Mice were sensitized on days 1 and 14 by i p injection of 20 μg

Mice were sensitized on days 1 and 14 by i.p. injection of 20 μg OVA (Sigma-Aldrich, LY294002 datasheet St. Louis, MO, USA) emulsified in 1 mg of aluminum hydroxide (Pierce Chemical, Rockford, IL, USA) in a total volume of 200 μL, as previously described with some modifications 9, 48. On days 21, 22, and 23 after

the initial sensitization, the mice were challenged for 30 min with an aerosol of 3% (weight/volume) OVA in saline (or with saline as a control) using an ultrasonic nebulizer (NE-U12, Omron, Japan). OVA-treated mice are defined throughout the manuscript as OVA-sensitized and OVA-challenged mice. BAL was performed 48 h after the last challenge as described previously 9. Total cell numbers were counted with a hemocytometer. Smears of BAL cells were prepared with a cytospin (Thermo Electron, Waltham, MA, USA). The smears were stained with Diff-Quik solution (Dade Diagnostics of P. R., Aguada, Puerto Rico) in order to examine the cell differentials. Murine tracheal epithelial cells were isolated under sterile conditions as described learn more previously 48. The epithelial cells were seeded onto 35-mm collagen-coated dishes for submerged culture. The growth medium, DMEM (Invitrogen Life Technologies, Carlsbad, CA, USA), containing 10% fetal bovine serum, penicillin, streptomycin, and amphotericin B was supplemented with insulin,

transferrin, hydrocortisone, phosphoethanolamine, TCL cholera toxin, ethanolamine, bovine pituitary extract, and bovine serum albumin. However, DMEM without antibiotics was used as the growth medium for the transfections of siRNA. The cells were maintained in a humidified 5% CO2 incubator at 37°C until they adhered. RNA interference was performed with Stealth RNA interference

(Invitrogen Life Technologies). We transfected primary cultured tracheal epithelial cells in third passage with siRNAs in six-well plates, but not coated with collagen. Stealth siRNA targeting HIF-1α or negative control siRNA was transfected to the cells grown until 30–50% confluence. After the transfections, the cells were incubated for 72 h and then harvested. For transfections, siRNA duplexes were incubated with Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instruction. The sequences of Stealth siRNA were as follows: mouse HIF-1α, 5′-AAGCAUUUCUCUCAUUUCCUCAUGG-3′ (sense); corresponding negative control, 5′-AAGACCUUUAUCUCUUACUCCUUGG-3′ (sense); mouse HIF-2α, 5′-GUCACCAGAACUUGUGCAC-3′ (sense); corresponding negative control, 5′-UAGCGACUAAACACAUCAA-3′ (sense). Cells were seeded in culture dishes and grown until 70% confluence. The medium was then replaced with a new medium containing vehicle (0.1% DMSO), 2ME2 (50 or 100 μmol/L, Calbiochem-Novobiochem, San Diego, CA, USA) for 24 h at 37°C, or IC87114 (2 or 10 μmol/L) for 2 h at 37°C, respectively 40.

Virus-derived siRNAs (vsiRNAs) are generated in the host during i

Virus-derived siRNAs (vsiRNAs) are generated in the host during infection by RNA viruses in both Drosophila click here and mosquitoes. The biogenesis of these vsiRNAs has been the focus of much research to discover the identity of the viral RNA precursor targeted, and to provide insight into how the RNAi pathway mechanistically responds to infection against distinct classes of viruses [1]. Figure 1A diagrams the potential RNA precursors of vsiRNAs generated during RNA virus infection, bearing in mind that these precursors must be in the form of dsRNA.

