In summary, a total of at least 15 unique GAD65 epitopes elicited CD4+ T-cell responses in subjects with HLA-DR0401 haplotypes that could be visualized using the corresponding DR0401 tetramers. Although 15 unique antigenic sequences within GAD65 were capable of eliciting T-cell responses in vitro, some of Luminespib these may not be processed and presented from intact protein. To identify the subset of peptides that correspond to processed and presented epitopes, the proliferation of tetramer-positive T-cell lines

was measured after stimulation by monocytes loaded with whole, recombinant GAD65 protein. As shown in Fig. 3, 11 of these 13 T-cell lines responded to the GAD65-protein-primed monocytes in a dose-dependent manner (whereas irrelevant control T-cell lines did not). Therefore, the peptides recognized by these cell lines (GAD105–124, GAD113–132, GAD201–220, GAD265–284, GAD273–292, GAD305–324, GAD353–372, GAD369–388, GAD433–452, GAD545–564 and GAD553–572) contain epitopes that are processed and presented by autologous monocytes. GAD321–340 was not evaluated in this assay, as this cell line was unavailable. However, this epitope was subsequently confirmed as being recognized in the primary T-cell

assays using intact protein (described in the Materials and methods section). The FDA approved Drug Library remaining peptides (GAD73–92 and GAD473–492) appear to contain cryptic epitopes. The magnitude of T-cell responses to a given epitope are determined

by various factors, including the efficiency of presentation and the frequency of the responding T cells in circulation. To estimate the relative O-methylated flavonoid prevalence of T cells that recognize various GAD65 epitopes, we stimulated CD4+ T cells from eight subjects with DR0401 haplotypes (four healthy and four diabetic) with CD14+ monocytes pulsed with recombinant GAD65 protein using four replicate wells for each subject. After 14 days of in vitro expansion, T cells from each well were stained with each of the 15 tetramers identified as putative DR0401-restricted GAD65 epitopes. For 10 of these 15 peptides, antigen-specific T cells were detectable after direct protein stimulation, further confirming these as peptides that contain DR0401-restricted epitopes that can be processed and presented. A representative staining for each of these is shown in Fig. 4(a). As shown in Fig. 4(b), the prevalence of responses to these epitopes varied. Among the 10 peptides that elicited responses, response rates ranged from six of eight subjects (GAD265–284) to one of eight subjects (four epitopes, including GAD321–340, which had not been previously confirmed by proliferation assay). In these protein-stimulated assays, the GAD265–284, GAD273–292, GAD305–324 and GAD553–572 epitopes were detected in multiple subjects, suggesting that these could be immunoprevalent.

All animal experiments described in the paper were done under UK Home Office Project Licence numbers 70/5791 and 70/6724 and were approved by the in-house ethics committee of the Institute of Cancer Research. All antibodies for flow cytometry were purchased from eBioscience or BD Biosciences. The following fluorescently labeled or biotin-conjugated anti-mouse antibodies were used: CD4 (GK1.5), CD69 (H1.2F3), CD8α(53-6.7), CD8β (CT-CD8b), TCR-β find more (H57-597), CD5 (53-7.3), Bcl-2 (3F11), IL-7Rα (B12-1). Staining by biotin-conjugated antibodies was visualised using streptavidin-conjugated

fluorophores. Immunofluorescence data were collected using a Becton Dickinson FACSCalibur, or a Becton Dickinson LSRII using CellQuest software and analysed Palbociclib order using Flowjo software (Treestar). Cells were sorted on a FACS Aria (Becton Dickinson) or a MoFlo (DakoCytomation), with DAPI staining used to exclude dead cells. Total RNA was

isolated using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesised using Invitrogen M-MLV Reverse Transcriptase. Each reaction contained 200 U enzyme, in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 and 10 mM DTT, 1 mM dNTP, 500 ng oligo (dT)15 primer (Promega), and 40 U RNAsin ribonuclease inhibitor (Promega) and was incubated for 50 min at 37°C, prior to heat inactivation at 70°C for 15 min. For qPCR, gene-specific primer/probe sets were purchased from Applied Biosystems as “Gene Expression Assays”, and reactions were performed with Taqman Universal PCR Mastermix (Roche/Applied Biosystems) on an ABI 7900 Real Time PCR System, using Hprt or Rps16 as a comparator. Standard curves were created using standards (usually serial dilutions of total thymus cDNA) for relative quantitation of the data. To assay the kinetics of Egr2 upregulation, MHC° thymocytes were cultured with 10 ng/mL PMA and 1 μg/mL Ionomycin. Signaling inhibitors were included at mafosfamide the following concentrations: U0126 (Promega) 10 μM, PD98059 (Promega)

