Moreover, even different strains or mutants of particular Lactobacillus species stimulated very different immunological outcomes in mice [16,17]. Recent evidence demonstrates that colonization of germ-free mice with complex microbiota orchestrated a broad spectrum of Th1, Th17 and Treg responses. Whereas most tested individual bacteria failed to stimulate intestinal T cell responses efficiently, a

restricted number of individual bacteria can control the tonicity of the gut immune system [18]. The key commensal organisms in immune system development have been identified very recently as segmented filamentous bacteria [18,19]. A further reflection of how the make-up of the intestinal flora can impact upon systemic responses is found in studies of non-obese diabetic (NOD) mice, which succumb spontaneously Selleckchem PD98059 to type 1 diabetes (T1D); it has been known for some time that higher microbial exposure militates against development of this autoimmune disease [20], but it was shown recently not only that conventionally housed myeloid differentiation primary response gene 88 (MyD88)−/− mice are resistant to T1D, but that resistance to disease is due to the distinct microbial

combination with which they are colonized. Hence, MyD88−/− mice develop T1D under germ-free conditions, while wild-type mice given the microbial population from MyD88−/− animals had reduced susceptibility to disease [21]. It is tempting

to speculate that alteration of Treg homeostasis mediated by TLR signalling, either because of selleck chemical genetic polymorphism or because of changes in gut flora composition, could also have consequences on development of gut inflammatory disorders. Indeed, gut flora bacteria are not equal in their capacity to stimulate TLR-9 and do so with various levels of efficiency that correlate with the frequency of cytosine–guanine dinucleotides. Thus, control of the Treg ratio and effector T cell function in the GI tract is likely to be regulated differentially by specific gut flora species. An illustration of how the presence of defined bacterial species can influence the outcome of an infection comes from the observation that mice fed Bifidobacterium Ceramide glucosyltransferase infantis are protected from the pathogenic effect and translocation of Salmonella[22]. Activation of Tregs by the probiotic microorganism contributed to this protective effect. The proposition that certain commensal species may act in a counterinflammatory manner has led to extensive investigation of potential probiotic regulation of immunopathology. Promising results have been obtained with probiotics in the treatment of human inflammatory diseases of the intestine and in the prevention and treatment of atopic eczema in neonates and infants, but mechanism(s) of action remain to be elucidated [23].

g. mild bronchitis vs. severe pneumonia check details requiring intubation). Therefore, further analysis of more strains coupled with clinical observations are required in order to define these phylogenetic clades described by Erwin et al. (2008) as well as to identify potential clones that may possess unique invasive properties. However, this type of study requires prospectively enrolling patients into study cohorts and careful planning. Another limitation of our study is the relatively small number of isolates examined. Analysis with more isolates collected from the two groups of patients (respiratory tract infection vs. systemic disease) may allow us to confirm if there are clones that may be mainly

associated with invasive diseases such as clones identified as clusters 7 and 8 in Table 2. In summary, our results showed the NT Hi that caused invasive disease were not necessarily different from the NT Hi isolates recovered from the respiratory tract based on phenotypic (biotype) and genetic (MLST) selleck compound traits. This supports earlier findings by other investigators (Saito et al., 1999) that the source of invasive NT Hi originates from the respiratory tract of carriers. Furthermore, we have demonstrated that the emergence of NT Hi as a cause of invasive disease was not due to virulent capsular strains

that have undergone genetic mechanisms to shed or switch their capsules. Finally, the burden of invasive Hi disease, which used to be mainly a childhood disease, has now shifted to involve both adults and the very young. We wish to thank the staff at the DNA Core Facility of the National Microbiology Laboratory for the DNA sequencing work. RSW Tsang had received funding from Health Canada’s Biotechnology-Genomics Research and Development Fund for studies on vaccine preventable bacterial diseases. This study made use of the Hi MLST website (http://haemophilus.mlst.net), developed and maintained by David Aanensen at the Imperial

College, London, UK, and funded by the Wellcome Trust. The site is currently curated http://www.selleck.co.jp/products/U0126.html by Daniel Godoy. “
“Sepsis and type 2 diabetes exhibit insulin resistance as a common phenotype. In type 2 diabetes we and others have recently provided evidence that alterations of the pro-inflammatory wnt5a/anti-inflammatory sFRP5 system are involved in the pathogenesis of insulin resistance. The aim of the present study was to investigate whether this novel cytokine system is dysregulated in human sepsis which may indicate a potential mechanism linking inflammation to metabolism. In this single-centre prospective observational study, critically ill adult septic patients were examined and pro-inflammatory wnt5a and wnt5a inhibitor sFRP5 were measured in serum samples by ELISA at admission to the intensive care unit (ICU) and 5 days later. 60 sepsis patients were included and 30 healthy individuals served as controls.