Small RNA sequencing of virus-infected cells or animals has revealed that the dsRNA replication intermediate of RNA viruses is a common target of the antiviral machinery [4, 7-9] (and Sabin and Cherry, unpublished observations). In addition, as RNA viruses have limited coding capacity, they often encode highly structured cis elements (structured viral RNA) with double-stranded character that direct transcription, replication, and packaging. Therefore, it is perhaps not surprising that the antiviral BGB324 price RNAi machinery is capable of targeting those regions with double-stranded character within the highly structured viral transcripts. Viruses such as Flock House virus, Drosophila C virus, and West Nile virus, appear

to expose such structures during infection; the majority Acyl CoA dehydrogenase of the small RNAs generated during their replication derive from only the genomic RNA strand [10-12] (and Sabin and Cherry, unpublished observations). This suggests that double-stranded structures within single-stranded RNAs can be processed into siRNAs during infection. Genetic studies have indicated that robust antiviral RNAi requires not only vsiRNA biogenesis by Dicer-2, but also the action of the core siRNA RISC effector, Ago2; however, only a fraction of vsiRNAs are specifically bound to Ago2 in infected cells [13, 14] with a large proportion of vsiRNAs being stable, but not bound to Ago2. Whether the

“free” vsiRNAs are loaded onto another RISC, such as Ago1 RISC, which normally binds miRNAs, or whether the vsiRNAs are stabilized elsewhere remains unknown. Furthermore, while some reporters that bear viral RNA target sequences can be silenced by vsiRNAs produced during infection, this is not always the case [8, 13, 15]. Altogether, these findings raise questions regarding which vsiRNAs reflect the active pool for viral silencing, and whether viral sequences are indeed generally targeted by Ago2-RISC. Additional studies of the effector step of antiviral RNAi are necessary to resolve these issues. Since viruses co-evolve with their hosts, one hallmark of an important antiviral pathway is the development of robust countermeasures against the host-encoded antiviral immune factors by viruses.

047; Fig 4B) Therefore, IL-7 secretion by leukemic cells contri

047; Fig. 4B). Therefore, IL-7 secretion by leukemic cells contributes to the survival of CML-specific CTL. Our results in a murine CML model

using LCMV-gp33 as model leukemia antigen suggested that IL-7 signaling maintains CML-specific CTL and may contribute to disease control. LCMV-gp33 is a foreign antigen, which is expressed in the H8 transgenic mice under a relatively strong promoter. Therefore, the model leukemia antigen used has many similarities to the junction peptides derived of BCR/ABL, which are similarly selleckchem expressed under a strong promoter and are novel antigens without pre-existing self-tolerance. Nevertheless, the H8-CML model might overestimate the contribution of IL-7 signaling and CD8+ T-cell control. To test the physiological role of IL-7 in CML control, IL-7-deficient bone marrow or C57BL/6 bone marrow was transplanted to irradiated C57BL/6 recipient mice. IL-7−/−-CML mice died within 30 days after bone marrow transplantation (Fig. 5A). On the contrary, JNK inhibitor C57BL/6-CML mice survived significantly longer

(p=0.02). A similar retroviral transduction efficiency of IL-7-deficient and C57BL/6 donor bone marrow cells was confirmed by FACS analysis 3 days after spin-transfection (Fig. 5B). Taken together, these results indicate that IL-7 production by leukemic cells improves the immunological control of CML, in the absence of model antigen gp33. Specific CTL participate in the control of CML without eradicating the disease completely 6, 7, 20. In fact, CML disease is characterized by a chronic phase of 3–5 years during which a specific CTL response coexists with the CML and probably controls the disease. This is followed by the transition to blast crisis. The mechanisms which control this delicate balance between the immune system and the leukemia are largely unknown. Adoptive transfer

experiments revealed that a large fraction of specific CTL disappeared from the circulation and from the lymphoid organs. This process has also been documented for chronic viral infections, and is referred to as exhaustion19, 21–26. The phenotype of CTL that resist physical for deletion in the presence of a chronic infection has been analyzed before. These CTL were characterized by varying degrees of functional impairment, such as the lack of cytotoxic activity and a reduced capacity to produce IFN-γ 21, 22. However, if partially exhausted and dysfunctional T cells still contribute to disease control is less clear and is often difficult to assess in the presence of a chronic infection. Indications that partially exhausted CTL are of importance for disease control come from experiments with rhesus macaques infected with SIV. Animals which were depleted of CD8+ T cells by monoclonal antibody had significantly higher viral loads 27. We now analyzed the relevance of partially exhausted CTL in the control of CML.

