In this case–control

study, we present novel data from a large group of CF patients with bacterial sinus colonizations treated with EIGSS combined with an intensive peri- and postoperative treatment regimen intending to eradicate the bacteria and prevent recolonization. We found significantly lower levels of IgA and IgG BPI-ANCA after surgery both compared with the individual values before surgery and compared with CF patients Galunisertib molecular weight without EIGSS and LTX. We also confirmed the previous finding [5] of decreased IgA and IgG BPI-ANCA levels following double LTX. The decrease in the level of BPI-ANCA following LTX was more pronounced than after EIGSS. This could be ascribed to the immunosuppressive treatment given to

Selleck Lapatinib the LTX patients as well as the lungs being larger organs with more infected tissue than the sinuses. Our results strongly suggest that the surgical procedure of EIGSS and LTX with removal of the chronically infected tissue results in decreased BPI-ANCA levels. Our findings of unchanged antibody levels in the EIGSS group indicate that the BPI-ANCA decrease is not caused by a general decrease in immune response. As the CF treatment protocol basically has been unchanged throughout the period of observation, the pre- and postoperative treatment is not expected to influence the results [15]. However, the intensive postoperative local antibiotic treatment regimen in the EIGSS group is presumed to play a role in preventing

recolonization. There is limited knowledge regarding the mechanisms that determine the levels of BPI-ANCA in patients with CF. As BPI-ANCA is strongly correlated with colonization by P. aeruginosa and lung damage in patients with CF [5, 8], and as BPI-ANCA may be produced due to costimulation of the immune system with a complex of BPI and P. aeruginosa surface antigens, this could explain our findings and supports the theory that BPI-ANCA may be a useful surrogate marker of the Gram-negative bacterial load in patients with CF. Our findings in the 14 patients cultured from the sinuses during and HSP90 after EIGSS, showing that the sinus bacterial load in the majority of cases was eradicated or reduced postoperatively, further support this theory. Apart from reducing/eliminating the bacterial load in the nose and sinuses, it is also possible that our observation, that EIGSS can reduce the frequencies of not only upper but also lower airway cultures positive for Gram-negative bacteria in intermittently colonized patients [16], will contribute to decreasing BPI-ANCA due to the reduction in the bacterial load in the lungs, because intermittent colonization also stimulates an inflammatory response in patients with CF [17, 18].

In each of the outbreaks there was high sequence identity between the strains isolated within each individual outbreak. Panobinostat price The strain causing the outbreak in November of the same year had the closest

sequence identity to the Gulu 2000 outbreak strain [20]. The first recorded outbreak caused by BDBV, representing the species Bundibugyo ebolavirus, occurred in Uganda in 2007 [7] (Table 3). The virus was found again in a 2012 outbreak in Isiro in the DRC: this was the first identification of BDBV in the DRC. The BDBV isolate showed 98.6% full genome sequence identity with the prototype BDBV isolated in the 2007 outbreak in Bundibugyo, Uganda [20]. While FHF outbreaks have been reported in few countries in Africa (Fig. 1, Tables 2

and 3), the geographical distribution of filoviruses may be wider than previously thought. A feature of recent outbreaks is new strains/species in new locations, as has been the case with the MVD outbreak in Angola, the discovery of BDBV in Uganda and the DRC, and the current EBOV infection in West Africa [7, 20, 29, 35]. Using ecological niche modeling, filovirus distribution was generally predicted to occur across the Afro-tropics, with ebolaviruses occurring in the central and western African rain forests PLX4032 in vivo and marburgviruses in the drier and less forested central and eastern Africa [3]. Countries like Tanzania, Mozambique, Madagascar and Mauritania have had no reported outbreaks of filovirus infections, but do fall within the ecological niche of