Next, T-cell proliferation and polarization were investigated by mixed

leucocyte reaction to determine whether the effect HSP inhibitor induced by IFN-β on A. fumigatus-infected DC maturation resulted in an enhanced capacity in promoting the expansion of Th1-oriented CD4+ T cells. As shown in Fig. 5(a), A. fumigatus-infected DCs induced the proliferation of naive allogeneic cord blood CD4+ T cells, which was not significantly modified when infected DCs were primed with IFN-β. Interestingly, IFN-β priming of A. fumigatus-infected DCs highly enhanced the production of IFN-γ, as observed by the analysis of supernatants obtained from mixed leucocyte reaction cultures (Fig. 5b). Conversely, no induction of IL-4 was found when T cells were co-cultured with A. fumigatus-stimulated DCs in the presence or absence of IFN-β (data not shown). Type I IFNs, originally identified for their see more ability to induce cellular resistance to viral infections, are key immunomodulators of the innate and

adaptive immune responses.29 By acting on DC differentiation and maturation, these cytokines can induce cross-priming of CD8 T cells19 and stimulate a Th1-oriented T-cell response.21,22 Accordingly, our recent findings showed that IFN-β potentiates DC immunological functions following bacillus Calmette–Guérin infection, pointing to the importance of IFN-β in promoting a protective Th1 immune response against Mycobacterium tuberculosis.30 Based on this evidence, the use of type I IFN constitutes a promising immunotherapy for infectious diseases.13,15 Invasive aspergillosis is a serious opportunistic fungal infection in immunocompromised hosts. Advances

in more potent and less toxic antifungal agents have reduced Bcl-w the mortality rate of IA and represent a promising area of research and development to cure invasive fungal infections. Moreover, novel strategies for immunotherapy and vaccine are also currently designed on the knowledge of the immunopathogenesis of fungal infections.31 Although clinical evidence points to a crucial role for the Th1 reactivity in the control of IA, more recently regulatory T cells and Th17 cells could display important functions in the scenario of the immune response against A. fumigatus.32 However, if the role of IL-17-producing T cells in protection versus pathology in fungal infections is still controversial,33–35 it is generally accepted that a defective differentiation of regulatory T cells may cause an unacceptable level of tissue damage.3 Several studies in human and murine models have, however, confirmed the central role of IFN-γ released by interstitial lung lymphocytes in controlling IA through the stimulation of phagocytosis and intracellular antifungal killing mechanisms of neutrophils and macrophages.

The latter may explain in part why the most commonly used vaccine cannot Ixazomib cost prevent a tuberculosis epidemic worldwide. Other reasons for the variability in the protective efficacy of BCG, which varies from 0% to 80%, include host population genetics, different strains of BCG and the interference of environmental mycobacterium (Behr & Small, 1997; Brandt et al., 2002). After entering the human body through the aerosol route, Mtb successfully survives immune-mediated destruction within the endosome of macrophages by utilizing a range of intriguing evasion mechanisms including

preventing fusion with the lysosome, acidification of the phagosomal contents, subversion of the host immune response through selleck chemical decoy antigens, and dampening of functional Th1 immune responses (Russell, 2001; Doherty & Andersen, 2005). In the initial phase of tuberculosis infection, Mtb proliferates rapidly and stimulates a Th1-type immune response that is predominantly targeted toward secreted bacterial antigens. The most important cytokine is interferon (IFN)-γ, which synergizes with tumor necrosis factor-α. Together, these cytokines activate macrophages to initiate the production of effector molecules such as nitric oxide and the development of characteristic granulomas that isolate and control pathogen replication without

killing it. At later stages, granulomas are surrounded by a fibrotic wall and lymphoid follicular structures, and in addition to Th1 cytokines, there is both an interleukin (IL)-4 response

