The authors are grateful to Prof. Kathleen Reilly for comments and critical reading. This study was supported by a grant from the National Natural Science Foundation of China (No. 30872788) and Beijing Municipal Science Technology

Commission (No. Z09050700940903). J.X., L.S. and H.Y. contributed equally to this learn more study. “
“The objective of this study was to determine whether there was any association between the peripheral blood CD4+ CD25+ Foxp3+ regulatory T cells (Treg cells) and implantation success in patients undergoing in vitro fertilization (IVF) treatment. Prospective observational study of 101 randomly selected women who underwent IVF treatment for tubal factor from May 2011 to June 2011. The percentage of peripheral blood Treg cells and the expression levels of Foxp3

and CTLA4 mRNA in peripheral blood mononuclear cells (PBMCs) were recorded and their relations to IVF treatment outcomes were analyzed. Treg cells were significantly elevated in the pregnant group (P = 0.03). The expression level of Foxp3 mRNA in PBMCs from pregnant group also significantly increased (P = 0.02). A receiver operating characteristic analysis (area under curve = 0.631) found that those women with Treg cells >0.6%, the pregnancy rate and live birth rate were much higher as compared to women with Treg cells below this level (P < 0.05). An increase of Treg see more cells in the peripheral blood was associated with a

better IVF treatment outcome (OR 4.3, 95% CI = 1.76–10.48), with a sensitivity of 64%, specificity of 71%. An elevated level of circulating Treg cells was associated with increased rates of pregnancy and live birth in IVF treatment. “
“This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term iNKT derives from the fact that a large fraction of murine NKT cells recognize the MHC class I-like CD1d protein, are CD4+ or CD4-CD8- (double negative), and use an identical “invariant” TCRα chain, which is generated by precise Vα14 and Jα281 (now renamed Jα18) rearrangements with either no N-region diversity or subsequent trimming to nearly identical amino-acid sequence (hence, ‘iNKT’). Basic Protocol 1 and Alternate Protocol 1 use multi-color Resveratrol FACS analysis to identify and quantitate rare iNKT cells from human samples. Basic Protocol 2 describes iNKT cell purification. Alternate Protocol 2 describes a method for high-speed FACS sorting of iNKT cells. Alternate Protocol 3 employs a cell sorting approach to isolate iNKT cell clones. A Support Protocol for secondary stimulation and rapid expansion of iNKT cells is also included. Basic Protocol 3 explains functional analysis of iNKT. Curr. Protoc. Immunol. 90:14.11.1-14.11.17. © 2010 by John Wiley & Sons, Inc. “
“IL-23 is absolutely crucial for the development of T-cell driven autoimmune disease in mice.

As previously described 54, immunoprecipitations were performed with an anti-CD16 mAb (clone 3G8, mice IgG1, BD biosciences) or an anti-EGFR mAb (mice IgG1, Santa Cruz, Heidelberg, Germany) and sera of

non-immunized mice (Dako) used as the negative control. Immunoblotting was performed using Nupage selleck chemicals system (Invitrogen) and L1 proteins from VLPs were detected using CAMVIR antibody (Abcam, Cambridge, UK) and Clean Blot IP detection reagent (Thermo Fisher). The assay to detect activated GTPase proteins was carried out as previously described 55. Briefly, cells were lysed by addition of 200 μL of ice-cold lysing buffer. Lysates were centrifuged for 5 min at 16 000×g. Supernatants were immediately frozen in liquid nitrogen and stored at −80°C until use. For pull-down assays, supernatants were incubated for 30 min with 30 μg of GST-PBD protein containing the Cdc42 and Rac1 binding regions of PAK-1B, affinity linked to glutathione-sepharose beads. The beads were washed in ice-cold washing buffer and boiled in SDS-PAGE lysis buffer. The amount of Rac1 and Cdc42 in the samples was CCI-779 cost determined by immunoblotting with antibodies specific to Rac1 (23A8, Upstate Biotechnology,

Waltham, USA) and Cdc42 (BD Biosciences). Prism 4.0 (GraphPad Software) was used for data handling, analysis and graphic representation. Statistical analysis was performed using Student’s t-test or the Mann–Whitney test. The authors thank Dr. S. Ormenese from the GIGA-Imaging and Flow Cytometry platform for her support with flow cytometry and confocal microscopy and Prof. N. Antoine for the preparation of electron microscopy grids. They are also grateful to Dr. P. Coursaget for the provision of baculovirus expressing HPV16 and HPV31 L1, Dr. L. Bousarghin for providing electron microscopy grids with DCs containing HPV-VLPs, Prof. N. Christensen for providing

