Baboons (Papio anubis, from the CNRS Primatology Center, Rousset, France) were negative for all quarantine tests, including a tuberculin skin test. Animals were housed at the large animal facility of our laboratory following the recommendations of the Institutional Ethical Guidelines of the Institut National de la Santé Et de la Recherche Médicale, France. All experiments were performed under general anaesthesia with Zoletil (Virbac, Carron, France). Pharmacokinetic and pharmacodynamic

studies were performed during DTH experiments on five baboons receiving an i.v. bolus of either 1 mg/kg or 0·1 mg/kg of chimeric A9H12. Chimeric A9H12 was quantified in baboon sera using a specific sandwich ELISA. LAG-3-Ig (Immutep, Orsay, France) was immobilized on plastic at pH 9·5 overnight at a concentration of 5 µg/ml. After saturation with

5% gelatin at 37°C for 2 h, serum diluted PD98059 in PBS-0·05% Tween 20 were incubated for 4 h at room temperature, washed and revealed with a mouse anti-human IgG kappa chain Compound Library price antibody (EFS, Nantes, France) at a 1:2000 dilution, followed by peroxidase-labelled goat anti-mouse antibody (Jackson Immunoresearch, Westgrove, PA, USA) at a 1:5000 dilution. Optical density was recorded at 450 nm after a tetramethylbenzidine (TMB) revelation period of 10 min at room temperature in the dark and addition of 25 µl 1 N sulphuric acid/well. Baboons were immunized intradermally (i.d.) twice with a bacillus Calmette–Guérin (BCG) vaccine (0·1 ml; 2–8 × 105 UFS; Sanofi Pasteur MSD, Lyon, France) in the upper region of the leg, 4 and 2 weeks before the DTH skin test. To investigate antigen-specific T cell immunity before

DTH skin testing, successful immunization was confirmed by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay (non-human primate IFN-γ ELISPOT kit; R&D Systems, Minneapolis, MN, USA) on freshly isolated Adenosine triphosphate PBMC, according to the manufacturer’s instructions. Intradermal reactions (IDR) were performed with duplicate intradermal injections of two doses (2000 UI or 40 UI) of tuberculin-purified protein derivative (PPD; Symbiotics Corporation, San Diego, CA, USA) in 0·1 ml in the skin on the right back of the animals. Saline (0·1 ml) was used as a negative control. Dermal responses at the injection sites were measured using a caliper square. The diameter of each indurated erythema was measured by two observers from days 3–8, and were considered positive when > 4 mm in diameter. The mean of the reading was recorded. Skin biopsies from the DTH or control (saline) site were performed at day 4 on one duplicate and placed in Tissue Tek optimal cutting temperature (OCT) compound (Sakura Finetek, Villeneuve d’Ascq, France) for immunohistochemical analysis. A second IDR was performed after a 3-week washout period and animals received one i.v. injection of either 1 mg/kg or 0·1 mg/kg of chimeric A9H12 1 day before this second challenge with PPD.

Both groups showed a significant novelty preference only for the no-delay condition. On day

two, event-related potentials (ERPs) were recorded while infants viewed the VPC familiar face, a more recently familiarized face, and a novel face, and mean amplitude for components thought to reflect memory (positive slow wave, PSW) and attention (negative central, Nc) were computed. In temporal regions, HII showed a diminished Nc and enhanced PSW to the recently familiarized face, while CON showed a similar trend for the PSW only. Overall, infants showed the largest PSW over left scalp regions. Finally, a positive correlation between VPC novelty preference after 24 h and PSW was found in CON, and preliminary results suggest that this association differs as a function of group. Therefore, selleck inhibitor in comparison with CON, HII showed both similarities and differences on individual

