When the animals were deeply anaesthetized blood was obtained by cardiac puncture of the right ventricle. Bronchoalveolar lavage (BAL) was performed by instilling 0·25 ml PBS through the tracheal cannula, followed by gentle aspiration and repeated with 0·2 ml PBS. Finally, one femur was cut at the epiphysis and the BM cells were flushed with 2 ml PBS. Bronchoalveolar lavage fluid and bone marrow.  Samples of BALF and BM were centrifuged at 300 g for 10 min at 4°. The BAL supernatant

was saved for eotaxin-2 measurement and stored at − 80° until analysis. The cells were resuspended with 0·03% BSA in PBS. The total cell numbers in BAL and BM were determined using standard haematological procedures. Cytospins GSK-3 beta pathway of BAL and BM were prepared and stained with May–Grünwald–Giemsa for differential cell counts by counting 300–500 cells using a light microscope (Zeiss Axioplan 2; Carl Zeiss, Jena, Germany). The cells were identified using standard morphological criteria, and BM mature and immature eosinophils were determined by nuclear morphology, check details cell size and cytoplasmic granulation.23 Lung tissue cells.  The pulmonary circulation was perfused with ice-cold PBS and lungs were removed from the thoracic cavity. The lung tissue was thinly sliced and suspended

RPMI-1640 (Sigma-Aldrich) complemented with 10% fetal calf serum (FCS), collagenase (5·25 mg/ml) and DNAse (3 mg/ml; Roche). After 90 min incubation in a shaking water bath (37°), any remaining intact tissue was disrupted by repeated passage through a wide-bore Pasteur pipette and filtered through a 40-μm nylon mesh (BD Biosciences, Erembodegem, Belgium). The parenchyma lung cells were diluted in Percoll (density 1·03 g/ml; Amersham Bioscience, Uppsala, Sweden) and layered on a discontinuous gradient,

centrifuged at 400 g for 20 min. The cells in the top layer, mainly macrophages, dead cells and debris, were discarded. Cells at the Percoll interfaces were collected and washed in PBS complemented with 10% FCS. Total cell numbers were determined using standard haematological procedures. Antibodies.  Fluorescein isothiocyanate (FITC) -labelled anti-mouse CD34 (clone RAM 34; BD Bioscience), phycoerythrin (PE) or FITC-labelled anti-mouse CCR3 (clone 83101; R&D systems, Oxymatrine Abington, UK), biotinylated anti-mouse stem cell antigen-1 (Sca-1)/Ly6 (clone 177228; R&D Systems) followed by peridinin chlorophyll protein (PerCP) -labelled streptavidin, PE-labelled anti-mouse IL-5Rα (Clone 558488; BD Bioscience), PercP-labelled anti-mouse CD45 (clone 557235; BD Bioscience), FITC-labelled BrdU (BD Bioscience) and rabbit anti-mouse major basic protein (MBP) polyclonal antibody in combination with goat anti-rabbit PE or with biotinylated swine anti-rabbit followed by streptavidin-FITC were used. Animals were sensitized and exposed to OVA or PBS as described above.

No recommendation. The imaging of kidneys prior to donor nephrectomy can be accomplished by several means, including: ultrasound (US); conventional angiography Protein Tyrosine Kinase inhibitor (CA); digital subtraction angiography (DSA); computed tomography (CT) and magnetic resonance imaging (MRI), each of which has inherent limitations, strengths and weaknesses. A single modality to assess vasculature, renal parenchyma and urinary drainage is preferred. The pre-nephrectomy anatomy which most anticipates complications during the transplant procedure

is the presence or absence of variant arteries. Numerous studies have assessed the sensitivity, specificity and accuracy of each imaging technique Ruxolitinib in relation to surgical anatomy. The objective