and an expansion of regulatory T cells (Guyot-Revol et al., 2006; Ribeiro-Rodrigues et al., 2006). These changes may play a role in inhibiting the production of T-cell IFN-γ, which both limits the pathology and suppresses cellular immune responses in patients with tuberculosis. The granuloma can persist for decades, and despite being deprived of oxygen and P-type ATPase nutrients, Mtb survives in a state of dormancy. The outcome is a latent infection with minimal bacterial replication and a characteristic set of differentially expressed genes (Sherman et al., 2001; Park et al., 2003; Rogerson et al., 2006). The first Mtb gene that was identified as being induced by hypoxia and potentially involved in latency was hspX (Rv2031), also known as α-crystallin. hspX encodes a 16-kDa heat shock protein (HspX) that is required for mycobacterium persistence within macrophages. HspX is also produced abundantly during static growth (Yuan et al., 1998). Many studies have revealed that antigens such as ESAT6, Ag85 and other secreted antigens are strongly recognized in patients with active disease (Boesen et al., 1995; Ravn et al., 1999). Recent research demonstrated that HspX-specific IFN-γ responses were significantly higher in Mtb-exposed individuals than in Mtb-unexposed BCG-vaccinated individuals, but no differences were observed for Ag85B-specific responses (Geluk et al., 2007).

To distinguish irradiated allogeneic stimulator PBMC from

effector cells they were labelled with PKH26 (Sigma-Aldrich). Effector–stimulator cell combinations were chosen on the basis of a minimum of four HLA mismatches. MLR were set up in the absence or presence of MSC (1:10; MSC/effector cells) and belatacept (1 μg/ml). After a 7-day incubation period, cells were restained with mAbs against CD3 (AmCyan), CD4 (APC), CD8 (FITC), CD28 (PerCP-Cy5·5) and analysed on the BD FACSCanto II flow cytometer using the BD FACSDiva software (BD Biosciences). MLR were set up in the absence of MSC. To track cell proliferation, effector PBMC were labelled with VPD450. After 7 days, cells were restimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma-Aldrich) and ionomycin (1 μg/ml; Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences). Following a 4-h incubation period, cells were Saracatinib concentration Selleckchem Idasanutlin stained with mAbs against CD3 (AmCyan), CD4 (APC), CD8 (FITC),

CD28 (PerCP-Cy5·5), tumour necrosis factor (TNF)-α [pyycoerythrin (PE)], interferon (IFN)-γ (PE; all BD Biosciences) and granzyme B (PE; Sanquin). Intracellular staining for TNF-α, IFN-γ and granzyme B was performed according to protocol B for staining of intracellular antigens for flow cytometry (eBioscience, San Diego, CA, USA) using the described buffers. For the identification of extracellular CTLA-4 expression and the expression of programmed death ligand-1 (PD-L1) in proliferating

CD8+CD28− T cells, MLR were set up as described above, but cells were not restimulated. After 7 days, cells were harvested and stained with monoclonal antibodies (mAbs) against CD3 (AmCyan), CD4 (PE), CD8 (FITC), CD28 (PerCP-Cy5·5), CTLA-4 (APC) (all BD Biosciences) and PD-L1 (PE-Cy7; eBioscience). Fluorescence minus one (FMO) controls were used to determine negative expression. Flow cytometric analysis was performed using the BD FACSCanto II flow cytometer using the BD FACSDiva software (both BD Biosciences). MLR were set up in the absence or presence of MSC (1:10; MSC/effector cells). Effector PBMC were labelled with VPD450 (BD Biosciences) and γ-irradiated, allogeneic stimulator PBMC were the labelled using the PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich). Cells were incubated for 4 or 7 days. Apoptotic cells were identified using the annexin V PE Apoptosis Detection Kit I (BD Biosciences), according to the manufacturer’s instructions, in combination with mAb labelling against CD3 (AmCyan), CD8 (APC), CD28 (PerCP-Cy5·5). Flow cytometric analysis was performed using the BD FACSCanto II flow cytometer and BD FACSDiva software (both BD Biosciences). Statistical analyses were performed by means of paired t-tests using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). A P-value lower than 0·05 was considered statistically significant. Two-tailed P-values are stated.