this virus and its reservoir(s). It is possible that there have been misdiagnosed and undiagnosed cases in countries with no FHF outbreak history. In some areas with no recorded outbreaks of EVD, EBOV seroprevalence in humans and some species of nonhuman primates has been found to be unexpectedly high [32, 36]. This suggests either Thalidomide the presence of non-pathogenic variants of EBOV or unknown filoviruses antigenically similar to EBOV, but with lower pathogenicity, causing high seropositivity [32, 37-39]. This also implies high exposure of these populations to the virus [36]. Wider filovirus distribution, even into the Eurasian continent, has been suggested by recent studies that have reported the discovery of RESTV in domestic pigs in China [40]; identification of a new filovirus, LLOV in Spain [41] and detection of antibodies to filoviruses or unknown filovirus-related viruses in Indonesian orangutans [42] and fruit bats in Bangladesh [43]. Apart from R. aegyptiacus, the only bat species from which infectious marburgviruses have been isolated, other bat species in which filovirus genome RNAs have been detected are Epomops franqueti, Hypsignathus monstrosus and Myonycteris torquata for EBOV [44]; Miniopterus inflatus and Rhinolophus eloquens for MARV [45], and Miniopterus schreibersii for LLOV [41]. Many more bat species have been found to have antibodies to various filoviruses [46].

Before HPLC analysis, exopolysaccharide polymers were hydrolyzed into monomers by adding 1 mL of TFA 4 M to 1 mL of exopolysaccharide sample. The reaction was carried out for 2 h at 120 °C and TFA was removed by SpeedVac. The final exopolysaccharide sample was resuspended in 1 mL of dH2O. 1D and 2D NMR spectra of the exopolysaccharide in D2O (1 mg in 0.5 mL) were recorded at 70 °C on a Bruker AVANCE III 700 MHz spectrometer and on a Bruker AVANCE 500 MHz spectrometer, both equipped with 5 mm TCI Z-Gradient CryoProbes. 1H chemical shifts were referenced to internal TSP (δH 0.00) and Lumacaftor 13C chemical shifts

were referenced to external dioxane in D2O (δC 67.40). The 1D 1H,1H-TOCSY experiments were carried out with five different mixing

times between 10 and 120 ms. The 1H,13C-HMBC Decitabine order experiment was performed with a 65-ms delay for the evolution of long-range couplings. Data processing was performed using vendor-supplied software. Measurement of the translational diffusion coefficient of the exopolysaccharide was carried out as described previously (Eklund et al., 2005). We used 50 mM Tris-HCl pH 7.5 and borate–10%NaCl (in some animals, up to 10% NaCl is necessary for the IgG to precipitate with the Brucella O-chain or NH, and borate buffers often help in the diffusion of polysaccharides). Exopolysaccharide was used at 5 mg mL−1 and tested with a pool of cattle sera that yields good precipitin bands with S Brucella polysaccharides (as a reference, with this pool of sera, B. melitensis lipopolysaccharide precipitates at about 1 mg mL−1, the O-PS down to 100 μg mL−1, and the pure NH down to 5 μg mL−1). Other sera were PAK6 also tested from rabbits infected with B. melitensis (109 CFU intravenously) bled 3 months later, and from a rabbit infected with B. abortus 544 bled 6 months later. We also tested the exopolysaccharide in double-gel diffusion

with a serum from a rabbit hyperimmunized with B. melitensis 115 (rough) that yields several precipitin lines with soluble proteins. Brucella melitensis were grown for 20 h in 2YT medium at 37 °C. Cultures were then supplemented with 50 μg mL−1 DNaseI (Roche), incubated at 37 °C for different times and examined immediately by an agarose pad at appropriate times. For DIC imaging, cell populations of B. melitensis strains were placed on a microscope slide that was layered with a pad of 1% agarose containing PBS (agarose pads) (Jacobs et al., 1999). Samples were observed on a Nikon E1000 microscope through a differential interference contrast (DIC) × 100 objective with a Hamamatsu Orca-ER LCD camera. Images were taken and processed with Simple PCI (Hamamatsu). Brucella were grown for 20 h in 2YT medium at 37 °C. Cultures were adjusted at the same OD600 nm before centrifugation to separate the supernatants from the cell pellets.

