In the murine system, immune synapses can be analyzed in vitro us

In the murine system, immune synapses can be analyzed in vitro using TCR transgenic

T cells and supported planar bilayers (e.g. 2, 18), or APCs loaded with the cognate antigen Histone Methyltransferase inhibitor 19–21. Intravital microscopy in mice even allows analysis of T-cell/APC contacts in their physiological environment, e.g. in LNs 22–24. However, with regard to L-plastin regulation and function, clear differences exist between mice and human. Thus, the activation-induced upregulation of the surface receptors CD25 and CD69 is dependent on L-plastin phosphorylation in human T cells 8, but expression of this protein seems to be dispensable for activation-induced CD69 upregulation in murine T cells 25. The analysis of immune synapses of T cells of human origin has also been performed in Jurkat T-lymphoma cells 26–28. However, Jurkat T cells display strong distinctions from primary human T cells in their signaling pathways. For example, PI3K is a downstream effector of Ras in primary human T cells 29,

but not in Jurkat T cells 29, 30, which might, e.g., be due to a defective expression of the phosphatase PTEN in Jurkat T cells 31. Therefore, all experiments presented here were performed with primary human T cells. This research is challenging because primary human T cells represent a heterogeneous cell population in which only a small fraction of cells reacts on a given antigen, which limits the number of T-cell/APC couples to be evaluated 5, 26, 32, 33. Recent proceedings in high-throughput imaging significantly improved the statistical

evaluation click here of receptor clustering in the immune synapse of primary T cells 5, 16. Using such a high-throughput ImageStream™, we show here for the first time that dexamethasone interferes with actin stabilization and prevents the formation of the immune synapse of untransformed human T cells. Immune synapse formation requires Urocanase a dynamic reorganization of the actin cytoskeleton. During the reorganization process, G-actin is polymerized to F-actin by the Arp2/3 complex. The existing actin fibers are then dynamically depolymerized by cofilin or stabilized by actin crosslinking or bundling proteins (for review, see 6, 17). Here, we show that the phosphorylation of the actin-bundling protein L-plastin on Ser5, which represents a costimulatory signal 8, 9, is inhibited by dexamethasone. Nevertheless, although it was known that glucocorticoids can modify the actin cytoskeleton in endometria cells 34 or stabilize F-actin in AtT-20 cells 35, the targets of glucocorticoids that are involved in actin cytoskeleton changes were unknown. The inhibition of L-plastin phosphorylation upon costimulation of primary human T cells is therefore the first known event that may explain the effects of dexamethasone on the actin cytoskeleton. In order to get fully activated, T cells require costimulation, i.e.

The molecular identification of clinical mucorales using the ITS

The molecular identification of clinical mucorales using the ITS region has been successfully demonstrated in recent years.[9, 14, 18, 19, 21, 22] However, ITS sequencing failed with the strains of

the genus Syncephalastrum. This is in concordance with Walther et al. [21] who reported that direct ITS sequencing could not be achieved in strains of genera Syncephalastrum and Absidia. Furthermore, S. racemosum isolates characterised by LSU region in this study revealed at least two distinct clades. Further studies based on the multilocus sequence typing may suggest different genotypes in S. racemosum strains. Therefore, the need of detailed taxonomic studies for this genus can hardly be emphasised. The problem of overlapping click here of S. racemosum with other species of Syncephalastrum was also pointed out by Vitale et al. [14]. Notably, the type strain of S. racemosum is not yet available. Rhizopus was the most common mucorales identified from mucormycosis cases

involving lungs, sinuses, cutaneous and other sites. Currently accepted Rhizopus species have been shown to be well recognisable in the ITS tree.[18] The three strains of R. stolonifer in the present study originated from two cases of cutaneous and one from rhino-cerebral mucormycosis. Abe et al. [18] used genealogical concordance phylogenetic species recognition see more (GCPSR) to reclassify R. oryzae and proposed division of R. oryzae into R. arrhizus and R. delemar. The ITS tree in the present study clearly subdivided varieties

