Strips were rinsed briefly with 25% 1.5 M pH 8.0 Tris before SDS–PAGE was performed using Criterion 12.5% Tris-HCl Precast gels (Bio-Rad), run at 200 V for approximately 45 min. For each sample, two gels were run simultaneously, one for silver staining and another for

immunoblotting. Gels for silver staining were fixed individually in 0.1 L fixing solution [50% (v/v) methanol, 10% (v/v) acetic acid] for find more a minimum of 1 h, and were subsequently stained using a sensitive ammoniacal silver method based on silver nitrate. Gel images were acquired using the UMAX Powerlook 1000 flat-bed scanner. Proteins from unstained gels were transferred electrophoretically onto polyvinylidene fluoride (PVDF) membranes using the Trans-blot cell transfer system (Bio-Rad Laboratories). Tigecycline cost To visualize total proteins, membranes were stained with a Sypro Ruby blot stain (Bio-Rad

Laboratories). To detect immunoreactive proteins, membranes were destained and subsequently probed according to the Immun-Star™ WesternC™ kit protocol (Bio-Rad Laboratories). Membranes were immunolabeled with patients’ sera at a 1 : 250 dilution, and goat anti-human IgG antibodies coupled to HRP (1 : 2000; Bio-Rad) were used as a secondary antibody. The immunoreactive protein spots matched using both the Sypro Ruby stained membrane and the silver-stained gels were identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Briefly, spots were washed twice for 10 min in 200 μL of 100 mM NH4HCO3, reduced at 37 °C for 1 h with 50 μL of 10 mM DTT, alkylated for 1 h in 50 μL of 10 mM iodoacetamide, washed for 10 min with 200 μL of 10 mM NH4HCO3, dehydrated in acetonitrile, and trypsin-digested with 10 ng μL−1 of trypsin (Promega, Annandale, NSW, Australia). After digestion for 14 h at 37 °C, peptides were extracted by washing the gel slice for 15 min with 25 μL 1% formic acid, followed by dehydration in acetonitrile. Digests were then dried in vacuo, resuspended in 10 μL 1% formic acid and submitted for

a Quadrupole-TOF analysis on a Micromass instrument which generated collision-induced dissociation. Results were analyzed using the Mascot MS/MS ion search (Matrix Science, Boston, MA), and searches were performed on the National Centre for Biotechnology Phosphoglycerate kinase Information non-redundant (NCBI nr) database (specifically against the available genome sequence of C. concisus BAA-1457). This study was approved by the Research Ethics Committees of the University of New South Wales and the South East Sydney Area Health Service-Eastern Section, Sydney (Ethics No.: 03/163, 03/165 and 06/164). Recently, an association between the presence of C. concisus DNA and newly diagnosed CD was reported in two case–control studies using intestinal biopsies and fecal samples (Zhang et al., 2009; Man et al., 2010c). In addition, significantly higher levels of C. concisus-specific IgG antibodies were detected in children with CD as compared with controls (Zhang et al.

CD4+ T cells were depleted AZD0530 solubility dmso from PBMCs and the frequency of LAP (TGF-β1)-producing cells per 1·5 × 105 cells was determined using an ELISPOT assay. The results demonstrate that over 50% of GPC81–95-induced LAP (TGF-β1)-producing cells were CD4+ T cells (Fig. 1d; 210 responders per 1·5 × 105 total PBMCs versus 99 responders per 1·5 × 105 CD3+-depleted PBMCs). Given the important

role that CD4+ T cells play in modulating an immune response, we focused this study primarily on the effects of GPC81–95 on CD4+ T cells. The percentages of LAP (TGF-β1)+ CD4+ T cells in PBMCs of donors 1–4 after stimulation with GPC81–95 are shown using flow cytometry (Fig. 2a). The release of LAP (TGF-β1) was also analysed in the PBMCs of donors 5–8 (Fig. 2b). The results demonstrate that all the individuals tested in this experiment responded to GPC81–95 peptide but not an irrelevant peptide (AFP365–373) and expressed LAP (TGF-β1). To clarify whether