4A–D). Since phenotypic analysis of NK cells (including CD56brightCD16± and CD56dimCD16+ NK-cell subsets) from PTLD patients has identified PD-1 up-regulation (Fig. 3), we next investigated whether disrupting PD-1 receptor binding during NK-cell stimulation may result in NK-cell function restoration in this cohort. To test the mechanism of PD-1

regulation, we incubated NK cells with autologous LCL in the presence or absence of PD-1 blocking mAb (or isotype control). This HM781-36B supplier treatment restored the IFN-γ response by CD56brightCD16± (Fig. 5A) NK cells, while CD107a release by CD56dimCD16+ (Fig. 5B) was only partially increased in PTLD patients. Interestingly, similar experiments performed on NK cells from LVL patients, who displayed low levels of PD-1 expression but maintained high NKp46 and NKG2D expression, have showed that blocking PD-1 resulted in increased IFN-γ and CD107a expression (Fig. 5A and B). NK cells, as part of innate Carfilzomib ic50 immunity, play an important role in the initial immunologic defense against viral infections 6, 7. However, the role of NK-cell surveillance during EBV latency, or chronic EBV infection with increased viral loads after Tx, or during PTLD remains elusive. Overall, our results show that NK-cell

phenotype and function are profoundly impaired in pediatric Tx PTLD patients (with a similar trend for chronic HVL carriers), indicating a possible NK-cell contribution to the Demeclocycline immunopathogenesis of EBV complications in the Tx setting. Here, we have identified for the first time significant differences in NK-cell subset distribution between EBV seropositive HC and pediatric Tx patients carrying, or not, an EBV load. On one hand, the CD56brightCD16± subset was increased in asymptomatic

Tx patients, suggesting possible differences in the NK functional (IFN-γ) requirements in pediatric Tx recipients versus HC. In contrast, PTLD patients showed decreased CD56brightCD16± and CD56dimCD16+ subset levels with an accumulation of CD56dimCD16− and CD56−CD16+ NK subsets. These changes in the NK-cell subset levels may be a consequence of high EBV challenge of NK cells seen with PTLD patients, leading to the possible CD56 receptor down-modulation on the conventional “functional” NK-cell subsets. Interestingly, recent studies have also described unusual accumulation of circulating dysfunctional CD56dimCD16− and CD56−CD16+ NK-cell subsets in patients with complications of chronic HIV and HCV infections, indicating a direct correlation between NK-cell subset defective function and chronic viral uncontrolled challenge 19–21. Early protection against EBV replication and against proliferation of EBV-infected targets was shown to rely on NK-cell ability to release IFN-γ and to mediate cytotoxicity in response to cytokine milieu instructions and to triggering receptor ligation by molecules on EBV-infected target cells 15, 16.

However, RCDII IELs lack CD8 and surface CD3-TCR complex [21-24], and whether ACD IELs express CD8αα was not indicated [21]. Freshly isolated RCDII and ACD IELs express higher Bcl-XL but lower Bcl-2 compared with IELs from healthy donors [21]. Therefore, these IEL lines likely do not resemble normal primary CD8αα+ IELs, and the IL-15-mediated

survival signals in normal CD8αα+ iIELs remain elusive. Here, we delineated the IL-15-induced survival signals in primary murine CD8αα+ iIELs in vitro, and confirmed their role in vivo. IL-15 supports CD8αα+ iIEL survival through the activation of the Jak3-Jak1-PI3K-Akt-ERK pathway to upregulate Bcl-2 and Mcl-1. Furthermore, this signaling axis does not affect the level of Bim, but promotes the dissociation of Bim from the Bim-Bcl-2 complex and maintains the dissociated Bim in a phosphorylated state. These results ERK inhibitor suggest a new mechanism by which IL-15 ABT-888 cell line modulates the members of the Bcl-2 family to support cell survival. We previously found that IL-15Rα supports the survival of CD8αα+ iIELs in vivo, and that exogenous IL-15 maintains live CD8αα+ iIELs

