It was then shown that culture of T cells from IL-1R1-deficient mice which cannot

respond to IL-1β, exhibited substantially less IL-17 bias than WT T cells when co-cultured with R258W CD11b+ cells. Similar results were obtained when T cells were co-cultured with supernatants of R258W KI APC. Taken together, these findings indicate that the KI APCs act on differentiating CD4+ T cells to favor Th17-cell differentiation via IL-1β, providing that the T cells have undergone initial Th17-inductive steps. It should be noted, however, that as there was residual Th17-cell bias in the studies using IL-1R1−/− cells, other factors secreted by APC from R258W KI mice may also play a role in inducing check details Th17-cell differentiation 9. Parallel studies of T-cell differentiation directed by APC from A350V and L351P KI mice were

conducted with antigen-specific T cells. It was found that these APC exhibited a normal capacity to induce T cells to differentiate into any type of T-cell lineage under subset-specific conditions, and exhibited only a modest bias toward IL-17 under neutral conditions. This result was consistent with the fact that skin inflammation in these mice did not show an IL-17 cytokine bias. This discrepancy may be due to the fact that these in vitro studies were not conducted under conditions that allowed initial Th17-cell induction and thus did not assess IL-1β effects at an appropriate phase of T-cell differentiation 9, 10. The mechanism underlying the Th17-cell NVP-LDE225 in vitro bias in the inflammasome-associated inflammation noted above for R258W KI mice is not fully understood. Previous studies have shown that IL-1β together with TNF-α can augment TGF-β/IL-6-induced Th17-cell differentiation and that in fact IL-6 induces IL-1R1 expression on T cells 24, 25. In addition, IL-1β has been shown to upregulate factors that induce/enhance IL-17 transcription, such as RORγt and IRF-4 24, 26; however, isometheptene the molecular mechanism underlying this upregulation is not known. As for the fact that the inflammasome-associated

inflammation is marked by decreased IFN-γ as well as increased IL-17 production, it may be due to the fact that IL-1β downregulates IL-6-induced STAT-1 activation and thereby inhibits T-bet transcription 27. Additionally, it was observed that the inflamed tissue of the KI mice exhibited decreased IL-12Rβ2 expression and that treatment of mice with anti-IL-1R1 reversed this effect. Thus, IL-1β may inhibit IL-12p70 induction of STAT-4 activation, the essential initial step in Th1-cell development 28. Given the well-known propensity of IL-17 to induce a neutrophil-rich inflammation 29, 30, the Th17-cell bias inherent in inflammasome activity may be a major reason why neutrophils are a major component of autoinflammation in CAPS.

Similar to Australia/New Zealand, in Canada 63% of alternative HD patients also undertake NHD at home, although 27% carried out SDHD at home and no patients were undertaking NHD in-centre. In the USA, the majority of patients on alternative HD regimens (85%) received in-centre dialysis with

only a small percentage (5%) undertaking NHD at home. For the overall IQDR population, 66.3% of home NHD patients dialysed 3–4 nights per week and 33.7% dialysed 5–7 nights per week.6 This compared with those receiving NHD in-centre who were almost exclusively dialysed 3–3.5 nights per week. The average treatment session lengths for home and in-centre NHD were comparable at 420 ± 70 min in-centre and selleck chemical 426.5 ± 67.5 min at home. Although the type of dialysers for alternative HD regimens is similar to conventional HD (preferably being high-flux), the selleck dialysate concentration should vary between schedules4,26 (Table 3). Initial dialysate composition for SDHD is similar to that for conventional HD (Table 3),

but there are variations in NHD as listed below. The concentration of sodium in the dialysate may be similar or slightly higher for NHD. Potassium dialysate concentrations in NHD are usually similar to conventional HD and SDHD, although often not as low with most patients dialysing against 2.0 mmol/L baths. Patients often have more freedom in their diet with reduced dietary potassium restriction. Phosphate is cleared by dialysis in a time-dependent manner, and therefore SDHD and NHD result in increased phosphate removal compared with conventional HD. For SDHD, improvements in serum phosphate levels will result if the duration of dialysis is >2 h per session, although phosphate supplementation is rarely required.27,28 Phosphate removal in NHD is about two times greater than for conventional HD; and patients are often able to discontinue phosphate binders and may have less dietary phosphate restriction.9,20 Hypophosphatemia ALOX15 can occur with NHD schedules involving 5–7 nights per week;

