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1.
Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL–DR4 in live cells in real‐time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4‐CFP and DR4‐YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4‐CFP and DR4‐YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegel's YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4‐CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4‐CFP/YFP reporter cells exhibited a colocalized expression of DR4‐CFP and DR4‐YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi‐fluorescence microscopy for FRET analysis. The real‐time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time‐lapse fluorescence microscopy. Therefore, DR4‐CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti‐cancer drug screening to identify compounds that are capable of activating TRAIL pathways. Biotechnol. Bioeng. 2013; 110: 1396–1404. © 2012 Wiley Periodicals, Inc.  相似文献   

2.
Initiation of T lymphocyte responses to most Ags requires concurrent stimulation through the TCR and costimulatory receptors such as CD28. Following initial activation, secondary receptors are up-regulated that can costimulate T cells in concert with TCR engagement. One such receptor is the TNFR family member CD30. In this study, we report that unlike CD28, ligation of CD30 on normal effector T cells induces IL-13 production in the absence of concurrent TCR engagement. TCR-independent CD30-mediated IL-13 release correlated with activation of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase (MAPK), and NF-kappaB, and was completely inhibited by the expression of a TNFR-associated factor 2 (TRAF2) dominant-negative transgene (TRAF2.DN-Tg), but not by that of an I-kappaBalpha dominant-negative transgene. In parallel, expression of the TRAF2.DN-Tg selectively prevented the induction of c-Jun N-terminal kinase and p38 MAPK, but not that of NF-kappaB. Furthermore, IL-13 production was reduced in a dose-dependent manner by the p38 MAPK inhibitor SB203580. Together, these results suggest that TCR-independent CD30-mediated production of IL-13 is triggered by association of CD30 with TRAF family members and subsequent activation of p38 MAPK. Inasmuch as IL-13 can promote airway inflammation and cancer progression, production of IL-13 in a TCR-independent manner has important pathological implications in vivo.  相似文献   

3.
M cells are specialized epithelial cells mediating immune surveillance of the mucosal lumen by transepithelial delivery of Ags to underlying dendritic cells (DC). At least three M cell phenotypes are known in the airways and intestine, but their developmental relationships are unclear. We used reporter transgenic mouse strains to follow the constitutive development of M cell subsets and their acute induction by cholera toxin (CT). M cells overlying intestinal Peyer's patches (PPs), isolated lymphoid follicles, and nasal-associated lymphoid tissue are induced by distinct settings, yet show convergent phenotypes, such as expression of a peptidoglycan recognition protein-S (PGRP-S) transgene reporter. By contrast, though PP, isolated lymphoid follicle, and villous M cells are all derived from intestinal crypt stem cells, their phenotypes were clearly distinct; for example, PP M cells frequently appeared to form M cell-DC functional units, whereas villous M cells did not consistently engage underlying DC. B lymphocytes are critical to M cell function by forming a basolateral pocket and possible signaling through CD137; however, initial commitment to all M cell lineages is B lymphocyte and CD137 independent. CT causes induction of new M cells in the airway and intestine without cell division, suggesting transdifferentiation from mature epithelial cells. In contrast with intestinal PP M cells, CT-induced nasal-associated lymphoid tissue M cells appear to be generated from ciliated Foxj1(+)PGRP-S(+) cells, indicative of a possible precommitted progenitor. In summary, constitutive and inducible differentiation of M cells is toward strictly defined context-dependent phenotypes, suggesting specialized roles in surveillance of mucosal Ags.  相似文献   

4.
CD4+ T cells with their growing list of effector and regulatory subpopulations have vital functions within the immunohematopoietic system. We report here on the first mouse lines that allow temporally and quantitatively controlled expression of transgenes specifically in CD4+ thymocytes and T cells. These were constructed using the Tet-on system. The rtTA2(S)-M2 version of the reverse tetracycline-dependent transactivator was placed under control of all known CD4 regulatory elements. Reporter transgene expression in mice expressing these constructs is highly specific for CD4+ cells, is strictly dependent on the tetracycline derivative doxycycline, and can be regulated by up to five logs depending on the doxycycline concentration. Moreover, we demonstrate that these mice can be used for noninvasive in vivo imaging of a coexpressed luciferase reporter. These new mouse lines should be highly valuable for studying and manipulating numerous aspects of CD4+ T cell development, biology, and function.  相似文献   