of this guideline is to outline the best means of assessing donor kidney anatomy prior to surgery. Databases searched: MeSH terms and text words for kidney transplantation were combined with MeSH terms and text words for angiography, X-ray computed tomography and magnetic resonance angiography. The search was carried out in Medline (1966 – September Week 1, 2006). The Cochrane Renal Group Trials Register was also searched for trials not indexed in Medline. The Register searches all major medical electronic databases, including Embase. Date of searches: 19 September 2006. Update search: Databases searched: MeSH terms and text words for kidney transplantation were combined with MeSH terms and text words for living donor and combined with MeSH terms and text words for open and laparoscopic nephrectomy. The search was carried out in Medline (1966 – March Week 1, 2009). The Cochrane Renal Group Trials Register was also searched for trials not indexed in Medline. Date of searches: 9 March 2009. Six studies published from 1978 to 2000 compared operative findings with angiographic findings.1–6

The sensitivity in detecting accessory renal arteries ranged from 67%–100% (mean 86%). This method is useful for the detection of fibromuscular dysplasia. Seven studies published from 1985 to 2006 compared operative findings with selleck screening library digital subtraction angiography (DSA) findings.7–13 The sensitivity in detecting accessory renal arteries ranged from 60%–91% (mean 81%). This method is useful for the detection of fibromuscular dysplasia. Twenty-nine studies published from 1995 to 2006 compared operative findings with CT angiographic findings.3,5,6,8,9,12–35 The sensitivity in detecting accessory renal arteries ranged from 40%-100% (mean 84%). In studies with more than 100 participants, the mean sensitivity was 86%. This technique detects early branching with a mean sensitivity of 81%, but may miss fibromuscular dysplasia (incidence uncertain). Sixteen-slice machines are considered to be superior to 4-slice machines. Tombul et al.

Overall, despite the limitations as the result of serology and the possibility of natural selection acting on this system, the analysis of the GM polymorphism has been very useful in revealing the effects of both geographic and cultural differentiations on the genetic structure of modern human populations, and has provided noteworthy examples of the usefulness of this immunogenetic complex for

anthropology. The HLA molecules are peptide-binding molecules encoded by genes in the HLA complex on chromosome 6 (see ref. 37 for a review). They are divided into two classes, class I and class II, which both present peptide fragments of antigens to T cells. Some class I molecules also interact with natural killer (NK) cells. The HLA class I molecules consist of a polymorphic α heavy chain that is non-covalently Stem Cell Compound Library manufacturer bound to a small non-polymorphic β chain (β2m, encoded by a gene on chromosome 15). The α chain includes three extracellular domains, two of which (α1 and α2) form a peptide-binding cleft. The classical HLA class I molecules encompass the A, B and C series of molecules, encoded by three different corresponding α chain loci. They are extremely polymorphic (see next section) and expressed in almost all nucleated cells. They bind short peptide fragments (8–10 amino acids long) derived selleckchem primarily from endogenous proteins and present them at the cell membrane.

Here CD8+ T cells with appropriate T-cell receptors will interact with the peptide–HLA complex. Some class I molecules also interact with NK cells. The non-classical HLA class I molecules encompass the E, F and G molecules, which are much less polymorphic and which primarily function as ligands for NK cells. Two HLA class 1 α-related chains, MICA and MICB, are polymorphic but do not have a peptide-binding cleft nor do they bind β2m. They are stress

molecules that are up-regulated under certain conditions and function as ligands for the NKG2D activating receptor on NK cells. The HLA class II molecules consist of two heavy chains, α and β, which both include two extracellular domains. Their peptide-binding cleft is formed by their α1 and β1 domains. The class II molecules encompass the DR, DQ and DP series of molecules, encoded by corresponding α and β chain loci in the HLA complex. The DRβ, DQα, DQβ, DPα and DPβ Baf-A1 chains are extremely polymorphic (see next section), whereas the DRα chain is essentially monomorphic. Four different DRβ chains are expressed; DRβ1, DRβ3, DRβ4 and DRβ5. The class II molecules are expressed in specialized antigen-presenting cells such as dendritic cells, where they pick up longer peptide fragments (8–15 amino acids long) primarily from endocytosed exogenous proteins and present them at the cell membrane. Here CD4+ T cells with appropriate T-cell receptors will interact with the peptide–HLA complex. The 4-Mb DNA region of the short arm of chromosome 6 (6p21.