V5 antibodies, C1GALT1 M. Lebrun for her assistance with confocal microscopy, Dr D. Begon for her advice on co-immunoprecipitation and Prof. G. Thibault for helpful discussion. They thank GlaxoSmithKline Biologicals for providing polyclonal antibodies used to assess the quality of L1-VLPs by ELISA. This study was supported by the Belgian National Fund for Scientific Research (FNRS), C. D., A. C. and N. J. are supported by the FNRS. V. R., B. B. and I. L. are supported by a Télévie grant from the FNRS. 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.

17% CD8+ T cells) and triple (0.29–5.37% CD4+ T cells and 0.54–6.91% CD8+ T cells) cytokines in both ltLTBIs and PPD− donors (data not shown). Interestingly, the IFN-γ+TNF-α+

CD8+ T-cell population consistently was the most frequent multiple cytokine-producing T-cell subset identified (Fig. 1B, D and F). To assess the memory phenotype of these cells, we measured expression of memory markers CCR7 and CD45RA by Mtb antigen or peptide responsive cells from the ltLTBI population (Fig. 2A and B). T-cell subsets were classified according to the model described by Seder et al. 29. Only a minor fraction of the IFN-γ+TNF-α+ CD8+ T cells appeared to be “naïve” Dorsomorphin cost (CCR7+CD45RA+) or central memory T cells (CCR7+CD45RA−), while most were found to be effector memory (CCR7−CD45RA−) T cells, followed by effector (CCR7−CD45RA+) T cells (percentages ranged between 36 and 62% (SD±0–35) for effector memory T cells and 22–51% (SD±2.8–32) for effector T cells). Taken together, our results show the presence of Mtb DosR-regulon-encoded

antigen-specific double- and monofunctional CD4+ and CD8+ T-cell responses in ltLTBIs. IFN-γ+TNF-α+ CD8+ T cells were the most prominently present multiple cytokine-producing T cells, and comprised mainly effector memory and effector T cells. Next, we analyzed single peptide-induced responses in PPD positive (PPD+) individuals in order to identify immunogenic Mtb DosR antigen epitopes. In view of the number of cells required for these analyses, click here we used buffy coat-derived PBMCs. PBMCs of PPD+ individuals were incubated

with each single peptide of Mtb DosR Rv1733c, Rv2029c and Rv2031c and the control protein Ag85B. Proliferative responses were measured using CFSE labeling, an assay that we have described previously 27, 30. Figure 3 demonstrates typical proliferation profiles of CD4+ and CD8+ T cells in response to Mtb antigens and control conditions in one PPD+ donor. Following stimulation of PBMCs with PPD, Rv1733c or its corresponding peptides, significant CD4+ and to a lesser extent CD8+ T-cell proliferation were observed (Fig. 3A and B, respectively). No proliferation was observed to the irrelevant Paclitaxel mouse control peptide HIV-gag77–85 or for medium only (data not shown). A relative proliferation (see Materials and methods for calculation) of 10% was considered positive in this assay, in line with previous studies 27, 30. Responses to previously published HLA class I and class II restricted epitopes of Ag85B 31 and Rv2031c 17, 28, 32–34 could be confirmed, validating this approach (Fig. 3A and B). Results for CFSE-labeled PBMCs from all 15 PPD+ donors in response to PPD, Mtb DosR-regulon-encoded proteins Rv1733c, Rv2029c and Rv2031c and Ag85B protein and all respective single peptides from each of the four antigens are given in Fig. 4A and B, showing comprehensive epitope maps for CD4+ (Fig. 4A) or CD8+ (Fig. 4B) T cells.