10 μM, cyclosporin A (Calbiochem) 50 nM, FK506 (Sigma) 1 nM. For Erk phosphorylation in response to TCR ligation, thymocytes were treated with anti-CD3 diluted 1/100 in PBS, then warmed to 37°C. Goat anti-Armenian Hamster IgG (75 μg/mL; Jackson ImmunoResearch) was added and the cells were left for 2 min at 37°C before addition of paraformaldehyde to a final concentration of 2%, and incubation on ice for 10 min. Following centrifugation, cells were resuspended in 90% methanol and incubated for 30 min. Permeabilised cells were stained with Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mAb (Alexa Fluor 488 Conjugate) from Cell signaling Technology, using PBS-0.5% BSA as the staining buffer, in accordance with the manufacturer’s instructions. For anti-CD3 stimulation, 48-well tissue culture plates were coated with 150 μL of 2 μg/mL anti-mouse CD3ε (145-2C11) in PBS and incubated overnight at 4°C.

It has been proposed that pregnancy is a vascular “stress test”, GSK126 ic50 and because CVD and preeclampsia share many risk

factors (e.g., obesity and diabetes), failure results in preeclampsia and an “unmasking” of cardiovascular risk which may have otherwise gone unnoticed until later in life (reviewed in [30]). In addition, preeclampsia itself may induce changes to the maternal vasculature which may be irreversible and lead to increased cardiovascular risk under the stress of aging. In either case, understanding the cascade of events leading to vascular endothelial dysfunction and its feed forward progression to preeclampsia is critically important for the prevention and/or treatment of this disorder that has serious consequences to the mother, her offspring, and her own later-life cardiovascular health. Preeclampsia is a multifaceted disorder which threatens the health of millions of women each year and contributes to lifelong cardiovascular

risk. The maternal syndrome is characterized by systemic vascular dysfunction instigated by circulating factors released as a consequence of placental ischemia/hypoxia. Regorafenib molecular weight An imbalance in pro- and antiangiogenic factors, excessive inflammation, and the induction of oxidative stress within the endothelium are among the changes that contribute to endothelial dysfunction. An understanding of the mechanisms of dysfunction and its role in preeclampsia is critically important Megestrol Acetate for the prevention and/or treatment of this disorder. Dr. S.T. Davidge is a Canada Research

Chair in Women’s Cardiovascular Health and is an Alberta Innovates-Health Solutions funded Scientist. The laboratory is funded by grants from the Canadian Institutes of Health Research, Heart and Stroke Foundation of Canada and the Women & Children’s Health Research Institute. Lesley J. Brennan: Lesley Brennan received her Ph.D. from the Department of Biological Sciences at University of Alberta in Edmonton in 2011, where her work focused on the cellular interactions between symbiotic bacteria and their eukaryotic hosts. She then joined the laboratory of Dr. Sandra Davidge in the department of Medicine and Dentistry at the University of Alberta as a postdoctoral fellow. Dr. Brennan currently studies the long-term cardiovascular effects of a preeclamptic pregnancy on a woman’s health using animal models. Jude S. Morton: Jude Morton, Ph.D. received her doctoral training at the University of Glasgow, Scotland in the area of autonomic pharmacology. Her work focused on the investigation of vascular function in female sexual arteries. She then trained as a postdoctoral fellow continuing on to her current position as a research associate at the University of Alberta, Canada.

All tonsils had a negative culture test (except normal oral flora). Blood samples were obtained from all participants for a phadiatop test. If positive, it was followed by specific RAST for pollen

(birch, timothy and Artemisia). Patients included in the allergic group (n = 20) were classified as class 3 or higher on the RAST scale and had a history of allergic rhinoconjunctivitis. Patients included in the control group (n = 20) had a negative phadiatop test and no symptoms of allergy. Directly after surgery, one piece of tonsillar tissue (2–4 mm) was placed in RNA-later (Qiagen, Hilden, Germany) for 24 h and then kept at −80 °C until use. Another piece was fixed in a 4% solution of formaldehyde in 0.1% phosphate buffer (pH 7.0), thereafter embedded in paraffin, cut in 3 μm sections, mounted on glass slides and stored at −80 °C until use. None of the subjects Angiogenesis inhibitor displayed Vemurafenib in vitro any signs of acute infection at the time of surgery, or received antibiotic treatment for at least 1 month prior to surgery. Apart from the tonsillar symptoms, all subjects were healthy