1b shows H and E-stained tissue sections of NALT from normal BALB/c mice before and after teasing. NALT cells were readily isolated, Fulvestrant purchase yielding approximately 2.5 × 105 viable cells per palate. Because we had exsanguinated the mice from the inferior vena cava, we noted few erythrocytes; thus more than 96% of the cells were the following immune cells: CD3+ cells (53.5

± 3.8%; mean ± SD; n =3); CD4+ cells (38.6 ± 2.6%; mean ± SD; n =3); CD8+ cells (17.5 ± 2.5%; mean ± SD; n =3); B220+ cells (40.0 ± 3.7%; mean ± SD; n = 3); Mac-1+ cells (1.5 ± 0.4%; mean ± SD; n =3); CD11c+ cells (0.6 ± 0.0%; mean ± SD; n =3); and Ly-6G+ cells (0.3 ± 0.1%; mean ± SD; n =3). The cell yield from NALT and their phenotypic composition were essentially the same as those reported previously (17, 18), showing that they had been accurately prepared. Figure 2 shows the time-dependent Wnt antagonist changes in the total number of cells in NALT or submandibular lymph nodes of BALB/c mice after one i.n. injection of cedar pollen. The total number of NALT cells did not change significantly from days 0–14 after

i.n. injection of the allergen (Fig. 2a); and the percentages of B220+, CD3+, Mac-1+, CD11c+, and Ly-6C+ cells were also unchanged (data not shown). In contrast, the total number of submandibular lymph node cells started to increase on day 3 after i.n. injection of the allergen, reached a peak (≈ threefold that of the PBS-injected C-X-C chemokine receptor type 7 (CXCR-7) control) on day 10, and declined to the basal level by day 14 (Fig. 2b). Of particular interest, the percentage of B220+ cells on day 0 (≈ 36%) started to increase from day 3 (≈ 49%), reached a plateau on days 5–10 (54–55%), and decreased to the basal level by day 14 (≈ 42%). In contrast, those of CD3+ cells, Mac-1+, CD11c+, and Ly-6C+ cells decreased time-dependently and returned to the basal level by day 14 (data not shown), suggesting that B220+ cells (e.g., B or pre-B cells) in the submandibular lymph nodes might be the cells that respond to i.n. injections of allergen. Bulk cells from submandibular lymph

nodes from mice that had been treated once i.n. with allergen produced a significant amount of IgE Ab on day 7 (mean ± SE, 3.8 ± 1.0 ng/mL; n= 30) with a peak on day 10 (7.8 ± 1.6 ng/mL; n =30). The concentrations then decreased to the control level by day 14 (0.1 ± 0.1 ng/mL; mean ± SEM; n= 30), demonstrating time-dependent changes in the amount of IgE Ab similar to the changes in total cell numbers. In contrast, the bulk cells from the NALT from mice that had been treated once i.n. with allergen did not produce significant amounts of IgE (n =12) on days 0–14. The bulk cells of the axillary lymph nodes, Peyer’s patches, inguinal lymph nodes, and mesenteric lymph nodes produced 1.8 ± 0.3 (mean ± SEM; n =15), 1.3 ± 1.4 (mean ± SD; n =9), 0.5 ± 0.3 (mean ± SD; n =9), 0.1 ± 0.3 (mean ± SD; n =9) ng/mL IgE on day 10, respectively (data not shown).

