Early indications from clinical studies suggest vitamin D treatment of patients enhances T-cell expression of IL-10 in vivo, although data on the impact on Foxp3+ Treg cell frequencies in human peripheral blood are less clear [12, 23-26]. Here, we demonstrate that the active form of vitamin D3 increases the frequency of both IL-10+ and Foxp3+ cells

in cultures of human peripheral blood derived CD4+ T cells. The two Treg cell subsets promoted by 1α25VitD3 are distinct cell populations that are optimally induced by different concentrations of 1α25VitD3 in culture. Both Foxp3+ and IL-10+ 1α25VitD3-promoted T cells exhibited comparable regulatory activity in a conventional in vitro suppression assay. However, more than one inhibitory mechanism appears to exist. Inhibition by T cells generated under Saracatinib conditions that optimally promoted IL-10 was reversed upon addition of an antibody that blocked IL-10 signaling to the co-culture suppression assay. In contrast, the suppressive activity of Foxp3+ cells, generated in the presence of high-dose 1α25VitD3, was not reversed by neutralization of IL-10. A number of additional mechanisms of suppression by Foxp3+ Treg cells have been reported [27]. To investigate how vitamin D modulates the frequency of Foxp3+

cells in culture, initial studies focused on the capacity of 1α25VitD3 to maintain expression of Foxp3 by existing Treg cells. 1α25VitD3 maintained the levels of Foxp3 expression in human CD4+CD25high Treg cells, which otherwise were selleck compound lost upon in vitro culture. This observation was reproduced

using Foxp3GFP CD4+ cells from reporter mice. Using the CellTrace together with Foxp3 staining, we further demonstrated that 1α25VitD3 allowed the preferential expansion of Foxp3+ T cells over Foxp3− (effector) T cells and this could provide a contributory or additional mechanism by which 1α25VitD3 promotes Foxp3+ Treg cells. These data, together with earlier studies suggesting that vitamin D increases Foxp3 expression in human naïve T-cell cultures [10, 28], indicate that vitamin D acts through the several different mechanisms to enhance Foxp3 expression. IL-2 plays a central role in the maintenance of a functional Treg cell compartment [29, 30]. Interestingly, our data suggest that one mechanism by which 1α25VitD3 may act to maintain Treg cells is via the observed increased expression of the alpha chain of the IL-2 receptor, CD25, and this could be relevant to all of the pathways proposed above. An unprecedented finding of the present study is the reciprocal regulation of Foxp3 and IL-10 by 1α25VitD3. The phenotype of the Treg cell population generated is likely to depend not only upon the level of vitamin D available, but also the local cytokine milieu.

Interestingly, using tetramers with enhanced CD8 binding (CD8hi) revealed cross-reactivity for the Flu-NA peptide. This poor-quality response ABT-737 supplier is therefore measurable, although functionally the Flu-NA peptide was unable to trigger IFN-γ release. In further experiments it was possible to enhance the sensitivity of the T cell response by using a modified peptide derived from genotype 4. Here, increased sensitivity to peptide was accompanied by loss of dependence on CD8 for binding (i.e. binding of a CD8 null tetramer). Thus, overall,

this examination in detail of a case of heterologous reactivity has revealed some of the limits of T cell cross-reactivity and its dependence on T cell sensitivity. The ability to define T cell sensitivity readily using polyclonal responses independently of function may allow further examination of the importance of heterologous immunity in man. Advances in

understanding of the basic biology of TCR interactions with pMHCI have led to the development of new tools and assays for determining the quality of the T cell response. Conceptually, the presence of highly sensitive Talazoparib in vitro T cells should be of benefit in control of viral infections, although the twin threats of immune escape and immune exhaustion act to diminish the power of anti-viral responses. However, although there are some data to support the model that TCR avidity is a key determinant SPTLC1 of outcome, a casual link is not established fully. We suggest that there are two models which might be considered in trying confirm such a link (see Fig. 6). On one hand, different individuals may mount responses of different quality for the same epitope (depending upon a number of factors including site, duration and dose of antigen, as well

as host genetics). The variation in such responses might be linked to the suppression of viraemia or the induction of immune escape (‘private avidity’). Alternatively, all individuals may make responses of similar quality against specific epitopes, i.e. the quality of the response is essentially a fixed property of the epitope (‘public avidity’). In this case, the overall picture will be determined by the choice of epitopes available to the individual, which is driven in turn largely by MHC. In this respect, the overall role of TCR avidity in determining the striking protective effect of HLA B27 and B57 in the outcome of both HIV and HCV has not yet been explained fully. However, it has been suggested that avidity plays some role [9]. Overall, we have a large number of new tools at our disposal to dissect further the impact of changes in TCR avidity or quality on the outcome of virus infection. Further work is required in man, using carefully defined clinical cohorts studied ideally from acute infection onwards.

