapy using memory T cells is a highly successful therapeutic approach to eradicate a persistent viral infection. Understanding precisely how therapeutically administered memory T cells achieve clearance should improve our ability to terminate states of viral persistence in humans. Mice persistently infected from birth with lymphocytic choriomeningitis virus are tolerant to the pathogen at the T-cell level and thus provide an excellent model to evaluate immunotherapeutic regimens. Previously,we demonstrated that adoptively transferred memory T cells require recipient dendritic cells to effectively purge an established persistent viral infection. However,the mechanisms that reactivate and sustain memory T-cell responses during clearance of such an infection remain unclear. Here we establish that therapeutic memory T cells require CD80 and CD86 costimulatory signals to efficiently clear an established persistent viral infection in vivo. Early blockade of costimulatory pathways with CTLA-4-Fc decreased the secondary expansion of virus-specific CD8(+) and CD4(+) memory T cells as well as their ability to produce antiviral cytokines and purge the persistent infection. Late costimulation blockade also reduced virus-specific T-cell numbers,illustrating that sustained interactions with costimulatory molecules is required for efficient T-cell expansion. These findings indicate that antiviral memory T cells require costimulation to efficiently clear a persistent viral infection and that costimulatory pathways can be targeted to modulate the magnitude of an adoptive immunotherapeutic regimen. View Publication

The Msh2 mismatch repair (MMR) protein is critical for class switch recombination (CSR) events that occur in mice that lack the Smu tandem repeat (SmuTR) region (SmuTR(-/-) mice). The pattern of microhomology among switch junction sites in Msh2-deficient mice is also dependent on the presence or absence of SmuTR sequences. It is not known whether these CSR effects reflect an individual function of Msh2 or the function of Msh2 within the MMR machinery. In the absence of the SmuTR sequences,Msh2 deficiency nearly ablates CSR. We now show that Mlh1 or Exo1 deficiencies also eliminate CSR in the absence of the SmuTR. Furthermore,in SmuTR(-/-) mice,deficiencies of Mlh1 or Exo1 result in increased switch junction microhomology as has also been seen with Msh2 deficiency. These results are consistent with a CSR model in which the MMR machinery is important in processing DNA nicks to produce double-stranded breaks,particularly in sequences where nicks are infrequent. We propose that double-stranded break paucity in MMR-deficient mice leads to increased use of an alternative joining pathway where microhomologies are important for CSR break ligation. Interestingly,when the SmuTR region is present,deficiency of Msh2 does not lead to the increased microhomology seen with Mlh1 or Exo1 deficiencies,suggesting that Msh2 might have an additional function in CSR. It is also possible that the inability to initiate MMR in the absence of Msh2 results in CSR junctions with less microhomology than joinings that occur when MMR is initiated but then proceeds abnormally due to Mlh1 or Exo1 deficiencies. View Publication

As a member of the class III histone deacetylases,Sirtuin-2 (SIRT2) is critical in cell cycle regulation which makes it a potential target for cancer therapeutics. In this study,we identified a novel SIRT2 inhibitor,AC-93253,with IC(50) of 6 microM in vitro. The compound is selective,inhibiting SIRT2 7.5- and 4-fold more potently than the closely related SIRT1 and SIRT3,respectively. AC-93253 significantly enhanced acetylation of tubulin,p53,and histone H4,confirming SIRT2 and SIRT1 as its cellular targets. AC-93253 as a single agent exhibited submicromolar selective cytotoxicity towards all four tumor cell lines tested with a therapeutic window up to 200-fold,comparing to any of the three normal cell types tested. Results from high content analysis suggested that AC-93253 significantly triggered apoptosis. Taken together,SIRT2 selective inhibitor AC-93253 may serve as a novel chemical scaffold for structure-activity relationship study and future lead development. View Publication