Remarkably, the finding that PstS1 stimulates memory T cells specific for TT, suggests the potential exploitation of PstS1 immunomodulatory properties in other infections. Although effects on other APCs cannot be excluded, our study shows that the immunomodulatory properties of PstS1 are linked to its ability to activate DCs in vitro and in vivo. In particular, PstS1 promoted

the expression of IL-6, IL-1β, and, to a minor extent, IL-23. These cytokines were recently reported to drive a fine balance of CD4+ T-cell differentiation in the effector phase of the immune response to Candida albicans and Staphylococcus aureus [44]. Of interest, other cytokines pivotal for the homeostasis of memory T cells, such as IL-7

and IL-15 for CD8+ T cells [45], or IL-12p40 for Th1 Small molecule library purchase response [46], were selleck screening library not modulated by PstS1 (data not shown). The ability to stimulate DCs was peculiar to PstS1, since other immunodominant Mtb Ags such as Ag85B, Esat-6, or HBHA were unable to activate DCs (Fig. 4 and data not shown) and it was directed preferentially toward CD8α− DCs. The two major DC subsets of mouse spleen, CD8α+ and CD8α−, trigger distinct T-cell responses against pathogens. While CD8α+ DCs are thought to be specialized in antiviral response due to their unique cross-priming ability, CD8α− DCs have been involved in CD4+ T-cell immunity, particularly during bacterial infections [47]. CD8α− DCs efficiently induce CD4+ PTK6 T-cell responses through in vivo targeting of Ag via C-type lectin receptors, such as dectin-1 and DCIR-2 [30, 48]. The preferential ability of CD8α− DCs to prime CD4+ T-cell responses has been correlated with their superior capacity to process Ags via MHC class II molecules [30]. Accordingly, we report that PstS1 endowed CD8α− DCs with a strong ability to simulate CD4+ T cells. In particular, CD8α− DCs stimulated by PstS1 were found to produce much higher amounts of IL-6, IL-1β, and IL-23 with respect

to CD8α+ DCs. Moreover, PstS1-pulsed CD8α− DCs were far superior at inducing IFN-γ, IL-17, and IL-22 release by Ag85B-specific memory T cells, compared with CD8α+ DCs. The mechanisms by which PstS1 activates DCs remain to be established. Our data on DCs deficient for TLR2, the main PRR recognized by Mtb components, suggest that this receptor is dispensable. We envisage that the TLR2-independent pathway of DC maturation induced by PstS1 strongly differs from that triggered by the Mtb Ags Rv0577, Rv1196, Rv0978c, and Rv0754, which all recognize TLR2 and induce maturation of DCs leading to either Th1 or Th2 polarization, but not to IL-17 secretion by memory CD4+ T cells [14-18].

Orf2 was proposed to be formyltransferase for synthesis of dTDP-d-Qui3NFo from dTDP-d-Qui3N. Therefore, we suggested that orf3, orf4, orf5, and orf2 are involved in the synthesis of dTDP-d-Qui3NFo and named them rmlA, qdtA, qdtB, and qdtF, respectively. Orf11 shares 76% Selleckchem CHIR99021 identity or 89% similarity to UDP-glucose 6-dehydrogenase (Ugd) of Edwardsiella ictaluri, which is responsible for the synthesis of UDP-d-GlcA from UDP-d-Glc (Stevenson et al., 1996). Therefore, orf11 was proposed to be responsible for the synthesis of UDP-d-GlcA and named ugd. Both Orf13 and Orf14 belong to the NAD-dependent epimerase/dehydratase family (Pfam01370, E value = 3× e−23

and 3 × e−45, respectively). Orf13 shares 78% identity to UDP-N-acetylglucosamine 4-epimerase (Gne) of P. mirabilis. In Providencia (Ovchinnikova et al., 2012), as in most other Enterobacteriaceae members studied (Valvano, 2011), the O-unit synthesis is likely initiated LY294002 nmr by transfer of GlcNAc-1-phosphate or GalNAc-1-phosphate to the undecaprenol phosphate (UndP) lipid acceptor. Recent

biochemical studies showed that Gne from E. coli O157 is capable of interconverting GlcNAc-P-P-Und and GalNAc-P-P-Und rather than functions as a UDP-GlcNAc/UDP-GalNAc epimerase (Rush et al., 2010). As GalNAc is evidently the first monosaccharide of the P. alcalifaciens O40 O-unit (Ovchinnikova et al., 2012), it is not excluded that Orf13 is responsible for the synthesis of GalNAc-P-P-Und from GlcNAc-P-P-Und too. The fourth sugar component ID-8 of the O-unit is d-galactose. Seventy-four percent identity was observed for Orf14 compared to UDP-galactose 4-epimerase (GalE)