tasks of memory as well as potentially disparate relations between the www.selleckchem.com/products/epacadostat-incb024360.html behavioral and neural mechanisms underlying memory performance. The capacity to transform a new experience into a lasting memory is essential to human learning and development. The study of memory in infants can provide an early window into this process of cognitive development. Although infants are nonverbal, their memory can be evaluated through the use of both behavioral and electrophysiological measures. Visual paired comparison (VPC) is the behavioral task that is most often used to evaluate nonverbal visual recognition memory in infants. This task involves familiarizing the infant to a visual stimulus for a fixed period of time and subsequently testing the infant by showing the familiarized stimulus next to a novel stimulus such that the infant simultaneously PJ34 HCl views both the familiar and novel stimuli. Memory is inferred if the infant

shows preferential looking, greater than is expected by chance, to one stimulus over the other, typically a preference toward the novel stimulus (Bauer, San Souci, & Pathman, 2010). Prior studies have used the VPC task to demonstrate visual recognition memory across time delays at various infant ages. Geva, Gardner and Karmel (1999) demonstrated novelty preference after a short delay in 4-month-olds, Pascalis, de Haan, Nelson and de Schonen (1998) demonstrated novelty preference after a 24-h delay in 6-month-olds, and Morgan and Hayne (2011) demonstrated novelty preference in 12-month-olds when tested immediately but not after 24-h delay. Through use of the VPC task, all of these studies demonstrated the presence of visual recognition memory in infants from ages 4 to 12 months, and although the overall trend is toward retention over progressively longer time delays after shorter periods of familiarization with increasing age, the precise retention intervals at various ages during infancy remain to be identified (Rose, Feldman, & Jankowski, 2004).

Filters were applied based on absent calls in Ribociclib all replicates for both conditions (untreated versus MSU treated) and for detecting very low maximum signals (≤95th percentile of the global

Absent calls distribution). The Limma method [44] was used to define a set of genes differentially expressed between conditions, and a Benjamini–Hochberg multiple test correction of the false discovery rate was applied [45] (adjusted p-value ≤0.05). Functional analysis was performed on nonredundant probe sets using GeneGo MetaCore™ software to select the most significantly enriched pathways and biological processes (FDR ≤ 0.05). The comparison of gene expression patterns between conditions was conducted using hierarchical clustering with MultiExperiment Viewer software [46, 47], setting Euclidean distance as the dissimilarity measure and average linkage as the linkage method. For each selected pathway or biological process, the heat-maps show the Log2 (Ratio) average expression signal for each gene in the MSU-treated buy SAHA HDAC condition (WT and Nlrp3−/−) versus their respective untreated controls. The microarray data from this publication have been submitted to the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) and assigned the identifier E-MEXP-3858. DNA damage was quantified by single-cell gel electrophoresis (also known as the comet assay, R&D) according to the manufacturer’s instructions. DNA fragmentation was visualized by epifluorescence microscopy

using a FITC filter. At least 100 comets were analyzed on duplicate slides. Data were analyzed using Comet ScoreTM (TriTek Corporation). DNA damage was Tacrolimus (FK506) quantified by three observers in a blinded fashion based on the distribution of DNA between the head and the tail according to the following formula: Tail% DNA = 100 − (Head% DNA). Damage was also assessed using the Olive Tail Moment: (Tail mean − Head mean) × (Tail% DNA)/100. Total cellular extracts were prepared by lysing cells in ice-cold RIPA buffer (10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 355 mM EDTA, protease inhibitor cocktail (Complete Mini protease inhibitor cocktail, Roche), phosphatase

inhibitor cocktail (PhosStop, Roche), 1 mM β-mercaptoethanol). Equivalent protein extracts (40–60 μg) were denatured by boiling in SDS and β-mercaptoethanol before being separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories). The blots were then blocked and probed with the following antibodies: phospho-histone H2AX (Ser139) (#2577, 1:1000), phospho-ATR (Ser428) (#2853, 1:1500), phospho-p53 (Ser15) (#9284, 1:800), phospho-p53 (Ser20) (#9287, 1:800), and total p53 (1C12, #2524, 1:800) from Cell Signaling; phospho-ATM (Ser1981) (10H11.E12, 05-740, 1:1500) and GAPDH (MAB374, 1:20 000) from Millipore; and a-tubulin (sc-5286, 1:1000) from Santa Cruz. The protein complexes were detected using Western Lightning Enhanced Chemiluminescent Substrate (PerkinElmer Inc.

