In total we analyzed ten donors, of which five showed M1-specific responses. In all cases the responding T cells reacted against both peptide and recombinant

protein pulsed APC, showing that the M1-specific T cells recognize naturally processed epitopes. Moreover, the responses were accompanied by both IFN-γ and IL-10 (Fig. 1B). To characterize the influenza-specific IL-10-producing T cells at the single-cell level, the IL-10-producing influenza-specific T-cell population selleck compound was enriched by magnetic cell sorting (Fig. 2A). The bulk cultures from three different donors were enriched for IL-10-producing cells. The mean percentage of IL-10-producing T cells before enrichment was 0.33%. After enrichment the mean value was 49% and ranged between 18 and 90%. In total, EPZ015666 cost 125 T-cell clones were isolated from these enriched cultures by limiting dilution. The isolated T-cell clones displayed a CD3+CD4+CD8− phenotype and were assessed for clonality by analysis of their TCR-Vβ using flow cytometry. Consistent with findings in mice 15, most of the IL-10-producing

clones (79/83) produced both IFN-γ and IL-10 upon cognate peptide stimulation (Fig. 2B), indicating that the M1-specific T-cell clones are representative of the unsorted population. Furthermore, the isolated influenza-specific T-cell clones recognized their cognate epitope when naturally processed from M1 protein (Fig. 2C and D). D1.6 recognized M1 peptide 31–60, D1.52 and D1.4 recognized M1 peptide 1–30, D4.6 recognized M1 peptide 46–75, D1.68, D1.50 and D4.11 recognized M1 peptide 91–120. Moreover, the clones specifically proliferated when stimulated with live virus-infected monocytes, as one would expect from influenza-specific CD4+ T cells (Fig. 2E). Few clones did not respond to viral challenge, and is likely due from to differences in amino acid sequence between the synthetic M1 peptides (based on A/PR/8/34) and the virus used (A/Wisconsin/67/2005),

which share 96% amino acid sequence identity. Analysis of the clones on a single-cell level using cytokine capture assay revealed that the same cell produced both IFN-γ and IL-10 at high concentrations of cognate peptide. However, in some cases (D4.6 and D4.11) T-cell clones produced only IL-10 in the lower antigen range, but co-produced IFN-γ when stimulated with increasing concentrations of M1 peptide (Fig. 3). A number of isolated M1-specific clones did not produce IL-10 upon antigen challenge (e.g. D4.18, which recognized M1 peptide 196-225; Fig. 3), which could be explained by the fact that the T-cell clones were isolated from IL-10-enriched, but not pure M1-specific T-cell cultures of which not all M1-specific T cells produced IL-10. Subsequently, the expression of FOXP3 in these clones was examined.

The novel use of a known therapy – fecal microbiota transplantation has shown promise in recurring and refractory cases, with minimal complications in this susceptible population, as we illustrate in this case of a renal transplant recipient. Case description: We report the case of a 62yr deceased donor renal transplant Opaganib ic50 recipient on standard immunosuppression, who had multiple hospital admissions either as a result of, or complicated by CDAD. She was treated with specific antibiotics (vancomicin, metronoidazole, rifaximin and fidaxomicin; multiple courses) but proved to be refractory to medical therapy. She had a total of 20 hospital admissions across the health district in the period

from October 2011 to February 2014, resulting in a total of 397 days spent in hospital, during which she always developed CDAD. click here She underwent a fecal microbiota

transplant, which resulted in resolution of diarrhea, improvement in well being and has kept her out of hospital. Discussion: Clostridium difficile is more prevalent in immunocompromised patients, resulting in significant patient morbidity and strain on health care resources. This novel therapy has the potential to decrease hospitalization rates and length of stay in future especially with early application. To date there are only very few reported cases of the use of this therapy in solid organ transplant patients. 299 POST PARTUM POSTERIOR REVERSIBLE ENCEPHALOPATHY SYNDROME (PRES) SECONDARY TO EPIDURAL ANAESTHESIA R SUD1, S BHASKARA1, G LEE1, M SURANYI1, M DOWLA2, S LIM3, A HENNESSY3, A MAKRIS1,3 1Renal Department, Liverpool Hospital, Sydney, NSW; 2Neurology Thymidylate synthase Department, Bankstown Hospital, NSW; 3Heart Research Institute, Sydney, Australia Background: Posterior reversible encephalopathy syndrome (PRES) is a neurological disorder that has