These tasks are fulfilled by Treg cells and so-called tissue signaling leukocytes, respectively (reviewed in [43]). In addition, the specificity of bystander Th cells is still unclear, but it seems at least in allergen-specific eczema a substantial proportion, in particular of Th17 cells, is specific for staphylococcal antigens [12, 29] rather than for the eliciting allergen [8, 36]. Furthermore, increasing evidence exists that Th cells recognizing autoantigens may differentiate during the immune reactions in atopic eczema [44], lupus

erythematosis [45], or psoriasis [46]. It can be hypothesized that these autoreactive Th cells migrate into the tissue as bystander cells, encounter their antigen and serve as amplifiers Lumacaftor in vitro of inflammation. In summary, recruitment of antigen-specific Th cells into tissues initiates a cascade of immune events in the skin that is mediated by the majority of bystander T cells that in parallel migrate to the site of inflammation. Once a Th cell reaches its target organ and

is fully activated, it exerts its function via cell contact dependent mechanisms as well as secretion this website of soluble mediators such as chemokines and cytokines. Roughly, T-cell functions in inflamed tissue are (i) inflammation aimed at clearing the potentially harmful antigen, (ii) limitation of the immune response to prevent a cytokine storm with massive collateral tissue damage, and (iii) regeneration of tissue homeostasis after inflammation. Importantly, all three functional arms have to be in homeostasis,

as imbalance of any of these may have negative outcomes (Fig. 2). A simplified view to functionally categorize Th cells would be that IFN-γ-, TNF-α-, and IL-17-producing subtypes are mainly inflammatory, IL-10- and TGF-β-producing T cells are mainly limiting, PAK6 and IL-22 secretion is mainly associated with coordinating regeneration (Fig. 1). However, most cytokines have overlapping functions and are not exclusively attributable to the aforementioned functions. Furthermore, the function of a single cytokine critically depends on the context of the local microenvironment. Much progress has been made in understanding T-cell functions in a disease-specific context. This can be exemplified by three model diseases: psoriasis, atopic eczema and ACD that will be discussed separately in the following section. The pathogenesis of psoriasis is dominated by the Th17 cytokines IL-17, IL-21, IL-22, and TNF-α [30, 47-50]. IL-17 and IL-22 [51] as well as IL-22 and TNF-α [4, 52] co-operatively induce the secretion of antimicrobial peptides by epithelial cells such as human beta defensin 2 and S100 proteins, which prevent microbial colonization. Overrepresentation of IL-22 turns its positive role in tissue regeneration into a pathologic one through the induction of acanthosis, or thickening of the skin [53]. IL-21 has been shown to co-operate with IFN-γ in inducing epidermal hyperplasia [54].

Flow cytometry data were collected and analysed using CellQuest software. As IL-10R1 labelling displays with monophasic distribution, the data are presented as the mean fluorescence intensity (MFI) within each cell subset. For the detection of IL-10R signals after IL-10 stimulation, PBMCs were isolated from 10 ml of venous blood by Ficoll-Hypaque (TianJin Hao Yang Biological Manufacture Co., TianJin, China) density gradient centrifugation. Cell viability was determined, and cells were adjusted to 5 × 105 cells/ml in HyClone RPMI-1640 culture medium with l-glutamine (Thermo Fisher Scientific, Waltham, MA, USA), supplemented

with 10% heat-inactivated fetal calf serum (TianJin Hao Yang Biological Manufacture Co.). After culture at 37°C in a humidified 5% CO2 atmosphere for 1 h, cells were stimulated with recombinant buy NVP-LDE225 human IL-10 (Spodoptera frugiperda, Sf 21-derived; R&D Systems, Minneapolis, MN, USA), followed by phosphorylation analysis by flow cytometry. For dose–response experiments, cells were stimulated with increasing doses of recombinant human IL-10 (rhIL-10) (2, 5, 10, 20 and 40 ng/ml). For time–courses, PBMCs were stimulated with rhIL-10 (10 ng/ml) or left unstimulated and collected at different

times (5, 15 or 30 min). Phosphorylation of STAT-1 and STAT-3 Roxadustat mouse was detected by flow cytometry according to the manufacturer’s protocol (BDTM Phosflow protocol III for human PBMC). The

