IL-27 levels in astrocytes co-cultured with EAE lymphocytes were increased significantly compared to levels produced following culture with selleck screening library lymphocytes isolated from CFA-treated mice or by astrocytes cultured alone (P < 0·05). IFN-γ treated astrocytes showed no significant differences in IL-27 secretion regardless of whether they were cultured alone or in the presence of other cells (Fig. 2a,b). Production of IFN-γ, IL-17, IL-4 and TGF-β were detected in the supernatants

of astrocyte and lymphocyte co-cultures by ELISA (Fig. 1c,d). High levels of astrocyte-derived IL-27 were observed when co-cultured with EAE lymphocytes (Fig. 2a,b). Therefore, we examined what effect of neutralization of IL-27 would have on lymphocyte cytokine production by administration of anti-IL-27 neutralizing antibodies to astrocytes. Lymphocytes from EAE mice were restimulated with astrocytes for 72 h in the absence (astrocytes + anti-IL-27) or presence (astrocytes + goat IgG) of IL-27. Lymphocytes restimulated with astrocytes in the presence of IL-27 neutralizing antibodies expressed significantly elevated IFN-γ (P < 0·001), IL-4 (P < 0·01) and TGF-β (P < 0·001) expression levels compared to lymphocytes restimulated with astrocytes plus control antibody (Fig. 2c). Mice were killed during the course of the different EAE development phases. Spinal cords and

draining lymph node MNCs were harvested and the production of IL-27 and IFN-γ were evaluated by real-time PCR. Production of IL-27 p28 and IL-27 Kinase Inhibitor Library EBI3 were increased significantly in spinal cords at 7 dpi compared to levels observed in spinal cords at 16 and 28 dpi (P < 0·001). IL-27 p28 and IL-27 EBI3 levels in lymph nodes were almost undetectable (Fig. 3a,b). IFN-γ production in spinal cords peaked at 16 dpi relative to other time-points examined (P < 0·001). In the lymph nodes, IFN-γ production peaked at the beginning of disease (P < 0·001), decreased during the peak phase of EAE and was increased slightly during the remission phase (Fig. 3c). Astrocytes in culture were exposed to different concentrations of IFN-γ (ranging from 0 to 200 U/ml)

for 24 h. Total RNA was extracted Sodium butyrate and MHC-II mRNA expression was detected by RT–PCR and real-time PCR. MHC-II expression levels were elevated after stimulation with 100 U/ml IFN-γ, compared to levels observed following culture with either 0 or 50 U/ml IFN-γ (P < 0·001). However, culture in the presence of 200 U/ml IFN-γ down-regulated MHC-II expression levels slightly compared to levels observed following culture with 100 U/ml IFN-γ (Fig. 3d,e). The local microenvironment played a critical role in the development of immune responses [16]. CNS antigen presentation is also necessary for pathogenic lymphocytes reactivation and disease progression [41], so we characterized MHC-II expression levels in the spinal cord. mRNA levels were measured by RT–PCR and real-time PCR (Fig. 4).

The former group showed the same symptoms of septicaemia as pigs infected with Salmonella alone, whereas the latter thrived without any visible symptoms of enteritis or systemic disease. PR4 counts were lower in the colon (P < 0·001) of di-associated pigs (Fig. 1a). The differences between EcN counts in the gut of mono-associated (EcN) and di-associated pigs (EcN+LT2) were not significant (Fig. 1b). Both EcN as

well as PR4 reduced Salmonella counts in the ileum (P < 0·01), and also PR4 in the colon (P < 0·05). S. Typhimurium bacteria were present in blood and all organs examined from animals infected with LT2 (Fig. 2). In contrast, neither PR4 nor EcN bacteria were found in blood find more 24 h after oral administration. EcN also interfered with translocation of S. Typhimurium into mesenteric lymph nodes (P < 0·01) (Fig. 2): S. Typhimurium was absent in blood, liver and lungs of EcN-di-associated