and intradialytic phosphate supplementation may be required with the addition of sodium phosphate to dialysate.9 In Australia, the addition of Fleet®enema solution (C.B. Fleet Company, Inc., Virginia, USA) to the acid concentrate is recommended; and 30 mL can be added to 5 L of dialysate to increase the serum phosphate by 0.25 mmol/L. Titration of phosphate is according to pre- and/or post-dialysis levels, which should be maintained in the normal range. In alternate-night NHD, post-dialysate phosphate levels are often low but rebound quickly after a few hours of completing a dialysis session and phosphate supplementation is less often required. One of the more important minerals in dialysate requiring adjustment for alternative HD regimens is calcium.

Here we report a rare case of IgG4RD that developed during chronic hemodialysis. Case Report: A 61-year-old male with polycystic kidney disease who had been on hemodialysis for seven years was referred

to our hospital because of nausea, cough and asthma that recently appeared during hemodialysis RXDX-106 session. The symptoms continued even after dialyzers were changed to other ones. He had been having submaxillary gland swelling for five years. The blood tests showed eosinophilia (8000/ml), hypergammaglobulinemia (serum IgG 5462 mg/dl) with a rise in IgG4 concentration (1540 mg/dl). The biopsy of the gland revealed an

infiltration of plasma cells more than 50% of which being IgG4 positive without evidence of tumor, thus he was diagnosed as IgG4RD. No involvement was found in other organs including pancreas. Oral prednisolone (30 mg/day) was begun and the symptoms during hemodialysis immediately disappeared together with gradual improvement of eosinophilia and submaxillary gland swelling. Disussion and Conclusion: We should consider the possibility of IgG4RD when we see such patients on chronic hemodialysis showing episodic asthma and eosinophilia. EDAMATSU TAKEO, FUJIEDA AYAKO, EZAWA ATSUKO, ITOH YOSHIHARU Pharmaceutical Division, Kureha Corporation Introduction: Protein-bound

check details retention solutes, which are known to be accumulated in the body of chronic kidney disease patients, are considered to have deleterious Racecadotril effects on disease progression. In fact, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), two representative molecules of such solutes, have been extensively studied to have harmful impacts related to renal and vascular function. Although considerable amount has been detected in hemodialysis patients, little study on other molecules, such as phenylsulfate (PhS), indoleacetic acid (IAA) and hippuric acid (HA), has been performed to date. Here we conducted a comparative study for such molecules to see how similar or dissimilar these compounds are. Methods: We evaluated effects of these compounds in LLC-PK1, a porcine renal tubular cell line. Effect on viable cell number was determined using WST-8, a water-soluble version of MTT. Effect on cell cycle progression was determined using propidium iodide (PI), after appropriate synchronization. Apoptotic cells were detected with Annexin V-FITC and PI. Protein and gene expression were determined by western blotting and real-time PCR, respectively. Results: All these compounds reduced cell number after 2 day incubation.

To assess changes in the amount of inflammation-induced leucocytes, 5 × 106 washed spleen cells were stained with the following fluorescence-coupled monoclonal antibodies anti-CD11b-phycoerythrin (PE) or -allophycocyanin (APC), granulocyte-differentiation antigen-1 (Gr-1)-PE, B220-fluorescein isothiocyanate (FITC), anti-CD4-PE, anti-CD25-FITC

and biotinylated anti-CD3ε followed by incubation with streptavidin-PE-Cy5 (PharMingen Canada for conjugated monoclonal antibodies, and Cedarlane, Hornby, Ontario, Canada for streptavidin) for flow cytometry according to published procedures. The remaining splenic lymphocytes were placed into the wells of 96-well plates at a concentration of 2 × 105 cells per well. Cultures were stimulated with either sterile sonicates