The core structure of the ligand recognized by NOD-1 is the pepti

The core structure of the ligand recognized by NOD-1 is the peptidoglycan-specific dipeptide, γ-D-glutamyl-meso-diaminopimelic selleck kinase inhibitor acid (iE-DAP) and NOD-2 recognizes the muramyldipeptide (MDP), representing the minimal motif of bacterial peptidoglycan able of activating NOD2 [15]. Given the significance of TLR and NLR in immunity and cell differentiation, in this study we explored the expression of NLR in MSC, the transcriptional response of MSC to NOD-1 and TLR-2 ligands and the ability of galectin-3, an identified candidate gene, to affect the inhibitory function of MSC on T-cell proliferation to alloantigens. The peptidoglycan-specific dipeptide, γ-D-glutamyl-meso-diaminopimelic acid

(iE-DAP, a NOD1 ligand) and control peptide (iE-Lys) were purchased from InvivoGen (Toulouse, France) Pam3CS(K)4, and a TLR2 ligand was purchased from Calbiochem (La Jolla, CA, USA). Conjugated anti-CD14, anti-CD4 were purchased from DakoCytomation (Copenhagen, Denmark). Conjugated anti-CD34, anti-CD105, anti-CD106 and anti-NOD2 monoclonal antibody (2D9) were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-NOD1 polyclonal antibodies were purchased from Cell Signalling (Danvers, MA, USA). Total RNA isolation kit Trizol and cDNA synthesis kit were purchased from Invitrogen (San Diego, CA, USA) and GE Healthcare AS (Oslo, Norway), respectively.

SYBR Green PCR Master Mix was purchased from Cisplatin cell line Applied Biosystems (Foster City, CA, USA). An Illumina TotalPrep RNA Amplification Kit was purchased from Ambion (Austin, TX, USA). Expression arrays were purchased from Illumina (San Diego, CA, USA). Human VEGF monoclonal antibody (clone 26503, capture antibody), human VEGF 165 biotinylated affinity purified polyclonal antibodies (detection antibody) and the galectin ELISA kit were purchased from R&D systems (Abingdon, UK). MSC were isolated and expanded from bone marrow (BM) taken from iliac crest of adult volunteers with informed consent.

Heparinized BM was mixed with double volume of phosphate-buffered saline, and mononuclear cells were prepared by gradient centrifugation PIK3C2G (Lymphoprep). Subsequently, the cells were cultured in 75-cm2 flask at a concentration of 30 × 106 per 20 ml Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (FCS). Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2. After 48- to 72-h incubation, non-adherent cells were removed and adherent cells constituted the MSC cell population that was expanded. Cells were detached by a treatment with trypsin and EDTA (GibcoBRL, Grand Island, NY, USA) and replated at a density of 106 cells/75 cm2 flask. These cells were verified for positive staining for CD105 and CD106, and are negative for CD14, CD34 and CD4 markers. MSC were detached using trypsin/EDTA, resuspended in complete medium and placed at 37 °C for 2 h. Subsequently, cell aliquots (5 × 105) were incubated on ice with conjugated monoclonal antibodies against CD34, CD14, CD4, CD105 and CD106.

Cells were fixed and permeabilized with Perm/Fix solution (eBiosc

Cells were fixed and permeabilized with Perm/Fix solution (eBioscience, San Diego, CA, USA), and intracellularly stained with anti-IL-17, anti-FoxP3, anti-tumour necrosis factor (TNF)-α and anti-interferon (IFN)-γ (all from BD Biosciences, San Jose, find more CA, USA, except anti-IL-17; eBioscience). Flow cytometric analysis was performed on a fluorescence activated cell sorter (FACS)Calibur cytometer. Data processing was performed with CellQuest software (Becton Dickinson, San Jose, CA, USA). CD4+CD25- and CD4+CD25+ T cells were isolated from peripheral blood mononuclear cells

and tumour-infiltrating lymphocytes by sorting with the FACSCalibur system after staining with anti-CD4 and anti-CD25 monoclonal antibodies (mAbs). The purity of the isolated CD4+CD25- and CD4+CD25+ T cells was greater than 97%. FoxP3 mRNA expression was quantified by real-time PCR using ABI PRISM 7700 Sequence Detector Ulixertinib mw (Applied Biosystems, Foster City, CA, USA). The human housekeeping gene β-actin primers and probe set was used as a reference for sample normalization. Total RNA isolated from CD4+CD25high T cell was reverse-transcribed into cDNA using random hexamer primers. The primer set for FoxP3 was 5′-TTCGAAGAGCCAGAGGACTT-3′ and 5′-GCTGCTCCAGAGACTGTACC-3′. The probe for FoxP3 was 5′-FAM-CTCAAGCACTGCCAGGCGGACCATC-TAMRA-3′. The primer set for β-actin was 5′-ATCTGCTGGAAGGTGGACAGCGA-3′