POSH, JIP-1, MLK3, MKK7, JNK1, JNK2, NF-κB p65, Rac1, T-bet, and p-cJUN antibodies (Santa Cruz Biotechnology). pSAPK/JNK, p-p38 MAPK, JNK2, cleaved caspase-3, and pSAPK/JNK antibodies (Cell Signaling). Perforin and Eomes antibodies (eBioscience). Granzyme B was from Invitrogen. TNF-α, IFN-γ, and Ki-67 (BD Pharmigen). Rac1 was from Millipore. β-actin was from Sigma. Tat-POSH (NH2-GRKKRRQRRRPPRPRKEDELELRKGEMFLVFER-amide), selleck products Tat-scrambled (NH2-GRKKRRQRRRPPRPDRKLEVFEKEFLRMELGER-amide), and Tat (NH2-GRKKRRQRRRPP-amide) peptides were synthesized by New England Peptides to a purity

of >70 and >90%, respectively. Peptides were used at 20 μM. None of the peptides exhibited nonspecific toxicity

at any concentration tested. Tat and Tat-scrambled gave similar results and were used interchangeably throughout and labeled as Tat-control. SP600125 (Calbiochem) was used at 33 μM. All inhibitors were added 30 min before stimulation and cultures were maintained in constant presence of fresh inhibitor except where indicated otherwise. For IP-FCM and immunoblot experiments, naïve T cells from OT-I Rag−/− mice were stimulated with OVA-Tet plus α-CD28 (1 μg/mL) or 50 ng/mL PMA plus 500 ng/mL ionomycin (Sigma). For all other experiments, OT-I splenocytes were stimulated with 0.2 nM OVA-peptide. GW572016 IL-2 was used at 50 μ/mL. Where indicated, OT-I T cells were labeled with 10 μM CFSE. When required, cells were restimulated with OVA-Tet in the presence of 3 μg/mL Brefeldin A (Sigma) for 6 h. Cells were lysed in buffer containing 10 mM Tris, 1% Triton X-100, 0.5% Igepal CA-630 (Sigma), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and freshly added protease and phosphotase inhibitors. Following lysis for 20 min on ice, samples were spun to clear lysates of cellular debris and the cleared supernatant was

used for immunoblot or IP-FCM analysis. Standard IP with Rac1 and GST-PAK were performed as previously described [51]. IP-FCM was performed using α-Rac1, α-JIP-1, and α-POSH CML beads as previously Buspirone HCl described [33-35]. In brief, antibodies were covalently coupled to polystyrene latex beads, then incubated with cell lysates overnight at 4°C, extensively washed in lysis buffer, and stained with the appropriate primary and highly crossabsorbed secondary antibodies (Invitrogen) and analyzed by FCM. Singlet beads were identified on the basis of forward and size scatter. A minimum of 1500 bead events was collected for each experiment and analyzed using FlowJo (TreeStar). Graphs depicting relative secondary analyte were generated by normalizing the geometric MFI of the secondary analyte to the geometric MFI of the primary analyte (to control for potential variations in IP efficiency (loading control)) to Tat-cont.-treated cells.

reported that LAR was dispensable in T-cell MG-132 datasheet development 17. In their study, they compared LAR−/− mice with LAR+/− heterogenic

mice, whereas we used WT mice as a control. As they showed, surface expression level of LAR in LAR+/− mice was about half of that in WT mice. This subtle difference between LAR+/− and WT mice could make them difficult to find the effect of LAR deficiency on thymocyte differentiation. Because LAR is expressed in various kinds of cells, tissues and organs, including neurons in the brain, LAR deficiency influences a variety of cellular functions. Further analysis of LAR signaling may clarify its ability to modulate TCR signaling and might contribute to understanding the role of LAR in pathological T-cell differentiation. All animal experiments have been approved by the Committee on Animal Experiments at the University of Toyama. Mice deficient in the LAR phosphatase domain (LAR−/−)