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6.
Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction   总被引:42,自引:0,他引:42  
TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-kappaB (NF-kappaB) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-kappaB-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-kappaB-mediated gene expression in cells transfected with the TLR4 gene in a dose- and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dose-dependent manner (IC50 approximately 30 nM). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-kappaB activation and subsequent cellular events.  相似文献   

7.
Similar to murine models with compromised CD22/SHP-1 function, flaky skin (fsn) mutant mice exhibit lymphocyte hyperactivation and an autoimmune phenotype characterized by circulating autoantibodies to dsDNA and glomerulonephritis. Immunophenotyping of fsn/fsn splenic B cells was performed to determine if abnormalities in CD22 expression contributed to the phenotype. We identified an expansion of an IgM(bright) CD22lo population consistent with immature B-lymphocytes. While normal B-lymphocytes require IL-4 to achieve down-modulation of CD22 expression in response to BCR cross-linking, culture with anti-IgM alone led to reduced CD22 expression in fsn/fsn mice. Furthermore, when IL-4 was added to fsn/fsn cultures, no further reduction in CD22 expression was observed. This suggested that fsn/fsn B cells were pre-activated in vivo by chronic IL-4 exposure. A portion of these CD22lo cells expressed the B-1 surface marker CD11b. We contend that decreased activation thresholds among CD22lo B-lymphocytes contributes to the expansion of immature and B-1 B cell populations and to the development of autoimmune pathology in fsn/fsn mice.  相似文献   

8.
By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.  相似文献   

9.
Analysis of the NK cell developmental pathway suggests that CD2 expression may be important in regulating NK maturation. To test this hypothesis, we developed mice containing only an inhibitory CD2 molecule by linking the extracellular domain of CD2 to an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) motif. Mice containing the CD2 Tg(ITIM) transgene, introduced into a CD2 KO background, have no morphologically detectable lymph nodes, although development of the thymus appears normal. In addition, these mice had major loss of both NK and NKT subsets in peripheral organs, while T and B cell frequencies were intact. Expression of CD2 was low on T cells and lacking on B cells and functional defects were observed in these populations. NKT cells expressing CD4 were absent, while the CD8+ and double negative NKT cells were retained. Small subsets of NK cells were detected but expression of CD2 on these cells was very low or absent, and their maturation was impaired. Based on the phenotype described here, we believe that these mice represent a unique model to study lymphoid organ and lymphocyte development.  相似文献   

10.
利用荧光抗体标记和流式细胞术检测喘可治对刀豆蛋白A(ConA)诱导的T细胞CD69和CD25表达的影响,研究喘可治是否具有促进CD4 CD25 调节性T细胞升高的作用.结果发现喘可治对ConA诱导的T细胞活化标志分子CD69的表达具有抑制作用,但对CD25的表达具有促进作用.说明喘可治对T细胞活化具有抑制作用,CD25表达的上调并不是由活化引起的,而很可能是CD4 CD25 Tr水平升高的标志.  相似文献   

11.
Experimental autoimmune thyroiditis (EAT), a model for Hashimoto's thyroiditis, is a T cell-mediated disease inducible with mouse thyroglobulin (mTg). Pretreatment with mTg, however, can induce CD4+ T cell-mediated tolerance to EAT. We demonstrate that CD4+CD25+ regulatory cells are critical for the tolerance induction, as in vivo depletion of CD25+ cells abrogated established tolerance, and CD4+CD25+ cells from tolerized mice suppressed mTg-responsive cells in vitro. Importantly, administration of an agonistic CD137 monoclonal antibody (mAb) inhibited tolerance development, and the mediation of established tolerance. CD137 mAb also inhibited the suppression of mTg-responsive cells by CD4+CD25+ cells in vitro. Signaling through CD137 likely resulted in enhancement of the responding inflammatory T cells, as anti-CD137 did not enable CD4+CD25+ T cells to proliferate in response to mTg in vitro.  相似文献   