Next, we set out to determine the phenotypic characteristics of NKG2C+CD56dim NK cells from the present patient cohort. Because our data suggested that expansion of NKG2C+ NK cells was dependent on HCMV infection, we choose to perform an aggregate analysis of NKG2C+ NK cells in patients with HCV and HBV. Significantly fewer click here NKG2C+ CD56dim NK cells expressed NKG2A, CD161, Siglec-9, and NKp30 compared with NKG2C− CD56dim NK cells (Fig. 1C). In contrast, NKG2C+

NK cells more commonly expressed ILT2, CD57, and CD2. Percentages or MFI of CD62L, CD8, NKG2D, CD16, and DNAM-1-positive cells were indistinguishable when comparing NKG2C+ and NKG2C− NK-cell subsets (Fig. 1C and Supporting Information 2). The expression pattern of cytolytic molecules in the granules of CD56dim NK cells revealed that both Granzyme A and perforin were expressed at equivalent levels in NKG2C+ and NKG2C− NK cell subsets. In contrast, expression of Granzyme B was higher and Granzyme K lower in NKG2C+, compared with NKG2C− NK cells (Fig. 1D). Importantly, the phenotype of NKG2C+ NK cells was identical in HCV- and HBV-infected individuals (data not shown). Together, these data show that NKG2C+ NK cells have a full cytolytic arsenal and a highly differentiated phenotype, as defined by the

high expression of CD57. To examine the functionality of the NK cells and its relation to their expression of NKG2C, we separated them into three subsets: NKG2A+NKG2C−, NKG2A−NKG2C−, and NKG2A−NKG2C+ NK cells. We simultaneously assessed these subsets Dactolisib in the presence of various target cells for multiple functional responses. NKG2C+NKG2A− NK cells derived from patients with HBV or HCV infection displayed

stronger and more diverse functional responses than NKG2C− NK subsets following stimulation with targets expressing HLA-E, and against RAJI cells in the presence of anti-CD20 mAb (Fig. Etomidate 2A). In agreement with the prominent role for NKG2A in NK cell education 8, 29, NKG2A+ NK cells responded better than NKG2A− NK cells, regardless of their NKG2C expression, against both MHC class-I-negative K562 and 721.221 target cells. Furthermore, NKG2A+ NK cells produced high levels of IFN-γ in response to stimulation with IL-12/IL-18 (Fig. 2B), while IFN-γ production was almost undetectable in the NKG2C+CD56dim subset. Together, these results demonstrate that NKG2C+ NK cells display a functional profile similar to highly differentiated NK cells, shown to have a high responsiveness via ADCC but poor ability to respond to exogenous cytokines 30, 31. Extending previous results, we here show that differentiated NKG2C+ NK cells are polyfunctional and respond strongly to specific stimulation by HLA-E expressing target cells. Of note, NKG2C+ NK cells were also present in the liver (Supporting Information 3A). NKG2C+ NK cells in the liver were mostly NKG2A− and responded to stimulation with HLA-E expressing 721.221 target cells but not against control 721.

In many disorders resulting from a lack of iron, hemoglobin synthesis is deeply suppressed, resulting Mitomycin C order in iron-deficient anemia (IDA). IDA is characterized by small erythrocytes (microcytic) that contain less hemoglobin (hypochromic). IDA is mainly caused by a low dietary intake of iron, but can also be caused by chronic intestinal hemorrhage associated with hookworm infestation or by vitamin A deficiency, which is critical for iron metabolism. Both are common in

developing countries 1. Nearly half of the children living in developing countries are estimated to suffer from IDA; twice the number in industrialized countries. Iron deficiency adversely affects cognitive performance, behavior and physical growth, and IDA patients experience impaired gastrointestinal function and altered patterns of hormone production and metabolism 1. Moreover, morbidity

due to infectious diseases is increased in iron-deficient populations because of its adverse effects on the immune system HM781-36B 1, 2. Based on this, the World Health Organization recommends iron supplementation for children and pregnant women to treat IDA. Malaria is still a major health problem, resulting in more than 200 million infections and around a million deaths annually 3. Almost all victims of malaria are children under 5 years of age living in sub-Saharan Africa 3, whose geographical and age distribution completely overlap those of IDA. Thus, the coexistence of IDA and malaria seems common, and IDA may modulate the course of malaria. In Kenya, however, clinical malaria is significantly less frequent among iron-deficient children 4. In infants from Papua New Guinea, iron supplementation increased the prevalence of parasitemia 5. In the largest study, involving Zanzibari children, routine supplementation with iron and folate was found to increase the risk of severe malaria and death 6. Taken together, these findings suggest that routine supplementation with iron, or iron plus folate, increases childhood morbidity and mortality from malaria. Recently, one study assessed the effect of iron supplementation on the intermittent preventive treatment of malaria 7;

however, the mechanisms involved are still not fully understood. Here, we addressed the mechanisms underlying decreased susceptibility to malaria in IDA individuals crotamiton using a mouse malaria model. We found that macrophages preferentially sensed and engulfed parasitized erythrocytes from IDA mice, resulting in rapid clearance of the parasite from the circulation. One possible reason for this rapid clearance may be increased phosphatidylserine (PS) exposure at the outer leaflet of parasitized IDA erythrocytes. C57BL/6 mice were fed with a chemically defined iron-deficient diet to mimic IDA, the most prevalent form of anemia observed in endemic areas of malaria. The effect of this diet on hematopoiesis was assessed by measuring a number of hematological variables (Table 1).

