In the

first paper to describe the use of an in vitro system for assaying the suppressive Selleck Talazoparib function of Tregs it was demonstrated that Tregs suppress production of IL-2 by effector T cells and that the provision of exogenous IL-2 could overcome Treg-mediated suppression [40]. A recent study revisited this theme, demonstrating cytokine deprivation-induced apoptosis in effector T cells co-cultured with Tregs[118]. Although IL-2 is important in supporting the expansion of Th1 cells and the differentiation and survival of iTregs[27], it is now recognized that, at least in mice, IL-2 acting via signal transducer and activator of transcription 5 (STAT5) constrains the development of Th17 responses [119]. In this sense, a mechanism acting to suppress the development of a Th1 response could facilitate simultaneously the expansion of a Th17 response, which is supported Enzalutamide molecular weight further by the findings that IFN-γ blockade promotes Th17 responses [120,121]. Furthermore, exposure to IL-2 during T cell activation is known to predispose cells for activation-induced cell death (AICD) [122] via the up-regulation of Fas and FasL expression [122–124]. Sensitivity to AICD is enhanced by IFN-γ[125], which may underlie the increased sensitivity of Th1 cells to AICD compared

to their Th2 counterparts [126]. The fate of ‘suppressed’ effectors and the comparative sensitivity

of Th17 effectors to AICD deserve further study. It is clear that Tregs can modulate both Th1 and Th2 effector responses during infection [41,127,128] as well as in models of autoimmunity and allergy [43,85,86]. However, the impact of Tregs on Th17 responses in autoimmunity MTMR9 and infection requires more detailed study. This may be because many of our infectious and autoimmune models were constructed and characterized during the tenure of the Th1/Th2 dichotomy and have been described consequently in its limited parlance. Even in those diseases in which Th17 cells are now considered key players (for example, CIA and EAE [129]), many experiments looking at the effects of Tregs on immune responses in vivo and in vitro were carried out before the full significance of the emerging Th17 subset was realized, and have not been revisited in its new light. Finally, and perhaps most significantly, the apparent lack of data on the regulation of Th17 cells by FoxP3+ Tregs may be due to our increasing recognition that these two subsets share overlapping pathways of differentiation, and it is at this level that we have focused upon Treg/Th17 interplay. A full examination of the Th17/Treg developmental relationship is reviewed elsewhere in this series [130,131]; however, the central observations are pertinent to the topic considered here.

Paradoxically, IFN-γ-producing cells were more prevalent than IL-17-producing cells in CNS mononuclear fractions from CXCR3−/− and CXCL10−/−, as well as WT, mice with MOG-induced EAE (Fig. 1D and H). Enrichment of IFN-γ producers within the CNS could be secondary to preferential trafficking, survival,

and/or expansion of Th1 over Th17 cells. If so, the data in Figure 1 would insinuate that MOG-specific Th1 cells cross the blood–brain barrier and are retained in the brain and SC by a CXCR3/CXCL10-independent mechanism. Alternatively, the majority of CNS-infiltrating IFN-γ-producing T cells could represent transformed Th17 cells that PD98059 datasheet acquire Th1-like characteristics within the CNS microenvironment (the so-called “ex-Th17” cells) [28]. Th17 cells have been shown to access the CNS via a CCR6-CCL20-dependent pathway, which could explain the dispensability of CXCR3-CXC chemokine interactions for the development of IFN-γ-rich neuroinflammatory infiltrates in MOG-immunized mice [26]. In support of the latter hypothesis, mRNA for IL-17A, RORγt, and

CCL20 was upregulated in the CNS of CXCR3−/−, CXCL10−/−, and WT mice with EAE (Fig. 1I and J). Next, we sought to directly compare the relative dependence of MOG-specific Th1 and Th17 cells on CXCR3/ELR− CXC chemokine interactions for their encephalitogenicity. MOG-primed CXCR3−/− T cells exhibited similar cytokine profiles to their WT counterparts following culture under either Th1- or Th17-polarizing selleck products conditions (Fig. 2A). As expected, MOG-primed, IL-23-polarized CXCR3−/− Th17 cells were