were obtained from McGill University (Montreal, Que., Canada) with permission from Dr. W. Hendriks (University Medical Center Nijmegen, The Netherlands) 16. Transgenic mice expressing a transgene that encodes a TCR recognizing a male-specific peptide presented on H-2Db (HY-TCR-Tg mice) were obtained from Dr. M. Kubo, Riken, Japan. HY-TCR-Tg mice deficient in LAR were generated by crossing HY-TCR-Tg mice and LAR−/− mice in our animal facility. The antibodies recognizing HY-TCRα (T3.70) Wnt inhibitor 21 and LAR/IMT-1 18, 19 were purified from culture supernatants of hybridoma cells. The FITC-, PE- or biotin-conjugated CD4-specific antibodies, the cychrome- or biotin-conjugated CD8-specific antibodies, the FITC-conjugated CD25-specific

antibody, the PE-conjugated CD44-specific antibodies and the PE- or Cychrome-conjugated streptavidin were purchased from Pharmingen (San Diego, CA, USA). Cells were incubated with PE-, FITC-, cychrome- or biotin-conjugated antibodies followed by fluorophore-conjugated streptavidin in PBS containing 0.1% BSA and 0.05% NaN3 for 20 min on ice as described previously 18. The stained cells were analyzed using a FACSCanto with FACSDiva software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). The authors Fenbendazole thank W. Hendriks (University Medical Center Nijmegen, The Netherlands) for providing the LAR−/− mice, M. Kubo (Riken, Japan) for the HY-TCR-Tg mice, Sanae Hirota for technical assistance and Kaoru Hata for secretarial work. The project was supported by Grants in Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Citation Ticconi C, Giuliani E, Veglia M, Pietropolli A, Piccione E, Di Simone N. Thyroid autoimmunity and recurrent miscarriage.

5A). No interaction of other mutants was partly because the mutant V proteins did not accumulate in the infected cells as revealed by immunoblotting selleck chemicals llc using anti-Vu antibody (Fig. 5A, IB: αVu). The amounts of V proteins synthesized for 30 min in the presence of [35S]Cys and [35S]Met were almost equivalent to each other, although the wild-type V protein

band was faint in this gel (Fig. 5A, [35S]Cys, Met). Thus, some V mutant proteins are presumed to be unstable and easily degraded in cells. The V-R320G and V-W336G proteins appear to be stable and to accumulate in cells, while the V-W336G protein failed to interact with FL-MDA5 different from the V-R320G protein (Fig. 5A). 293T cells were transfected with p-55C1B together

with MDA5 and one of the V mutant plasmids. Cells were further transfected with poly(I:C), and proteins were metabolically labeled with [35S]Cys and [35S]Met. After 24 hr, cells were lysed and luciferase activity in the cell lysates was investigated. V proteins were then analyzed by immunoprecipitation, SDS-PAGE and an imaging analyzer, and the V protein amounts and IRF3 activation were plotted on a graph (Fig. 5B). V protein expression was almost equivalent in V-WT, V-R320G, and V-W336G. The V-WT protein suppressed IRF3 transcription activation, but V-R320G and V-W336G proteins did not. check details These are results of one of three experiments, and results of the other two experiments showed a similar tendency. The V-R320G protein was unique in its high stability and binding capacity with the V protein. However, the binding of V-R320G