of P. mirabilis. Therefore, orf14 was named galE. However, it should be noted that in most other Providencia strains studied, galE is located at the 3′ end of O-antigen gene clusters between cpxA and yibK independently of the presence of galactose in the O-unit (Ovchinnikova et al., 2012). Orf10 shares 28% identity or 48% similarity to a putative galactoside acetyltransferase of Bacteroides thetaiotaomicron, and the corresponding gene was named wpaC. The presence of this gene is consistent with partial O-acetylation of the O-unit; however, the position of O-acetyl group on the Gal residue was not confirmed chemically. The transfer of a 2-acetamido sugar 1-phosphate to UndP is mediated by WecA, which also takes part in the enterobacterial common antigen (ECA) synthetic pathway. The wecA gene encoding this enzyme is located in the ECA biosynthesis gene cluster (Alexander & Valvano, 1994). Therefore, three individual glycosyltransferases were expected to assemble the UndPP-linked tetrasaccharide O-unit of P. alcalifaciens O40. Both Orf7 and Orf12 belong to the glycosyltranferase group 2 family (Pfam00535, E value = 2 × e−16 and 9 × e−33, respectively).

These cells also regulate the immune response through secretion of IL-10 and TGFβ, and it is possible that they are involved in immunoregulation in spirocercosis. One weakness of the current study is that tissue sampling

was not standardized. Unfortunately, this is the reality when utilizing clinical cases, especially in a retrospective study. The cell counting was also limited to a single section. However, because this is primarily a descriptive study, we believe the results are valid. Moreover, in the search for Tregs, we tried to augment the chances for finding them by limiting the count to areas with high CD3+ cells presence (based on the lymph node findings and pilot observations), Palbociclib Selleckchem Erlotinib and yet, we met with limited success. Therefore, the lack of FoxP3+ cells in most of the S. lupi nodules seems reliable. The study also provides unique in situ morphologic picture of the FoxP3+ infiltrate, in which no dog study has reported. The key question in spirocercosis remains:

What is the trigger for the transformation from the chronic inflammatory, fibroblastic nodule to sarcoma? This transformation may be triggered by the inflammatory response or, alternatively, via worm excretory/secretory (ES) products. Recent studies have shown that ES products from O. viverrini, a helminth that induces cholangiocarcinoma in humans, increased fibroblast cell proliferation in cell cultures (37). However, the theory of stimulation of cells in the nodule by the worm does not completely exclude the inflammatory mediation hypothesis, because other studies have shown that O. viverrini ES products up-regulate the expression of TGFβ, which may represent an indirect carcinogenic effect via immunosuppression (38). Many studies have elucidated the role played Farnesyltransferase by helminth ES products in the modulation of the immune response, especially via the inhibition of innate cell functions and induction of a Th2 response (39). Such mechanisms clearly warrant further

investigation whether we are to understand the pathogenesis of S. lupi-induced sarcoma. This study was funded by Petplan Charitable Trust. The authors would like to thank Jeanie Finlayson, Dr Julio Benavides and the Histopathology laboratory at Moredun Research Institute, and Neil McIntyre at the Royal (Dick) School of Veterinary Studies, for assistance with immunohistochemical staining and analysis. “
“The proto-oncogenes Myc and Pim1, which are deregulated in many types of cancers, are known to cooperate in B lymphoma development. Here we show that overexpression of retrovirally transduced, doxycycline-inducible Myc alone in IL-7-deprived, growth-arrested pre-B cells enhanced cell cycle entry without impairing apoptosis. Overexpression of Pim1 decreased apoptosis, but had no effect on cell cycle entry.