In cases with diffuse traumatic axonal injury the microglial reaction is particularly pronounced in the white matter. These results demonstrate that prolonged microglial activation is a feature of traumatic brain injury, but that the neuroinflammatory response returns to control levels after several years. “
“Cerebral selleck products amyloid angiopathy (CAA) predisposes to symptomatic intracerebral hemorrhage (sICH) after combined thrombolytic and anticoagulant treatment of acute myocardial infarction. However, the role

of CAA in stroke thrombolysis has not been established. Here, we describe a confirmed case of CAA-related hemorrhage in a patient receiving thrombolysis for acute ischemic stroke. On autopsy, immunohistochemistry revealed amyloid-β positive staining in thickened cortical and meningeal arteries at sites of hemorrhage. Further research is urgently needed to determine

the hemorrhage risk related to CAA in stroke thrombolysis and develop better diagnostic tools to identify CAA in the emergency room. “
“Sphingosine-1-phosphate receptor (S1PR) modulating therapies are currently in the clinic or undergoing investigation for multiple sclerosis (MS) INCB018424 price treatment. However, the expression of S1PRs is still unclear in the central nervous system under normal conditions and during neuroinflammation. Using immunohistochemistry we examined tissues from both grey and white matter MS lesions for sphingosine-1-phosphate receptor 1 (S1P1) and 5 (S1P5) expression. Tissues from Alzheimer’s disease (AD) cases were also examined. S1P1 expression was restricted to astrocytes and endothelial cells in control tissues and a decrease in endothelial cell expression was found in white matter MS lesions. In grey matter MS lesions, astrocyte expression was lost in active lesions, while in quiescent lesions it was restored to normal expression levels. CNPase Atezolizumab cost colocalization studies demonstrated S1P5

expression on myelin and both were reduced in demyelinated lesions. In AD tissues we found no difference in S1P1 expression. These data demonstrate a differential modulation of S1PRs in MS lesions, which may have an impact on S1PR-directed therapies. “
“C. Billingham, M. R. Powell, K. A. Jenner, D. A. Johnston, M. Gatherer, J. A. R. Nicoll and D. Boche (2013) Neuropathology and Applied Neurobiology39, 243–255 Rat astrocytic tumour cells are associated with an anti-inflammatory microglial phenotype in an organotypic model Aim: Microglia form a high proportion of cells in glial tumours but their role in supporting or inhibiting tumour growth is unclear. Here we describe the establishment of an in vitro model to investigate their role in astrocytomas. Methods: Rat hippocampal slices were prepared and, after 7 days to allow microglia to become quiescent, rat C6 astrocytic tumour cells were added.

Heligmosomoides polygyrus antigens variably activated NF-κB

of MLN cells of uninfected and infected mice. The growing activity of p50 was observed after infection, and additionally, complete antigen and F9 enhanced p50 activity in the cytoplasm and nucleus; p50 as a homodimer is a repressor of κB site transcription in the cytoplasm [41], but it also participates in target gene transactivation by forming heterodimers with p65 [42]. In our studies, the level of NF-κB subunits, for example, p50 and p65 both in the cytoplasm and nuclei were distinct, and elevated activity of p50 was observed both in the cytoplasm and nuclei. After infection and after restimulation with H. polygyrus antigen fractions, expression learn more of p65 in cytoplasm rather decreased. As heterodimer p50/p65 translocated, increasing

activity of p50 was observed in nucleus both after infection and also when cells were treated with the nematode antigens. F9 like F17 up-regulated p50 and F17 strongly reduced activation of p65 in nucleus. Activation of NF-κB is essential for both cell proliferation and resistance of cells to apoptosis [43]. Infection with filarial parasites transitorily activated the factor with degradation of the cytoplasmic inhibitor protein IκΒ, and ES-62, a molecule secreted by filarial nematodes selectively blocks signal transduction events including NF-κB activation [44, 45]. The mechanism by which the parasite triggers and regulates activation of NF-κB is unspecified [46] and more detailed studies with recognition of receptor pathway induction with separate molecules CP-690550 manufacturer of H. polygyrus antigens might be promising. Selectively enhanced p50 activity and antiapoptotic activity of antigen fractions support an observation that cells might be hyporesponsive. DEX-induced apoptosis also requires protein synthesis