of R. arrhizus into two groups viz. R. arrhizus var. delemar in group 1 and R. arrhizus var. arrhizus in group 2. Furthermore, AFLP clearly revealed marked genotypic diversity within the Indian isolates of R. arrhizus and demarcated five distinct subgroups (group I–V), suggesting that AFLP could be explored in future studies to examine the relatedness of varieties within R. arrhizus isolates from different sources. In the present study 3.7% of cases of mucormycosis were due to Lichtheimia species which is in concurrence with Roden et al. [34] who reviewed 25 well documented cases of Lichtheimia and reported that 5% of the cases of mucormycosis are caused by this fungus. According to Alastruey-Izquierdo et al. [11] the genus Lichtheimia contains five species. Of Roflumilast these only L. corymbifera and L. ramosa have been reported from human infections. However, L. ramosa was more common in the previous studies and similar dominance of this species was observed in our settings.[11] The three isolates of L. ramosa identified in the present study originated from pulmonary (n = 2) and cutaneous (n = 1) mucormycosis cases. The previous studies based on sequence analysis of ITS, LSU, translation elongation factor 1α have established L. ramosa as separate species from L. corymbifera.[35, 36] Mucor is the polyphyletic genus and is the most clinically relevant genus after Rhizopus.

17–19 However, several studies suggest that nTreg do not universa

17–19 However, several studies suggest that nTreg do not universally suppress all T helper cell subsets to the same extent. In newborns, human thymus-derived nTreg strongly suppress Th1 cells but not Th2 cells, and similar properties have been ascribed to nTreg in mice.20,21 Additionally, nTreg isolated from peripheral human blood have been shown to strongly suppress the production and secretion of interferon-γ (IFN-γ), IL-2

and IL-4, but not that of IL-10, in an allogenic model.22 Thus, diurnal changes in the Th1/Th2 balance could also be find more regulated by the diurnal rhythm of nTreg-suppressive activity. We previously demonstrated that the suppression of CD4+ CD25− T-cell proliferation by nTreg followed a sleep-dependent rhythm.23 However, whether

this suppressive rhythm of nTreg affects the proliferation and cytokine secretion of Th1, Th2 and Th17 cells to the same extent is not yet clear. Furthermore, the signal-transduction mechanisms by which nTreg mediate their suppressive function in responder T cells (Tres) are largely unknown in humans. One possible mechanism of diurnal changes in the Th1/Th2/Th17 balance could be the hormonal priming of T cells and/or nTreg in vivo through HTS assay the diurnal secretion of hormones with known immunomodulatory effects, such as prolactin, growth hormone, cortisol, noradrenalin and melatonin.8,24–31 To address the vital question of whether nTreg or hormones regulate diurnal changes in the Th1/Th2/Th17 balance, and whether Th1, click here Th2 and Th17 cell activity follows a diurnal rhythm, we investigated the activity of the Th1/Th2/Th17 cells and their regulation by nTreg. We were able to demonstrate that nTreg suppressed IFN-γ, IL-2 and tumour necrosis factor-α (TNF-α), but not IL-4, IL-6, IL-10, or IL-17A. The suppression of IL-2 was reduced if nTreg-associated CD25 was inhibited. Highly purified nTreg secreted IL-6, IL-10 and IL-17, but not IL-2, IL-4, IFN-γ or TNF-α. Furthermore, we observed that secretion

of the cytokines IL-2, IFN-γ, TNF-α and IL-10 by naïve CD4+ T cells follows a diurnal rhythm. Multiple regression analysis, as well as subsequent in vitro experiments, suggested that serum levels of cortisol and prolactin contribute to the underlying mechanisms. Taken together, our findings imply that hormones and nTreg contribute to the diurnal secretion of cytokines from T helper cells. Cytokine secretion, and suppression of cytokine secretion by nTreg, was analyzed for Th1 (IFN-γ), Th2 (IL-4, IL-6) and Th17 (IL-17) cytokines, as well as for the cytokines IL-2, IL-10 and TNF-α. Furthermore, the proliferation of cytokine (IL-2, IL-4, IL-10, IL-17A, IFN-γ, TNF-α)-producing CD4+ CD25− Tres was investigated. For these analyses, T cells were isolated from blood samples taken from healthy male donors at 08:30 hr.