In this study, we demonstrate that semi-allogeneic DC, which share half of the genes of the recipient, are more effective when used via the intratumoural (i.t.) injection route, rather than the

subcutaneous (s.c.) injection route, for the induction of efficient antitumour effects and the generation of a significant tumour-specific CD8+ T-cell response. The i.t. route has the advantage of not requiring ex vivo pulsation with tumour lysates or tumour antigens, because the i.t.-injected DC can engulf tumour antigens in situ. Allogeneic bone marrow transplantation (BMT) models, which permit us to separately assess the three factors described previously, show that while all three factors are important for efficient antitumour effects, the control of the alloresponse to Nutlin-3a in vitro injected DC is the most crucial for host-derived pAPC to function

well when DC are administered intratumourally. This information may be useful for DC-based cancer immunotherapy under circumstances that do not allow for the use of autologous DC. Dendritic cells (DC), the most potent antigen-presenting cells (APC), play a central role in the presentation of antigens to naive T cells and the induction of the primary immune response [1]. In active and specific immunotherapy for cancer, DC are the preferable professional APC p38 MAPK inhibitor (pAPC) for priming TAA-specific CD8+ T-cell responses [2], and recent developments in ex vivo generation Mannose-binding protein-associated serine protease systems enable the use of large numbers of DC for immunotherapy [3, 4]. In DC-mediated cancer immunotherapy, effective priming of TAA-specific CD8+ T cells is the most important concern because the frequency of functional TAA-specific effector CD8+ T cells is positively correlated with the clinical response or survival [5, 6]. A number

of clinical trials of anticancer immunotherapy using DC are now ongoing [1, 7]. To induce efficient antitumour immune responses, the injection dose, maturation status and route of administration of DC are crucial in DC-based antitumour immunotherapy [3, 8]. Currently, the consensus opinion is that adequate maturation signals are required for the induction of antigen-specific T-cell responses; otherwise, immature DC, without the provision of danger signals, will be tolerogenic for the immune system [1]. Although there are controversial reports regarding the best administration route for DC [9–11], it may be preferable to inject DC into lymphatic vessels, lymph nodes or cutaneous sites where tumour-draining lymph nodes exist [9, 10, 12, 13]. Our group and others have reported that the intratumoural (i.t.) route is an alternative route for DC-based immunotherapy that can yield efficient antitumour responses [14–19]. The i.t. route has the advantage of not requiring ex vivo pulsation with tumour lysates or tumour antigens, because the i.t.-injected DC can engulf tumour antigens in situ [15].

Therefore, the escape of T cells bearing TCRs with some degree of affinity toward TAPAs is probable. Furthermore, differences in the presentation of certain antigens, resulting from variable gene expression [26] and instability within the peptide MHC complex [27], may also contribute to thymic escape. The clear difference in binding parameters between VA- and TAPA-specific TCRs has implications

for therapeutic approaches. buy EPZ-6438 Vaccines rely on the activation of preexisting T cells to target tumors; however, since TAPA-specific T cells possess TCRs with relatively low affinities for antigen, vaccines may be largely ineffective in eliciting an effective antitumor CTL response. This may provide one explanation for the limited success of such approaches [10, 11]. A more promising strategy, for modulating the immune system to target tumors is through adoptive therapy [28], especially if this is combined with genetically engineered TCRs designed to have a “VA-TCR-like” affinity. Indeed, T cells carrying these enhanced affinity TCRs have been shown to recognize tumor antigens with high avidity [29]. BMN 673 ic50 The construction of enhanced affinity TCRs is also central to emerging

cancer therapies comprising soluble, bispecific proteins, such as the recently described ImmTACs. These molecules combine a genetically engineered, picomolar affinity, soluble TCR, with a humanized anti-CD3 antibody, capable of redirecting Protein kinase N1 non tumor-specific T cells [30, 31]. Similar fusions that rely on monoclonal antibody binding to redirect the CTL response have been applied with success [32]. However, the antigens targeted by antibodies are limited to those produced as integral membrane proteins; TCRs meanwhile can recognize the larger pool of intracellular-derived peptides presented in the context of the MHC. Therefore therapeutic agents exploiting enhanced affinity TCRs hold substantial promise. Immune tolerance to tumors is a critical issue to overcome in the development of effective immunotherapies against cancer. By comparing the binding

parameters of individual TCRs to their respective pHLAs, the data presented here provide an enhanced understanding of the role of TCR affinity in tumor immune evasion, informing on the most appropriate strategies for successful therapeutics. CD8+ T cells from donors were enriched from freshly prepared peripheral blood by negative selection using microbeads according to the manufacturer’s instructions (Dynal). DCs and activated B cells were generated as described in [20, 33]. Purified CD8+ cells were cultured in CTL medium: IMDM (Invitrogen), 10% human AB serum (Sera Laboratories Int.), 100 U/mL penicillin, 100 μg/mL streptomycin, 1% glutamine (Invitrogen), supplemented with IL-7 at 10 ng/mL and autologous peptide pulsed irradiated DCs were added in a 5:1 ratio (T cells: DCs).