with MDA5 did not inhibit the signal induced by MDA5. SeV V protein is essential for efficient virus growth in mouse lungs and for viral pathogenicity. The V protein counteracts innate immunity that is exerted through activation of IRF3 (13). We therefore next investigated the possibility of involvement of multiple molecules in the target of SeV V protein. A search for V-interacting molecules revealed some IRF3-activating molecules including MDA5, RIG-I, IKKɛ and IRF3 as interacting partners in a co-immunoprecipitation assay. However, the V protein only interacted with MDA5 at the Vu region, depending on the conserved cysteine residues of the Vu region. We thus focused on the interaction of the V protein with MDA5. Almost all of the SeV V mutants used in this study have 10–200-fold lower pathogenicity than that of the wild-type SeV (12). The V proteins derived from such SeV mutants did not interact with MDA5 except for V-R320G. This was due to inability of the V proteins to bind with MDA5 and, in some cases, due to instability of the V proteins in virus-infected cells. The V-R320G mutant protein was stable and interacted with MDA5 but did not inhibit IRF3 activation induced by overexpression of MDA5 and poly(I:C).

Murine NKP are lineage(lin)−CD122+NK1.1−CD49b−. NKP differentiate into immature NK (iNK) cells,

which exhibit a lin−CD122+NK1.1+CD49b− phenotype. Although iNK cells display CD94 and in some cases Ly49 receptors, most of them are not yet functional 17, 18. iNK cells differentiate further into lin−CD122+NK1.1+CD49b+ mature NK (mNK) cells. mNK cells migrate to the periphery and are located in spleen, liver, lungs and blood and to a lesser extent in BM, lymph nodes and GDC-0973 research buy thymus 19. mNK cells gradually up-regulate CD43 and CD11b expression, two receptors involved in cell adhesion and cell activation 20, 21. Interestingly, Hayakawa and Smyth 22showed that within the TCR β−NK1.1+ gated NK cell pool there is a CD11blow subpopulation, including both iNK and early mNK cells, which is homogenously CD27high (referred to as subset 1), whereas the CD11bhigh population of late mNK cells consists of two functionally distinct subsets: i.e. CD27high (referred to as subset 2) and CD27low (referred to as subset 3). NK cells from subset 1 are the first NK cell population detected after BM transplantation and they give rise to subset 2 after adoptive transfer. Subset 2 consists of functional active NK cells, which can differentiate into the resting NK cell population of subset 3. Both mature subsets 2 and 3 are present in spleen and liver,

whereas only subset 3 is observed in lungs and peripheral blood. NK cells of subset 2 are not only characterized by CD27 expression PS-341 clinical trial and stronger effector functions compared with subset 3, but also by their Ly49lowKLRG1− phenotype, which is the exact opposite of that of subset 3 22. CD27 is a disulphide-linked 120-kD type I transmembrane protein belonging to the TNF receptor (TNFR) family 23. The TNFR family is involved in diverse immunological processes such as proliferation, differentiation, survival and Baf-A1 datasheet migration 24, 25. CD70, the ligand of CD27,

is a type II transmembrane protein of the TNF family transiently up-regulated on activated lymphocytes 26. Interestingly, down-modulation of CD27 expression is witnessed in T cells upon in vitro incubation with CD70+ B-cell lines 27 as well as in BM progenitor cells and peripheral T cells in CD70-Tg mice 28, 29. Also, progressive differentiation of naïve T cells into effector-memory T cells is evidenced in CD70-Tg mice 30. As these effector T cells produce high amounts of IFN-γ, BM located B-cell development is declined in CD70-Tg mice 29. However, until now, only few studies report on the interaction of CD70 with CD27 expressed on NK cells. Cross-linking of CD27 on NK cells stimulates their proliferation and IFN-γ production. There is also an IFN-γ-dependent effect of CD27 stimulation on NK cell cytotoxicity 31. This indicates that CD27 and CD70 are tightly linked with NK cell biology.