as efficient as WT Th17 cells in trafficking to the CNS and inducing clinical EAE following adoptive transfer into naïve syngeneic WT hosts (Supporting Information Fig. 1 and Fig. 2B). Surprisingly, IL-12-polarized CXCR3−/− Th1 cells showed no defect in EAE induction (Fig. 2C). In fact, recipients of CXCR3−/− Th1 cells underwent a prolonged disease course with attenuated remission compared to recipients of WT Th1 cells. CXCR3−/− Th1 cells accumulated in the CNS in comparable numbers to WT Th1 donor cells, and the majority of Ceramide glucosyltransferase CXCR3−/− donor T cells in the SC were IFN-γ+ (Fig. 2D). CXCL10 is a dominant CXCR3 ligand in the CNS of the EAE models employed in our studies; C57BL/6 mice do not produce functional CXCL11 protein and CXCL10 is significantly upregulated in the inflamed CNS of MOG-immunized mice (Fig. 2E). In parallel experiments, CXCL10−/− and WT hosts exhibited a comparable degree of susceptibility to EAE mediated by WT Th1-polarized, MOG-specific effector T cells (Fig. 2F). Similar to WT recipients of CXCR3−/− Th1 cells, CXCL10−/− recipients of WT Th1 cells experienced a relatively severe disease course. Mice that are genetically deficient in an immunological molecule can develop compensatory pathways as they mature.

The immunomodulatory properties of the selected Lactobacillus bacteria were assessed by measuring the induction of innate and adaptive cytokine production, proliferation and cell death of unstimulated, polyclonal stimulated and allergen-specific stimulated hPBMC. The Lactobacillus strains studied showed an overall stimulating effect on IL-10, decreased prototypical Th2 cytokines and differentially stimulated signature Th1 cytokine induction. Blood was collected from five birch pollen-allergic patients, two grass pollen-allergic patients

and one adult healthy control. All birch- and grass-allergic patients reported having rhinoconjunctivitis during the birch or grass pollen season, respectively, and had serum-specific IgE to birch

or grass pollen Selleckchem Fluorouracil of at least class 4 (except for one person who had class 3), measured by ImmunoCAP selleck kinase inhibitor (Phadia AB, Uppsala, Sweden). The healthy donor displayed no birch or grass pollen-specific IgE in his sera (<0.35 kU L−1/class 0). Blood was obtained outside the pollen season in September, and none of the patients showed allergic symptoms at the time of investigation. Furthermore, none of the patients had received allergen-specific immunotherapy or used antihistamines or corticosteroids in the month before the blood drawing. All participants gave their informed consent and the performed experiments were approved by the local ethical committee (Commissie Mensgebonden Onderzoek, regio Wageningen). Six Lactobacillus strains (Table 1) of the species Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus fermentum were selected from our culture collection on the basis of high survival rates under conditions of low pH and/or the presence of bile, isolation from gastrointestinal tract, or were strains from species that are among the predominant Lactobacillus populations in the human gut. Further selection, including 70 strains, was based on IL-10-inducing capacities in 24-h hPBMC cultures of a healthy donor according to standardized procedures in our laboratories. Furthermore,

a mixture of strains B2261 and B633 was included, further referred to as a mixture of B2261 and B633. The choice for this mixture was based on combining the highest IL-10-inducing strain (B633) and the highest Non-specific serine/threonine protein kinase IL-12-inducing strain (B2261), of the 70 strains included in this initial screening. Strains were cultured for 24 h at 37 °C in Man Rogosa Sharpe (MRS) broth (Merck, Darmstadt, Germany), after which fresh broth was inoculated with 1% (v/v) overnight culture. After an additional 24 h of incubation at 37 °C, bacterial cells were harvested by centrifugation at 1000 g, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS. The bacterial cell numbers were determined by plate counting on MRS agar, and OD was measured at a wavelength of 600 nm.

5–7 A small number of phase I/II clinical studies have been completed and have confirmed that sufficient numbers of genetically

modified T cells can be generated ex vivo, that TCR-transduced autologous T cells can persist after adoptive transfer and that anti-tumour activity in melanoma patients was feasible.8 However, further improvements are required to optimize the efficacy of TCR gene transfer in the clinical setting. this website The efficiency of TCR gene transfer, and the subsequent function of the TCR-transduced T cell, is influenced by the vector delivery system, the TCR transgenes and the transduction conditions. To date, most TCR gene-transfer protocols have utilized gamma-retroviral vectors. Stable genomic integration of retroviral vectors requires full T-cell activation and proliferation during the transduction process. This process requires stimulation through the TCR complex using antibodies against CD3, with or without anti-CD28, in order to stimulate progression through the cell cycle, followed by Selleckchem CH5424802 a period of in vitro expansion in the presence of interleukin (IL)-2. During this in vitro activation process, T-cell differentiation occurs and cell-surface molecules important for homing to secondary lymphoid organs (i.e. CD62L) or costimulation (i.e. CD28) are down-regulated. There are theoretical advantages to redirecting the antigen