and did not receive any medications. Additional tonsils were obtained for in vitro experiments and lymphocyte isolation. These were not characterized according to infectious or allergic status of the donor. The study was approved by the local Ethics Committee, and an informed consent was obtained from all participants. Fresh tonsils were cut into small pieces of ~1.5 mm and placed in complete RPMI 1640 supplemented with 0.3 g L−1 l-glutamine (PAA, Pasching, Austria), 10% FBS (PAN, Aidenbach, Germany), 100 U mL−1 penicillin/100 μg mL−1 streptomycin (Gibco, Grand Island, NY) and 50 μg mL−1 gentamicin (Gibco). The tonsillar pieces were cultured at 37 °C in a humidified FER 5% CO2 air atmosphere in the absence or presence

of recombinant human IL-4, IL-5, IL-13 (R&D Systems, Minneapolis, MN) or histamine (Sigma-Aldrich, St. Louis, MO). After 24 h of culture, the cells were examined for the expression of HBD1-3 using real-time RT-PCR, and levels of HBD1-3 in the supernatants were analyzed by use of ELISA. Fresh tonsils were minced in complete RPMI 1640 medium. Mixed tonsillar lymphocytes were isolated from the cell suspension after density-gradient centrifugation using Ficoll-Paque (Amersham Bioscience, Uppsala, Sweden) as previously described (Petterson et al., 2011). The lymphocyte-enriched interphase fraction was recovered and resuspended in complete RPMI 1640 medium and cultured (1 × 106 cells mL−1) for 4, 16 and 24 h with or without IL-4, IL-5 and histamine. Thereafter, the supernatants were collected and analyzed for levels of HBD1-3 using ELISA. Fresh tonsils were minced in complete RPMI 1640 medium. The cell suspension was incubated with neuraminidase-activated sheep red blood cells (SRBC) followed by density gradient centrifugation with Ficoll-Paque. T cells were obtained from the pellet after lysing the SRBCs with dH2O and 1.

Missense mutations in the csrS gene were detected in 11 strains (Table 2). One emm1 strain having 9.7, one of the M protein-high producers, carried two amino acid substitutions

(I332V, E428G), whereas three other emm1 strains carried only one amino acid substitution each (I332V). To further confirm the association between prolific M protein production and the csrS mutation, we performed TaqMan RT-PCR on the emm1 strains, including the M protein-high and -low producers, in order to determine the degree of transcriptional of the emm gene (Fig. 4). The M protein-high producer expressed more emm gene (6.1 ± 4.1, t-test P > 0.05) buy Pexidartinib than did the M protein-low producer (1.8 ± 0.5). SF370 ΔcsrS expressed more emm gene (22.4 ± 7.2, click here t-test P = 0.0089) than did the WT strain (2.6 ± 0.3). Next, to investigate whether the csrS (I332V, E428G) gene was substantially non-functional, we analyzed the complemented M protein-high producer with csrS (I332V, E428G), comparing it to that with csrS (I332V). The amount of M protein produced by the M protein-high producer was 9.3 ± 0.53, whereas the complemented strain with the control pLZ12-Km2 vector alone produced 9.0 ± 0. On the other hand, M protein production of the pLZ12-Km2-csrS

(I332V)-complemented strain tended to be less than that of the M protein-high producer (8.0 ± 0, t-test P = 0.0572). As for the mga gene, which is a positive regulator for the emm gene (21), no mutations were found in the two emm1 strains belonging to M protein-high and -low producers. As for the emm6 strains, one strain with 10, an M protein-high producer, harbored two substitutions for the amino acids in its CsrS protein (Table 2). In contrast, out of 11 strains (emm4–102 in Table 2) producing medium and low amounts (5.3–2.7) of M protein, a single amino acid substitution occurred in each of five strains, one with emm4, one with emm75, one with emm77, and two with emm28. Nonsense mutations, resulting in no csrS mutations, occurred in the

remaining six strains. On the other hand, no CsrS mutations were found see more in the eight strains with either emm3 or 12, in spite of the fact that one of the four emm3 strains was an M protein-high producer. These results suggest that the CsrS mutation may contribute, at least in part, to the prolific production of M protein seen in several emm genotypes. Streptococcus pyogenes strains of the OF+ lineage, except for emm 1, 3, and 6, carry Enn or Mrp, a member of the M family of proteins. These proteins are similar not only in terms of function–for example, both bind to fibrinogen or IgA–but also in terms of amino acid alignment to the M protein–for example, the amino acids between the Enn protein in M28 or EnnX in M49 and the recombinant M protein are often identical.