pigs. In contrast, all PR4-di-associated pigs suffered from septicaemia. The concentrations of IL-8, TNF-α and IL-10 click here were measured in plasma, ileum and colon lavages of germ-free pigs, gnotobiotic pigs mono-associated with LT2 strain of S. Typhimurium, gnotobiotic pigs di-associated with EcN and LT2 and gnotobiotic pigs di-associated with PR4 and LT2. No inflammatory cytokines were found in samples from germ-free pigs (Fig. 3a–c). Plasma cytokines.  IL-8 was not found in any plasma sample (Fig. 3a). LT2 induced significant IL-10 and TNF-α responses in circulation. There was no significant difference between the levels of both cytokines in plasma samples from pigs infected with LT2 alone and those from pigs associated with PR4 and LT2. Bacteraemia in piglets infected with Salmonella (Fig. 2) was

correlated highly with plasma IL-10 (r = 0·909, Fig. 4a) and TNF-α (r = 0·769, Fig. 4b) levels. A marked decrease was observed Rapamycin in pigs di-associated with EcN and LT2 compared to LT2 alone: IL-10 was absent in their plasma and TNF-α levels were significantly lower (Fig. 3a). Ileum cytokines.  IL-8 was present in all samples infected with Salmonella, but there were no significant differences between the groups (Fig. 3b). IL-10 was not found at all. TNF-α levels were lower (P < 0·01) in pigs di-associated with EcN and LT2 than in the pigs infected with LT2 alone. In contrast, TNF-α levels in the ileum of pigs associated with PR4 and LT2 were similar to these in the pigs infected with S. Typhimurium alone. Colon cytokines.  IL-8 was detected in all samples infected with Salmonella while IL-10 was not found in any sample, as in the ileum (Fig. 3c). The pre-association of pigs with commensal bacteria decreased dramatically (P < 0·01) the levels of IL-8 in Salmonella-infected pigs.

A specific point mutation p.Arg246Gln in LMXB1 has recently been reported in a family with isolated FSGS, and no ultrastructural abnormalities of the GBM or extrarenal manifestations. Case Report: We report the same LMXB1 mutation in a family with two affected members. The index case is a twelve year old boy, who presented with acute appendicitis and was noted to have mild lower limb oedema, significant proteinuria (5.93 g/L), hypoalbuminemia (albumin

29 g/L) and normal renal function. Additional investigations for the cause www.selleckchem.com/products/NVP-AUY922.html of proteinuria were negative. Renal biopsy showed variable glomerular basement membrane (GBM) thickening

and electron microscopic findings of a focally wrinkled GBM, and scattered aggregates of collagen fibrils and STI571 solubility dmso small cellular blebs. The patient’s mother had a history of childhood failure to thrive and nephrotic syndrome and had progressed to end stage renal failure. She had undergone a deceased donor renal transplant which failed secondary to recurrent FSGS. Mutation testing for NPHS1 and NPHS2 were negative. Whole exome sequencing was undertaken at the Beijing Genomics Institute and identified a heterozygous mutation of LMX1B (NM_001174146:c. 737 G>A:p.Arg246Gln). Conclusions: Whole exome sequencing of patients with genetic disease of unknown aetiology is allowing for rapid genetic diagnoses and should be considered in steroid resistant patients with nephrotic syndrome.

This patient adds to the genotype/phenotype variability associated with LMXB1. 196 EVALUATION OF VALIDITY OF DATA COLLECTION IN ANZDATA N AUNG, S MAY Tamworth Base Hospital, New South Wales, Australia Aim: To evaluate the validity of pathology data collected for ANZDATA using one result (December) from a 12 months period of data collection. Background: Each year, ANZDATA surveys are sent out to participating renal units across Australia for collection of pathology data at one time point only. Methods: We randomly select 20 patients from our renal unit and compared their range of monthly phosphate, hemoglobin Carbohydrate and ferritin level over 12 months with the data entered for ANZDATA. Results: The finding shows significant differences in all 3 parameters we conducted. With phosphate level, maximal individual difference between data range and data entry is 2.04 mmol/L (70%); the difference from mean is 0.628 mmol/L (24%) and median is 1.255 mmol/L (59%). With hemoglobin level, maximal individual difference between data range and data entry is 63 g/dL (41%); the difference from mean is 18.42 g/dL (14%) and median is 19.5 g/dL (15%).