6% and 44.4% of patients in the TSP and ST groups, respectively, achieved CR. Cox proportional hazards models revealed that CR was achieved about six-fold more effectively by TSP than SP (HR for CR; 5.85, p = 0.028). Conclusion: TSP is a potential modality for inducing CR in patients with IgA

nephropathy and mild proteinuria. MUTO MASAHIRO1, SUZUKI YUSUKE1, SUZUKI HITOSHI1, JOH KENSUKE2, IZUI SHOZO3, HUARD BERTRAND3, TOMINO YASUHIKO1 1Division of Nephrology, Juntendo University Faculty Selleck CP690550 of Medicine, Tokyo, Japan; 2Division of Pathology, Sendai Shakaihoken Hospital, Sendai, Japan; 3Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland Introduction: A proliferation-inducing ligand (APRIL) is a critical mediator for antibody-producing plasma cell survival. Recent works suggest that APRIL is involved in autoimmune diseases such as SLE, and lymphoid malignancies. However, the pathogenetic roles of APRIL in IgA nephropathy (IgAN) are unclear. Since immunological disorders in mucosal immunity are recently discussed in the pathogenesis of IgAN, we investigated the clinical impact of mucosal APRIL expression in IgAN patients. Methods: In addition to clinical background before and after tonsillectomy, the expressions of APRIL and its receptors (TACI; transmembrane activator and calcium modulator cyclophilin ligand interactor, BCMA; B-cell

maturation antigen) in check details tonsils from IgAN patients (n = 56) and control patients (chronic tonsillitis without renal diseases, n = 12) many were evaluated by real-time PCR, immunohistochemistry (IHC)

and flow cytometric analysis (FCM). For IHC and FCM, polyclonal rabbit anti-APRIL antibody specifically recognizing APRIL-producing cells (Stalk-1) was used. Results: Tonsillar transcript levels of APRIL and its receptors in IgAN were significantly higher than those in control patients (P < 0.05). IHC revealed that Stalk-1+ cells in IgAN were detected not only in the subepithelial area but also germinal centers (GC) much more than those in control. Percentage of Stalk-1+ GC (27.4 ± 21.3%) in IgAN patients was significantly higher than that in control (7.2 ± 6.81%, P = 0.0005) and correlated with amount of proteinuria (P = 0.0017) and treatment responses, such as decrease of proteinuria (P = 0.0003). Furthermore, percentage of Stalk-1+ GC was correlated with the serum levels of IgG-IgA immune complex in patients with IgAN (P = 0.0304), but not the serum levels of Gd-IgA1. FCM showed that the percentage of Stalk-1+ CD19+ cells in tonsillar pan CD19+ cells was significantly higher in patients with IgAN than control (P = 0.0314). IHC revealed that majority of Stalk-1+ CD19+ cells was localized at GC. Conclusion: It appears that APRIL+ GC B cells in tonsils may determine the disease activity of IgAN, presumably via production of anti glycan or polyreactive antibody. YAMADA KOSHI1,2, HUANG ZHI-QIANG1, RASKA MILAN1,3, REILY COLIN R.

4- or 8.2-fold in CXCL4-stimulated cells, while in the same samples SphK2 (SPHK2), which is barely detectable in monocytes and macrophages, is down-regulated by 89 or 34%, respectively. S1P-degrading enzyme sphingosine-1-phosphate phosphohydrolase 2 (SGPP2) mRNA expression is rapidly up-regulated by 190-fold within 4 h of stimulation with CXCL4 and decreases thereafter (19-fold of unstimulated control), and sphingosine-1-phosphate lyase 1 (SGPL1) expression increases 1.6- Selleckchem AZD0530 or 1.3-fold in the presence of CXCL4 (Fig. 1, lower panels). These data clearly show that CXCL4 regulates expression of genes involved in S1P metabolism

in human monocytes. Next, we were interested in whether SphK1 is directly activated in CXCL4-stimulated monocytes. Activation of SphK1 was tested by its membrane translocation as well as by its ability to phosphorylate exogenous sphingosine in the presence of Triton X-100 14. Monocytes were stimulated for up to 30 min in the presence of 4 μM CXCL4. Subsequently, cytosol and membrane fractions were isolated and membrane fractions were tested for SphK1 by western blot analysis. As shown in Fig. 2, stimulation with CXCL4 provoked a rapid biphasic increase in membrane-bound SphK1 as well as SphK1 enzyme activity reaching