Most efforts in characterizing HLA-DQ binding specificities have been directed towards a few selected molecules, such as DQA1*05:01-DQB1*02:01 (also known as DQ2) or DQA1*03:01-DQB1*03:02 (DQ8) because of their association with disease.19–21 The data published by Wang et al.7 aim to be more comprehensive in terms of human population coverage, and they include binding data for the six most common allelic PLX4032 variants across different ethnicities. The HLA-DQ sequence motifs identified by NNAlign are shown in Fig. 2. In contrast to the DP variants, which appear

to share a common supertypical pattern, the DQ molecules show very little overlap in specificity. There do not appear to be common amino acid preferences, and the anchors are found at different positions within the 9-mer core. In particular, DQA1*01:01-DQB1*05:01 shows a strong preference for aromatic residues (F, W, Y) at P5, and secondary anchors at P6 and P7. The only previous report addressing the binding motif of this molecule8 also found a dominant

anchor characterized by a preference for W and F, but placed this anchor at P4, and is generally in disagreement with our findings on other positions. The binding motif for DQA1*01:02-DQB1*06:02 appears loose, with several amino acids allowed at most positions. Previous reports22,23 identified mainly a P4–P6–P9 anchor spacing, with small and hydrophobic PXD101 residues at P4, hydrophobic/aliphatic amino acids such as I, L, M, V at P6, and small residues like A and S at P9. Similar amino acid preferences are reflected in the binding motif detected Tideglusib by NNAlign, with additional anchors at P3 and P7. The only pair of molecules that appear to have a somewhat similar specificity is composed of DQA1*03:01-DQB1*03:02 and DQA1*04:01-DQB1*04:02. Both show a dominant anchor at P9, with preference for the acidic residues E and D. Additionally, they both show a preference for hydrophobic amino acids at P6, and mainly for A or E at P8. The strong acidic anchor at P9 was observed before.19,24 In the case of DQA1*05:01-DQB1*02:01, previous studies describe

a motif with P1 and P9 binding pockets with hydrophobic/aromatic preferences, and acidic residues in the centre of the core, particularly at P4, P6 and P7.8,24–28 Besides the hydrophobic/aromatic P1–P9, NNAlign places the strongest anchor at P7, but with preferences for glutamic acid (E) also at P6 and P8. Finally, the somewhat peculiar sequence motif of DQA1*05:01-DQB1*03:01 seems to just prefer small amino acids such as A, G and S, especially on the central positions of the core, in agreement with the motif previously suggested for this molecule.8 It is evident that the peptide-binding specificities for HLA-DQ variants are much more diverse than for HLA-DP variants. In particular, the strong hydrophobic/aromatic P1 anchor that generally characterizes all known HLA-DR and DP molecules is not observed here.

However, apart from the IFN-α-related effect on CD69 up-regulation, our study does not provide evidence that these activated NK T cells cross-react with and thereby activate antigen-presenting cells, conventional T cells and non-T cells, as we neither detected enhanced T or NK cell numbers, IL-12 expressing DC in situ nor enhanced IL-12, IL-7 or IL-15 plasma levels. Direct anti-tumour responsiveness by NK T cells in our two patients, as tested by IFN-γ responsiveness to tumours or tumour lysates, however, was not observed either. In vivo, this may be hampered by lack of CD1d expression on the tumours and lack of NK T cell infiltration into the tumour tissues.

Alternatively, NK T function may be influenced by Treg cells [36], Ibrutinib which are known to be elevated in cancer patients [37] and were found to be enriched, compared to normal individuals, in the peripheral blood

of the RCC patients, without any relationship to NK T frequency. To test whether NK T cell-mediated anti-tumour responsiveness might be induced in the absence of Treg cells, NK T cell lines were isolated from the cell populations, cultured in the presence of IL-2 and IL-15 and tested for anti-tumour reactivity. The cell line C1R-huCD1d, expressing human CD1d, was added to serve as antigen-presenting cell in this system. However, despite appropriate CD1d-ligand binding capacity and IFN-γ response to αGalCer by the isolated NK T cell lines, no consistent reactivity to tumours or tumour lysates was observed. Tumour lysates were Y-27632 even found to suppress the αGalCer response of the B7 NK T cell line. These data point to an intrinsic inability of the patient NK T cells to respond to the autologous tumour, even in an activated state and in the absence of Treg cells. Our observation of highly elevated levels of NK T cells in these RCC patients during an extended period of time bears resemblance to the observations of Chan et al. [38] on a healthy individual at risk for type 1 diabetes, and contrasts with the