and 5′-CCCAGCACAATGAAGATCAAGATCAT-3′. The probe for β-actin almost was 5′-FAM-TGAGCGCA AGTACTCCGTGTGGATCGGCG-TAMRA-3′. The primers and probes used in the real-time PCR were ordered from Sangon (Shanghai, China) and designed not to amplify genomic DNA. Standard curves were generated from serial dilutions of purified plasmid DNA encoding the respective genes with a linear regression R greater than 0·99 and used to quantify mRNA copy numbers for each sample. The amplification protocol used was described as follows: 1 µl of synthesized cDNA product was subsequently added into PCR mix containing

25 µl of TaqMan 2 × PCR master mix (Applied Biosystems), 30 pmol human FoxP3 primer with 10 pmol probe, 2·5 µl β-actin primer/probe set, and distilled water was added to make a total reaction volume of 50 µl. The PCR was programmed as an initial incubation for 10 min at 95°C followed by 40 thermal cycles of 15 s at 95°C and 1 min at 60°C. The normalized values in each sample were calculated as the relative quantity of FoxP3 mRNA expression divided by the relative quantity of β-actin mRNA expression. All reactions were confirmed by at least one additional independent run. The suppressor capacity of Treg was studied in a co-culture suppression assay. A 96-well U-bottomed plate was treated by coating with 10 µg/ml anti-CD3 (UCHT1) and 10 µg/ml anti-CD28 (clone 28·2) monoclonal antibodies in sodium hydrogen carbonate buffer (pH = 9·2) for 2 h. The buffer was washed off with PBS and the plates blocked using T cell media.

Although human NK cells can be either CD8α+ or CD8α−3, in most no

Although human NK cells can be either CD8α+ or CD8α−3, in most nonhuman primate species the bulk of NK cells express high cell surface levels of CD8α [1, 2, and our unpublished observations]. Recently, Rutjens et al. 4 described subsets of CD16+CD8α+ and CD16+CD8α− NK cells in the peripheral blood of chimpanzees. As has been observed in humans and macaque models, the CD16+CD8α+ FK228 in vivo NK-cell population expressed higher levels of activating NK receptors and responded to a classical NK stimulus, K562 cells. However, unexpectedly, the CD16+CD8α− NK-cell population was characterized

by high HLA-DR expression, dull expression of NK-specific markers and lack of responsiveness to NK-specific stimuli. Because the CD16+CD8α− cells were generally enriched in HIV-infected chimpanzees, and similar phenotypic alterations have been observed in NK cells in HIV-infected humans 5, the authors concluded that selleck chemicals llc the lack of responsiveness to NK-cell stimuli was indicative of functional anergy and the increased expression of HLA-DR on CD16+CD8α− cells was indicative of NK-cell activation. However, recent data suggest that CD11c+ myeloid DCs (mDCs) also express CD16 in humans and rhesus macaques 2, 6–8. Thus, using CD16 as an inclusive marker for

NK cells could confound analysis of NK cells by inadvertently including mDCs, which do not express CD8, but are HLA-DR+. To address this possible confusion, we sought to phenotypically and functionally characterize the CD3–CD16+ cell population in the peripheral blood of chimpanzees. In our analyses of peripheral blood NK cells in HIV-uninfected chimpanzees, we used phenotypic markers similar to those described by Rutjens et al. 4 and also Amylase identified a subset of CD3−CD16+ cells (Fig. 1A and Table 1). We found negligible expression of CD14 and CD20 on CD3−CD16+