7 Consequently, PTH and FGF-23 maintain normal calcium and phosphate levels in early stages of CKD,8 but progressive renal damage results in hyperphosphataemia, increasing Small molecule library FGF-23 levels (up to 1000 times the normal range) and the development of secondary hyperparathyroidism (SHPT) in many patients.9 Current management of disordered mineral homeostasis in CKD involves the control of hyperphosphataemia

by dietary modification or phosphate binders and the use of calcium, calciferol or active vitamin D compounds to maintain normal PTH levels in CKD stages 1–5. Calcimimetic agents may be added when patients are dialysis dependent and if PTH levels are high or patients have hypercalcaemia thought because of SHPT. Unfortunately, difficulties with phosphate control increase when patients reach CKD stage 5, or patients commence dialysis, and despite dietary restriction and phosphate binder therapy, patients often have poor phosphate control unless they advance to longer dialysis sessions. Patients with CKD have

an excessive burden of CVD and related mortality.10,11 Age-standardised rates of all-cause mortality and cardiovascular (CV) events are 5–20 times higher in people with CKD as compared with those with normal kidney function12 and a collaborative meta-analysis of general population PR-171 research buy cohorts, consisting of more than 1.2 million people, showed that an estimated glomerular filtration rate (eGFR) of <60 mL/min per 1.73 m2 was an independent predictor of all-cause and CV mortality.13 The risk of CV morbidity and mortality progressively worsen with decline in eGFR.

Traditional CVD risk factors (hypertension, older age, hyperlipidaemia and diabetes) are highly prevalent in patients with CKD although they do not explain the heightened CV risk in stages 4–5D. For these patients, ‘non-traditional’ factors, particularly relating to abnormal PtdIns(3,4)P2 mineral metabolism, are associated with the increased risk of CVD (Fig. 1).14,15 Recognizing the intimate associations between CVD and abnormalities of bone and mineral metabolism, the term ‘chronic kidney disease-mineral and bone disorder’ (CKD-MBD) was applied, encompassing the disturbances of mineral metabolism, renal bone disease and vascular calcification, together with patient-level outcomes of fracture, CVD and mortality in patients with CKD.16 Hyperphosphataemia, a key component of CKD-MBD, is strongly associated with adverse outcomes in CKD patients, including CVD, vascular calcification and increased arterial stiffness (Table 1).29,30 The relationship between phosphate and CVD may be explained by several putative mechanisms.31–34 The most plausible mechanism concerns the accelerated progression of vascular calcification, which is conceptually linked to the positive phosphate balance seen in CKD (as well as excessive doses of calcium-based phosphate binders).

RNA samples were resuspended in diethylpyrocarbonate-treated water and stored at −70 °C. The RNA concentration was determined

from the optical density using a micro-volume spectrophotometer (Nanodrop 1000, Nanodrop Technologies LLC, Wilmington, NC, USA). Real-time PCR reactions.  Reverse transcription total RNA was DNase treated (Turbo DNA-frees, Ambion Inc., Austin, TX, USA), and 1 μg was used for cDNA synthesis. The reaction was carried out using the First-Strand cDNA synthesis kit (Fermentas, Glen Burnie, MD, USA), following the manufacturer’s recommendations. Primer design.  Primers were designed using the Primer buy FK228 Express 3.0 probe design software (Applied Biosystem, Foster City, CA, USA). The primer sequences are presented in Table 1. PCR Reactions.  Quantitative real-time polymerase chain reaction (qPCR) was performed in the 7300 Real Time PCR (Applied Biosystem) using the SYBR Green PCR Master Mix (Fermentas). The reaction product was quantified with the Relative Quantification tool, using GAPDH as the reference