For example, activation of iNKT cells by administration of α-GalC

For example, activation of iNKT cells by administration of α-GalCer has been shown to protect against autoimmune diseases in IL-4- or IL-10-deficient mice.106,107 It has also been demonstrated that iNKT cells can prevent type I diabetes without driving a

Th2 shift in autopathogenic T cells.108 Thus, attention has focused on the role of iNKT cells in the induction of tolerizing or non-inflammatory NSC 683864 chemical structure DCs. At least three different pathways have been identified by which iNKT cells may promote the generation of regulatory DCs. These are illustrated in Fig. 2, and described in detail below. Repeated administration of cognate antigens can lead to an ‘exhaustion’ phenotype in MHC-restricted T cells, and a similar Ruxolitinib order effect appears to occur for iNKT cells with α-GalCer (Fig. 2a): after multiple exposures to α-GalCer in vivo, iNKT cells develop a functionally anergic phenotype that is associated with expression of the inhibitory receptor programmed death (PD)-1.109 When iNKT cells become exhausted in this way, their interactions with DCs change and instead of promoting the maturation of pro-inflammatory

DCs, they induce a regulatory DC phenotype that is characterized by lower expression levels of CD80, CD86 and CD40, with reduced IL-12 and increased IL-10 secretion.110,111 In autoimmune disease models, regulatory DCs that are generated through this pathway prevent the onset of autoimmunity and silence autopathogenic T cells.91,111 It is difficult to fully gauge the effects of self antigen-activated iNKT cells on DC phenotype in vivo; however, in vitro studies have suggested that this pathway can provide a maturation stimulus to immature DCs, but that the resulting DC phenotype is a comparatively non-inflammatory one (Fig. 2b). Vincent et al.65 showed that, in contrast to DCs that matured in response to α-GalCer-stimulated iNKT cells, those that matured in response to self antigen-activated iNKT cells showed up-regulation

of costimulatory Rho molecules such as CD86 but produced more IL-10 than IL-12. These DCs efficiently promoted T-cell proliferation, but did not stimulate marked T-cell IFN-γ production.65 DCs are known to develop from haematopoietic stem cells via multiple distinct differentiation pathways. Some develop directly into precursor DCs in the bone marrow, which then enter the bloodstream and continuously renew immature DC populations within the tissues.112 Other myeloid DCs arise from progenitors that reside in the periphery. Monocytes constitute one such precursor population. Every day about one-third of the blood monocytes are estimated to leave the bloodstream and enter the tissues.113,114 There, they can remain monocytic, become macrophages, or become DCs. Thus, understanding the types of signals that determine their choice of fate is an area of great interest.

Moreover, both morphotypes should be examined by strain biotyping

Moreover, both morphotypes should be examined by strain biotyping methods. Beta-N-hexosaminidase (HexNAcase) activity assessed by the api® ZYM test and on CHROMagar Candida® medium (Becton Dickinson, USA) is also discussed. “
“The mode of inhibitory action of Zataria multiflora Boiss. essential oil (EO) on the fungus, Aspergillus flavus, was studied by colony morphology examination, light microscopy, scanning electron

microscopy (SEM) and transmission electron microscopy (TEM). The EO at concentrations used in this study suppressed the size of the colony as well as sporulation. SEM of mycelia treated with given concentrations of EO showed morphological alterations ranging from loss of turgidity and uniformity of mycelia at low concentrations of EO to evident destruction of the hyphae at higher concentration of EO. Semi-thin LGK-974 in vitro sections of mycelia exposed to different concentrations of EO were analysed by light microscopy and revealed that the major change at level as low as 50 ppm of EO was limited to vacuolisation of cytoplasm resulting in cell swelling, while at higher concentrations, detachment of the cell membrane from the cell wall, deformation of mycelia and shedding the cytoplasm from the cell were the main alterations. These damages were well documented by TEM, which showed that the main sites