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is overexpressed in many types of tumors,including pancreatic cancer,and plays an important role in cell adhesion and survival signaling. Pancreatic cancer is a lethal disease and is very resistant to chemotherapy,and FAK has been shown recently to assist in tumor cell survival. Therefore,FAK is an excellent potential target for anti-cancer therapy. We identified a novel small molecule inhibitor (1,2,4,5-Benzenetetraamine tetrahydrochloride,that we called Y15) targeting the main autophosphorylation site of FAK and hypothesized that it would be an effective treatment strategy against human pancreatic cancer. Y15 specifically blocked phosphorylation of Y397-FAK and total phosphorylation of FAK. It directly inhibited FAK autophosphorylation in a dose- and time-dependent manner. Furthermore,Y15 increased pancreatic cancer cell detachment and inhibited cell adhesion in a dose-dependent manner. Y15 effectively caused human pancreatic tumor regression in vivo,when administered alone and its effects were synergistic with gemcitabine chemotherapy. This was accompanied by a decrease in Y397-phosphorylation of FAK in the tumors treated with Y15. Thus,targeting the Y397 site of FAK in pancreatic cancer with the small molecule inhibitor,1,2,4,5-Benzenetetraamine tetrahydrochloride,is a potentially effective treatment strategy in this deadly disease. View Publication

Platelets have a crucial role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion,which underlie stroke and acute coronary syndromes. Anucleate platelets contain mRNAs and are capable of protein synthesis,raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs),which are known as key regulators of mRNA translation in other cell types. Further analyses revealed that platelets contain the Dicer and Argonaute 2 (Ago2) complexes,which function in the processing of exogenous miRNA precursors and the control of specific reporter transcripts,respectively. Detection of the receptor P2Y(12) mRNA in Ago2 immunoprecipitates suggests that P2Y(12) expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system. View Publication

Mesenchymal stem cells (MSC) have been extensively studied and gained wide popularity due to their therapeutic potential. Spontaneous transformation of MSC,from both human and murine origin,has been reported in many studies. MSC transformation depends on the culture conditions,the origin of the cells and the time on culture; however,the precise biological characteristics involved in this process have not been fully defined yet. In this study,we investigated the role of p53 in the biology and transformation of murine bone marrow (BM)-derived MSC. We demonstrate that the MSC derived from p53KO mice showed an augmented proliferation rate,a shorter doubling time and also morphologic and phenotypic changes,as compared to MSC derived from wild-type animals. Furthermore,the MSC devoid of p53 had an increased number of cells able to generate colonies. In addition,not only proliferation but also MSC differentiation is controlled by p53 since its absence modifies the speed of the process. Moreover,genomic instability,changes in the expression of c-myc and anchorage independent growth were also observed in p53KO MSC. In addition,the absence of p53 implicates the spontaneous transformation of MSC in long-term cultures. Our results reveal that p53 plays a central role in the biology of MSC. View Publication

Retroviruses can establish persistent infection despite induction of a multipartite antiviral immune response. Whether collective failure of all parts of the immune response or selective deficiency in one crucial part underlies the inability of the host to clear retroviral infections is currently uncertain. We examine here the contribution of virus-specific CD4(+) T cells in resistance against Friend virus (FV) infection in the murine host. We show that the magnitude and duration of the FV-specific CD4(+) T-cell response is directly proportional to resistance against acute FV infection and subsequent disease. Notably,significant protection against FV-induced disease is afforded by FV-specific CD4(+) T cells in the absence of a virus-specific CD8(+) T-cell or B-cell response. Enhanced spread of FV infection in hosts with increased genetic susceptibility or coinfection with Lactate dehydrogenase-elevating virus (LDV) causes a proportional increase in the number of FV-specific CD4(+) T cells required to control FV-induced disease. Furthermore,ultimate failure of FV/LDV coinfected hosts to control FV-induced disease is accompanied by accelerated contraction of the FV-specific CD4(+) T-cell response. Conversely,an increased frequency or continuous supply of FV-specific CD4(+) T cells is both necessary and sufficient to effectively contain acute infection and prevent disease,even in the presence of coinfection. Thus,these results suggest that FV-specific CD4(+) T cells provide significant direct protection against acute FV infection,the extent of which critically depends on the ratio of FV-infected cells to FV-specific CD4(+) T cells. View Publication