[47]. Heligmosomoides polygyrus-originated factors may inhibit apoptosis inducing enzymatic pathway, which depends on glucocorticoid Methane monooxygenase receptor or that regulates the activity of NF-κB [48] and are potent to restore activation of protein kinase pathways and support survival of the cells. Our study identified H. polygyrus antigen factors with potential activity for regulation of surviving T-cell populations. These fractions can simultaneously target c-FLIP, Bcl-2 expression and increase p50 activity in mice infected with the parasite. Heligmosomoides polygyrus antigens contain many different proteins, which may have distinct activity in apoptosis. The content of protein fractions was compared with H. polygyrus-secreted proteins [13]. As we used somatic homogenate of adult stages, identified proteins were representative for cytoskeleton and metabolic pathways. There were also proteins which are specific for each fraction. The differences between F9, F13 and F17 fractions are their distinct antiapoptotic activity to different cell populations and also with distinct activation of NF-κB subunits.

CFSE labeling (1 μM) and flow cytometry analysis were performed as previously described [30]. We thank Stephen Cobbold for the kind gift of YTS 177.4 antibody, Corinne Cordier and Jérôme

Mégret for cell sorting. This work was supported by the Association Française contre les Myopathies and the Agence Nationale de la Recherche (ANR-11-JSV3). R428 cell line M. Carpentier, P. Chappert, and M. Lalfer were supported by the French Ministry of Research. C. Kuhn was supported by the Fondation pour la Recherche Médicale (FRM). The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. “
“Interleukin-21 (IL-21) exerts critical functions in T helper type 17 (Th17) cell development. However, the effect of IL-21 on the differentiation of IL-22-producing T cells is not clear. Here we showed that IL-21 induced the differentiation of human naive CD8+ T cells into Tc22 cells without the expression of IL-17. The addition of transforming growth factor-β inhibited the production of IL-22 but induced the production of IL-17. Both IL-15 and IL-2 induced interferon-γ production

but did not induce differentiation of Tc22, which suggests that common γ-chain signals are not specific to promote IL-22 synthesis. The IL-21 induced naive CD8+ Fulvestrant supplier T cells to produce IL-22 in greater amounts than memory CD8+ T cells. In addition, we demonstrated that IL-21 promoted the proliferation and increased the expression of IL-21 receptors on activated naive CD8+ T cells. Furthermore, IL-21 increased the expression of granzyme B molecules. Analysis of molecular mechanisms indicated that IL-21 induced phosphorylation of signal transducers and activators of transcription 1, 3 and 5 in CD8+ T cells. Overall, our data indicated that IL-21, Anacetrapib an effector cytokine produced by CD4+ T cells,

might mediate the cross-talk between CD4+ and CD8+ T cells through the production of IL-22. Interleukin 21 (IL-21) is a recently identified member of the common γ-chain (γc) -signalling family of cytokines that includes IL-2, IL-7 and IL-15.1 Interleukin-21 is an effector cytokine that is produced by various T helper cell subsets, including T follicular helper cells, T helper type 17 (Th17), Th2 and Th1 cells, and natural killer T cells.2,3 The functional IL-21 receptor consists of IL-21R and γc IL-21R is expressed on T cells, B cells, natural killer cells, dendritic cells, macrophages and epithelial cells, indicating roles of IL-21 in both innate and adaptive immune responses.4 Interleukin-21 signals via the janus kinase–signal transducers and activators of transcription (JAK-STAT) pathway.