or not the responsive CD4+ LAP (TGF-β1)+ fraction corresponds to the FoxP3+ regulatory T-cell population, GPC81–95-stimulated CD4 T cells were co-stained for intracellular Foxp3 and membrane-bound LAP (TGF-β1). The results demonstrate that the reacting CD4+ T cells do not express Foxp3 (Fig. 2c). To examine whether GPC81–95 can directly stimulate CD4+ T cells, we performed two sets of experiments. The ability of GPC81–95 to stimulate LAP (TGF-β1) was demonstrated c-Met inhibitor in purified primary CD4+ T cells (95% purity as determined by FACS) and Jurkat CD4+ T cells (data not shown). We used several

approaches to confirm that GPC81–95 has click here intrinsic ability to induce LAP (TGF-β1) on CD4+ T cells. First, we demonstrated that alanine substitution at positions 81, 82, 83, 84, 85 (alanine to serine), 86, 87, 88, 89, 92, 93 and 94 reduce the ability of GPC81–95 to stimulate LAP (TGF-β1) (Fig. 3a). This result suggests that the biological activity of the GPC81–95 depends on its amino acid composition. Second, we observed that GPC81–95 peptide with higher purity (> 90%) induced higher percentages of LAP (TGF-β1) expression than the lower purity peptide (70%) (data not shown), suggesting that non-GPC81–95 peptide derivatives produced during peptide synthesis (shorter peptides, peptides with amino acid deletions or substitutions) are not the bioactive components. We also found that none of the truncated 10-mer peptides or the reversed form of GPC81–95 (SQLLQEMNLRATLQY) induced LAP (TGF-β1) (Fig. 3b,c), indicating that the biological activity of the GPC81–95 also depends on its length. To confirm that the GPC81–95-induced LAP (TGF-β1) expression on CD4+ T cells is not the result of contamination with TLR ligands, we tested commercially available TLR1–9 ligands in a broad range of concentrations. None of these treatments had the ability to induce LAP (TGF-β1) expression (Fig. 3d).

multilocularis metacestode (i.e. the target of BZ treatment) displays Tyr residues at positions 200 and 167 and might thus represent a potentially BZ-resistant isoform (Table 2). Highly homologous

isoforms with Tyr at these two positions are also encoded by the genomes of E. granulosus and T. solium (Table 2), and in the respective LY2606368 molecular weight EST databases, transcripts for this isoform are particularly abundant (data not shown), indicating high expression in the metacestodes of these species as well. Hence, limited bioavailability of the drug at the site of infection, which is particularly an issue for the infiltratively growing E. multilocularis metacestode, combined with a potentially

reduced affinity of BZs to the major β-tubulin isoform of the metacestode, could be the main reasons for limited efficacy of BZ treatment in AE. Employing in vitro cultivation systems for the E. multilocularis metacestode stage and classical approaches of testing selected compounds for anti-parasitic activities, Andrew Hemphill’s laboratory and others (71) have recently identified several compounds such LBH589 datasheet as nitazoxanide, isoflavones or amphotericin B that could be used as drugs in AE treatment, mostly in combination with BZs (reviewed in 68). However, compounds that act not only parasitostatic but truly parasitocidal against E. multilocularis in vivo have not been discovered to date, indicating that new chemotherapeutic strategies against AE are urgently needed. With the availability of the E. multilocularis whole genome together with those of E. granulosus and T. solium, targeted drug design should be one of the most promising approaches for the development of anti-cestode drugs in the next years. On the one hand, comparative genomics

can be employed to identify factors Flavopiridol (Alvocidib) that are unique to cestodes or flatworms and could serve as targets for compound screening. The drawback of this approach is that the function and biochemical properties of parasite-specific factors are usually unknown, which severely hampers the design of efficient inhibitors. Furthermore, many of these parasite-specific proteins have redundant functions and are often not essential. An alternative and much more promising approach should rather concentrate on drug targets that are, to a certain degree, homologous between parasite and host, thus providing information on function and biochemistry, but that display sufficient functional modification between both species to allow the development of parasite-specific inhibitors. A highly promising group of factors in this regard are protein kinases (Table 3) that are crucially involved in the regulation of metazoan development and that mediate cell–cell communication by participating in cellular signalling systems (72).