been associated with numerous underlying causes. In the post partum period, pre-eclampsia is frequently assumed to be the cause. Case reports of postpartum PRES have been reported due to alternative aetiologies, including spinal anaesthesia. The ratio of soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) has been shown to discriminate between normal pregnancy and the hypertensive disorders of pregnancy (HDP). These markers may have a role in clarifying causes of post partum PRES. Case Report: We present a case of a 28 year old female presenting with seizures, severe headache, confusion and hypertension 48 hours after a normal vaginal delivery. The delivery was facilitated by an epidural anaesthetic – complicated by dural puncture. Anti-inflammatories were given for perineal pain. MRI findings were consistent with PRES. No proteinuria, liver, renal or haematological abnormalities were demonstrated at presentation. Serum was stored for later measurement of circulating angiogenic markers.

3a). However, there was no significant alteration in the CD4+ : CD8+ T-cell ratio when comparing the placebo group with the monoclonal anti-CD3 F(ab′)2-treated group as

a whole. By contrast, the percentage of CD4+ T cells in peripheral blood that were FoxP3+ (i.e. Treg cells) was markedly higher in the monoclonal anti-CD3 F(ab′)2-treated mice (23·0% ± 1·4%) compared with placebo mice (8·1% ± 1·0%, P < 0·001). buy Small molecule library Given the transient decline in total lymphocyte numbers in the peripheral blood, and the increased percentage of CD4+ FoxP3+ T-cells at the end of dosing, we hypothesized that CD4+ FoxP3+ T cells were either selectively maintained or expanded as a result of treatment with monoclonal anti-CD3 F(ab′)2. At the 12-week end-point, flow cytometric analysis of peripheral blood showed that CD4+ and CD8+ T-cell populations had significantly recovered but remained below baseline levels, and that the CD4+ FoxP3+ T-cell population had diminished (from elevated post-dosing levels) to slightly above baseline levels (Table 2). While significant changes in the proportion of various T-cell subsets in peripheral blood were detected during the dosing period, long-term follow-up of peripheral blood PD parameters did not reveal

any long-term changes. Potential differences in the T-cell compartments sequestered learn more at the site of inflammation (e.g. the pancreas) were not assessed. The PD parameters observed at completion of dosing were also analyzed according

to the monoclonal anti-CD3 F(ab′)2 dose regimen and whether the mice had entered remission or remained diabetic after treatment. Reductions in the proportions of CD4+ and CD8+ T cells, and increases in the proportions of CD4+ FoxP3+ T cells tended to be greater at higher doses (Fig. 3b). Also, at the higher doses, reductions in CD4+ T-cell proportions were greater than that observed in CD8+ T cells, resulting in a temporary decrease in the CD4+ : CD8+ T-cell ratio. At the 12-week end-point, the CD4+ : CD8+ T-cell ratio returned to baseline, as both CD4+ and CD8+ T-cell populations had significantly recovered (Table 2). At the lower, but still efficacious, doses, a decrease in the CD4+ : CD8+ T-cell ratio was not observed. Ultimately, unlike the modulation patterns of the CD3–TCR complex that were elicited by varying doses of monoclonal PTK6 anti-CD3 F(ab′)2 (Fig. 1b), a strictly dose-dependent relationship for the alterations in proportions of T-cell subsets was not observed. Furthermore, within each dose regimen, proportions of circulating CD4+, CD8+ and CD4+ FoxP3+ T cells at completion of dosing were similar in responder and non-responder mice. However, it is possible that at local sites of inflammation, such as the pancreas and pancreatic lymph nodes, there may be significant differences between responder and non-responder mice in the proportions of these T-cell populations.

albicans check details were incubated on egg yolk agar to detect phospholipase activity. Virulence of C. albicans was assessed by the average survival time of infected mice. Expression of phospholipase B1 mRNA and protein were detected by RT-PCR and Western blot method. Significant differences between the two groups of Candida strains were observed in phospholipase activity and average survival time of infected mice. The expression of phospholipase B1 mRNA and protein

(both of secreted and intracellular forms) were higher in resistant strains than in susceptible strains. The results indicate that the phospholipase activity of C. albicans may be related to its resistance to antifungal drugs. “
“Widespread use of fluconazole has resulted in resistance in strains of Candida. The aim of our study was to investigate