In this report, we have demonstrated that IL-15 plays an important role in supporting FDC proliferation and in the production of certain chemokines by FDCs. These findings suggest that IL-15 is one of the key factors in the production of protective antibodies by stimulating rapid GC formation, offering a potential target for immune modulation. This study was initiated at the Laboratory of Cellular Immunology (Ochsner Clinic Foundation, New Orleans, LA) and completed at the Asan Institute for Life Science, Seoul. The reagents IL-15 and CD40L were the generous gift of Dr Richard Armitage (Amgen, Seattle, WA). The study was supported by a grant W06-408 from the Asan Institute for

Life Science, Seoul, and by a National Research Foundation grant from the Korean government A (R13-2008-023-01003). Quizartinib mw None of the authors have any potencial financial conflict of interest related to this work. “
“Invariant natural killer T (iNKT) cells are a distinct lineage of innate-like T lymphocytes and converging studies in mouse models have demonstrated the protective role of iNKT cells in the development of type 1 diabetes. Recently, a new subset of iNKT cells, producing high levels of the pro-inflammatory cytokine IL-17, has BAY 73-4506 concentration been identified

(iNKT17 cells). Since this cytokine has been implicated in several autoimmune diseases, we have analyzed iNKT17 cell frequency, absolute number and phenotypes in the pancreas and lymphoid organs in non-obese diabetic (NOD) mice. The role of iNKT17 cells in the development of diabetes was investigated using transfer experiments. NOD mice exhibit a higher frequency and absolute number of iNKT17 cells in the lymphoid organs as compared with C57BL/6 mice. iNKT17 cells infiltrate the pancreas of NOD mice where they express IL-17 mRNA. Contrary

to the protective role of CD4+ iNKT cells, the CD4− iNKT cell population, which contains iNKT17 cells, enhances the incidence of diabetes. Treatment with a blocking anti-IL-17 antibody prevents the exacerbation of the disease. This study reveals that different iNKT cell subsets play distinct roles in the regulation of type 1 diabetes and iNKT17 cells, which are abundant in NOD mice, exacerbate 4��8C diabetes development. Invariant natural killer T (iNKT) cells represent a distinct lineage of T cells that co-express a highly conserved αβ T-cell receptor TCR along with typical surface receptors for natural killer cells. The invariant TCRα chain of iNKT cells is encoded by Vα24-Jα18 gene-segments in humans and Vα14-Jα18 gene-segments in mice. The TCRβ chain is also strongly biased, encoded by Vβ11 gene-segment in humans and Vβ8.2, Vβ7 and Vβ2 gene-segments in mice. These lymphocytes recognize both self and microbial glycolipid antigens presented by the non-classical class I molecule CD1d.

, 2008). Modified Vaccinia Ankara (MVA) adenovirus, a recombinant-vector vaccine expressing the secreted mycobacterial antigens Ag85A and 85B, has been studied as a subunit vaccine, either as a prime vaccine or as a BCG-boosted vaccine (Williams et al., 2005; Santosuosso et al., 2006). Although this system has a potent adjuvant effect and can deliver vaccine antigens through mucosal tissues to induce strong T-cell stimulation, its drawbacks include increased reactogenicity and pre-existing immunity induced by exposure to natural antigens that are cross-reactive with vector components (McShane et al., 2005; Hoft, 2008). Phase I/II clinical trials have been completed for MVA-Ag85A in Oxford,

UK, and Gambia to assess vaccine safety, immunogenicity and dosage in individuals previously exposed to mycobacterial antigens. Tuberculosis vaccine development

has been progressing EPZ 6438 empirically for many years. Currently, increased understanding of the immune system and the development of advanced delivery and adjuvant systems are enabling the design of improved prophylactic vaccines. As a result, in the last 10 years, the international research community has developed more than 200 tuberculosis vaccine selleck inhibitor candidates currently being tested in mouse, guinea-pig and human primate models. These approaches are aimed at achieving a more potent and prolonged immunological memory, a goal of great global importance, given the rise of MDR-tuberculosis worldwide and the poor efficacy of the BCG vaccine against adult pulmonary tuberculosis. Despite a lack of relevant animal models that correlate