2A and BB shows that MxA protein expression was clearly observed in the epithelial layer of periodontal tissue. Epithelial MxA immunoreactivity seemed to be stronger in basal and spinous layers than outermost layer of oral epithelium. Using semiquantitative scoring, there

was a significantly higher score of epithelial MxA in healthy group than periodontitis group (Table 1) (p = 0.012), thus highlighting the role of MxA protein in healthy perio-dontal tissue. Since MxA protein is known to be induced by type I and type III IFN [[27-29]], we then investigated the presence of type I and type III IFN in periodontal tissue. The mRNA expression of IFN-α, IFN-β, and IFN-λ in healthy BYL719 mouse periodontal tissue was negligible (n = 10, data not shown). The findings led us to hypothesize that other local mediators may be responsible for the observed MxA protein expression in healthy periodontal

tissue. Antimicrobial peptides including α-defensin, β-defensin, and LL-37 are constitutively expressed in healthy periodontal tissue [[30]] and these mediators could conceivably play a role in MxA expression. Furthermore, a recent study described a fish homologue of MxA protein which was induced by human α-defensin [[31]]. FDA approved Drug Library Therefore, we stimulated primary HGEC cultures with nontoxic concentrations of α-defensin-1, -2, and -3, β-defensin-1, -2, and -3, and LL-37. Fig. 3A shows that α-defensin-1, -2, and -3 markedly induced MxA protein in HGECs. There seemed to be stronger MxA staining in HGECs treated with α-defensin-1 than in those treated with α-defensin-2 and α-defensin-3. In contrast, β-defensin-1, -2, -3 and LL-37 induced only negligible MxA protein expression. IFN-α was used as positive control and induced strong MxA protein expression. The results of MxA protein expression induced by α-defensin-1, -2, and -3, β-defensin-1, -2, and -3, and LL-37 agree with mRNA expression using real-time RT-PCR (Fig. 3B). α-defensin-1 was also able to stimulate MxA protein expression in other cells including normal human bronchial epithelial cells and primary

human microvascular endothelial cells (Fig. 3C). Addition of neutralizing antibodies against type I IFN (IFN-α and IFN-β) into the cultures of α-defensin-1-treated HGECs had no effect on MxA expression whereas these neutralizing antibodies markedly inhibited MxA expression in IFN-α-treated HGECs (Fig. MG-132 purchase 3D). The IFN-α-induced MxA protein expression was likely to be independent on α-defensins since no detection of α-defensin production was observed in cultures of IFN-α-treated HGECs (Supporting Information Fig. 1). In addition, no production of type I IFN (IFN-α and IFN-β) was observed at both the mRNA and protein levels in α-defensin-treated HGECs (data not shown). Collectively, these data suggest that α-defensin and type I interferon use different triggering pathways to induce MxA expression. The antiviral activity of MxA against influenza A virus is well recognized [[25]].

The heparinized Ruxolitinib price blood was layered carefully onto Ficoll (density 1·077 g/ml; Fresenius Kabi Norge AS for Axis-Shield PoC AS, Oslo, Norway) and centrifuged at 800 g for 30 min without brake to obtain a density gradient separation. After centrifugation, the mononuclear cell layer was recovered and washed twice with PBS; Sigma). Human CD4+ T cells were isolated from the PBMCs by positive selection using the Midi MACS CD4+ T cells magnetic isolation kit (Milteny Biotec), according to the manufacturer’s instructions. In order to evaluate the immunosuppressive activity of MSCs, these cells were isolated from both HC and SSc and plated in triplicate into 12-well plates. HC–PBMCs resuspended in 2 ml of RPMI-1640 (Invitrogen,