in vitro in an IL-15Rβ-dependent manner [2]. To dissect the IL-15-mediated survival signals using the in vitro system, we cultured CD8αα+ iIELs in 50 ng/mL of IL-15, as this amount of IL-15 stably maintained the percentage of live cells up to 64 h (Fig. 1A, top panels). Although 50 ng/mL of IL-15 induces proliferation of murine NK cells in vitro [25], it had little mitogenic effect on CD8αα+ iIELs as few PFKL cell in G2/S/M phase appeared by 64 h of culturing in IL-15 (Fig. 1A, lower panels). On the other hand, 50 ng/mL of IL-15 supported cell survival as shown by the relatively low percentage of cells in sub-G1 phase (Fig. 1A, lower panels). We investigated IL-15-triggered survival signals in CD8αα+ iIELs in vitro first by using inhibitors. Cells were treated with individual inhibitor for 1 h before the addition of IL-15. The inhibitor treatment did not alter the level of IL-15Rβγ on CD8αα+ αβ and γδ iIELs (Supporting Information Fig. 1A and B). Inhibitors of Jak3, PI3K (LY294002), protein kinase B/Akt (Akt) (Akt IV) and MEK (U0126) abolished IL-15′s

prosurvival, whereas inhibitors of p38 mitogen-activated protein kinase (SB203580) and mammalian target of rapamycin inhibitor (rapamycin) had no effect (Fig. 1B, line graphs). The effective inhibitors diminished IL-15′s prosurvival effect in a dose-dependent manner (Supporting Information Fig. 1C). As the αβ and γδ cell composition of CD8αα+ iIELs remained the same before and after culturing in medium alone, in IL-15, or in IL-15 plus each inhibitor (Fig. 1B, bar graphs), the IL-15-triggered survival signals are similar in the two subsets at the level of Jak3, PI3K, and ERK1/2 activation. Consistent with the inhibitors’ effects on CD8αα+ iIEL survival (Fig. 1B), IL-15 induced phosphorylation of Jak1, Akt, and ERK1/2 (Fig. 1C) with delayed kinetics for ERK1/2 phosphorylation.

Microglia and astrocytes are activated following tissue injury or inflammation and have been reported to be both necessary

and sufficient for enhanced nociception. Blood-borne monocytes/macrophages can infiltrate the central nervous system (CNS) and differentiate into microglia resulting in hypersensitivity and chronic pain. The primary aim of this study was to evaluate the proportion of the proinflammatory CD14+CD16+ monocytes as well as plasma cytokine levels in blood from CRPS Selleck Poziotinib patients compared to age- and gender-matched healthy control individuals. Forty-six subjects (25 CRPS, 21 controls) were recruited for this study. The percentage of monocytes, T, B or natural killer (NK) cells did not differ between CRPS and controls. However, buy Ceritinib the percentage of the CD14+CD16+ monocyte/macrophage subgroup was elevated significantly (P < 0·01) in CRPS compared to controls. Individuals with high percentage of CD14+CD16+ demonstrated significantly lower (P < 0·05) plasma levels on the anti-inflammatory cytokine interleukin (IL)-10. Our data cannot determine whether CD14+CD16+ monocytes became elevated prior

to or after developing CRPS. In either case, the elevation of blood proinflammatoty monocytes prior to the initiating event may predispose individuals for developing the syndrome whereas the elevation of blood proinflammatory monocytes following the development of CRPS may be relevant for its maintenance. Further evaluation of the role the immune system plays in the pathogenesis of CRPS may aid in elucidating disease mechanisms as well as the development of novel therapies for its treatment. Complex regional pain syndrome (CRPS) is a severe chronic pain disorder that often follows an injury to peripheral nerves [1,2]. CRPS demonstrates a 3:1 female to male preponderance and is characterized by pain that is out of proportion to the initial injury and does not respect a nerve or root distribution [3,4]. The signs and symptoms of CRPS cluster into four categories: (1) abnormalities in pain processing; (2) skin colour and temperature