Y132H and other mutations in the ERG11 gene in conferring fluconazole resistance to C. albicans isolates. Seven fluconazole-resistant (R)/susceptible dose-dependent (SDD)/trailing and 10 fluconazole-susceptible (S) isolates were included. Restriction enzyme analysis was performed on all isolates for Y132H mutation and sequence analysis was performed for other mutations in the ERG11 gene. None of our strains had Y132H mutation. One single mutation (D153E, E266D, D116E, V437I) was detected in isolates 348, 533, 644, selleck 1453, 2157, while the others had more than one nucleotide change. D116E and E266D, which were two mutations found

in fluconazole R/SDD/trailing isolates with the highest frequency, were also detected in azole S strains. K143R, G464S, G465S and V488I mutations were determined in three of the R/SDD isolates. S412T and R469K mutations were detected only in this group of strains by sequence analysis. Mutations such as K143R, G464S, G465S, V488I, S412T and R469K in the ERG11 gene were determined to be effective mechanisms in our fluconazole R/SDD C. albicans isolates. Other mechanisms of resistance, Dichloromethane dehalogenase such as overexpression of ERG11 and efflux pumps and mutations in the ERG3 gene should also be investigated. “
“Allergic bronchopulmonary aspergillosis (ABPA) is a complex immune hypersensitivity reaction to Aspergillus fumigatus, usually complicating the course of patients with asthma and cystic fibrosis. The common radiological manifestations encountered are fleeting pulmonary opacities, bronchiectasis and mucoid impaction. Uncommon radiological findings encountered in ABPA include pulmonary masses, perihilar opacities simulating hilar adenopathy, miliary nodules and pleural effusions. Herein, we describe a 22-year-old female patient who presented with acute hypoxaemic respiratory failure secondary to left lung collapse, which necessitated rigid bronchoscopy for management. On further evaluation, she was diagnosed to have ABPA. This is the first documented report of ABPA presenting as acute hypoxaemic respiratory failure secondary to lung collapse.

Recent evidence suggests that similar mechanisms may regulate the commitment of Thp between Treg and Th17. In human cells, FoxP3 exists in two separate but equally expressed isoforms: one (FoxP3), which is encoded by a full length mRNA and the other a truncated form lacking exon 2 (FoxP3Δ2), which is coded by a splice variant mRNA [104,109]. Tregs, perhaps unexpectedly, also express Th17-specifying transcription factors, notably RORα[110] and RORγt [111]. However, co-immunoprecipitation experiments have shown that FoxP3 binds to RORα and RORγt and inhibits their biological activity

in a dose-dependent fashion [110,111]. This interaction is mediated through a (LxxLL) motif in the FoxP3 second exon; as expected, the FoxP3Δ2 isoform is unable to bind RORα or Romidepsin order GS 1101 RORγt [110,111]. A similar interaction has subsequently been described, by the same group and others, in murine cells. Specifically, both FoxP3 and RORγt are co-expressed

in naive CD4+ T cells exposed to TGF-β, where FoxP3 inhibits RORγt directly through a physical interaction, repressing the Th17 programme [111]. In these experiments exposure of Thp to TGF-β leads to rapid induction of RORγt [92], but the binding of RORγt to the IL-17 promoter is suppressed by interaction with FoxP3 [112]. Upon addition of exogenous IL-6 or IL-21, the inhibitory effect of FoxP3 on IL-17 induction is circumvented [111] and FoxP3 levels are reduced [112]. The interaction between FoxP3 and RORγt

in murine cells is also dependent upon the second exon of FoxP3 [111,112]. These observations have also been confirmed by another, independent group [74]. These interactions can, in part, explain the conversion of Tregs to Th17, at least in mice. While TGF-β induces both FoxP3 and RORγt expression, IL-6 does not alter expression of RORγt but inhibits FoxP3. As a result, exposure of Tregs to IL-6 down-modulates FoxP3 preferentially and reduces the Tyrosine-protein kinase BLK physical inhibition of RORγt, permitting binding to the IL-17 gene promoter. In addition, very recent murine data suggest that IL-1 regulates expression of RORγt [79]. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is a receptor-coupled signal transduction mechanism linking cytokine–receptor interactions to gene expression. There are seven STAT (STAT1-4, 5A, 5B and 6) and four JAK [JAK1-3 and TYK2 (tyrosine kinase 2)] proteins in humans (reviewed in [113]). Specific JAKs are associated with the cytoplasmic tails of multimeric cytokine receptors, and are activated upon ligand-induced receptor oligomerization [113,114]. Activated JAKs phosphorylate specific tyrosine residues on cytoplasmic tails of their associated cytokine receptors, creating docking sites for the SH2 (Src-homology-2) domain of STAT proteins, and then activate the docked STATs through tyrosine phosphorylation.