prepared from pure strains of selected endogenous bacteria, as detailed in Sydora et al.[8], or with sterile lysates prepared from faecal material GSK1120212 molecular weight using glass beads as described in Sydora et al.[9]. Bacterial sonicates and faecal lysates were added at a protein concentration of 50 µg/ml, which was found to be optimal for cytokine production. Control stimuli included plate-bound anti-CD3ε clone 145-2C11 (PharMingen Canada) and medium alone. After 48 h of incubation at 37°C in a humidified Selleck BGJ398 incubator at 5% CO2, the plates were centrifuged, and the amounts of the indicated cytokines in the supernatants were quantified using standard ELISA techniques, as described above. Data are expressed as means ± standard error of the mean (s.e.m.) or means ± standard deviation (s.d.) dependent upon whether data were combined from both experiments of the same mouse strain or whether they were derived from only one experimental group, respectively. Differences between mean values were evaluated using analysis of variance or paired t-tests, where appropriate (SigmaStat; Jandel Corporation, San Rafael, CA, USA). In axenic mice, spontaneous release

of cytokines from colonic and caecal mucosal tissue was low (Fig. 1, day 0), similar to cytokine release in wild-type mice raised under conventional, non-pathogenic conditions in the presence of commensal intestinal bacteria [8]. However, inoculation of the axenic mice with faecal bacteria slurry resulted in a significant colonic and caecal immune response of proinflammatory cytokines, IFN-γ, TNF-α and IL-17 that peaked at Uroporphyrinogen III synthase 3–7 days after faecal slurry exposure (Fig. 1 and data not shown). Similarly, there was a significant increase in G-CSF 3 days post-faecal slurry feeding. In contrast, colonic and caecal immune response of anti-inflammatory cytokines, IL-4 and IL-10, followed that of the proinflammatory cytokines and peaked at day 7 (Fig. 1). While small increases in production of IL-6 were noted on days 3 and 7, these increases were not significant (data not shown). By day 14 following faecal slurry exposure, production of all cytokines was diminished and reached background levels by 28 days (Fig. 1 and data not shown).

RNA was prepared from purified B cells of Foxo1f/fCd19Cre and Foxo1f/f mice using Trizol reagent (Invitrogen) as described previously 1, 2. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA,

USA). Primers for genes of interest Cell Cycle inhibitor (Klf2, Klf4, Ccng2, Rbl2, Sell and Ltb) and housekeeping gene (β-actin) were optimized to amplify products between 75 and 200 nucleotides. Primer sequences are available on request. Quantitative PCR was performed with SyBr green as described previously 1, 2. A Student’s t-test was used for all comparisons. The specifics of each test (one versus two-tailed) are indicated in the figure legends. This study was supported in part by a Research Scholar Grant from the American Cancer Society (to D. A. F.) and by NIH grants AI057471 and AI061478 (to S. L. P.). The authors thank Craig Walsh and Aimee Edinger for helpful discussions, Lomon So for technical assistance and Christine McLaren for statistical analysis. Conflict

of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting click here Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4+ and CD8+ immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207+, CD11c+, CD4+, CD8+, iNOS+ cellular densities. almost Cytokine (IFN-γ, IL-4

and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207+ and CD11c+ were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4+ and CD8+ were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS+ was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-γ was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response. American cutaneous leishmaniasis (ACL) is a zoonotic protozoal disease caused by different species of Leishmania (1). In Brazil, L. (V.) braziliensis and L. (L.) amazonensis are considered the main pathogenic species causing human ACL (2). The human L. (L.

Rather, the ability of oxaliplatin to induce ROS production via the NADPH oxidase NOX2 in tumor-infiltrating myeloid cells was inhibited in antibiotic-treated mice [22] (Fig. 2). ROS production by myeloid cells was needed for oxaliplatin’s antitumor effect and oxaliplatin efficiency was decreased by inhibition of ROS by the antioxidant N-acetylcysteine,

in animals deficient for the gene encoding NOX2, or following depletion of myeloid-infiltrating cells [22]. Although ROS and particularly H2O2 production were previously shown to be required for the genotoxic effect of platinum compounds [171, 172], this was studied mainly in tumor cell lines in vitro, and ROS was thus expected to be endogenously produced in the tumor cells, either selleck chemicals llc as mitochondrial or NADPH oxidase generated ROS. However, in the tumor microenvironment in vivo, ROS produced by tumor-associated myeloid cells is required for oxaliplatin cytotoxicity, and the microbiota has been shown to regulate the ability of oxaliplatin to induce early cytotoxicity of tumor cells by systemically priming tumor-associated myeloid cells for ROS production [22].