of action of EO INK-128 were the plasma membrane and cell wall. In conclusion, morphological and structural changes observed in this study may be one of the mechanisms involved in growth inhibition of the fungi and reducing aflatoxin Obatoclax Mesylate (GX15-070) production. “
“Various studies have documented a shift in species distribution in Candida bloodstream infections (BSI), but there are little data from Southeast Asia. This study was performed to determine the species epidemiology and antifungal susceptibilities of Candida species BSI in Singapore. Candida spp. from BSI were collected from a tertiary and secondary referral hospital, and an obstetrics/paediatric hospital over a 3-year period. The most common isolates were Candida albicans (36%), Candida tropicalis (27%), Candida glabrata

(16%) and Candida parapsilosis (16%). Candida parapsilosis and C. albicans were predominant in the paediatric hospital, and C. albicans and C. tropicalis predominant in the other two institutions. Candida tropicalis temporarily replaced C. albicans as the predominant strain from BSI in 2006. Overall, 87.3% of Candida isolates were susceptible to fluconazole, and 10.4% classified as susceptible-dose-dependent. Fluconazole resistance was detected in C. tropicalis (3.6%), C. parapsilosis (2.1%) and C. glabrata (4.0%). Candida albicans is the predominant species isolated from BSI in Singapore. However, non-albicans species accounted for nearly two-thirds of all cases of candidaemia and the relative increase in C. tropicalis infections deserves further investigation.

The mutant strain additionally lacked the ability to adsorb Congo

The mutant strain additionally lacked the ability to adsorb Congo red, no longer fermented sugars AZD3965 mouse other than glucose and L-arabinose, did not harbor four known virulence-associated genes (iss, tsh, cvaA, papC), and was susceptible to many antimicrobials, with the exception of nalidixic acid. The lethal dose (LD50 value) of the mutant strain on intravenous challenge in chickens was approximately 10-fold higher than that of the parent strain. Additionally, the mutant strain was rapidly eliminated from chickens, being detected in the respiratory tract only on the first

day post-inoculation by fine spray. Administration of the mutant strain via various routes such as spray and eye drop for chickens, as well as in ovo inoculation for embryonated egg, evoked an effective immune response that protected against a virulent wild-type E. coli O78 strain. Specifically, after immunization with the mutant strain, chickens challenged intravenously with an E. coli O78 strain exhibited decreases in mortality, clinical scores, organ lesion scores, and recovery of the challenge strain from organs compared to non-immunized chickens. These findings suggest that AESN1331 is a suitable candidate for a

live vaccine strain to protect chickens from colibacillosis CH5424802 clinical trial caused by avian E. coli O78. Colibacillosis, a serious disease of poultry, is caused by APEC (1, 2). APEC is one of the most important causes of a number of extra-intestinal diseases in the poultry industry, including airsacculitis, pericarditis, perihepatitis, and cellulitis. Colibacillosis results in significant economic losses to the poultry industry each year. Traditionally, antibiotic agents have been used to control APEC infections (3–7), but the emergence of drug-resistant mutants (4, 5, 8–12) and the demand for chemical-free feeding

have led to increased interest in alternative methods of protecting flocks against APEC. Various types of vaccines for control of respiratory tract infections caused by APEC in poultry have been tested (13–20). However, these inactive vaccines have not found PtdIns(3,4)P2 widespread use in the poultry industry because, in broiler chicken farming, administration by injection is unappealing compared to administration by feeding. Recently, a disrupted whole-cell vaccine including lipid adjuvant was reported (21). Unfortunately, in Japan this mucosal vaccine was approved only for administration by eye drop, and not by coarse spray. Currently, live vaccines are preferred, because such vaccines can be used for mass immunization via aerosol, feed, or drinking water. Kwaga et al. demonstrated the immunogenicity of the carAB mutant strain of APEC O2 (22). Peighambari et al. reported that a ΔcyaΔcrp mutant of APEC O2 strain was moderately immunogenic, but a mutant bearing the same lesions in the APEC O78 background was not immunogenic for sprayed chickens (23, 24).