specificity of less-differentiated cells and this can be achieved using lentiviral vectors, which permit gene transfer into non-dividing T cells.9,10 These approaches are currently being explored by a number of research teams, together with TCR transfer into selected central memory or naïve T cells and co-transfer of specific homing molecules. A number of challenges remain, including: (i) to maximize the cell-surface expression of the introduced TCR; (ii) to minimize or eliminate the mispairing of introduced

TCR-α and TCR-β chains with endogenous TCR chains; (iii) to improve the association of the introduced TCR with molecules of the CD3 complex; and (iv) to enhance the functional avidity of the TCR-transduced T cells. The relevant steps in the generation of antigen-specific T cells by TCR gene transfer are Evodiamine indicated in a schematic representation (Fig. 1). TCR assembly and expression is a complex process.11 Before cell-surface expression, the TCR-α and TCR-β chains have to form a heterodimer. This process is influenced by the secondary and tertiary structures of both the variable and constant domains. The TCR-αβ then associates with the CD3 complex within the endoplasmic reticulum (ER), which involves interactions between the TCR constant domain (both intracellular and intramembrane portions) and the CD3 molecules. Finally, the TCR–CD3 complex is released from the ER and translocates to the cell membrane.

LDH activity was analyzed using the commercially available Cytotoxicity Detection Kit (Roche). For three-dimensional skin models, 1×106 human oral keratinocytes (TR146) were seeded on inert filter substrates (Nunc, polycarbonate filter, 0.4 μm pore size, 0.5 cm2) in antibiotic/antimycotic-free defined keratinocyte growth medium (Lonza) for 9 days. After 5 days inert filter substrates were lifted to the air–liquid interface and basal cells were fed through the filter substratum. Epithelium was treated with IFN-γ (300 U/mL), IL-17, IL-22, TNF-α (50 ng/mL each), IL-22/TNF-α combination or Th22 supernatant directly before infection

with 2×106 Candida yeasts for 12 h. Light microscopical studies ABT-199 cell line were performed as previously described using paraffin-embedded oral epithelium specimens 34, 35. Statistical analysis was done using One-way ANOVA and Bonferroni’s Multiple Comparison Test as post test. Statistically significant differences were defined as *p≤0.05, **p<0.01,

***p<0.001. This work was supported by the German Research Foundation selleckchem (DFG) EY97/2-1 and SFB Tr22. We thank Kerstin Holtz and Gaby Pleyl-Wisgickl for outstanding technical assistance. Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“Viral double-stranded RNA (dsRNA) mimetics have been explored in cancer immunotherapy to promote antitumoral immune response. Polyinosine–polycytidylic acid (poly I:C) and polyadenylic–polyuridylic acid (poly A:U) are synthetic analogs of viral dsRNA and strong inducers of type I interferon (IFN). We describe here a novel effect of dsRNA analogs on cancer cells: besides their potential to induce cancer cell apoptosis through an IFN-β autocrine

loop, dsRNA-elicited check details IFN-β production improves dendritic cell (DC) functionality. Human A549 lung and DU145 prostate carcinoma cells significantly responded to poly I:C stimulation, producing IFN-β at levels that were capable of activating STAT1 and enhancing CXCL10, CD40, and CD86 expression on human monocyte-derived DCs. IFN-β produced by poly I:C-activated human cancer cells increased the capacity of monocyte-derived DCs to stimulate IFN-γ production in an allogeneic stimulatory culture in vitro. When melanoma murine B16 cells were stimulated in vitro with poly A:U and then inoculated into TLR3−/− mice, smaller tumors were elicited. This tumor growth inhibition was abrogated in IFNAR1−/− mice. Thus, dsRNA compounds are effective adjuvants not only because they activate DCs and promote strong adaptive immunity, but also because they can directly act on cancer cells to induce endogenous IFN-β production and contribute to the antitumoral response.