Cergy, France) supplemented Dabrafenib in vitro with 10% inactivated human serum (from human male AB plasma; Sigma) were added to wells in a 1:1 ratio with BM–MSCs and cultured in the presence of 4 ug/ml phytohaemagglutinin (PHA) for 5 days, as described previously [20]. After PHA stimulation, PBMCs were pulsed with 1 uCi/well of [3H]-thymidine ([3H]-TdR)

(Amersham Pharmacia) for 18 h. Cells were harvested and thymidine incorporated in DNA was recovered on filters. [3H]-TdR incorporation was measured using a scintillation counter (KLB Wallac, Gaithersburg, MD, USA). Lymphocyte proliferation was quantified by means of an 18-h pulse with 1 mCi/well ([3H]-TdR) (Amersham, Bucks, UK) and expressed as counts per minute (cpm). CD4+ T cells were isolated from SSc and HC PBMCs, resuspended in 2 ml RPMI-1640 (Invitrogen) supplemented with 10% inactivated FBS (Gibco) and co-cultured with HC– and SSc–MSCs at a 1:5 ratio. To evaluate the role of MSCs and CD4+ T cells in our system, we planned a set of experiments in autologous and heterologous conditions: (i) HC–MSCs+HC–CD4; (ii) SSc–MSCs+SSc–CD4; (iii) HC–MSCs+SSc–CD4; and (iv) SSc–MSCs+HC–CD4,

to assess the specific activity of each cell subset. After 5 days, CD4+ cells were harvested and analysed for the expression of specific surface antigens by monoclonal antibody directed against CD3, CD4, CD25 (Beckman-Coulter), FoxP3 (BioLegend) and CD69 (Miltenyi Biotec, Ltd, Bisley, Surrey, UK). CD4+CD25brightFoxP3+ and CD4+CD25brightFoxP3+CD69+ cells were quantified by cytofluorimetric analysis (Cytomics FC500; Beckman-Coulter) within an initial fraction Glycogen branching enzyme of 1 × 106 CD4+ cells. Tregs were isolated further from each experimental culture by CD25 microbeads (Miltenyi Biotec). The suppressive capacity was established as follows: CD4+ cells were cultured in 96-well plates with PHA (4 μg/ml) alone and in the presence of enriched Tregs (the CD4+ T cell/Treg cell ratio was 10:1). After 4 days of co-culture, [3H]-TdR was added for a further 24 h. Cells were harvested into glass fibre filters and [3H]-TdR incorporation was assessed by a beta scintillation counter. The concentrations of both IL-6 and TGF-β released in the culture supernatants were measured by a specific ELISA.

Background: ATHOME enrolment is organised by treating physicians for patients after a minimum 12 AAG or 3 VAG in hospital infusions. Methods: The ATHOME Program Coordinator arranges for an IV administration trained registered nurse to deliver, prepare, administer and monitor infusion safety in the home or workplace. Physicians receive written reports after each infusion. Records of infusion timings, retention rates and patient numbers are collated by the nurses and managed by the ATHOME Coordinator. Results: ATHOME commenced in Australia July 2010 for AAG patients. In May 2013

it was extended to MLN0128 molecular weight VAG patients. Total enrolments to 28 February 2014 were 30 AAG and 12 VAG patients. Patient retention to ATHOME over the length of the program has been 86.7% and 75.0% with an adherence of 97.9% and 98.1% of planned infusions administered, 89.7% and 86.9% delivered within 2 days of due date for AAG and VAG respectively. Conclusions: ATHOME infusion service successfully offered enrolled patients the convenience and flexibility to receive their treatment in the home or workplace environment with high adherence. 227 COCA COLA? THE NEW TOBACCO