12.
Co-signal receptors provide crucial activating or attenuating signals for T cells. The B and T lymphocyte attenuator (BTLA/CD272) is a third member of co-inhibitory receptors, which belongs to the CD28 immunoglobulin-superfamily. Using monoclonal antibodies (mAbs) against human BTLA, we show that BTLA is constitutively expressed on most CD4+ and CD8+ T cells and its expression progressively decreases upon T cell activation. Polarized Th1 and Th2 cells contained both BTLA-positive and BTLA-negative populations, but the extended culture diminished BTLA expression. Cross-linking BTLA with an agonistic mAb inhibited T cell proliferation and the production of the cytokines IFN-gamma and IL-10 in response to anti-CD3 stimulation. BTLA-mediated inhibition of T cell activation occurred during both primary CD4+ T cell responses and secondary CD4+ and CD8+ T cell responses, suggesting that BTLA ligation sends a constitutive "off" signal to T cells and thus might play an important role in the maintenance of T cell tolerance.  相似文献   

13.
Manganese superoxide dismutase (MnSOD) is the only primary antioxidant enzyme in mitochondria that scavenges superoxide radicals. Overexpressing MnSOD in cancer cells by cDNA transfection suppresses tumor formation and reverses malignant growth. In this study, we examined the effect of recombinant human manganese superoxide dismutase (rhMnSOD) alone and in combination with adriamycin (ADR) against solid tumors of sarcoma 180 in Institute of Cancer Research (ICR) mice. Administration of rhMnSOD alone and in combination with ADR significantly inhibited tumor growth in a dose-dependent manner. The use of rhMnSOD in combination with ADR enhanced ADR’s anti-tumor potency without increasing toxicity. Histopathological examination provided evidence of the anti-tumor effect. In addition, we found lymphocyte infiltration of the tumors, with an increase in both CD4- and CD8-positive cells in the treated tumors. The expression of CD4 and CD8 was up-regulated with increasing dose of rhMnSOD, and the combination treatment with ADR further enhanced this up-regulation. Collectively, these data indicate that rhMnSOD may exhibit an anti-tumor effect by stimulating the immune system and promoting the recruitment of lymphocytes into the tumor to kill tumor cells. Thus MnSOD may constitute a potential new therapeutic agent to be exploited as an adjuvant in cancer therapy.  相似文献   

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16.
Triggering of integrins can deliver signals that will regulate T cell activation and proliferation when coupled with TCR/CD3 signaling. While co-activation stimuli can be achieved either with immobilized natural ligands or immobilized monoclonal antibodies specific for various integrin subunits, counterposing effects can be delivered by ligation of the integrin β1 chain (CD29) resulting in the downregulation of T cell proliferation. Thus, integrins may play a pivotal role in cell activation and are involved in both positive and negative regulatory pathways. In this report, anti-β1 mAb 18D3 was used to investigate the role of β1 in the negative regulation of T cell proliferation. T lymphocytes were stimulated to proliferate when activated with immobilized mAb to CD3 in conjunction with all of a panel of immobilized mAb to different α4 (CD49d) and β1 epitopes, except the anti-β1.1 mAb 18D3. In soluble form, mAb 18D3 inhibited the induction of DNA synthesis dependent on costimulation of CD3 and the integrin α4 subunit by a mechanism independent of anti-adhesive properties. In kinetic experiments, the addition of mAb 18D3 effectively inhibited the ultimate induction of DNA synthesis at all time points until the time coinciding with the onset of T cell proliferation, indicating that triggering the β1.1 epitope may only act to quench activation events prior to cellular commitment to synthesize DNA. MAb 18D3 did not induce cell death nor render cells incompetent for restimulation, but appeared to selectively inhibit IL-2 synthesis with little effect on the induction of IL-2 receptor expression.  相似文献   