Therefore, we believe that calcium and PKC signals are required for sufficient Nur77/Nor-1 mitochondrial localization and reversal of Bcl-2 pro-survival function. In this study, we report the biological activity of a synthetic DAG-lactone, HK434, in thymocytes. HK434, like the other synthesized DAG analogs, binds with Forskolin high potency to the phorbol ester/DAG binding site within the C1 domain of PKC 52. Using the crystal structure of the PKCδ C1b domain with pharmacophore and receptor-guided approaches, structurally

primitive DAG-lactone ligands were designed with binding affinities for PKCα in the low nanomolar range 39. These DAG-lactones exhibit 3–4 orders of magnitude higher affinity for PKC isozymes than natural DAG and phorbol esters. They have been characterized in other cell types and have phorbol ester-like effects 39, 53–56. Here, we report that DAG-lactone, HK434 and ionomycin signals are sufficient to induce Nur77/Nor-1 mitochondrial targeting in thymocytes. Furthermore, HK434, like phorbol esters can induce apoptosis in thymocytes. An interesting finding is that HK434 and PMA exert their regulation of Nur77 and their apoptotic activities through activation of different subsets of PKC isoforms (Fig. 6B). While the classical PKC isoform inhibitor

Gö6976 is sufficient in blocking HK434/ionomycin-induced Nur77 mitochondrial targeting and thymocyte apoptosis, no effect was observed with PMA/ionomycin-stimulated thymocytes. A correlation was found between Kinase Inhibitor Library cell assay PKC activation, induction of thymocyte apoptosis, either Nur77/Nor-1 phosphorylation, mitochondria translocation and exposure of the Bcl-2 BH3 epitope in stimulated thymocytes, further confirming the important role of Nur77/Nor-1 mitochondria translocation in TCR-induced thymocyte apoptosis. It is not clear if PKC acts directly or indirectly on Nur77/Nor-1. An interaction between PKC and Nur77 has been reported

before 57. Ser350 within the DNA binding domain of Nur77 was previously shown to be phosphorylated by protein kinase A and PKC in an in vitro kinase assay of stimulated PC12 neuronal cells 49. However, in another study, the association of Nur77 and PKCθ in T-cell hybridomas did not induce Nur77 phosphorylation 57. It is possible that a direct PKC regulation of Nur77 might be unique to immature T cells. Alternatively, phosphorylation of Nur77 may be indirectly regulated by PKC proteins. PKCθ has been initially suggested to be the PKC isoform crucial for negative selection. This notion was based on findings that during negative selection, PKCθ, but not other PKC isoenzymes, is recruited to the site of TCR aggregation 35. However, PKCθ−/− mice show no defects in negative selection 58. This suggests some functional redundancy among PKC family members and that a PKC isoenzyme distinct from PKCθ is involved in TCR signaling events in thymocytes.

The dose and orientation of the antigen FK506 cost towards HSP in the fusion gene may have clinical implications for the design and optimization of HSP-based vaccines [21, 29, 55, 56]. Regarding to the previous studies, the increasing amount of N-terminal fragment of gp96 leads to rise in the percentage of the peptide-specific T cells responses [21]. Therefore, higher dose of rE7-NT-gp96 protein might produce more effective immune responses. Many studies have been focused on applying different delivery systems and adjuvants to increase the immunogenicity of E7 expressing protein vaccines [57, 58]. SmithKline Beecham Biologicals have prepared vaccine formulations of a recombinant fusion protein

with a range of adjuvants based on combinations of the immunostimulants such as MPL and QS21 in different vehicles

like liposomes, oil-in-water emulsions or aluminium PCI-32765 cost salts. Formulations including immunostimulants MPL and QS21 leads to the induction of CTL responses and ultimately to tumour rejection [58]. Another study demonstrated that ISCOMATRIX adjuvant stimulates both cellular and humoral immune responses when co-administered with recombinant HPV16-derived E6E7 or E7GST fusion proteins [59]. In our study, it is suggestible to examine the effect of different adjuvants and delivery systems on the fusion protein vaccine potency enhancement. In summary, our result indicated that the recombinant E7-NT-gp96 without any adjuvant elicit efficient Epothilone B (EPO906, Patupilone) E7-specific immune responses. The fusion of NT-gp96 to E7 leads to Th1 directed immune responses. E7-NT-gp96