generally reduced NK T cell numbers in cancer patients [7,8,10,11]. In conclusion, Cyclic nucleotide phosphodiesterase despite the elevated and sustained levels of NK T cells in these patients, any functional role of the NK T cells in these patients thus remains elusive at present and it will be of interest to elucidate whether RCC aetiology is linked with conditions that stimulate NK T cell expansion. We greatly acknowledge Drs S. Horenblas and W. Meinhardt for providing patients, Dr H. Ovaa for providing αGalCer, Dr V. Cerundolo for providing C1R and C1R-huCD1d cell lines, the NIH Tetramer Facility for providing PE-conjugated PBS57 loaded CD1d tetramer, A. Pfauth, F. van Diepen and M. van der Maas for help with flow cytometry and Drs J. Borst and J. Coquet for carefully reading the manuscript. The authors declare that they have no conflict of interest.

Glucocorticoids are the sole drugs of clinical interest for DMD patients.

The mechanism for their beneficial action is not completely understood yet and may involve multiple effects, beside the classical anti-inflammatory and immunosuppressive ones. These include an improvement of regeneration and an increased expression of utrophin, the homologue-surrogate for dystrophin [20–22]. However, the clinical use of glucocorticoids in DMD children is limited by severe side effects over long-term use; this compels the search of safer drugs or of strategies to limit their side Angiogenesis inhibitor toxicity [23]. As for other complex disorders, one feasible strategy is to find compounds with relevant synergistic interactions: thus glucocorticoids in combination with a synergistic drug, may exert greater effects and/or have less side effects as a result of dose lowering. This rationale is reinforced by the anecdotal report that DMD patients often take various food and drink supplements or herbal remedies along with the classical glucocorticoids and it is important

to develop a more systematic preclinical evaluation of the outcome of drug combinations, both in vitro and in vivo[23,24]. For instance, the combination of deflazacort with the food supplement L-arginine has been reported to produce an improved functional benefit in dystrophic mdx Fluorouracil order mice [25]. We therefore aimed to investigate the effects of a combined treatment of α-methyl-prednisolone (PDN), a clinically used glucocorticoid, with taurine. Taurine is a sulphonic amino acid normally present in skeletal muscle, able to modulate sarcolemmal excitability and calcium homeostasis [26]. It is used as a soft-drink supplement for its claimed ability to stimulate metabolism and provide energy. Little, if any, toxicity has been reported for taurine

at the generally assumed quantities [27]. Complex Acesulfame Potassium fluctuations in tissue taurine content occur in mdx mouse in the different phases of muscle degeneration/regeneration, suggesting that the amino acid levels may be influenced by myofibre state and may in turn contribute to cellular and tissue dysfunction and/or repair; taurine increases seem to be generally associated with muscle regeneration and membrane stabilization [28–30]. In addition, taurine exerts anti-inflammatory and antioxidant actions [31], with potential beneficial outcomes on the pathology progression. We have previously found that taurine either applied in vitro or administered in vivo exerts beneficial effects on the altered excitation-contraction coupling mechanism of mdx myofibres [8,29]. Also the amino acid administration enhances mdx mouse strength impaired by a chronic exercise on treadmill, a protocol that is able to exacerbate in vivo and ex vivo markers of the murine pathology [2,8]. We have performed a chronic (4–8 weeks treatment) in vivo treatment with α-methyl-prednisolone (1 mg/kg i.p.

Over the next few years, both the recently identified lymphocyte lineages as well as the application of deep sequencing approaches will provide insight into the link between antigen specificity and phenotype

– and into how Th cells choose the appropriate phenotype to regulate adaptive immunity. HJvdH, AA and RdB wrote the manuscript. “
“The inhibitor PD-0332991 mouse of κB kinase ε (IKKε) is pivotal for an efficient innate immune response to viral infections and has been recognized as breast cancer oncogene. The antiviral function of IKKε involves activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB, thus inducing the expression of type I IFN. Here, we have identified two novel splice variants of human IKKε, designated IKKε-sv1 and IKKε-sv2, respectively. Interestingly,