17.
In T lymphocyte, activation of Kv1.3 channel, the major voltage-dependent K+ channel, is an essential step for cell proliferation in immune responses. Here, effects of anti-CD3 and anti-CD28 antibodies on Kv1.3 current were examined in three types of human T lymphocyte derived cell lines, Jurkat E6-1, p56lck-kinase deficient mutant JCaM.1, and CD45-phosphatase deficient mutant J45.01. Kv1.3 current was partly reduced by CD3 stimulation and more strongly by addition of anti-CD28 antibody in E6-1. In JCaM.1, Kv1.3 current responses to anti-CD28/CD3 antibodies were similar to those in E6-1. In J45.01, CD3 stimulation partly inhibited Kv1.3 current, but the additive reduction by CD28 stimulation was not significant. The inhibition of tyrosine phosphatase in E6-1 abolished the additional inhibition by anti-CD28 antibody in a similar manner as in J45.01. In conclusion, the stimulation of CD28 in addition to CD3 strongly inhibits Kv1.3 current and this additive inhibition is mediated by CD45 activation.  相似文献   

18.
Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3(+) T cells were activated and cultured in the presence or absence of MSCs. CD4(+) cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69(+) cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69(+) cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling.  相似文献   

19.
Thy-1 (CD90) expressed by mouse T cells is known to have signal transducing properties, but the ability of Thy-1 to enhance cytotoxic T lymphocyte (CTL) development is not well understood. Here we show that stimulation of mouse T cells with monoclonal antibodies (mAb) to CD3, CD28 and Thy-1 (clone G7), which were coimmobilized on polystyrene microbeads, resulted in a greater proliferative response than stimulation with only anti-CD3 and anti-CD28 mAb, indicating that Thy-1 cross-linking enhanced T cell receptor/CD28-driven T cell activation. Consistent with this finding, Thy-1 blockade with a soluble nonactivating anti-Thy-1 mAb (clone 30-H12) inhibited anti-CD3-induced proliferation of CD4+ and CD8+ T cells, and the induction of cytotoxic effector cells in a dose-dependent fashion. Interleukin-2 synthesis and CD25 expression were also impaired by Thy-1 blockade. The inhibitory effect involved a defect at or before the level of protein kinase C activation because the addition of phorbol ester ablated the anti-Thy-1-mediated inhibition of anti-CD3-induced T cell activation. The CTL that were induced in the presence of blocking anti-Thy-1 mAb adhered to target cells but showed reduced expression of granzyme B and perforin. In contrast, Fas ligand expression and function was not affected by Thy-1 blockade. We conclude that Thy-1 signalling promotes the in vitro generation of CTL that kill in a granule-dependent fashion.  相似文献   

20.
First-generation adenovirus vectors will have limited application in gene therapy for chronic diseases because of destructive host immune responses. Important immune effectors include CD8+ T cells, which mediate target cell destruction and ablate transgene expression, and B cells, which produce neutralizing antibodies that block effective readministration of vector. Previous studies indicated that activation of CD4+ T cells by virus capsid proteins is necessary for full realization of effector function of CD8+ T cells and B cells. In this paper, we present a strategy for preventing CD4+ T-cell activation by an adenovirus vector delivered to mouse liver and lung tissues which is based on interfering with T-cell priming via CD40 ligand-CD40 interactions. Adenovirus transgene expression was stabilized in mice genetically deficient in CD40 ligand (CD40L), and neutralizing antibody to adenovirus did not develop, allowing efficient readministration of vector. A transient blockade of T-cell activation with an antibody to CD40L infused into the animal at the time of adenovirus vector-mediated gene transfer led to stabilization of transgene expression and diminished production of neutralizing antibody, allowing readministration of vector. In vitro T-cell assays suggested that a block in the primary activation of CD4+ T cells was responsible for the lack of B-cell- and cytotoxic-T-cell-dependent responses. This suggests a strategy for improving the potential of adenovirus vectors based on administration of an antibody to CD40L at the time of vector administration.  相似文献   

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