following antibodies were used: AlexaFluor 488 mouse anti-pSTAT1 (pY701), clone: 4a; AlexaFluor 647 mouse anti-pSTAT3 (pY705), clone: 4/P-STAT3; and mouse IgG2a, mouse IgG1 isotype. Flow cytometry analysis was performed using a BD FACSCalibur cytometer (BD Biosciences). Flow cytometry data were collected in list mode and analysed using CellQuest software. To determine the cytokine profiles of SLE patients and controls, we detected several Th1/Th2 [interferon (IFN)-γ, IL-2, IL-6 and IL-10] cytokines simultaneously in the plasma of SLE patients and healthy controls using flow cytometric bead array (CBA) techniques. The human enhanced sensitivity Flex Set system (BD Biosciences) was used. Briefly, following the preparation of standards and dilution of the individual plasma samples, mixed capture selleckchem beads were incubated with the standards or plasma for 2 h, and then with added detection reagent for another 2 h. After washing the tubes, the enhanced sensitivity detection reagent was added and incubation was continued for an additional 1 h. After washing the tubes again, samples were analysed by a FACSAria cytometer (BD Biosciences) and data were analysed using FCAP Array software. Statistical analysis was performed using spss version 13·0 software. The MFIs of IL-10R1 expression levels were expressed as mean ± standard deviation (s.d.

Previously, it was shown that coculture of BMECs with astrocytes directly affects the maintenance of BBB function and is necessary for its tightness (Tao-Cheng & Brightman, 1988; Holash et al., 1993). Only limited numbers click here of pathogens are capable of penetrating physiologically impermeable biological barriers such as the BBB and the placenta. BMECs seem to be the primary site of pathogen traversal into the CNS. Pathogens may disrupt the BBB and traverse into the CNS via transcellular penetration, paracellular entry, and/or transmigration with infected leukocytes (‘Trojan horse’ mechanism) (Fig. 2). In the further part of this review, we have focused on transcellular and paracellular traversal of the microorganisms. Transcellular

passage involves penetration of the pathogens through

the BMECs. This pathway is initiated by adherence of the pathogen to the ECs leading to the entry of bacterium into the CNS across the BBB using pinocytosis or receptor-mediated mechanisms. Remarkably, some pathogens are able to mimic natural host ligand–receptor interactions that could facilitate interaction between ECs and microorganisms. Transcellular traversal of the BBB has been demonstrated for Escherichia coli (Kim, 2000), Group B Streptococcus (Nizet et al., 1997), Listeria monocytogenes (Greiffenberg et al., 1998), Mycobacterium tuberculosis (Jain et al., 2006), Citrobacter freundii (Badger et al., 1999), Haemophilus influenzae (Orihuela et al., 2009), Streptococcus pneumoniae (Ring et al., 1998), and Candida albicans (Jong et al., 2001). The paracellular route is defined as microbial infiltration between barrier cells. This traversal involves loosening of the TGF-beta inhibitor TJs or disturbing the supporting components of TJs, i.e. basement membrane and glial cells (Tuomanen, 1996). The paracellular transmigration of the BBB has been suggested for the Trypanosoma (Grab et al., 2004) and Treponema pallidum (Haake & Lovett, 1994). Either the transcellular and/or

the paracellular route may serve as possible modes of amoebae entry into the CNS (Khan, 2007). Both routes have also been suggested for Cryptococcus neoformans (Chang et al., 2004; Charlier et al., 2005), Neisseria meningitidis (Nassif et al., 2002; Coureuil et al., 2009), and Lyme disease buy Dolutegravir pathogen Borrelia burgdorferi (Comstock & Thomas, 1991). In addition, phagocyte-facilitated entry into the CNS using Trojan horse mechanisms has been suggested for L. monocytogenes and M. tuberculosis (Drevets et al., 2004; Join-Lambert et al., 2005). Transcellular migration mediated by adhesion is described without any evidence of microorganisms between the cells or of intercellular tight-junction destruction. On the other hand, paracellular penetration is characterized with and/or without evidence of tight-junction disruption. Because in vivo experiments in humans are difficult or impossible, suitable in vitro models of the BBB are essential to understand how pathogen crosses the human BBB.