We observed the same preferential usage of particular TCR Vβ subsets by CD8+ TEM cells regardless if the analyses were performed on the basis of absolute numbers of CD8+ T cells per liver or on the basis of percentages of CD8+ T cells per liver IHMC. Expansions in CD8+ TEM subsets

were observed in 13 of the 18 mice (72%), with either 1 (22%), 2 (39%) or 3 (11%) different TCR Vβ expanded in each mouse. The particular TCR Vβ expanded on CD8+ TEM cells varied between individual mice, Selleck MK 1775 with expansions seen for all TCR Vβ except Vβ3. The observed mouse to mouse variability in the TCR Vβ profiles makes it difficult to determine correlations between immune and immune/challenged TCR Vβ repertoires. Moreover, this type of analysis permits only a single sampling, learn more which may not reflect fully the changes that have taken place in the expression of the TCR repertoire during the immunization and challenge of a single mouse. To address this issue, we decided to examine the CD8+ T cell subsets in peripheral blood of immunized mice, which would provide us with information

regarding kinetics of any changes that occurred during the history of Pbγ-spz immunization and challenge. As we observed previously (30), in the current study, we also detected CD8+ TEM in the blood, concomitant with a decrease in CD8+ TN cells following immunization (Figure 4). CD8+ TCM expanded following the initial priming but returned to pre-immune levels and remained stable during the immunization protocol. Nonimmunized control mice were kept for the duration of the Liothyronine Sodium 5-week experiment, and the blood CD8+ T cells showed only a negligible increase in TEM (data not shown). Thus, the appearance of TEM in the blood was in response to immunization with γ-spz. Furthermore, the timing of the appearance of TEM in the blood was similar to that observed

in the liver [(30,31), data not shown]. To determine whether the TCR Vβ expression on CD8+ T cell subsets from liver and blood was consistent within an individual mouse, we compared the TCR Vβ expression on CD8+ subsets from liver, blood (Figure 5) and spleen (data not shown). In total, eight mice were analysed and the results from four representative mice are shown. The TCR Vβ repertoire of CD8+ TN and TCM cells was conserved between individual mice, in all organs examined. In contrast, the expression of TCR Vβ by CD8+ TEM varied between individual mice. However, the pattern of expression was the same in the blood, liver and spleen of each individual mouse. Thus, at the level of TCR Vβ expression, TEM in the blood reflect the population found in the liver, and the blood CD8+ T cells can be used as a surrogate of liver CD8+ T cells. To determine whether the repertoire of CD8+ TEM cells induced by immunization with Pbγ-spz changes after challenge, we followed the TCR Vβ profiles in the blood of individual mice. In all individual mice examined, the pre-challenge profile of TCR Vβ expression by CD8+ TEM remained the same after the challenge (Figure 6).

Wet tail-blood films of the infected mice were examined microscopically at 2-day intervals to estimate the parasitaemia (15). When the parasitaemia reached between 107 and 108 trypanosomes/mL, tail-blood was collected and diluted with Phosphate buffer Saline Glucose (PSG) to achieve a concentration of 105 parasites in a total

volume of 0·2 mL. This volume was injected Tanespimycin I.P. in six OF1 mice for each strain. A group of six mice, injected I.P. with 0·2 mL of PSG, was used as control. For each strain, the prepatent period (number of days between the inoculation and the first appearance of parasites in the blood) and the survival time were recorded up to 60 days post-infection. Mortality in infected and control mice was recorded daily. An animal was considered parasitologically

negative when no trypanosomes were detected in at least 50 microscopic fields. Animal ethics approval for the experimental infections was obtained from the Ethics Commission of the Institute of Tropical Medicine, Antwerp, Belgium (Refs DG001-PD- M-TTT and DG008-PD-M-TTT). The median mice survival time of the infected mice was estimated in parametric survival models using a log-normal see more hazard distribution in Stata 10. The strains for which none of the infected mice died during an observation period >60 days were discarded from the analysis. In a first model, the strains were used as discrete explanatory variables. In a second model, transmission cycle type (domestic or sylvatic) was used as explanatory variable. Data clustering in relation to the different isolates was taken into account using the frailty option (shared for strains). Strains were subsequently allocated to three virulence classes according to their estimated median survival time (<10 days, 10–50 days and >50 days). Strains for which none of the infected mice died during an observation period of more than 60 days were allocated

to the last class. An ordered Resveratrol multinomial regression was applied on the data using the cycle type as explanatory variable. The virulence of a total of 62 T. congolense strains was tested and compared. Median survival time of infected mice differed substantially between strains with mice infected with the most virulent strains having a median survival time of <5 days and mice infected with the least virulent strains surviving for more than 50 days. An overview of the median survival time (95% C.I.) of mice infected with 60 of the 62 strains (survival time could not be calculated for two strains because survival was more than 60 days) is presented in Figure 1. Based on the distinction made by Masumu et al. (9), strains were grouped into a high virulence (median survival time <10 days), a medium virulence (median survival time between 10 and 50 days) and a low virulence (median survival time between >50 days) category.