At present, the events that occur to facilitate leukocyte TEM aft

At present, the events that occur to facilitate leukocyte TEM after opening a VE-cadherin gap are unclear. These findings are reminiscent of reports of the effect of CD99 blockade 41, 42. CD99 appears to function at a point after the development of a gap in VE-cadherin to facilitate completion of the diapedesis step. Interestingly, we identify no change in the total distribution of endothelial CD99 following either IQGAP1 knockdown or ND treatment. Mamdouh et al. showed that monocyte and lymphocyte diapedesis is associated with MT dependent-targeted recycling of membrane vesicles in which PECAM-1 but not VE-cadherin

are components of this membrane vesicle compartment 19. Our data are compatible Target Selective Inhibitor Library cost with a model in which IQGAP1 is involved in the recycling of membrane vesicles that might facilitate lymphocyte diapedesis by increasing the membrane surface Trichostatin A research buy area or, alternatively, bringing more free junctional molecules such as CD99 to the surface. Future work will be needed to establish such a link. Our observation that VE-cadherin gap formation is not affected by loss of IQGAP1 or MT favors the model that VE-cadherin gap formation is regulated by a separate mechanism. In our experiments we found that only about a third of lymphocytes that are associated with a VE-cadherin gap are surrounded by a ring of PECAM-1. Previously, it was reported that PECAM-1 is enriched around lymphocytes

transmigrating through human microvascular EC 6. This discrepancy might be due to the subset of lymphocytes that were analyzed. We depleted naive T cells (CD45RA+), which have been shown to express PECAM-1, in order to be able to specifically analyze endothelial PECAM-1 enrichment 43. Alternatively, it may be that only the fraction of PECAM-1-enriched lymphocytes in our samples are actively undergoing diapedesis. This cannot be distinguished by imaging fixed co-cultures. Nevertheless, IQGAP1 does not seem to be required for PECAM-1 enrichment around lymphocytes. Our findings suggest a model of upstream regulation of IQGAP1 activation for interendothelial junction remodeling during lymphocyte

4��8C TEM. IQGAP1 is an effector of calcium signaling, tyrosine kinases, and Rho GTP-binding proteins 28. Previous work identified the participation of phosphatidylinositol 3-kinase activity in junction remodeling during paracellular TEM of lymphocytes 44. Phosphatidylinsositol-3,4,5-triphosphate, the product of phosphatidylinositol 3-kinase activity, enables recruitment of PH domain-containing molecules such as GDP/GTP exchange factors for Rho GTP-binding proteins. Future work to further define specific intermediates of this pathway will be required. In summary, our results indicate that endothelial IQGAP1 and MT are involved in remodeling interendothelial junctions to accommodate lymphocyte diapedesis under physiologic shear stress.

Among the TND-positive clones, only one nucleotide difference

Among the TND-positive clones, only one nucleotide difference

was noted in comparison to the transgene VDJ sequence, indicating a low PCR error rate. Next, we analyzed the sequences of the 29 TND-negative clones to estimate the number of possible V genes that can be amplified with the V-gene primer, L3RI. We determined that at least ten V genes, or 9% of the functional V genes (assuming that all of the functional 110 V genes click here that are available 33 are expressed), can be amplified with the L3RI primer. This analysis provides an approach to estimating the percentage of switch events in the stimulated B cells that lead to chromosomal translocations. This approach relies on assumptions that are described in the Discussion. The frequency of translocations is considered to be indicated by (total number of translocations)/(total

number of switch events) in the stimulated population. We calculate the total number of switch events as (100–27.5)×(110/10)+27.5=825 (in arbitrary units) and then the translocation frequency as 27.5/825=0.033 or 3.3%. Two-color FISH was used to label the 3′ region of the Igh locus using BAC199 (a gift from Fred Alt at Harvard Medical School, Boston, MA with permission from Barbara Birshtein, Albert Einstein College of Medicine, New York, NY), which encompasses the Igh 3′ enhancer and 100 kb downstream 34, and the Cμ gene using an 8 kb plasmid containing the VV29 R16.7 VDJ segment, the Igh intronic Eμ enhancer, and the Cμ gene (referred as the Cμ probe throughout this article). BAC199 was labeled this website with biotin and the 8 kb Cμ plasmid was labeled with digoxigenin by nick translation (Roche) as per the manufacturer’s instructions and as described previously 34. Metaphases