In contrast, adults with active pulmonary TB in a highly TB endemic area in Indonesia had significantly lower plasma granulysin concentrations than did controls, these concentrations increasing after 2 months of anti-TB therapy to values similar to those of controls, and having increased even further after completion of anti-TB therapy. These changes in granulysin concentrations occurred predominantly in patients Fulvestrant research buy in whom IFN-γ negative T cells were expressed, suggesting that in TB the cellular sources of IFN-γ and granulysin are partly non-overlapping (14). Similar findings have

been reported for Italian children, the lowest concentrations having been found in TB patients who were PPD negative at the time of diagnosis (15), indicating the involvement of granulysin and IFN-γ in curative immune learn more responses against Mtb. In chronic pulmonary TB, lung tissue biopsy has shown reduction in amounts of perforin and granulysin in relation to granzyme

A, while higher per cell expression of perforin and granulysin is associated with bacteriological control, suggesting that perforin and granulysin could be used as markers or correlates of immune protection in human TB (16). However, effective host mechanisms against Mtb infection are not well understood, this lack of understanding being a problem in regard to vaccine C-X-C chemokine receptor type 7 (CXCR-7) development and immunotherapy for TB. Moreover, so far there is limited information regarding the roles of IFN-γ and granulysin in recurrent TB. Therefore, the present study aimed to investigate whether granulysin and IFN-γ responses are associated with clinical disease in patients with newly diagnosed, relapsed and chronic pulmonary TB in northern

Thailand, where TB is endemic. One hundred and fifty-five pulmonary TB patients (aged 9 to 88 years) were recruited from the outpatient and inpatient clinics of Chiang Rai Hospital and Mae Chan Hospital, in the north of Thailand. These included 102 male and 53 female patients with newly diagnosed and previously treated pulmonary TB. Patients with extrapulmonary TB and pulmonary TB/HIV seropositive were excluded. All patients with pulmonary TB had clinical symptoms and a confirmed diagnosis on the basis of presence of acid-fast bacilli in sputum on microscopic examination, positive cultures of Mtb, medical history and chest radiographic findings. Patients were categorized according to World Health Organization criteria (1), which include ascertaining whether the patient has previously received TB treatment. The TB drug regimens were based on the recommendations of the National Tuberculosis Program, Ministry of Public Health, Thailand. Standard TB treatment drugs consist of streptomycin (S), isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E).

Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org/our-partners.php. “
“Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4+ T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide-binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II-mediated antigen presentation. Lysosome-associated PI3K inhibitor membrane

proteins such as LAMP-2 reside in mature endosomes Romidepsin in vitro and lysosomes, yet their role in exogenous antigen presentation

pathways remains untested. In this study, human B cells lacking LAMP-2 were examined for changes in MHC class II-restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4+ T cells was impaired in the LAMP-2-deficient B cells. Peptide-binding to MHC class II on LAMP-2-deficient B cells was reduced at physiological pH compared with wild-type cells. However, peptide-binding and class II-restricted antigen presentation were restored by incubation of LAMP-2-negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP-2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was Methamphetamine detected using LAMP-2-deficient B cells. Consequently, LAMP-2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation. Major histocompatibility complex (MHC) class II molecules

present antigenic peptides derived from exogenous proteins to CD4+ T cells.1 These MHC class II proteins are constitutively expressed on the surface of a number of professional antigen-presenting cells (APC) such as dendritic cells, B cells and macrophages. The MHC class II complexes consist of α and β subunits which are first assembled in the endoplasmic reticulum with the chaperone molecule invariant chain (Ii).2,3 The cytoplasmic tail of Ii contains a motif that targets the Ii–MHC class II complexes to endosomal/lysosomal compartments. Here, acidic proteases degrade Ii to a small fragment known as class II-associated invariant chain peptide (CLIP), which remains associated with the MHC class II peptide-binding groove.4,5 Antigens delivered into the endosomal/lysosomal network via receptor-mediated or fluid-phase endocytosis are also exposed to proteases and denaturing reactions, yielding peptide ligands for class II molecules.