Because these preparations were crude extracts, the contribution of other JNK inhibitor supplier proteins and lipocarbohydrate to cytokine production cannot be discounted.

In these experiments too, no interstrain differences were identified. This is perhaps not surprising as HSPs are the most highly conserved proteins in the biosphere, a property that also makes them highly immunogenic owing to immunological memory (Zügel & Kaufmann, 1999). In keeping with previous observations, culture supernatants collected during growth of five C. difficile strains were able to induce a strong pro-inflammatory response (Canny et al., 2006); the production of TNF-α, IL-1β and IL-8 was detected (Fig. 5). There MK-1775 was greater TNF-α and IL-1β production in response to the stationary phase (20 and 24 h) culture supernatants as compared to the late exponential phase (8 and 12 h) supernatants, which correlated with the levels of toxin A and toxin B in them. IL-8 production was similar for all the samples. Although a correlation between cytokine production

and toxin levels was observed, contribution of other cell wall components such as lipoteichoic acid cannot be ruled out. Interestingly, no significant differences were identified between historic, endemic or hypervirulent strains even though the culture supernatants of C. difficile ribotype 027 and strain VPI 10364 contained

approximately 10 times more total toxin. It is possible that the large amounts of toxin rapidly induced Liothyronine Sodium toxicity in the THP-1 macrophages during the 3-h treatment. It has been previously observed that exposure of monocytes to toxin B was lethal; 500 ng of toxin B was lethal and even 5 ng of toxin B resulted in the death of 75% of monocytes within 5 h (Flegel et al., 1991). Further, macrophages were found to be more sensitive to the toxic effects of C. difficile toxins than monocytes (Linevsky et al., 1997), suggesting that more and rapid cell death could have occurred during the toxin shock. It has been suggested that release of pro-inflammatory cytokines followed by cell death could render monocytes unable to carry out phagocytosis, which could foster inflammation (Flegel et al., 1991). It was curious to detect rather low levels of IL-8 production with all the supernatants, especially when compared to IL-8 production in response to the surface-associated proteins. This observation suggested that a toxic environment was generated either directly by the toxins themselves or by the large amounts of cytokines being produced. The data presented here identified the SLPs, flagella and HSPs expressed at 42 and 60 °C of C. difficile as possible mediators of the inflammation observed in CDI, along with C. difficile toxin A and toxin B.

CS responses were elicited on day 4 after sensitization by painting both sides of the ears with 10 μl of 0.4% TNP-Cl in acetone and olive oil (1:1). Non-immunized controls were challenged identically. Ear thickness was measured with Seliciclib a micrometre 1 day prior to challenge (baseline) and then 2 h (peak of the CS-initiating phase) and 24 h (peak of the CS-effector phase) following challenge. Ear swelling units were expressed in mm × 10−2. Each

bar represents the average response ±SE in a group of four mice. Hepatic lipid extraction from contact-sensitized mice.  Wild-type BALB/c mice were contact-sensitized or sham-sensitized as described earlier. Thirty minutes later, mice were killed by cervical dislocation. Livers were isolated and placed in 2 ml of water on ice for several minutes to allow for hypotonic cell lysis before homogenization with tissue tearor at 17 000 rpm for 1 min. Samples were then sonicated while on ice for 1–2 min. Lipids were subsequently isolated from the lysate by two serial cycles of chloroform and methanol extraction (10 volumes each per gram of tissue per cycle; incubations were 12 h followed by 4 h, each at 4 °C). We recognize that the extracts we obtained also contained