a first maximum after 30 s of stimulation. After 2 min amounts of membrane-bound SphK1 and SphK1 enzyme activity decreased again, while a second peak occurred after 10–30 min of stimulation. In summary, CXCL4 stimulates activation and membrane translocation of SphK1 in human monocytes. However, CXCL4-induced activation of www.selleckchem.com/products/BMS-777607.html SphK1 is not accompanied by the release of S1P into the extracellular medium. This was evident from experiments where monocytes (1×106 cells/mL) were activated with CXCL4 (4 μM) for 30 min, 4 and 18 h and release was determined by competitive ELISA. Under these experimental conditions, S1P concentrations in supernatants of CXCL4 stimulated monocytes never reached levels of detection limit of the ELISA (about 30 nM; data not shown). To test whether SphK signaling is involved in CXCL4-induced monocyte

functions, the cells were preincubated in the presence or absence of increasing concentrations of SKI 17. Subsequently, the cells were stimulated with 4 μM CXCL4 and production of ROS was recorded for 60 min. Preincubation of the cells with SKI resulted in a significant Depsipeptide manufacturer and dose-dependent reduction of CXCL4-mediated respiratory burst by 73% at 1 μM SKI to 98% at 27 μM SKI (Fig. 3A). These data provided first evidence that activation of SphK is involved in the generation of ROS in CXCL4-treated monocytes. To investigate whether the same pathway is involved in the control of CXCL4-mediated protection from spontaneous apoptosis in monocytes, the cells were pretreated with inhibitors as indicated in Fig. 3A and subsequently cultured for 72 h in the presence or absence of 4 μM CXCL4. To assess the proportion of apoptotic cells, the cultured monocytes were labeled with annexin V.

Isolated DNA was analyzed by quantitative PCR. EL4 and RLM11 cell lines were electroporated with pCMV6-neo vector, either empty or containing c-Jun cDNA, by using Amaxa L kit (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. Image processing was performed by Adobe Photoshop CS4 Version Lumacaftor purchase 11.0 (Adobe Systems, San Jose, CA, USA). Image analysis was performed by ImageJ 1.42q freeware (http://rsb.info.nih.gov/ij). MS Excel 2007 (Microsoft Corp., Redmond, WA, USA) was used for the statistical analysis and generation of graphs and histograms.

Student’s t-test was used for statistical analysis. Values of p < 0.05 with a 95% confidence interval were considered significant. We are grateful to H. Schäfer, S. Gruczek, and M. Ohde for animal husbandry; Drs. R. Baumgrass and T. Scheel for human blood samples; Dr. B. Malissen for FoxP3-IRES-GFP mice; members of the German Rheumatism Research Center Flow Cytometry Core Facility (T. Kaiser,

J. Kirsch, and K. Raba) for help with FACS analysis and sorting; and H. Hecker-Kia, H. Schliemann, T. Geske, and A. Peddinghaus for preparation of media and antibodies. Finally, we thank Drs. A. Rudensky, and A. Arvey for helpful advice and Prof. P. Cockerill for critical reading of the manuscript and fruitful discussion. This work Decitabine molecular weight was supported by the Deutsche Forschungsgemeinschaft (SFB/TR52) (to S.A.N.), PAK6 RFFI-ofi-m grant 11-04-12159, and MCB Program of the Russian Academy of Sciences (to S.A.N. and D.V.K.). The authors declare no financial or commercial conflict

of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Table S1. List of used antibodies. Table S2. Primers used in MNase accessibility assay. Table S3. Primers used in Pull-down assay. Table S4. Conditions of T-helpers polarization Figure S1A. DNase I hypersensitive elements of TNF/Lymphotoxin locus Mouse TNF/LT locus. Analysis performed using UCSC Genome Browser (http://genome.ucsc.edu/cgibin/hgGateway) with selected tracks from ENCODE [20] and GEO databases (naïve CD4+ and Th1 cells: GSE26550, [21]; BMDM: GSE33802 [22]). B. DNase I hypersensitive elements of TNF/Lymphotoxin locus Human TNF/LT locus. Analysis performed using UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway) with selected tracks from ENCODE database. Figure S2. A, B. TNF expression in various subsets of mouse CD4+ T cells. Q-RT-PCR (A) and ELISA (B) analysis of polarized Th cells.