Ratios and fold upregulation was compared to 1 and statistical significance was calculated by a Wilcoxon-signed rank test. Cytokine data between groups were compared using a Mann–Whitney U-test. Data shown are representative of two or more independent experiments, performed in duplicate, with n = 5. Graph-Pad Prism 4.0 was used for statistics

this website (GraphPad Software). Values of p < 0.05 were considered statistically significant. Data are given as mean ± SEM. The authors would like to thank Dr. R. de Swart for providing RSV A2, Dr. F. van Kuppeveld for providing HRV-14, Prof. R. Fouchier for providing H1N1, and Dr. J. Murk for providing Reo-3 and HAdV-3. We are also very grateful to Prof. M. Netea and Dr. J. Heldens for their critical comments on the manuscript. M. Vissers and G. Ferwerda are financially supported by the VIRGO consortium, which is approved and financially supported by the Netherlands Genomics Initiative (NGI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no financial or commercial conflict of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. "
“Acute graft-versus-host disease (GVHD) is the most important cause of mortality after allogeneic haematopoietic stem cell transplantation. LY2157299 clinical trial Allo-reactive

T cells are the major mediators of GVHD and the process is regulated by positive and negative regulators on antigen-presenting

cells (APC). Because the significance of negative regulators in GVHD pathogenesis is not fully understood, and having discovered that syndecan-4 (SD-4) on effector T cells mediates the inhibitory function of DC-HIL on APC, we proposed that SD-4 negatively regulates the T-cell response to allo-stimulation in acute GVHD, using SD-4 knockout mice. Although not different from their wild-type counterparts why in responsiveness to anti-CD3 stimulation, SD-4−/− T cells lost the capacity to mediate the inhibitory function of DC-HIL and were hyper-reactive to allogeneic APC. Moreover, infusion of SD-4−/− T cells into sub-lethally γ-irradiated allogeneic mice worsened mortality, with hyper-proliferation of infused T cells in recipients. Although there my be little or no involvement of regulatory T cells in this model because SD-4 deletion had no deleterious effect on T-cell-suppressive activity compared with SD-4+/+ regulatory T cells. We conclude that SD-4, as the T-cell ligand of DC-HIL, is a potent inhibitor of allo-reactive T cells responsible for GVHD and a potentially useful target for treating this disease. Allogeneic haematopoietic stem cell transplantation (HSCT) is a potentially curative option for patients with high-risk haematological malignancies, such as multiple myeloma and leukaemia.

Interestingly, in the present study, the VLP internalization mechanism was observed to be different for NK cells: VLPs entered rapidly within large macropinocytosis vacuoles, independently of the clathrin and caveolae pathways (Figs. 4 and 5). RhoGTPase assays suggest the involvement of filopodia during Volasertib cell line VLP uptake (activation of the Cdc42, Fig. 5B) and a concurrent reduction of lamellipodia (inhibition of Rac1 Fig. 5C) 34, as observed by electron microscopy (Fig. 4G and H), and not membrane blebbing, described for host-cell entry of other viruses 24. Interaction of NK cells with VLPs was correlated with CD16 expression and experiments

with CD16 blocking antibody or co-immunoprecipitation confirmed the importance of this receptor for this interaction (Fig. 6A–F). Moreover, VLP internalization induced transient down-modulation of CD16, but no change in NKp46 expression, a receptor involved in Newcastle disease virus binding 35 (data not shown). Our findings are in agreement with those showing that binding of HPV–VLPs is mediated by CD16 on DCs 36 and that uptake of HPV–VLPs by DCs from FcγRIII-deficient mice is strongly reduced compared with wild-type mice 17. CD16 has been shown to be involved in macropinocytosis in AP24534 molecular weight macrophages 37 and in γδ T cells 38. Moreover, transduction of CD16 into a CD8+ T-cell clone was sufficient to increase HPV–VLP entry into these cells (Supporting

Information Fig. 5B).

To exclude an interaction with CD16 mediated by antibodies, we checked the absence of antibodies reacting against VLPs in the human and bovine sera used in culture medium (data not shown). We also performed some experiments without serum and obtained similar results (data not shown). NK cells play a key role in immune responses by exocytosis of cytotoxic granules, and CD16 is a major receptor capable of triggering NK cytotoxicity 21. We showed that VLPs induced cytotoxic activity of NK cells expressing CD16 (Figs. 2A–C and 7A, B). In addition Thymidine kinase to killing infected cells, this process could liberate granulysin, present in cytotoxic granules, which works as an alarmin and activates DCs 39. Besides this degranulation activity, through binding of CD16, NK cells are able to activate adaptive immune responses by the secretion of soluble factors such as IFN-γ and TNF-α 40. We showed that VLP stimulation induced the secretion of these cytokines in NK and NK92 CD16+ cells but not in NK92 CD16− cells (Fig. 7). VLPs were produced in insect cells infected with baculovirus coding for HPV16 L1. Because insect baculovirus contaminants have been reported to play a role in the immunogenicity induced by VLPs 41, we used a lysate of insect cells infected with WT baculovirus as a negative control and did not observe cytotoxic activity or cytokine production in this culture condition.