were prepared from VV29 or C57BL/6 splenic B cells stimulated for 24 h with 25 μg/mL lipopolysaccharide (LPS) (Sigma) and 10 ng/mL interleukin-4 (IL-4) (PeproTech). Stimulated B cells were then frozen in metaphase by incubating with colcemid (KaryoMax, Invitrogen), then swollen in KCl, and fixed in 3:1 methanol/acetic acid as described previously 34. Metaphase images were captured using Olympus BX50 microscope with Isis v5.1.2 software (MetaSystems) at the Cytogenetics Laboratory at Tufts University Medical Center. Thirty-five metaphases were analyzed for VV29 transgenic strains and 15 metaphases Methisazone were analyzed for C57BL/6 strains. Splenic B cells were isolated by negative selection using B-cell isolation kits (Stemcell Tech). Two million B cells were stimulated with 25 μg/mL of LPS (Sigma) and 10 ng/mL IL-4 (Pepro Tech) in 4 mL cultures of RPMI-1640 (BioWhittaker) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals). The authors thank Peter Brodeur and Naomi Rosenberg for critical reading of the manuscript and providing advice during the course of this investigation. This work was supported by National Institutes of Health Grant AI24465 and by the Eshe Foundation and the W. M. Keck Foundation.

Because both activated

CD4+ T cells and DCs express Tim-1

Because both activated

CD4+ T cells and DCs express Tim-1, we first tested the effect of Tim-1 crosslinking on CD4+ T cells in an APC-free system. In an APC-free culture, activation with anti-CD3/anti-CD28 in the presence of 3B3 anti-Tim-1 increased the frequency of IL-4- and IL-10-producing CD4+ T cells, while the treatment did not significantly change IFN-γ+ or IL-17+ T cells (Fig. 3A). However, when naïve CD4+ T cells were cultured with syngeneic DCs plus antigen together with 3B3, the responding T cells produced more IFN-γ and IL-17, in addition to IL-4 and IL-10 (Fig. 3A). Interestingly, in the absence or presence of DCs, RMT1-10 increased only Th2 responses (IL-4 and IL-10 production) but had no obvious modification

on Th1 (IFN-γ) or Th17 (IL-17) responses, suggesting that the low-avidity anti-Tim-1 RMT1-10 does not modulate DC function selleck chemicals (Fig. 2). These data suggest that Tim-1 crosslinking with both high-avidity and low-avidity anti-Tim-1 promotes Th2 responses regardless of the presence or absence of DCs. However, only the high-avidity anti-Tim-1 enhances Th1 and Th17 responses when DCs are present in the cultures. To demonstrate that Tim-1 signaling in DCs is responsible for promoting Th1 and Th17 responses in vivo, PLP139–151-loaded/anti-Tim-1-treated DCs were subcutaneously transferred into syngeneic SJL mice. Draining LN cells were then isolated and antigen-specific T-cell responses were examined ex vivo. We found that immunization with 3B3-treated DCs enhanced the production