WE HOY1, D EDDY2, RW MANNING3, L TUNGATALUM4, PW HOY5, SA MOTT1, PA BALL6 1Centre for Chronic Disease, NVP-BEZ235 The University of Queensland, Brisbane, QLD; 2Formerly Nguiu Ullintjinni Association, Tiwi Islands, NT; 3RWM Consultancy, Darwin, NT; 4Tiwi Land Council, Tiwi Islands, NT; 5Formerly MSC, Darwin Ribose-5-phosphate isomerase Diocese, NT; 6Charles Darwin University, Darwin, NT, Australia Aim: To highlight volumes

of sales of Coca-Cola in remote Aboriginal communities. Background: Aboriginal people in remote areas are impoverished, poorly educated, poorly nourished, have limited choices and pay high prices for every commodity. Early life malnutrition enhances susceptibility to chronic disease, which is amplified by a diet of highly processed micronutrient-deficient calorie-dense foods. The WHO recommends that sugars constitute <10% (soon potentially <5%) of energy intake. Brimblecombe recently estimated, in three remote communities, that sugars constituted about 30% of energy intake. Our observations. In a 2011 store audit in a separate study community, with the highest CV death and renal failure rates in Australia, soft drinks, sweets and ice-creams accounted for 46% of spending on consumables, exclusive of alcohol and cigarettes. Specifically, 108,000 litres of Coca-Cola Amatil (CCA) softdrink were sold in six months, or >16 litres per month for everyone age 15+ years. On enquiry, CCA’s Board Chairman cited corporate resolve to provide a full range of choices to even the most disadvantaged Australians. In 2007, CCA’s website nominated the NT as the global leader in per capita Coke consumption.

Our recent studies demonstrated high avidity binding of RTLs to macrophages, dendritic cells and B cells, and such RTL “armed” myeloid cells (but not B cells) could tolerize T cells specific for the RTL-bound peptide 43. The current study clearly demonstrates that two-domain MHC-II complexes embodied by RTLs are distinct from the corresponding four-domain complexes, and these two-domain structures deliver

Dactolisib tolerogenic rather than activating signals through the cognate TCR. We believe that the RTL-armed APCs are tolerogenic through two possible mechanisms: (i) that the RTLs present on the APC surface can still ligate the TCR of cognate T cells suboptimally as partial agonists; and (ii) the RTLs induce inhibitory cell surface co-inhibitory molecules (e.g. PD-1 or PD-L1/2) and/or secreted inhibitory cytokines (e.g. IL-10) CP-868596 mouse that inhibit T-cell activation in concert with RTL ligation of the TCR, with or without prior processing and re-presentation of RTL-derived antigenic peptide and MHC determinants. Our TCRL Fabs

will be used to further elucidate the in vivo therapeutic pathways of RTL1000 in the humanized DR2-Tg EAE model. RTL342m idiotype-specific TCRLs can be used to both inhibit RTL binding to APC and block RTL association with the TCR, as would be predicted for Fab 2E4. A similar approach can shed light on the functionality of the novel native two-domain structures and address whether they constitute Ag-specific tolerogens that resemble RTLs regulatory pathways. By using our conformational sensitive Fabs we will test our hypothesis that natural RTL-like structures are degradation products of soluble four-domain MHC-II molecules that have undergone

partial enzymatic cleavage. In addition, we are in the process of isolating TCRL Fabs specific for the native DR2–MOG-35-55 complex. Such Fabs will enable us to monitor possible processing and re-presentation of RTL peptides by APCs. In recent years, with the advantage of fluorochrome-labeled MHC-II multimers, there is increased knowledge about specific CD4+ T cells in various inflammatory autoimmune conditions 14, 44–47. T1D patients and at-risk subjects were found to have a significantly higher prevalence of GAD-555-567-specific CD4+ T cells than control Tau-protein kinase subjects 48. Our novel TCRL to four- versus two-domain MHC-II–peptide complexes have the potential to selectively recognize APCs presenting disease-inducing or regulatory determinants, respectively, to islet cell-responsive CD4+ T cells during T1D. Similarly, Fabs to four- versus two-domain DR2–MOG-35-55 determinants may be invaluable in localizing and quantifying encephalitogenic versus tolerogenic APC in subjects with MS. RTL1000 and RTL340 constructs were modified for a biotinylated version. In these constructs, a Bir-A tag for biotinylation was introduced to the N-terminus using a 20-aa flexible linker.