The effects mediated by the commensal microbiota on early responses to therapy are likely https://www.selleckchem.com/products/XAV-939.html dependent on a systemic priming effect of the preexisting microbiota composition on myeloid cells. However, both chemotherapy and radiation therapy can also modify the composition of the microbiota and exert severe toxicity on the intestinal mucosa, allowing transmucosal translocation of bacteria

and further contributing to therapy-induced dysbiosis [173, 174]. One of the most promising anticancer therapeutic approaches is the adoptive transfer of expanded, tumor-specific cytotoxic CD8+ T cells. In this therapeutic approach, some level Ixazomib order of lympho- and myelo-ablation in the host is necessary for the survival of the incoming T cells and effectiveness of the transfer [175]. In both patients and in mice, total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8+ T cells and favors DC activation and the production of homeostatic cytokines [175, 176]. Also following TBI in mice, commensal gut bacteria have been isolated from the MLNs and elevated LPS levels were observed in the sera [175]. The beneficial effects of TBI on tumor regression was reduced by antibiotic treatment, neutralization of serum LPS using polymyxin B, or prevention of LPS signaling in mice genetically deficient for CD14 or TLR4. LPS administration to nonirradiated mice enhanced the number and function of the transferred CD8+ T cells, leading to long-term cure of mice with large transplanted tumors and enhanced autoimmune vitiligo [175].

The phenotype of the generated DCs was assessed by morphologic observation and detection of specific surface markers by flow cytometry (FCM). According to the manufacturer’s protocol, CD4+CD25− and CD4+CD25+ cell populations were separated from purified CD4+T cells using a mouse Treg isolation kit (Miltenyi Biotec, Auburn, CA, USA). As determined by FCM, the CD4+CD25+ populations were >95% pure, and the CD4+CD25− populations were 98% pure. Antigen presenting cells (APCs) used for T-cell proliferation

in vitro were obtained from pan-T-cell-depleted splenocytes of untreated, age-matched female BALB/c mice and treated with 25 μg/mL mitomycin C (Sigma) for 30 min in 5% CO2 at 37°C (22). For suppression assays, 1 × 105 CD4+CD25− T cells/well, 5 × 104 CD4+CD25+ T cells/well or both populations were cultured in 96-well U-bottom plates with Ivacaftor 1 × 105 APCs/well in triplicate for 72 h at 37°C in complete RPMI-1640 medium (0·2 mL/well). Cells in culture were stimulated with 1 μg/mL soluble anti-CD3 (BD PharMingen, San Diego, CA, USA) in the presence or absence of 0·5 μg/mL rSj16 or 0·5 μg/mL OVA (Sigma). Proliferation was determined after incubating with 0·5 μCi/well 3H-thymidine and measuring incorporation during the final 16–18 h of a 3-day culturing period. IL-10, IL-4, TGF-β and IFN-γ concentrations

in the supernatants of antigen-stimulated cells were quantified using an ELISA GDC-0941 nmr kit (Bender Med Systems, Vienna, Austria), according to the manufacturer’s protocol. Intracellular cytokines were detected by FCM as previously described (23). Briefly, 1 × 106/mL cells stimulated with PMA, ionomycin and Monensin (Sigma) in complete RPMI 1640 medium at 37°C in 5% CO2. After 4–6 h, cells were harvested and stained according to the manufacturer’s protocol. The Mouse Regulatory T Cell Staining Kit

C59 cell line (eBioscience, San Diego, CA, USA) was used for the analysis of CD4+CD25+Foxp3+ T-cell induction. Pooled splenic and lymph node cells from immunized mice or from cocultures were surface-stained with FITC anti-CD4 monoclonal (mAb) and APC anti-CD25 mAb. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm and then stained intracellularly with PE anti-Foxp3 mAb or PE IgG2a rat immunoglobulin (Ig) control antibody (Ab), according to the manufacturer’s protocol. Surface markers expressed by DCs were determined by FCM using the following mAbs: FITC anti-CD80 mAb, PE anti-CD86 mAb, PE anti-CD40 mAb and FITC anti-MHC II mAb (eBioscience). Cell staining was performed according to the manufacturer’s protocol. One-way anova and two-tailed Student’s t-tests were used in our statistical analysis; SNK method was used for multiple comparisons. A P-value <0·05 was considered statistically significant.