With

this report, we present a strategy for highly resolved mapping of serological specificities that allows assessing the range and specificities of immune responses to E. granulosus and, at the same time, to identify specific antigens. This strategy joins immunoblot immune screening with proteome technologies involving 2-DE-PAGE and mass spectrometry for the identification of the antigens. A comparison of the specificity patterns of sera from patients with different stage of the disease reveals great differences in the antigens targeted during development of CE infection. The identified HSP20 belongs to the family of highly Venetoclax conserved small HSPs that function as molecular chaperones and, preventing stress, induce aggregation of partially denatured proteins and promote their return

to native conformations when favourable conditions pertain (14). During transmission, E. granulosus undergoes a drastic change of environmental factors from the ambient temperature to higher temperature in the mammalian host. Given these circumstances, HSPs, in Echinococcus, play essential roles in the host–pathogen interaction. In theory, the unmistakable resemblance of parasitic HSPs to host homologous HSPs might render them identifiable to the immune system as self, thus obviating a response and providing a good example of ‘antigen mimicry’. AUY-922 Our results indicate that in CE, this tolerance does not occur and HSP20 derived from E. granulosus act as classical foreign antigen, and elicit immune response as several parasite HSPs. We have previously characterised Eg2HSP70 as an antigenic molecule inducing both B- and T-cell responses (15). Chemale et al. (2003) identified by proteomic analysis members of the heat shock protein family, HSP70 and HSP 20, in protoscoleces of E. granulosus (10). More

recently, Montero et al. (11) identified a HSP20-related protein among the intracellular proteins found in bovine hydatid fluid of E. granulosus. Serum derived from mice Sucrase infected with E. multilocularis also recognised putative HSP20-related protein, suggesting the potential of this protein as immunodiagnostic or vaccine candidate for alveolar echinococcosis infection (16). Our results here extend the current knowledge about the possible role of HSPs in the induction or modulation of the host immune response, and assign to HSP20 a crucial function in the host–parasite relationship. In particular, in this study, we observe that HSP20 induces a strong host immune response in the early stages of E. granulosus development (active disease) and a weak or undetectable host immune response in advanced stages of the disease (inactive disease).

To this end we used an NF-κB inhibitor (Bay11) and the mTOR inhibitor rapamycin. TLR-triggered IL-10 production was significantly reduced after treatment with Bay11 or rapamycin alone and nearly absent after combined inhibitor usage (Fig. 5C). As expected for NF-κB inhibition, TLR2/4-induced TNF and IL-12 secretion levels were decreased under Bay11 treatment, but only TNF production remained unaffected by rapamycin, thus, confirming its selective regulation via NF-κB (Supporting Information Fig. 2D). Altogether, these findings suggested

a possible involvement of the PKB/Akt and p38 MAPK pathways in LPS-induced IL-10 regulation learn more and provided the notion that IRAK4 might serve as a differential regulator of PKB/Akt and/or p38 EPZ-6438 clinical trial MAPK signaling and could thereby determine the IL-10/IL-12 ratio. Furthermore, IL-10 secretion is partially dependent on NF-κB, but is additionally driven by the PKB/Akt/mTOR pathway in an NF-κB-independent manner.

Based on these results we subsequently focused on the PKB/Akt pathway. Analysis of mRNA expression by quantitative real time RT-PCR showed that expression of IL-10 in response to LPS stimulation is markedly reduced in the presence of rapamycin, Akt inhibitor or wortmannin (Fig. 6A). This indicated that interference with PI3K/PKB/Akt/mTOR signaling negatively regulates- IL-10 synthesis at a transcriptional level. Confirming our hypothesis, western blot analysis demonstrated increased phosphorylation of the Akt kinase on Thr308 in IRAK4-silenced monocytes PD184352 (CI-1040) stimulated with LPS (Fig. 6B). This effect was specific as this was not observed under MyD88 knockdown conditions, which, by contrast, decreased phospho-Akt levels to those measured in unstimulated cells (Fig. 6B). Thus, this experiment

highlighted the selective role of IRAK4 in the quantitative regulation of PKB/Akt activation. Also in line with these findings we detected enhanced phosphorylation of the PKB/Akt-mTOR-dependent transcription factor FoxO3a in IRAK4-silenced monocytes (Fig. 6C). As a last step we wanted to assess the functional impact of IRAK4-silencing on T-cell responses. To this end we used co-cultures of monocytes and allogenic CD8+ or CD4+ T cells. The results demonstrated that IRAK4-silenced monocytes represent weaker inducers of CD8+ as well as CD4+ T-cell proliferation than monocytes transfected with control siRNA (Fig. 7A). Notably, flow cytometric analysis of expression of monocyte activation markers, for example, CD14, CD80, CD86, PDL-1, MHCII, and ICOS-L was not affected by IRAK4 knockdown (not shown). But, suppressive monocyte function was found to be IL-10-dependent, as full T-cell stimulatory capacity was restored via neutralization of IL-10 in the co-cultures (Fig. 7B).