Furthermore, this GAr-mediated function has been linked to its capacity to prevent EBNA1 synthesis14,15 and block proteasomal degradation.16,17 Although the role of the GAr domain on the stability/turnover of EBNA1 has only partially been clarified, it is

now evident that EBNA1 is immunogenic and capable of inducing CD8-mediated cells responses. As EBNA1 is the only antigen expressed in all EBV-associated tumours, and therefore represents an ideal tumour-rejection target for immunotherapy against EBV-associated malignancies, elucidation of the mechanisms by which EBNA1-specific CTLs recognize naturally EBNA1-expressing cells remains crucial.18,19 To explore target cell recognition by EBNA1-specific CTL cultures, CTLs specific for the C59 wnt cost EBNA1-derived HPVGEADYFEY (HPV), amino acids 407–417, presented by HLA-B35.01 and HLA-B53, were chosen as a model, as recognition of this immunodominant EBV epitope has been documented in the majority of B35-positive, EBV-seropositive donors, and during primary infection.9,20 Herein we demonstrate that the majority buy CT99021 of HLA-B35 positive donors do indeed respond to this epitope, thereby confirming the importance of EBNA1 as target of EBV-positive malignancies. We also show that HPV-specific CTLs recognize

and kill LCLs but not Burkitt’s lymphoma (BL) cells which, despite possessing proteasomes with much lower chymotryptic and tryptic-like activities than LCLs, were shown to degrade the HPV epitope. Interestingly, a partial sensitivity to HPV-specific CTLs was demonstrated in BL cells treated with proteasome inhibitors. In conclusion, our study suggests that antigen presentation in BL cells may be restored by the use of proteasome inhibitors, making them attractive candidates for inclusion in combined drug regimens against

EBNA1-positive malignancies. Lymphoblastoid cell lines were obtained by infection of lymphocytes from HLA-typed donors with culture supernatants of a B95.8 virus-producing cell line, cultured in the presence of 0.1 μg/ml cyclosporin A (Sandoz International GmbH, Holzkirchen, Germany). The LCLs and the BL cell lines (BJAB B95.8 and Jijoye) were maintained in RPMI-1640 supplemented with Phosphatidylinositol diacylglycerol-lyase 2 mm glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (HyClone; Thermo Fisher Scientific Inc., Waltham, MA). Phytohaemagglutinin (PHA) -activated blasts were obtained by stimulation of peripheral blood lymphocytes (PBLs) with 1 μg/ml purified PHA (Wellcome Diagnostics, Dartford, UK) for 3 days, and expanded in medium supplemented with human recombinant interleukin-2 (Proleukin, Chiron Corporation, Emeryville, CA) as previously described.3 Cell were washed in cold PBS and resuspended in buffer containing 50 mm Tris–HCl (pH 7·5), 5 mm MgCl2, 1 mm dithiothreitol (Sigma-Aldrich, St Louis, MO), 2 mm ATP and 250 mm sucrose.

We therefore hypothesized that the protective effect in our model could be due to transfer and survival of partially mismatched lymphocytes from pups to the mother during delivery. Despite the potential for such a mechanism in our model, we found no evidence of persistent chimeric CD4+ or CD8+ lymphocytes from paternal origin within the dams’ spleens to support this. As we examined spleens at the end of follow-up it is possible that such cells were transferred, but were not persistent. It is also possible that other cell types such as antigen-presenting cells

or cells in other organs are relevant in the process. An alternative hypothesis is that processing of paternal placental antigens within the maternal circulation leads to increases in the maternal regulatory T cell population [22,23] and that effects on diabetes development are mediated GSK126 nmr by such regulatory T cells. In summary, this study FGFR inhibitor demonstrates that gestation has no enhancing effects on pre-existent autoimmune destruction of islet beta cells, and that pregnancy via haploidentical male mates can delay the development of autoimmune diabetes in female NOD mice. The mechanism of this effect is unclear. This work forms part of the dissertation of Yannick Fuchs at the University of Technology Dresden and of Katharina Foertsch at the University of Technology Munich. Kerstin Adler received support from the NIH/DFG