(Cary, NC, USA). Differences between the two infection subgroups (suspected

and documented sepsis) and the control group for each parameter at the first and second study Poziotinib research buy periods were evaluated using the one-way anova test followed by the Fisher’s PLSD test, which was also used to estimate differences within the groups at the three study periods. Differences were considered significant at P < 0.05. Diagnostic accuracy of the inflammatory mediators was evaluated by estimating the sensitivity, specificity and the positive and negative predictive value at the first day of suspicion of infection, calculated using the optimal cut-off value. To quantify the overall ability of any study index to discriminate between neonates with infection and those without, infection

receiver operation curves (ROC) were constructed and the area under the ROC was calculated. This allowed comparison of the infection indices independently AZD3965 of the selected cut-off point for each of them. In the 25 neonates with a positive blood culture, the following organisms were isolated: Escherichia coli, (7 cases), Klebsiella (3), Staphylococcus aureus (2), Streptococcus group B (2), Corynobacter (1), Listeria (1), Streptococcus viridans (1), Staphylococcus coagulase negative (8). In 13 cases, the sepsis was classified as early (less than third day of life) and in other 12 cases as late (greater than third day). Comparisons between the three groups were made only for the first two study periods as the age of the neonates at the third study period was different (mean age 14, 7, 28 days for the sepsis, suspected infection and control groups, respectively). Table 1 shows the measurements of the parameters examined in the three study groups. At the first study period, CRP, IL1-b, IL6 and TNF-α were higher in the sepsis group than in the other two groups. At the second study period, IL1-b, IL-6 and

TNF-α had decreased in the sepsis group, but remained higher in the other two groups. IgM was Florfenicol higher in the sepsis group at the second study period, and IgG was lower in the sepsis and the suspected infection groups at both first and second study periods than in the control group. NK cells and B cells were higher in the sepsis group and in the suspected infection group at the first and second study periods while the CD3+/CD4+, CD3+/CD8+ and CD4+/CD8+ ratios did not differ between the groups at any study period (Table 2). The WBC count, differential count and platelet count in the three groups were similar at all three study periods. In the control group of healthy full-term neonates with no signs of infection, the cytokine levels were low and remained unchanged during the first month of life while the levels of IgA and IgM increased and IgG decreased (Table 1).

Furthermore, the association between SIRT1 and cortactin, an actin-binding protein, was investigated by immunostaining, WB, or immunopreciptation in vivo and in vitro. Results: Seven days after glomerular disease induction, u-alb/cre, BUN and the ratio of glomerular injury in SIRT1pod−/− mice were

significantly higher than those in wild-type mice. Consistently, significant decrease in podocyte-specific molecules was demonstrated in SIRT1pod−/− mice. Electron microscopy revealed the exacerbation of foot process effacement and actin cytoskeleton derangement this website in SIRT1pod−/− mice. Similarly, actin cytoskeleton derangement in H2O2 (as a mimic of anti-GBM antibody)-treated Angiogenesis inhibitor cultured podocytes became prominent when the cells were pretreated with SIRT1 inhibitors, while it was ameliorated by a SIRT1 activator. Furthermore, we assessed the link between SIRT1 and cortactin, which acts to polymerize and maintain actin cytoskeleton. While the cytoplasmic cortactin was colocalized with actin fiber, it was dissociated in association with cytoskeleton derangement. Importantly,

the increased actin cytoskeleton derangement by SIRT1 inhibition was correlated with an increase in the level of acetylated cortactin, which was detectable only in nucleus and co-precipitated with SIRT1. These results showed that SIRT1 deacetylated pentoxifylline cortactin in the nucleus and that the deacetylated