of IFN-γ and IL-17 as well as IL-4 and IL-10 in PLP139–151-responding T cells, whereas immunization with RMT1-10-treated DCs seemed not to significantly INCB024360 cost modulate any of these cytokines (Fig. 3B). LPS-treated DCs enhanced the production of IFN-γ and IL-17 but strongly inhibited IL-4 and IL-10 from T cells (Fig. 3B). There was no detectable clonidine production of these cytokines in the absence of antigen in any case (data not shown). These data further confirm that only the high-avidity anti-Tim-1 induces DCs activation, and Tim-1 signaling-activated DCs promote Th1 and Th17 as well as Th2 responses. TGF-β acts on naïve T cells to induce Foxp3 expression and these cells attain most of Treg properties. Addition of 3B3 anti-Tim-1 in the presence of either CD11b+ or CD11b− DCs to cultures where TGF-β was used to induce Foxp3+ Tregs led to the inhibition of Foxp3+ Treg generation. The frequency of Foxp3+ Tregs upon 3B3 treatment of CD11b− DCs was only about 4% compared with about 40% induction under control conditions (Fig. 3C). However, addition of 3B3 in APC-free cultures did not significantly change Foxp3+ Treg generation, with about 70% of Foxp3+ cells regardless of whether anti-Tim-1 was used. However, 3B3 treatment increased CD103 expression on both Foxp3+ and Foxp3− T cells (Fig. 3C). Furthermore, treatment with 3B3 increased the production of IL-17 from T cells in the presence of DCs (Fig. 3D).

As shown in Fig  1, αDC1s produced significantly higher amounts o

As shown in Fig. 1, αDC1s produced significantly higher amounts of the CXCR3 ligands CXCL9/MIG (P = 0.02), CXCL10/IP-10 (P = 0.02) and CXCL11/I-TAC (P = 0.03) (Fig. 1a–c), as compared with PGE2DCs. This chemokine production was not seemingly depressed by the number of contaminating CLL cells Forskolin solubility dmso in the cultures (Fig. 1D). Both

PGE2DCs, as well as αDC1s, showed a mature DC phenotype and morphology (Fig. 2). Importantly, loading with heat-stressed necrotic CLL cells had no significant impact on chemokine production or phenotype. Previously, it has been shown that PGE2DCs generated from healthy blood donors preferentially produced CCL22/MDC and attracted Tregs [17]. In line with this, we could show that monocyte-derived PGE2DCs from patients with CLL produced significantly higher levels of the Th2- and Treg-attracting chemokine CCL22/MDC as compared with αDC1 (P = 0.03). Regarding the production of CCL17/TARC, no statistical significant difference was found (Fig. 3A,B). Once again, tumour cell loading had no significant impact on chemokine production. To examine whether the high production of CXCR3-ligands by αDC1s could be translated into possible recruitment of NK and NKT cells, we used a transwell plate migration assay. Even though there were no differences in total number of recruited lymphocytes, we found that supernatants from tumour-loaded αDC1s induced a substantially higher recruitment of NK (P = 0.04) and NKT (P = 0.04) cells from PBMC in transwell

Kinase Inhibitor Library clinical trial experiments compared with supernatants from tumour-loaded PGE2DCs (Fig. 4A,B). When reaching the lymph node, antigen-loaded mature DCs undergo an additional activation step, termed ‘licensing’ in response to various stimuli, notably CD40 ligand that is expressed on cognate CD4+ T cells. Signalling through CD40 has multiple effects on DCs, inducing the upregulation of costimulatory molecules and the secretion of cytokines either and chemokines. Effective vaccine DCs should optimally mediate a CD4+ T cell-dependent guiding of rare tumour-specific CD8+ T cells to site of antigen-dependent DC–CD4+

T cell interactions by secretion of CCL3/MIP-1α and CCL4/MIP-1β chemokines [20]. We therefore considered whether differentially matured DCs were able to respond to subsequent CD40 ligation (mimicking CD4+ T cell interaction). To optimally mimic the situation in vivo, previously washed mature DCs were cultured in fresh medium for further 24 h (this being an estimation of the time required for the DCs to migrate to a draining lymph node) and subsequently washed before CD40 stimulation by cross-linked soluble CD40L. We found that tumour-loaded αDC1s, produced larger amounts of CCL3 (P = 0.02) and CCL4 (P = 0.04) after CD40 ligation, as compared with PGE2DCs (Fig. 5A,B). Finally, we could show, in accordance with Lee et al. [24], that tumour-loaded αDC1s were superior in producing the Th1-deviating IL-12p70 cytokine compared with PGE2DCs (P = 0.02) after CD40 ligation (Fig. 5C).