Research Career Transition Award Program (KO 3418/1-1). Yannick Fuchs is supported by a grant from the BMBF to the DZD e.V. (FKZ01GI0924) and the DFG Research Center and Cluster of Excellence–Center for Regenerative Therapies Dresden (FZ 111). The authors

have nothing to declare. Fig. S1. Schematic representation of the study design. Litter-matched female non-obese diabetic (NOD) mice were mated to syngeneic NOD, Adenosine major histocompatibility complex (MHC) haploidentical CByB6F1/J and fully mismatched C57BL/6J male mice at (a) 10 weeks and (b) 13 weeks of age. The number of females mated and the number of males used for mating are provided in parentheses. Unmated litter-matched female NOD mice were used as control groups. The total number of offspring and the number of NOD dams that had productive litters are also indicated. Fig. S2. Screening for fetal microchimeric cells in splenocytes from non-obese diabetic (NOD) dams after pregnancy from haploidentical CByB6F1/J mates. Fluorescence staining of major histocompatibility complex (MHC) H-2Kb (ordinate) molecules on CD4+ and CD8+ T cells was analysed by flow cytometry. The left column shows all viable cells additionally stained for H-2Db molecules. The column in the middle shows cells gated for CD4+, and the right column shows cells gated for CD8+. The numbers represent the percentage of H-2Kb-positive cells within the gated area of each graph. (a) To control the staining experiments, splenocytes of one C57BL/6J and one unmated NOD mouse were stained and analysed individually as well as in mixtures of 1:100 and 1:1000.

This may suggest that while high levels of FoxP3 expression are required to prevent Th2 differentiation, a reduced level of FoxP3 expression is still sufficient to prevent the emergence of Th1 and potentially Th17 responses. Indeed, mature Tregs

in which FoxP3 expression has been ablated (due to an induced cre-mediated deletion of a floxed FoxP3 allele) develop a capacity to produce considerable amounts of IL-2, tumour necrosis factor (TNF)-α, IFN-γ and IL-17 [36]. Furthermore, upon transfer to lymphopenic hosts, Tregs in which FoxP3 had been deleted failed to show suppressive function, but rather contributed to inflammation and predominated among tissue infiltrating lymphocytes. Any scientific readout is only as robust as the assay used to achieve it, and the assays used to measure suppressive potential in vitro and in vivo have different strengths and weaknesses. PD0332991 This must be borne in mind because, like many biological phenomena, Treg activity in vivo cannot always be predicted accurately from their behaviour in vitro and vice versa [37–39]. The techniques used to interrogate Treg activity Mitomycin C concentration have changed over time, reflecting our changing understanding of how Tregs function. The initial identification of the role of Tregs in preventing autoimmunity came from observations of autoimmune pathology in mice lacking CD25+ T cells [13]. Subsequently, assaying the capacity of CD25+

Tregs to suppress the proliferation of their CD25– counterparts in vitro became the gold standard measurement of suppressive potential (see below [40]) and antibody-mediated depletion of CD25+ T cells in vivo was adopted as an imperfect but practical strategy to assess the role Teicoplanin of Tregs in models of infection, allergy and autoimmunity [41–44]. These in vitro and in vivo experiments identified many of the suppressive pathways utilized by Tregs– IL-2 deprivation [40], expression of CTLA-4 and glucocorticoid-induced TNF receptor-related protein

(GITR) [45,46], cell contact-dependent suppression [40], production of anti-inflammatory cytokines such as IL-10, TGF-β and IL-35 [31,47–51] and the expression of enzymes promoting tryptophan catabolism and adenosine production [52–54]. Throughout this time the role of Tregs was seen primarily as preventing the activation and differentiation of autoreactive T cells and the main arena for suppressive activity was considered to be the draining lymph node during naive T cell priming [39,55,56]. Their potential to modulate ongoing responses, or to display suppressive activity at sites of inflammation, was harder to address using such assays, although promising findings have been reported [57–59]. On this point, it is important to remember that Tregs can have controlling effects on inflammation through actions on a range of immune cell populations, not simply T cells.