gene. Negative controls with SYBR Green PCR Master Mix and water were performed for all reactions. Statistical analysis.  The statistical analysis was performed using a software program (GraphPad Prism 4.0, La Jolla, CA, USA). Data were first examined for normality by the Kolmogorov-Smirnov DMXAA purchase test and, since the data achieved normality, parametric method was employed. The percentages of sites with visible plaque accumulation, BoP, SUP, the means PD, CAL were

computed for all teeth. Clinical parameters, mRNA data, the levels of cytokines and IgA were averaged into both groups. The differences in clinical parameters, age, mRNA levels, IgA, and cytokines levels between groups were compared using Student’s t-test. The level of significance was set at 5%. Table 2 summarizes the demographic characteristics and the clinical parameters of the study population. There (-)-p-Bromotetramisole Oxalate were no differences in the mean age and gender distribution between groups (p > 0.05). As expected, the levels of all periodontal parameters were lower in the control group when compared to chronic periodontitis group considering full-mouth and the teeth selected for gingival biopsies levels (P < 0.05). Salivary levels of antibody were normalized by comparing the IgA antibody in ELISA to the total protein (Bradford method) found in the saliva. The mean level of total protein found in the saliva of the periodontal disease individuals was 1471.60 ± 438.09 μg/ml, and from healthy individuals was 1056.79 ± 381.13 μg/ml. The normalized mean levels of IgA (pg/ml) in total saliva are presented in Figure 1. The total IgA antibody levels were significantly higher in the chronic periodontitis group compared to periodontally healthy ones (P < 0.05). As observed in Fig. 2A, the gingival mRNA levels for IL-21 was significantly higher (P < 0.05) in the chronic periodontitis group when compared to the healthy group.

Candida albicans was the predominant yeast isolated [30 patients (62.5%)], followed by C. parapsilosis [6 (12.5%)] and C. dubliniensis 5 (10.4%). Aspergillus fumigatus was the most common filamentous fungus [5 (10.4%)] and non-fumigatus Aspergillus species were isolated from four (8.3%) patients. Staphylococcus aureus was the most frequently detected bacterium in C. Obeticholic Acid ic50 albicans

positive samples (53.57%). A. fumigatus and Pseudomonas aeruginosa or S. aureus were detected together in 75% of A. fumigatus positive samples each. No statistically significant relationship was detected between growth of yeast and moulds and age, gender, the use of inhaled corticosteroids or tobramycin. No significant correlation was found between the isolation of C. albicans, A. fumigatus and P. aeruginosa, Stenotrophomonas maltophilia BGB324 concentration or S. aureus, and the isolation of C. albicans and Haemophilus influenzae. Other factors which may be responsible for the increased isolation of fungi in CF need to be investigated. “
“Patients with acute myelogenous leukaemia (AML) and neutropenia after chemotherapy are at high risk for life-threatening invasive fungal disease (IFD), in particular, invasive aspergillosis (IA). The aim of the study was to evaluate data on characteristics, risk factors, complications and additional

antifungal treatment of patients with AML receiving posaconazole prophylaxis (PP) after chemotherapy in an actual clinical setting. A retrospective single-centre observational study on 40 patients with AML, median age 66 years, was conducted. PP 200 mg three times daily was given routinely. After 76 cycles of remission induction chemotherapy followed by PP, median duration of 31 days (range 6–61 days), no fatal case occurred. Acyl CoA dehydrogenase The majority of patients had at least one additional risk factor for IFD and during 32 cycles (42.1%), three risk factors were present. During 40 therapy cycles (52.6%), fever of unknown origin occurred. Pneumonia was diagnosed after 23 cycles