changes; (3) sudomotor abnormalities and oedema; and (4) motor dysfunction and trophic changes [5,6]. Although the pathophysiology of CRPS is not completely understood, there is evidence demonstrating that neurogenic inflammation plays a significant role [7,8]. Fossariinae Furthermore, neuroinflammation and neuroimmune activation have been shown to act in concert in persistent pain states [9]. Following injury, mast cells, neutrophils and macrophages are recruited to the involved area and can invade the nerve through a disrupted blood–nerve barrier [10,11]. These cells produce a variety of proinflammatory cytokines that have been implicated in the generation of neuropathic pain either by direct sensitization of nociceptors or indirectly by stimulating the release of agents that act on neurones and glia [12,13].

n., submandibular lymph nodes, but not NALTs, were consistently www.selleckchem.com/products/Adrucil(Fluorouracil).html clearly stained (Fig. 3, inset). These results taken together demonstrate that the submandibular lymph nodes are the main organ that responds to i.n. injected allergens. To explore the mechanisms of IgG Ab production and compare them with those of IgE Ab production in submandibular lymph nodes, we injected the allergen with or without complete Freund’s adjuvant i.n. once into BALB/c mice (Fig. 4). A significant amount of serum IgE (465.4 ±111.6 ng/mL; mean ± SD; n =9) was induced by one i.n. injection of allergen alone. In contrast, one i.n. injection

of the allergen with adjuvant induced a much smaller amount of serum IgE (172.5 ± 74.7ng/mL; mean ± SD; n =9). This was greater than that (57.6 ± 32.2 ng/mL; mean ± SD; n =9) in mice treated with adjuvant alone or that (40.8 ± 14.8 ng/mL; mean ± SD; n =9) in PBS-injected mice. In contrast, a large amount of serum IgG (1585.4 ± 161.0 μg/mL; mean ± SD; n =9) was induced by one i.n. injection

of the allergen with adjuvant into mice; this amount of serum IgG was greater than C59 wnt mouse that obtained after one i.n. injection of adjuvant (1018.2 ±33.2 μg/mL; mean ± SD; n =9) or allergen (904.9 ± 51.2 μg/mL; mean ± SD; n =9) alone, both of which were greater than that (514.7 ± 161.8 μg/mL; mean ± SD; n =9) in PBS-injected mice. These results indicate that one i.n. injection of allergen alone or with adjuvant is suitable for induction of serum IgE or IgG Ab, respectively. To explore which population of cells in the submandibular lymph nodes is involved in the production of IgE Ab in response to the allergen, we separated the cells into macrophage-, lymphocyte-, and granulocyte-rich populations by Percoll density-gradient centrifugation. The yield of cells from the submandibular lymph nodes

was 78–89% (n =9). Fraction 3 (rich in lymphocytes) was the major (93.5 ± 7.2%; mean ± SD; n =9) population, Non-specific serine/threonine protein kinase followed by fraction 2 (rich in macrophages; 1.2 ± 0.1%; mean ± SD; n =9) and fraction 4 (rich in granulocytes; 0.3 ± 0.1%; mean ± SD; n =9) in that order. Fraction 1 (rich in somewhat damaged cells) contained a small number of cells. As we obtained the macrophage-, lymphocyte-, and granulocyte-rich fractions, we incubated various combinations of these cells for 6 days and then assessed the amounts of IgE Ab in the culture media (Fig. 5). Bulk submandibular lymph node cells from mice that had been treated with allergen once i.n. produced a significant amount of IgE Abs (6.2 ± 3.4 ng/mL; mean ± SD; n =9); whereas the lymphocyte-rich (fraction 3) fraction of the lymph node cells did not (1.5 ± 0.8 ng/mL; mean ± SD; n =9). The macrophage-rich (fraction 2) fraction was also inactive (1.1 ± 0.9 ng/mL; mean ± SD; n =9). Of particular interest, IgE Ab production (4.6 ± 2.8 ng/mL; mean ± SD; n =9) was restored by addition of the macrophage-rich fraction to the lymphocyte-rich fraction.