with protection in humans and the lack of markers capable of demonstrating the efficacy of an antigen/adjuvant combination nearly (needed for a faster acceptance of new adjuvants), promising vaccines from the Fifth Framework Program FP5 (Mtb72F/AS01B, H1 in IC31 and CAF01; MVA-Ag85A) have been developed and tested in preclinical and clinical trials, and the optimized formulations and adjuvant combinations have been produced using good manufacturing practices. Further improvement of these adjuvants through combination with other delivery systems or recently identified mycobacterial immunomodulators is underway in the context of FP7 (from 2007 to 2013). It is clear that more research is required on adjuvants’ effects on antigen presentation, APC activation, long-lived memory T-cell induction and Th-1/Th-2 cell polarization to avoid undesirable effects. Efforts directed toward the development of postexposure vaccines against latent tuberculosis are also needed. Thus, the development of new adjuvants and delivery methods is as important as the search for antigens that allow discrimination between latent and active disease. Also, special attention to several candidate nonprotein antigens (sulphoglycolipids, phosphoantigens, etc.) is required, due to their potential usefulness in subunit vaccines and/or adjuvants capable of stimulating CD1-restricted γ-δ or NKT cells.

T-cell clones were expanded every 2–3 wk using a mix of IMDM supplemented with 10% FBS and 10% TCGF, irradiated PBMC from five different donors and irradiated autologous B-LCL selleck chemicals loaded

with 5 μg/mL cognate peptide. T-cell cultures (25 000–50 000 cells/well) were tested on pulsed autologous APC (monocytes or irradiated autologous B-LCL) for the recognition of M1 peptides (5 μg/mL) and protein (10 μg/mL) in triplicate in a 3-day proliferation assay 38. For generation of monocytes, PBMC were seeded in flat bottom 96-well plates (Greiner bio-one, The Netherlands) and adherent PBMC were cultured for 3 days in X-vivo medium (BioWhittaker) containing 800 IU/mL GM-CSF (Invitrogen, UK) before use. For experiments with influenza virus, autologous monocytes were infected at a MOI of 1 with A/Wisconsin/67/2005 for 5 h before addition of M1-specific T-cell clone. After 48 h supernatant was harvested and stored at −20°C for cytokine analysis. During the last 16 h of culture 0.5 μCi/well [3H]thymidine (Perkin Elmer, USA) was added to measure proliferation 17. Antigen-specific IFN-γ and IL-10 production was measured by ELISA according to manufacturer protocol (Sanquin,

The Netherlands). The cut-off of the ELISA was based on the start of linearity of the standard curve, which was 100 pg/mL for IFN-γ and 50 pg/mL INCB024360 manufacturer for IL-10. Specific responses were positive when they were at least twice the level of control antigen and above the cut-off level. For the analysis of cytokine production on a single-cell level T-cell clones were stimulated for 4 h with peptide-loaded autologous monocytes and were subsequently stained for IL-10 and IFN-γ according to manufacturer protocol (IL-10 and IFN-γ secretion

assay; Miltenyi Biotech) and analyzed by flow cytometry. For anti-CD3-based suppression assays responder CD4+CD25− cells were isolated from PBMC as described before 5. CD8+ lymphocytes were isolated using magnetic Dynal beads (Invitrogen, USA) and used as CD8+ responder cells where indicated; 1×105 responder cells were cultured with M1-specific T-cell clone at different ratios in the presence of 1×104 irradiated B-LCL and 1 μg/mL next agonistic anti-CD3 antibody (OKT-3, Ortho Biotech, USA). Proliferation and cytokine production was determined as described above. Cell surface activation markers were stained 24 h after stimulation and analyzed by flow cytometry. For antigen-dependent suppression experiments CD4+CD25− responder cells were stained with 5 μM CFSE (Invitrogen) for 15 min at 37°C. M1-specific T-cell clone was stained with PKH26 according to the manufacturer’s protocol (Sigma), treated with Mitomycin C (50 μg/mL; Kyowa, Japan) for 1 h and irradiated (2000 Rad) to prevent proliferation of the clone.