(30.3%), thereof one case of proven IA (1.3%). PP was interrupted in 25 cycles (32.9%) and was followed by systemic antifungal therapy with different agents, with a median duration 15 days (range: 6–32 days). PP appears to be an effective and well-tolerated protection against IFD for AML patients under natural clinical conditions. “
“Data on the epidemiology of invasive Candida infections in paediatric patients in Europe are still limited. The aim of this retrospective study was to analyse the epidemiology of candidaemia in a tertiary paediatric hospital in Poland from 2000 to 2010. Using microbiological records, a total of 118 episodes of candidaemia were identified in 114 children, with an annual incidence of 0.35 episodes/1000 discharges. The highest incidences were found in the medical intensive care unit (5.28), and in neonatal intensive care (1.47). The mortality rate was 8.5%. Candida albicans and C. parapsilosis were the most prevalent species (39.8% and 35.6% respectively).

IL-10 increases host susceptibility to extracellular bacteria such as Streptococcus pneumoniae, Klebsiella pneumoniae and Pseudomonas aeruginosa in models of primary infections. In addition, IL-10 has a complementary role to IL-4, another macrophage-deactivating cytokine, in the increased susceptibility of mice to murine leishmaniasis [53, 54]. Based on the above, the low level of IL-10 in the mice immunized with pBKTcSPR could improve resistance to infection with T. cruzi. Furthermore,

compared with pBKTcSP and pBKTcSPA, pBKTcSPR does not induce selleck chemical IL-2 and IL-5 and promoted lower concentrations of IL-6. Although we do not know exactly why mice immunized with the recombinant protein die after infection with T. cruzi, high serum levels of IL-10 before infection could be considered to be the reason, along with the mixed Th1/Th2 T-cell induced immune response. To better understand the antigen-specific cellular immune responses induced by immunization with the recombinant proteins

and naked DNA before parasite challenge, we are currently conducting experiments to investigate the cytokine production by splenocytes harvested from immunized animals. It is also necessary to implement experiments using anti-IL-10 mAbs or IL-10 KO mice to determine the role that IL-10 plays in the protection-death of vaccinated mice. Some determining factors that favour Th1 vs. Th2 T-cell immune response BAY 80-6946 in pathogen infections have been proposed, including the nature of the antigen (intracellular antigens favour Th1 and extracellular antigens favour Th2) and the concentration of antigen (low concentrations favour Th1 and high concentrations favour Th2) [55]. Another factor that affects the immune response is the use of adjuvant; in the present work, we use Freud’s adjuvant for protein immunization, which has the ability to elicit both Th1 and Th2 T-cell immune response. Incomplete Freund’s adjuvant (IFA) was used in human trials; however, it was discontinued as a vaccine adjuvant in humans due to several safety concerns that were determined in animals [56]. Based on this, we are conducting experiments in mice using adjuvants that have been developed

for human use. In these protection isothipendyl assays, low concentrations of recombinant proteins of TcSP domains are being used to study whether they are able to protect against T. cruzi infection. Alum, CpG and liposomes were selected because they are able stimulate the production of antibodies and cytokines but differ in their mechanism of action: alum acts through APC death, CpG acts through TLR9, and liposomes act through antigen delivery [56]. None of the mice immunized with PBS/adjuvant survived; however, 50% of those immunized with empty plasmid did survive – despite parasitemia being similar (97 × 104 vs. 91 × 104). The survival may be due to the response induced by immunostimulatory sequences in the plasmid that trigger innate immunity in the host [57].

More importantly, DN T cells may prevent GVHD in hematopoietic stem cell transplantation patients [[19]]. CD4+ and CD8+ T cells play central roles for rejection of MHC-mismatched allografts. However, the innate immune response, including NK cells and macrophages together with the cytokines and chemokines that

they produce, also participates in graft rejection [[20-23]]. In our recent study, we found that donor-derived DN Treg cells can suppress NK cell-mediated allogeneic BM graft rejection in an irradiated condition [[24]]. In this study, we determined if we could develop a strategy by administering DN Treg cells with optimal immune suppressive treatment to help establish-mixed chimerism in an irradiation-free nonmyeloablative condition. Our results Selleck PI3K inhibitor indicated that adoptive transfer of DN Treg cells could induce nonmyeloablative BM chimerism by inducing T-cell clonal deletion and suppressing NK-cell function. To Decitabine research buy develop a suitable clinical method, we tried to establish mixed chimerism with an irradiation-free protocol by transferring DN Treg cells and using clinically available immune suppressive drugs. Cyclophosphamide (CY), cyclosporine A (CyA), FK506, and rapamycin (RAPA) were tested in this study. Recipient BALB/c mice were treated with the immunosuppressive agents before and after BM transplantation. CY: 200 mg/kg on day 0 and 100 mg/kg on day 3; CyA:

15 mg/kg from day 0 to 9; FK506: 16 mg/kg from day 0 to 9; RAPA: 2 mg/kg from day 0 to 9; phosphate-buffered saline (PBS): 0.3 mL/mouse from day 0 to

9. DN Treg cells were purified from C57BL/6 mice and were activated by plate-coated anti-CD3 in presence of IL-2. P-type ATPase The purity was confirmed by anti-CD3, CD4, CD8, TCRγδ, and NK1.1 (Fig. 1A). DN Treg cells (4 × 106 /mouse) were intravenously (i.v.) injected to BALB/c mice on day 0. 30 × 106 C57BL/6 BM cells were depleted of CD4+ and CD8+ T cells before being injected to BALB/c mice on day 6. Busulfan (30 mg/kg) was given to all mice 1 day before BM transplantation to enhance efficiency of BM engraftment [[25-27]]. Peripheral blood was collected 60 days after to detect donor-derived lymphocytes by staining with antidonor MHC H-2b antibody. As shown in Fig. 1B, donor-derived cells were found in the CY-treated group in combination with DN Treg cells treatment (mean ± SD = 41 ± 19%, p < 0.01), and barely detectable in CyA, FK506, and RAPA-treated groups, as well as in CY alone or DN Treg-cell alone treated groups (Fig. 1B). Expression of donor and recipient MHC class I antigens were determined using antidonor H-2b antibody in combination with staining cells for CD3+ and CD19+ expression. As shown in Fig. 1C, 34 ± 17% (mean ± SD) donor-derived H-2b+CD19+ B cells and 19 ± 10% donor-derived H-2b+CD3+ T cells were identified in spleens of chimeric mice after 100 days, indicating multilineage and stable-mixed chimerism. Next, we studied whether mixed chimerism would lead to graft tolerance.

3A). Five chemokines, CCL2, CCL7, CCL8, CXCL9 and CXCL10, were present at high levels in the supernatants of co-culture spheroids, but almost absent or significantly lower in the supernatants of both tumour spheroids and monocyte cultures, Daporinad mouse indicating that co-culturing with tumour cells stimulated the production of these chemokines by the TAMs. When the supernatants of co-culture spheroids of other cancers (prostate, ovarian and breast) were assessed, CCL2, CCL7, CXCL9 and CXCL10 were present at significantly lower levels compared with that of colorectal

cancer (Supporting Information Fig. 5). This implies that TAMs in colorectal cancers secrete more chemokines to attract T cells than TAMs in other cancers Selleck SRT1720 in which TAMs promote tumour growth. To ascertain that the chemokines present in the supernatants of the colorectal tumour

model were functionally capable of attracting T cells, we performed Transwell assays using two supernatants: supernatants from co-culture spheroids to mimic microenvironment of a tumour with macrophage infiltration, and supernatants from tumour spheroids to mimic microenvironment of a tumour without macrophage infiltration. Indeed, the supernatants of co-culture spheroids attracted significantly more of both CD4+ and CD8+ T cells than the supernatants of tumour spheroids (Fig. 3C), showing that the chemokines in the supernatants of co-culture spheroids were functionally able to attract T cells. As the TAM genes indicated that the