cells, indicating that this gate was not contaminated with B cells or monocytes (data not shown). However, the CD3−CD16+ cell population could be broken down into three subpopulations: a dominant CD8α+ population that was negative for CD11c and HLA-DR (I); and two smaller CD8α– subpopulations that could be further subdivided into CD11c−HLA-DR− (II); and CD11c+HLA-DR+ (III) cells. Both subpopulations I and II had phenotypic features of NK cells, expressing high cell surface levels of the NK-specific marker, NKp46, and high intracellular expression of the cytolytic enzyme, perforin (Fig. 1B). In stark contrast, subpopulation III expressed neither NKp46 nor perforin but did express high levels of BDCA-1, an mDC marker 6, 8. High-level expression of CD11c, HLA-DR, and BDCA-1, none of which were found on populations I and II, is consistent with phenotypic definitions of mDCs in multiple primate species, including humans and rhesus macaques 2, 6–8.

As shown in Fig 3A, the expression levels of FOXP3 and IFN-γ in

As shown in Fig. 3A, the expression levels of FOXP3 and IFN-γ in expanded E3-Th17 cells were not significantly altered even after culture for 9 days. However, the number of IL-17-producing cells significantly decreased during the culture, from above 60% to approximately 40%. Recent studies have shown that the stable expression of FOXP3 in

naturally occurring Tregs involves epigenetic regulations, including DNA methylation and histone modification 41, 43. Furthermore, these studies demonstrated that human FOXP3 contains several highly conserved demethylation regions that are exclusive for Tregs. Thus, we next investigated whether expanded Th17 cells expressing FOXP3 exhibited FOXP3 DNA demethylation. We designed the human FOXP3 methylation-specific primers based on the Treg-specific demethylated region (TSDR) within the NVP-BGJ398 ic50 FOXP3 CpG island 43–45, and then compared the FOXP3 methylation levels in expanded Th17 cells, CD4+CD25+ naturally occurring Tregs and OKT3-activated naïve CD4+ T cells. As expected, the TSDR within FOXP3 of CD4+CD25+ Tregs was almost completely demethylated

compared with that of CD4+CD25– T cells (Fig. 3B). In contrast to CD4+CD25+ Tregs, FOXP3 methylation levels of two OKT3-activated naïve T cells were similar to levels in CD4+CD25– T cells (100% methylation), although approximately 15% of these activated cells expressed FOXP3. However, Th17 clones derived from different rounds of expansion displayed partial methylation in AZD8055 purchase TSDR within FOXP3, and this decreased significantly with increasing stimulation and expansion cycles. In addition, demethylation

levels of FOXP3 in Th17 clones at different expansion cycles were correlated positively with FOXP3 expression (Fig. 3B). These results indicate that epigenetic modification of FOXP3 occurred in Th17 cells following multiple cycles of in vitro TCR stimulation, resulting in increased Metalloexopeptidase and stable expression of FOXP3 in expanded Th17 cells. It is well known that TCR–ligand interactions are critical for T-cell lineage commitment, including FOXP3 induction and Treg lineage differentiation 3, 16. Given that Th17 clones differentiate into IFN-γ-producing and FOXP3+ T cells after in vitro expansion, we next investigated whether TCR stimulation is the primary determinant for this process. E1-Th17 clones were expanded in vitro with allogeneic PBMCs in the presence or absence of OKT3 and then evaluated for the IL-17, IFN-γ, and FOXP3 expression. As shown in Fig. 4A, the proportions of IL-17-producing cell populations in Th17 clones were significantly decreased after in vitro expansion, regardless of whether the system included OKT3 or not. Notably, the Th17 clones contained higher percentages of IL-17-producing cells when cultures included both PBMCs and OKT3 than those in the absence of OKT3.

As shown in Figure 1A, apomorphine-induced rotations reached seve

As shown in Figure 1A, apomorphine-induced rotations reached seven turns per min at 2 weeks and increased gradually from week 2 to week 5 after a RGFP966 price unilateral injection of 6-OHDA into the right MFB. No rotations (0 turns per 30 min) were observed during 5 weeks of testing in the sham group. To evaluate alterations of dopaminergic neurones in substantia nigra, immunostaining and Western blot