RT-PCR revealed quantitatively different isoform expression in PBMC from different individuals. Moreover, we found cell type- and stimulus-specific protein expression of the various splice variants. Overexpression of full-length wt IKKε (IKKε-wt) leads this website to the activation of NF-κB- as well as IRF3-driven luciferase reporter genes. Although none of the splice variants activates IRF3, IKKε-sv1 still activates NF-κB, whereas IKKε-sv2 is also defective in NF-κB activation. Both splice variants form dimers with IKKε-wt and inhibit IKKε-wt-induced IRF3 signaling including the antiviral activity in a dominant-negative manner. The lack of IRF3 activation is

likely caused by the failure of the splice variants to Resveratrol interact with the adapter proteins TANK, NAP1, and/or SINTBAD. Taken together, our data suggest alternative splicing as a novel regulatory mechanism suitable to shift the balance between different functions of IKKε. Viral infections are recognized by the innate immune system, which is essential for the subsequent initiation of adaptive immunity. Invading viruses are sensed by pattern-recognition receptors (PRR) recognizing pathogen-associated molecular patterns such as single- or double-stranded RNA. These PRR comprise TLR with endosomal/lysosomal localization like TLR3 and cytoplasmic receptors such as the retinoic acid-inducible protein I and melanoma differentiation-associated gene 5. Activation of these PRR engages intracellular signaling cascades leading to the secretion of type I IFN, which are important anti-viral cytokines ultimately facilitating viral clearance 1, 2. The signal transduction pathways leading to type I IFN expression involve activation of the serine/threonine kinases TANK-binding kinase 1 (TBK-1), also known as NF-κB activating kinase NAK 3, and inhibitor of κB kinase ε (IKKε), also known as IKKi 4.

Such materials are peer reviewed and may be re-organized www.selleckchem.com/products/ulixertinib-bvd-523-vrt752271.html 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. Figure 1. Gating strategy, abundance of TAM subsets and expression of Gr-1 and Ly6C in MMTVneu tumors. Figure 2. Expression of M1, M2 and functional macrophage markers in CD11bloF4/80hi and CD11bhiF4/80lo TAMs. Figure 3. Localization of TAMs in tumors.

Figure 4. Flow cytometry gating strategy for detection of CD64 and MERTK in TAM populations. Figure 5. Long-term in vivo BrdU labeling of blood monocytes and TAMs. Figure 6. Efficacy of monocyte depletion with Clodronate-loaded liposomes. Figure 7. Population definitions applied in the bone-marrow transfer experiment. Figure 8. Time-course of blood leukocyte chimerism after bone marrow transfer. Figure 9. Level of chimerism within lung macrophages. Figure 10. Presence

ABT-888 of eFluor670+ grafted macrophages in recipient tumors. Figure 11. Differentiation of adoptively transferred monocytes in circulation of recipient animals. Figure 12. Anti-BrdU and 7AAD staining of MMTVneu tumors, BrdU incorporation in bone marrow and blood monocytes. Figure 13. Blockade of cell cycle progression in TAMs by doxorubicin. Figure 14. Influence of CSF-1R blockade on blood monocyte populations. Figure 15. In silico promoter analysis of murine Csf1 gene. Table 1. Characteristics of Innsbruck and TCGA breast cancer patient PFKL cohorts. Table 2. Antibodies applied in flow cytometry and immunofluorescence. Table 3. Primers used in quantitative Real-Time PCR (qPCR). Table 4. Primers used for PCR after Chromatin Immunoprecipitation (ChIP). “
“Cross-presentation is an important mechanism by which DCs present exogenous antigens on MHC-I molecules, and activate CD8+ T cells,

cells that are crucial for the elimination of tumors. We investigated the feasibility of exploiting the capacity of the mannose receptor (MR) to improve both cross-presentation of tumor antigens and Th polarization, processes that are pivotal for the anti-tumor potency of cytotoxic T cells. To this end, we selected two glycan ligands of the MR, 3-sulfo-LewisA and tri-GlcNAc (N-acetylglucosamine), to conjugate to the model antigen OVA and assessed in vitro the effect on antigen presentation and Th differentiation. Our results demonstrate that conjugation of either 3-sulfo-LewisA or tri-GlcNAc specifically directs antigen to the MR. Both neo-glycoconjugates showed, even at low doses, improved uptake as compared with native OVA, resulting in enhanced cross-presentation. Using MR−/− and MyD88-TRIFF−/− bone marrow-derived DCs (BMDCs), we show that the cross-presentation of the neo-glycoconjugates is dependent on MR and independent of TLR-mediated signaling.