DNA and RNA, but herein for convenience we refer to them as ‘lipid extracts. Isolation of iNKT cell-containing liver mononuclear cells (LMNC).  Liver mononuclear cells isolation was performed as described previously [9]. LMNC were obtained from wild-type BALB/c mice Tangeritin except as otherwise indicated in the text. Viability selleck chemicals llc was >90%, and ∼0.5−1 × 106 LMNC were obtained per mouse. iNKT cells constitute approximately 70% of wild-type LMNC; hepatic iNKT cells have previously been shown to play a key role in CS [9]. For simplicity, iNKT cell-containing LMNC will be referred to as ‘iNKT cells’ in the text. In vitro treatment of iNKT cells with lipid extracts.  Naïve wild-type iNKT

cell-containing LMNC were incubated in vitro with α-GalCer or hepatic lipid extracts from wild-type mice (after either contact sensitization or sham sensitization), with or without anti-CD1d antibody (at a concentration of 10 μg/ml for 1 h at 37 °C). Lipid donors and LMNC donors were age-, sex- and size-matched. The ratio of number of lipid donors to number of LMNC donors was 1:1 in incubations. Isolation of peritoneal B-1 B cells.  Peritoneal cells of wild-type CBA/J were harvested by lavage with 4 ml of cold 1% foetal bovine serum (Gibco BRL, Carlsbad, CA, USA) containing heparin (10 U/ml; Sigma) in PBS, washed three times and resuspended in RPMI 1640 containing 10% FBS, 25 mm Hepes, 100 units/ml penicillin and 100 μg/ml streptomycin; 5 × 106 peritoneal cells were obtained per mouse. Peritoneal cells contain approximately 20% B-1 B cells; the vast majority of murine B-1 B cells reside in the peritoneum.

The MFI of the ice control cells was subtracted from that of cells incubated at 37° with OVA per treatment or control. Data were analysed using the FlowJo Software (Tree Star). Endocytic behaviour and morphology of DCs treated with chemokines and/or subsequent LPS were examined by confocal laser scanning microscopy. Briefly, DCs were collected on Day 1 and Day 2 post-treatment and resuspended in medium (without phenol red) at 1 × 106 cells/ml. Then, each sample was incubated with 5·8 μg/ml of

fluorescent Alexa Fluor 488-Ovalbumin (OVA) (a model antigen) (Invitrogen) or 0·5 mg/ml Lucifer Yellow (LY) (Invitrogen) for 30 min at 37°. OVA is known to be internalized by DCs by a combination of receptor-mediated endocytosis and fluid-phase macropinocytosis[17] whereas Idelalisib manufacturer LY is internalized by only fluid-phase macropinocytosis.[34] RAD001 mouse After incubation,

any excess fluorochrome bound to cell surfaces was quenched for 3–4 min on ice using 0·5% Trypan Blue/2% FBS/1× PBS solution. After two sequential quenching steps, cells were washed three times using 1% BSA/PBS solution, resuspended in complete medium (without phenol red) at 1 × 106 cells/ml, then the cell suspension was used to submerge a glass cover slip and allowed to incubate for 4 hr at 37° to induce cell attachment to the cover glass. After incubation and another washing, cells were fixed with 2% paraformaldehyde for 10 min at room temperature, and permeabilized with 0·05% Triton-X 100 (Sigma) for 15 min at room temperature. Then, cells were washed three times, and incubated with Adenosine Texas red-X phalloidin (Invitrogen) at 0·165 μm in 1% BSA/PBS solution for 20 min at room temperature. Cells were then washed and permanently mounted using Fluoromount G (SouthernBiotech, Birmingham, AL). Microscopic images were acquired with a Zeiss 510 META confocal laser scanning microscope (Zeiss, Thornwood, NY) using 100× /1·4 NA oil objective. For this analysis, at least seven cells were examined per treatment condition. Each cell was ‘optically sectioned’ by collecting x–y plane images or slices

at 12–14 different z-direction altitudes through the cell (x-y slices were collected every Δz = 507 nm). A single x-y slice was selected from the middle of the z-stack of images (middle of the cell) for reporting here. To measure expression levels of DC surface markers, cells were resuspended in FACS buffer, blocked with anti-mouse Fcγ III/II receptor monoclonal antibody (clone 2.4G2; IgG2bκ) (BD Pharmingen), and stained with saturating concentrations of fluorescently conjugated rat or mouse anti-mouse monoclonal antibodies against CD86 (clone GL1; IgG2aκ), MHC Class I (H-2Kb) (clone AF6-88.5; IgG2aκ) and MHC Class II (I-2Ab) (clone AF6-120.1; IgG2aκ) (all from BD Pharmingen) for 30 min at 4° in the dark. After staining, cells were extensively washed three times using ice-cold FACS buffer and then, analysed immediately with 10 000 events per sample using FACS Canto (BD Biosciences).