The mRNA levels of IL-4, IL-10, IL-21, IL-21R, CD40L in the gingival biopsies were evaluated by quantitative real-time polymerase chain reaction. The salivary levels of IgA and the levels of IL-4 and IL-10 in the gingival biopsies were analyzed by ELISA. The mean levels of

IgA were significantly ABT-263 purchase higher in the chronic periodontitis compared to periodontally healthy group (P < 0.05). The mRNA levels for IL-21 was higher (P < 0.05) in the chronic periodontitis when compared to the healthy group. However, the expression of IL-21R and CD40L did not differ between groups. The IL-10 was significantly elevated at mRNA and protein levels in chronic periodontitis when compared to periodontally healthy group (P < 0.05). Conversely, the mRNA levels as well as the protein amount of IL-4 were significantly lower (P < 0.05) in chronic periodontitis than healthy ones. In conclusion, the upregulation of IL-21 and KU-60019 research buy IL-10 and downregulation of IL-4 in periodontitis tissues may be collectively involved in the increased levels of salivary IgA in chronic periodontitis subjects. The mucosal immune system generates frontline immune protection at the interface between the host and the environment by forming

a highly integrated system of lymphoid organs collectively known as mucosa-associated lymphoid tissue, which play a crucial role in antibody formation [1]. The antibody immunoglobulin (IgA) is the predominant immunoglobulin secreted by oral mucosal sites, considered one of the most important protein contributing to microbial defence from toxins, viruses, and bacteria by means of direct neutralization or prevention of microbial binding to the mucosal surface [2]. Previous studies have long demonstrated that the humoral immune response, especially Cell Penetrating Peptide mediated by secreted IgG and IgA, plays protective role in the pathogenesis of periodontal diseases, including

gingivitis, chronic and aggressive periodontitis. It was previously showed that the levels of salivary IgA directed to Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were significantly higher in subjects with deeper periodontal probing depth (PD) compared to healthy subjects [3]. Moreover, the serum IgA- and IgG-class antibody levels against A. actinomycetemcomitans and P. gingivalis were higher in the pathogen carriers compared with the non-carriers, and clearly higher in the carriers with periodontal pockets compared with the carriers without pockets [4]. Also, it was reported that the level of specific salivary IgA antibodies against mycobacterial heat shock protein (HSP) 65 was significantly increased in patients with gingivitis compared to healthy and periodontitis subjects [5].

3A and B). The polyfunctional CD4+ T-cell response peaked 28 days after vaccination in most adolescents, and 84 days after vaccination in most children. However, in some children this response peaked 7 days after vaccination (Fig. 3A and B and Supporting Information Fig. 4). The polyfunctional CD4+ T-cell population was long-lived in both age groups as frequencies detected at 168 days after vaccination still exceeded pre-vaccination levels (Fig. 3A and B and Supporting Information Fig. 4). A novel population of polyfunctional CD4+ T cells that co-expressed all four of IFN-γ, IL-2, TNF-α and IL-17, which we have termed Th1/Th17 cells, was induced by MVA85A vaccination in adolescents

(Fig. 3A and D). In contrast, the find more Autophagy signaling pathway inhibitors frequency of this population in children was much smaller (Fig. 3C and F). In children, we also assessed expression of GM-CSF; the majority of Ag85A-specific IFN-γ-, IL-2- and TNF-α-expressing cells co-expressed this cytokine (Fig. 3B and E and Supporting Information Fig. 3). Overall, >50% of Ag85A-specific CD4+ T cells were polyfunctional (i.e. the cells expressed ≥3 cytokines) at all time points following vaccination,

both in adolescents and in children (Fig. 3D–H). In children, the proportion of cytokine-producing T cells that were polyfunctional increased over time; by 168 days post-vaccination >60% of Ag85A-specific cells expressed ≥3 cytokines (Fig. 3E, F and H). Interestingly, in contrast to the Ag85A-specific response, the BCG-specific response was markedly less polyfunctional throughout the follow-up period;