analyses were performed to examine TH expression. In the sham group, TH expression remained relatively stable at several time points after operation; therefore, we utilized saline-lesioned rats at 2 weeks after lesion as a control for the PD group. FEZ1 and GFAP performed similar comparison. Western blot analysis showed that in 6-OHDA-lesioned rats, TH immunoreactivity gradually decreased, in a statistically significant manner, from week 2 to week 5 in striatum (Figure 1B,C) and in substantia nigra (Figure 1D,E) FK506 mouse of the lesioned hemisphere when compared with the TH immunoreactivity of the sham-operated rats. Relative density of TH/β-actin in 6-OHDA-lesioned rats (0.39 ± 0.02 in striatum and 0.35 ± 0.04 in substantia nigra) at 5 weeks after injury was markedly lower than it in sham-operated rats (0.87 ± 0.07

in striatum and 1.06 ± 0.05 in substantia nigra). The decrease in TH immunoreactivity was further confirmed by Nissl staining (Figure 1F) and TH immunostaining (Figure 1G). 6-OHDA lesions induced a dramatic unilateral loss of dopaminergic neurones in substantia nigra pars compacta of PD rats (F″ and G″) but not in sham-operated rats (F′ and G′). The mRNA expression of FEZ1 in rat striatum and substantia nigra was analysed by real-time PCR. Compared with the sham-operated rats (relative value was 0.033 ± 0.002), the FEZ1 mRNA level in striatum was markedly increased during the initial post-injury period, reached peak level (0.038 ± 0.002) at 2 weeks after

injury, and then gradually decreased (Figure 2A). However, in substantia nigra, FEZ1 mRNA expression levels were significantly enhanced at 2 weeks after injury, peaked at 3 weeks after injury (0.63 ± 0.002 compared with 0.46 ± 0.004 in the sham-operated rats), and then decreased (Figure 2B). FEZ1 mRNA expression level next was higher in striatum of the PD group at 2–3 weeks after lesion than in striatum of the sham group. However, in substantia nigra, FEZ1 expression levels were higher in the PD group at 2–5 weeks after lesion when compared with the sham group. We next examined FEZ1 protein expression by Western blot analysis. As shown in Figure 2C,D, FEZ1 protein levels mirrored mRNA levels, as FEZ1 protein levels were significantly increased in striatum at 2 weeks (0.82 ± 0.07 compared with 0.75 ± 0.06 in the sham-operated rats) after surgery compared with the sham group and then decreased to normal levels at 4 weeks after surgery (0.62 ± 0.03).

Taken together, IC pretreatment can significantly inhibit LPS or

Taken together, IC pretreatment can significantly inhibit LPS or CpG ODN-induced maturation of DCs in a FcγRIIb-dependent manner. Mature DC-induced Th1 and Th17 responses are involved in the pathogenesis of some autoimmune Metabolism inhibitor diseases, whereas immature DCs contribute to tolerance induction by downregulation of T-cell response and subsequently attenuate the pathogenesis of some autoimmune diseases. Next we investigated whether IC pretreatment could enhance tolerogenecity of immature DCs. OVA-pulsed immature DCs, which were pretreated with IC/Ig and then stimulated with LPS or CpG ODN, were incubated with OVA323–339-specific CD4+ T cells in vitro. We found that IC pretreatment reduced the

ability of LPS or CpG ODN-stimulated DCs to induce the proliferation and IL-17, IFN-γ secretion of antigen-specific CD4+ T cells (Fig. 1C and D). In contrast, IC/Ig pretreatment could not reduce the ability of FcγRIIb−/− DCs to induce proliferation and IL-17 secretion of antigen-specific CD4+ T cells. Altogether, the data suggest that IC pretreatment could enhance tolerogenecity of immature DCs in FcγRIIb-dependent manner. We previously showed that IC can induce massive amount of PGE2 from macrophages, which is responsible for the inhibition of TLR4-triggered inflammatory response. Similar

to macrophages, immature www.selleckchem.com/products/LBH-589.html DCs produced large amount of PGE2 once stimulated with IC. LPS or CpG ODN could not further promote IC-induced PGE2 production of immature DCs (Fig. 2A). Also, immature FcγRIIb−/− DCs released some PGE2 in response to IC stimulation, but less than the PGE2 secreted by WT DCs in response to IC stimulation (Fig. 2B). To investigate whether PGE2 was responsible