TAMs were involved in antigen presentation (Fig. 3A), and chemokines that attract T cells were present in the co-culture supernatants, we assessed colorectal TAMs for the expression of cell surface molecules involved in interaction with T cells. The TAMs expressed molecules for antigen presentation (HLA-DR, CD74), T-cell co-stimulation (CD40, CD80, CD86) and CD54 (or ICAM-1), an adhesion molecule that stabilises cell contact during T-cell co-stimulation (Fig. 4A, top panel) 15. To obtain an idea of the level of expression of these molecules on colorectal TAMs, we compared them with in vitro differentiated macrophages and freshly isolated monocytes. The median fluorescence intensity (MFI) of the expression of the molecules (Fig. 4A, middle panel) as well as the percentage Vitamin B12 of cells that expressed the molecules (Fig. 4A, bottom panel) were studied. Colorectal TAMs exhibited higher expression of all the molecules compared with in vitro MCSF-differentiated macrophages, and up-regulated the expression of all molecules except CD74 compared with freshly isolated monocytes. In addition, a significantly larger percentage of TAMs (than macrophages or monocytes) expressed CD74, CD40, CD80 and CD86. This observation indicated that co-culturing with colorectal tumour cells promoted the differentiation of monocytes to TAMs with enhanced expression of antigen presentation and T-cell co-stimulation molecules.

Since influenza infections are characterized by acute onset and lack a chronic phase 12, our data reveal that virus-specific Treg are also induced by viruses that are cleared by the immune system. These influenza-specific Treg may come in two flavors, Foxp3+ and Foxp3−

and are readily isolated from the population of IL-10-producing influenza-specific T cells. It is envisaged that these influenza-specific Treg are induced PD98059 manufacturer to prevent immunopathology, which may occur otherwise as a result of an uncontrolled anti-viral immune response during viral clearance. Anonymous healthy blood bank donors participated in this study after written informed consent. PBMC were prepared by Ficoll-amidotrizoate density gradient Compound Library manufacturer centrifugation. Peptides spanning the whole M1 protein consisted of 16 peptides with a length of 30 amino acids, and an overlap

of 15 amino acids (C-terminal peptide with an overlap of 18 aa), the sequence was derived from influenza A/PR/8/34. Recombinant M1 and HPV16 E6 protein (the latter served as control protein) were produced in E. coli as described previously 17. Live influenza A/Wisconsin/67/2005 was kindly provided by W.M. Liu (NVI, Bilthoven, The Netherlands). Fluorescent-labelled antibodies used were CD4-PE (Clone SK3), CD4-APC (Clone RPA-T4), CD8-PERCP-CY5.5 (Clone SK1), CD25-APC (Clone M-A251), CD69-FITC (Clone L78), CD137-APC (Clone 4B4-1) and IL-2-PE

(Clone MQ1-17H12) and were obtained from Becton Dickinson (USA). FOXP3 was stained using the FOXP3-PE staining kit (clone PCH101) according to manufacturer Adenosine triphosphate protocol (eBiosciences, USA). The previously described clones C148.31 and C271.9 were used as reference to determine the cut-off level 1, 5. Samples were analyzed by flow cytometry using FACS Calibur (Becton Dickinson) and data was analyzed using Cell Quest pro (Becton Dickinson) and FlowJo software (Tree Star). To generate M1-specific T-cell lines PBMC were cultured in IMDM (BioWhittaker, Belgium) supplemented with 10% human AB serum (PAA laboratories, Austria) and 10% T-cell growth factor (TCGF, Zeptometrix, USA) and were stimulated with 5 μg/mL peptide pools containing the first eight or the last eight overlapping peptides. After 2 wk of culture the reactivity against M1 peptides and recombinant protein was assessed. Positive cultures were stimulated for 4 h with pooled M1 peptide-loaded autologous monocytes and were subsequently enriched for IL-10-producing cells according to manufacturer protocol (IL-10 secretion assay; Miltenyi Biotech, Germany). Directly after enrichment T-cell clones were isolated by limiting dilution as described before 38. After limiting dilution, T-cell clones were tested for their specificity and maintained in IMDM supplemented with 10% FBS and 10% TCGF.