fusion protein could delay tumour occurrence and growth in comparison with E7 protein alone. Considering the efficient immune-enhancing effects provided by E7-NT-gp96, it is worth to determine the effect of fusion direction of NT-gp96 towards E7 in this vaccine modality. EM thanks Pasteur Institute of Iran for the grants supporting her PhD studentship. The authors wish to thank Mr. A. Javadi (Pasteur Institute of Iran, Department of Immunology) and also Mr. Sh. Alizadeh (Pasteur institute of Iran, Molecular Immunology and Vaccine Research Laboratory) for their technical assistance. “
“Natural Treg cells acquire their lineage-determining transcription factor Foxp3 during development in the thymus and are important in maintaining immunologic tolerance. Here, we analyzed the composition of the thymic Treg-cell pool using RAG2-GFP/FoxP3-RFP dual reporter mice and found that a population of long-lived GFP− Treg cells exists in the thymus. These long-lived Treg cells substantially increased with age, to a point where they represent >90% of the total thymic Treg-cell pool at 6 months of age. In contrast, long-lived conventional T cells remained at ∼15% of the total thymic pool at 6 months of age.

p.m. versus 3000 c.p.m.; P < 0·03). From these data, along with

those shown in Figs 2 and 3, we speculate that eosinophils not only present antigens to CD4+ T cells in an MHC class II pathway, but also present antigens to CD8+ T cells by using their MHC class I molecules. To test this hypothesis, experiments were performed to determine whether the induction of C. neoformans-primed T-cell proliferation was caused by the presentation of FDA-approved Drug Library cost antigens by eosinophils in conjunction with MHC class I and MHC class II molecules. C. neoformans-pulsed eosinophils were treated with anti-MHC class I or anti-MHC class II mAbs before incubation with C. neoformans-primed CD4+ and CD8+ T cells. The blocking of MHC molecules on the eosinophil surface was found to suppress the ability of C. neoformans-pulsed eosinophils to stimulate C. neoformans-primed T-cell proliferation (Fig. 6d). Moreover, the suppression seen Epigenetic Reader Domain inhibitor in the lymphocyte proliferation was more pronounced with anti-MHC class II, which coincided

with the higher proliferation of CD4+ T cells shown in Fig. 6c. In conclusion, C. neoformans-pulsed eosinophils stimulated C. neoformans-primed MSCs and T cells (CD4+ as well as CD8+) in an MHC class II- or class I-dependent manner. This stimulation of proliferation, however, was not observed for naive T cells or when C. neoformans-pulsed Mφ were used as APCs. To characterize and differentiate the T-cell profile seen after co-culture with C. neoformans-pulsed eosinophils, C. neoformans-primed purified T cells (CD4+ and CD8+) were analyzed Palmatine by flow cytometry to determine the intracellular expression levels of IFN-γ and IL-4 after 4 days of culture with C. neoformans-pulsed eosinophils or medium alone. Figure 7 shows a significant increase in the percentage of IFN-γ-producing cells when T cells were incubated with C. neoformans-pulsed eosinophils compared with T cells cultured in medium alone (6·56% versus 1·61%; P < 0·02). With regard to the IL-4-producing T-cell population, the percentage

with C. neoformans-pulsed eosinophils (2·42%) was similar to that for medium alone (2·35%). These results allowed us to conclude that C. neoformans-pulsed eosinophils were able to induce the expansion of IFN-γ-producing Th1 cells, but not of IL-4-producing Th2 cells. To analyze the production of cytokines by CD4+ and CD8+ T cells in supernatants, the concentrations of IFN-γ, TNF-α, IL-4, IL-10 and IL-13 were measured after 4 days of culture. The results presented in Fig. 8(a,b) show that there was a significant increase in the production of IFN-γ and TNF-α generated by C. neoformans-primed T cells cultured with C. neoformans-pulsed eosinophils compared to the cytokine production by T cells cultured in medium alone, with fixed yeasts of C. neoformans or with unpulsed eosinophils. In contrast, no differences in the levels of IL-4, IL-13 or IL-10 were detected in supernatants of C. neoformans-primed T cells cultured with C.