for the hyporesponsiveness of T cells induced by DCs pretreated with IC, we first observed the direct effect of PGE2 on the proliferation of CD4+ T BCKDHA cells by anti-CD3/CD28. As expected, PGE2 inhibited the proliferation of T cells in a dose-dependent manner (Supporting Information Fig. 2). Next, OVA323–339-pulsed DCs were incubated with celecoxib, an inhibitor of COX2, 30 min prior to treatment with IC and TLR ligands. The hyporesponsiveness of OVA323–339-specific T cells disappeared when PGE2 secretion was inhibited, and addition of exogenous PGE2 could restore the inhibitory effect on T-cell proliferation in this system (Fig. 2C). Altogether, these data confirmed that IC-induced PGE2 from DCs was responsible for the downregulation of T-cell response by immature DCs that were pretreated with IC and then stimulated with TLR ligands. The data in the previous sections indicated that IC could downregulate DC-initiated T-cell response by inducing PGE2 production from DCs via FcγRIIb. To investigate whether IC could also inhibit in vivo T-cell response triggered by TLR agonists, we i.v. injected mice with OVA323–339-specific CD4+ T cells 24 h and OVA together with IC before i.p. administration of LPS or CpG ODN.

Again, neutralizing TNF-α did not cause a decrease in TRAF2 expre

Again, neutralizing TNF-α did not cause a decrease in TRAF2 expression levels in activated WT cells, presumably because the TNFR2-mediated degradation of TRAF2 opposes this effect of TNFR1 (Fig. 5D). These data indicate that signaling through TNFR1 is required for maintaining high TRAF2 levels in TNFR2−/− CD8+ T cells. They also provide further support for the hypothesis that in TNFR2−/− CD8+ T cells, TNFR1 functions as a survival receptor by ALK inhibitor cancer regulating TRAF2 levels in these cells. NF-kB is a key transcription factor that regulates many pro-survival

genes in activated T cells 18, 19. To provide further evidence that TNFR1 functions as a pro-survival receptor in TNFR2−/− CD8+ T cells, we measured the level of NF-κB activation in these cells by quantifying the level of phosphorylated IκBα in these cells. We found that the AICD-resistant TNFR2−/− CD8+ T cells expressed higher levels

of phosphorylated IκBα compared with similarly activated WT CD8+ T cells (Fig. 6A). Consistent with the idea that TNFR2 signaling opposes NF-κB activation, we found that blocking TNFR2 in WT cells also led to increased levels of phosphorylated IκBα (Fig. 6A). As expected, the anti-TNFR2 antibody had no effect on phosphorylated IκBα levels in TNFR2−/− CD8+ T cells. We also determined that the effect of neutralizing endogenously produced TNF-α on the levels of phosphorylated IκBα in activated WT and TNFR2−/− CD8+ cells. In TNFR1+/+ TNFR2−/− CD8+ T cells, blocking TNF-α signaling Amrubicin led to Dorsomorphin nmr a decrease in the levels of phosphorylated

IκBα (Fig. 6B). Independent evidence for increased NF-κB activation in anti-CD3+IL-2-activated TNFR2−/− CD8+ T cells was obtained with the TransAM p65 Transcription Factor Assay. In this assay, an oligonucleotide containing an NF-κB consensus-binding site is immobilized to a 96-well plate. Activated NF-κB homodimers and heterodimers contained in nuclear extracts specifically bind to this consensus oligonucleotide. Binding of the p65 (RelA) subunit is detected by specific antibodies and the amount of binding is quantified by ELISA. We found that the nuclear extracts of activated TNFR2−/− CD8+ T cells possessed significantly more p65 binding activity relative to similarly activated WT CD8+ T cells (Fig. 6C). The specificity of the p65 binding to the NF-κB consensus site is indicated by complete abrogation of p65 binding with a WT oligonucleotide but not a mutated form of the oligonucleotide (Fig. 6C). Furthermore, blocking activated WT CD8+ T cells with anti-TNFR2 antibodies increased p65 binding to that observed in activated TNFR2−/− CD8+ T cells and neutralizing TNF-α decreased p65 binding in activated TNFR2−/− CD8+ T cells to WT levels (Fig. 6C).