[10, 12, 13] Despite their

unquestionable impact on functions of myeloid and lymphoid cells of the innate and adaptive immune system, little is known about the regulation of these important mediators by particular local conditions in specific organ systems. In the present study we aimed to get further insight into the regulation of eicosanoid metabolism by n-butyrate in human monocytes. Based on insights from a multigene signature approach evaluating a broad range of inflammation-related genes we focused here on the modulation of the expression of eicosanoid pathway-related genes after microbial activation and concomitant interference with n-butyrate. We found that in bacterially activated human monocytes activated by Toll-like receptor 2 (TLR2) and TLR4 ligation n-butyrate potentiated the expression of cyclo-oxygenase 2 (COX-2) along with increased PGE2 expression. selleckchem The implications

of these findings are discussed. RPMI-1640, supplemented with 2 mm l-glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin and 10% fetal calf serum were purchased from PAA (Pasching Austria). The sodium salt of n-butyric acid, TLR4 ligand LPS from Escherichia coli 0111:B4 and TLR2 ligand Staphylococcus aureus Cowan strain A cells were purchased from Sigma (Deisenhofen, Germany). The dose of LPS used in our assays was 100 ng/ml and the n-butyrate dose was 1 mm if not indicated differently. Palbociclib supplier Human peripheral blood mononuclear cells were isolated from buffy coats (provided by the Austrian Red Cross) by density gradient centrifugation with Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). Subsequently, monocytes were isolated from peripheral blood mononuclear cells by magnetic cell sorting using anti-CD14-conjugated magnetic beads purchased from Miltenyi Biotec (Bergisch-Gladbach, Germany). The purity of the monocytes was verified via FACS analysis on a FACSCalibur. Purity of isolated monocytes in all experiments was > 95% (data not shown). We here used a validated multigene signature approach to investigate transcriptional programmes triggered by n-butyrate and LPS alone or in combination.

Based on the knowledge-driven approach of innate immune cell biology and inflammatory process data mining, a signature of immunity/inflammation-associated LY294002 genes was assembled. TaqMan® array covering immunity/inflammation-related genes (pre-designed; Applied Biosystems, La Jolla, CA) were used as part of the self-designed 180-gene signature. This signature contained targets involved in immune response and inflammation, and included many upstream signalling molecules (kinases and phosphatases in hierarchical levels), transcription factors, and the downstream chemokines and cytokines. PTGS2 (also known as COX-2), a key enzyme in the biosynthesis of prostanoids, and other molecules central to eicosanoid signalling were also included on the array.

Taken together, our studies indicate that IL-13 production is more widespread than previously appreciated and that blocking this cytokine may have therapeutic benefits even in settings where traditional IL-4-driven Th2-type responses are not evident. Naive CD4+ “helper” T cells can differentiate into multiple effector subsets, each defined by the transcription factors (TFs) that they employ, the cytokines they secrete, and ultimately, the functions selleck compound they execute. The Th2 subset was among the first to be recognized and is characterized by STAT6 and Gata-3, production of IL-4, its role in combating helminth infections, and its association with inflammatory disorders like

asthma and allergy [1, 2]. Traditionally, Th2 differentiation is thought to be driven by IL-4 and its ability to activate STAT6, a potent inducer of hallmark Th2-type genes, including IL-4, IL-13, c-Maf, and Gata-3. However, despite a dominant role for IL-4 and STAT6 in many settings, Th2 responses can be generated in their absence and other factors are known to promote Th2 differentiation, including