more than 75% of BCG-specific CD4+ T cells expressed one or two cytokines only (Fig. 3G and H). We also compared the magnitude of the Ag85A-specific T-cell response between adolescents and children 7 days after vaccination (Supporting Information Table 2). The frequencies selleck chemicals llc of IFN-γ-expressing T cells, whether measured by ELISpot or flow cytometry, did not differ between the two groups. IL-2-expressing CD4+ T-cell frequencies were also not different. However, when total cytokine+ CD4+ T-cell frequencies, TNF-α-expressing, or IFN-γ, IL-2 and TNF-α co-expressing polyfunctional CD4+ T-cell frequencies were compared, lower frequencies were observed in children. Because lymphocyte and CD4 counts are highest in infants and decrease with age 26, 27, we hypothesized that adjustment for cell counts would negate these differences. However, absolute lymphocyte and CD4 counts for the vaccinees were not available. We therefore classified the subjects into different age categories, and adjusted the corresponding lymphocyte or CD4 counts for median cell counts reported in Ugandan children 26. Adjustment of these T-cell response data for age-specific CD4 counts did not negate the differences observed for total cytokine+ and TNF-α levels (Supporting Information Table 2).

Thus, we provide further evidence for the impairment of induced Treg (iTreg)-mediated immunoregulation by TLR7 ligands which is in accordance with the previous results 19, 34. Furthermore, we identify additional mechanisms for the reduction of Treg-mediated suppression by TLR7 activation, which are not mediated by resistance of responder T cells to Tregs. Our study shows ABT-888 cost that TLR7-mediated activation of DCs reduces immunoregulation by Tregs at the levels of Treg generation as well as suppressive function thus contributing to the breakdown of peripheral tolerance and development of autoimmunity, for example, in SLE, where activation of TLR7 by endogenous ligands was shown to play

a role in the pathogenesis. Therapeutic approaches aiming to reverse Foxp3 downregulation by interfering with TLR7 activation or by blocking downstream

effector cytokines such as IL-6 are therefore promising strategies for the treatment of SLE. C57BL/6 and BALB/c mice were purchased from Harlan Winkelmann (Borchen, Germany). TLR7−/−35, DEREG 23.2 (both on the C57BL/6 background) 36, DO11.10/Rag2−/−, OTII/Rag2−/−/DEREG, and CD45.1 congenic mice were bred in our animal facility Z-IETD-FMK research buy under specific pathogen-free conditions. Experiments were performed in accordance with the German animal care and ethics legislation and had been approved by the local government authorities. CD11c+ DCs were isolated from splenocytes after digestion with DNAse I and collagenase D (Roche Applied Science, Mannheim, Germany) using anti-CD11c-coupled magnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany, purity 90–98%). CD4+CD25− T cells Tenoxicam were isolated using the CD4+ T-cell isolation kit (Miltenyi Biotec) supplemented with biotinylated anti-CD25 antibody (eBioscience, San Diego, CA, USA, purity 90–95%). Naïve T cells were stimulated with 5 μg/mL anti-CD3 antibody (eBioscience) coated to the surface of a 96-well round-bottom plate together with CD11c+ splenic DCs at a ratio of 2:1 (80 000 T cells and

40 000 DCs) or 5 μg/mL soluble anti-CD28 antibody (eBioscience) in 200 μL/well complete medium (RPMI1640, 10% FBS, 1% glutamax, 1% penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate, 50 μM β-mercaptoethanol) with TGF-β (3–5 ng/mL, Peprotech, Hamburg, Germany) and IL-2 (200 U/mL, PromoKine, PromoCell GmbH, Heidelberg, Germany). The following TLR ligands were used: TLR7 ligand S-27609 (3 μM, imiquimod analogue, 3 M Pharmaceuticals, St. Paul, MN, USA), TLR9 ligand CpG 1668 (0.5 μM, MWG Operon, Ebersberg, Germany) and TLR4 ligand LPS (100 ng/mL, Sigma-Aldrich, St. Louis, MO, USA). Where indicated, 40 μg/mL U1snRNP (gift of Bertold Kastner, Berlin, Germany) complexed with 12.5 μg/mL cationic lipid DOTAP (Roth, Karlsruhe, Germany) was used to stimulate the cells 5. IL-6 was neutralized by anti-IL-6 (5 μg/mL) together with anti-IL-6R antibody (2 μg/mL).