cortactin was transported to the cytoplasm for maintenance of actin cytoskeleton. Conclusion: SIRT1 regulates the functional state of cortactin by deacetylation, and thereby maintains actin cytoskeleton integrity, indicating that SIRT1 is a critical factor for podocyte homeostasis, especially structure of slit diaphragm. TANAKA ERIKO1,2, ASANUMA KATSUHIKO1,3, TAKAGI MASATOSHI2, KOYANAGI AKEMI4, MIZUTANI SHUKI2, YAGITA HIDEO5, TOMINO YASUHIKO1 1Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine; 2Department of Pediatrics and Developmental Biology, Graduate School of Medicine, Tokyo Medical and Dental University; 3Medical Innovation Center, Laboratory for Kidney Research(TMK project), Kyoto University Graduate School of Medicine; 4Division of Cell Biology, Biomedical Reseach Center, Juntendo University Graduate School of Medicine; 5Department of Immunology, Juntendo University School of Medicine Background: Notch signaling pathway is an evolutionarily conserved intracellular signaling pathway that regulates cell fate. Activation of Notch1 and Notch2 has been recently implicated in human glomerular diseases and Notch1 reactivation is reported to correlates with glomerulosclerosis. However, the role of Notch2 reactivation remains unclear.

Little is known about the role of the NF-κB family member c-Rel in the development and function of TH17 and Treg. In this study, we show that while conversion of naive CD4+ T cells into both iTreg and nTreg requires c-Rel, this transcription

factor is not required for differentiation of TH17 cells. While our manuscript was prepared, Gerondakis and colleagues have shown that c-Rel is essential for the development of CD4+Foxp3+ T cells in the thymus and peripheral lymphoid organs 31. These authors also demonstrated that despite their lower frequency, c-Rel-deficient see more Treg suppressed effector T-cell function at normal levels. We here confirm reduced frequencies of CD4+Foxp3+ T cells in thymus, spleen and LN of c-Rel-deficient mice. In addition, we mechanistically extend this novel finding by examining the effect of c-Rel deficiency on differentiation of iTreg in vitro and show that c-Rel directly mediates upregulation of IL-2 production which is a prerequisite for iTreg development. WT C57BL/6 mice were purchased from Jackson Laboratory

(Bar Harbor, USA). c-Rel−/− mice were bred at the animal facility of the Biomedical Research Fulvestrant mouse Center, University of Marburg (Marburg, Germany). CD4+ and naive CD4+CD62L+ TH were purified from WT and c-Rel−/− mice by disrupting spleens and LN of 8- to 12-wk-old mice. All cells were cultured in Clicks medium supplemented with 10% fetal bovine serum, 2 mM glutamine and 2 μM β-mercaptoethanol. CD4+ and naive CD4+CD62L+ T cells were enriched by magnetic cell sorting with a Mouse CD4+ Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated naive CD4+ T cells (purity routinely >95%) were activated by plate-bound

anti-CD3 (5 μg/mL; 145-2C11) and soluble anti-CD28 (1.5 μg/mL; 37.51) for 3 days (unless stated otherwise) and cultured either under neutral “TH0” conditions: with anti-IL-4 (10% culture supernatant of clone 11B11), anti-IFN-γ Thymidylate synthase (5 μg/mL, XMG1-2) in the presence of recombinant human IL-2 (50 U/mL, Novartis (Nürnberg, Germany)); under TH17 culture conditions: recombinant human TGF-β1(ng/mL, R&D Systems (Wiesbaden-Nordenstadt, Germany)), recombinant murine IL6 (10 ng/mL, Peprotech (Hamburg, Germany)), anti-IL-4, and anti-IFN-γ; under iTreg conditions: TGF-β1(2 ng/mL, R&D Systems), anti-IL-4, and anti-IFN-γ. Where indicated, human IL-2 (50 U/mL, Novartis) or anti-murine IL-2 (50 μg/mL, S4B6.1) was added to the cell culture. After 3 days in culture, the T cells were washed and restimulated with PMA (50 ng/mL, Sigma (München, Germany)) and ionomycin (750 ng/mL, Sigma (München, Germany)) in the presence of brefeldin A (10 μg/mL, Sigma) for 4 h. Stimulation was terminated by fixing cells with paraformaldehyde.