技术资料

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

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

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

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

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 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

In chronic lymphocytic leukemia (B-CLL),aberrations along the p53 axis lead to decreased overall survival and therapy resistance. Recent studies identified microRNA-34a (miR-34a) as a major downstream target of p53. We monitored the expression of miR-34a during disease development in a murine B-CLL model. miR-34a was up-regulated more than 20-fold during the leukemic but not during the preleukemic phase. In the human system,B-CLL cells also had 4.6-fold higher miR-34a expression compared with B cells of healthy controls. In B-CLL cells of patients with p53 aberrations,miR-34a expression was consistently low. The broad distribution of miR-34a levels in p53 wild-type patients prompted us to study the correlation between single nucleotide polymorphism 309 (SNP309) in the intronic promoter of MDM2 and miR-34a expression. B-CLL cells of patients with the SNP309 GG genotype had significantly lower miR-34a expression levels compared with patients with the TT genotype (P = .002). Low miR-34a levels were able to predict shorter time to treatment (P = .003) and were associated with an abbreviated lymphocyte doubling time. Further,overexpression of miR-34a in primary B-CLL cells induced apoptosis. These findings suggest miR-34a as a possible therapeutic avenue and a sensitive indicator of the activity of the p53 axis in B-CLL. View Publication

Small intestinal CD103(+) dendritic cells (DCs) have the selective ability to promote de novo generation of regulatory T cells via the production of retinoic acid (RA). Considering that aldehyde dehydrogenase (ALDH) activity controls the production of RA,we used a flow cytometry-based assay to measure ALDH activity at the single-cell level and to perform a comprehensive analysis of the RA-producing DC populations present in lymphoid and nonlymphoid mouse tissues. RA-producing DCs were primarily of the tissue-derived,migratory DC subtype and can be readily found in the skin and in the lungs as well as in their corresponding draining lymph nodes. The RA-producing skin-derived DCs were capable of triggering the generation of regulatory T cells,a finding demonstrating that the presence of RA-producing,tolerogenic DCs is not restricted to the intestinal tract as previously thought. Unexpectedly,the production of RA by skin DCs was restricted to CD103(-) DCs,indicating that CD103 expression does not constitute a universal" marker for RA-producing mouse DCs. Finally� View Publication

PURPOSE: B-cell receptor signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However,blocking B-cell receptor signaling with dasatinib,an inhibitor of SRC kinase,produced variable results in preclinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. EXPERIMENTAL DESIGN: Fresh CLL B cells were treated with dasatinib,and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of B-cell receptor signaling molecules,as well as with molecular and cytogenetic prognostic factors. RESULTS: Among 50 CLL cases,dasatinib treatment reduced cell viability by 2% to 90%,with an average reduction of 47% on day 4 of culture. The drug induced CLL cell death through the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly,phosphorylation of SRC family kinases was inhibited by dasatinib in good,as well as poor,responders. As opposed to SRC family kinases,activities of two downstream molecules,SYK and phospholipase Cgamma2,correlate well with the apoptotic response of CLL cells to dasatinib. CONCLUSIONS: Thus,SYK inhibition predicts cellular response to dasatinib. SYK,together with phospholipase Cgamma2,may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective,our study suggests the existence of alternative mechanisms or pathways that activate SYK,independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment. View Publication

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases,although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology,we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus. 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

BACKGROUND: Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate large numbers of highly purified primary B lymphocytes from tonsils in a short and cost-effective single step,using a commercially available reagent designed for purifying cells from whole blood (RosetteSep). This technique relies on the presence of the large excess of red blood cells in whole blood for the formation of immunorosettes,whereas single cell suspensions from tonsils contain relatively few red blood cells. RESULTS: B cell enrichment from tonsils was achieved using RosetteSep with no modification to the whole blood procedure; however,the degree of purity depended on the extent of red blood cell contamination of the starting tonsil cell suspension. Addition of a 50-fold excess of allogeneic human red blood cells,but not sheep red blood cells,reproducibly resulted in high levels of purity. Depletion of mononuclear cells from the donor red blood cells eliminated potential contamination with allogeneic B cells. CONCLUSION: RosetteSep reagent can be used in combination with allogeneic human red blood cells to reproducibly isolate tonsil B lymphocytes to high levels of purity with no change in phenotype or loss of cells. This method provides considerable time and cost savings compared to other methods. View Publication