IL-2, IL-25, IL-33, TSLP, Notch, STAT3, STAT5, and IRF4 [1-4]. Because many of these are associated with alternative T-cell subsets, such as Th1 (STAT5) and Th17 (STAT3, IRF4), it is now understood that some of the TFs involved in Th2 differentiation are not subset-specific and that there is a degree of plasticity within the Th2 program. Consistent with this latter point, studies have shown that Th2 cells can be “re-programmed” to exhibit characteristics of other T-cell AZD6244 subsets, like production of IL-9 (Th9) or IFN-γ (Th1), and that IL-4+ memory Th2 cells can produce IL-17 [5-8]. Thus, while originally viewed as a terminal, IL-4-driven fate, current models posit that Th2 cells result from the integration of multiple signals, and that they are adaptable, at times

able to acquire the functions of related subsets. Along with IL-4, Th2 cells are known to produce IL-13 that is located adjacent to the il4 locus on mouse chromosome 11 (human chromosome 5). Due to this genomic Thiamet G proximity, it is was initially believed that IL-4 and IL-13 are regulated by the same upstream signals and since they share a common receptor (IL-4Rα) and signaling pathway (STAT6), it was also thought that they have analogous functions, an idea bolstered by studies showing that Th2-type inflammation is depressed in mice lacking either cytokine [2, 9]. However, despite the similarities, it is now understood that IL-4 and IL-13 are not always coexpressed, that they can act on different cell types, and that IL-13 can drive IL-4-independent inflammation in various settings [3, 9-13]. Based on these latter findings, IL-13 has become an attractive therapeutic target for Th2-mediated disease, creating a need to better define the source and function of this cytokine.

We found Tem cells highly expressing OX40, even if at lower level than Treg cells (Fig. 3C); thus intratumoral OX86 injection could directly target Tem cells at this site. Conversely, Tem cells obtained after immunization check details of naïve BALB/c mice with two consecutive injections of BM-derived dendritic cells (BMDCs) activated with LPS, as previously described 17, expressed low or absent OX40, even after in vitro activation (Supporting Information Fig. 3). In BMDC-injected animals, Tem cells were shown to constitutively express CD40L at sufficient levels to induce DC activation 17. The CD40/CD40L interaction is crucial for DC activation,

survival and proliferation 26. Many data suggest that this axis is involved in inducing protective anti-tumor immune response 18, 19, 27, 28 and that activation of this pathway may represent a strategy for tumor treatment. To investigate whether OX86-induced tumor rejection was dependent on the CD40/CD40L axis, WT and CD40−/− mice were inoculated subcutaneously

with CT26 cells and treated intratumorally with OX86. As shown in Fig. 4A, OX86 treatment was ineffective in CD40-deficient mice, while inducing tumor rejection or impaired tumor growth in CD40-sufficient mice. Such failure to induce anti-tumor response in the absence of CD40 could be either due to defective licensing of DC reactivation and migration from the tumor or due to impaired T-cell priming at the dLNs by DCs licensed, but not competent, for optimal T-cell costimulation. Methane monooxygenase To discriminate between these

two possibilities, an in vivo DC migration assay was performed. Tumors Selleckchem Trichostatin A growing in WT and CD40−/− mice were treated with OX86 or rat IgG co-injected with green fluorescent microbeads. Only DCs that have up-taken the beads at the tumor site can be detected as fluorescent in dLNs. Although OX86 rescued DC migration from tumor to dLNs in WT mice, the same treatment was ineffective in CD40−/− mice (Fig. 4B), a finding implying that in absence of the CD40/CD40L axis tumor-associated DCs cannot be reactivated. We also confirmed that, in CD40−/− mice, the OX40 expression by tumor-infiltrating Tem cells was not defective, indicating that the CD40/CD40L system did not affect the T-cell activation status at the tumor site or Tem-cell responsiveness to OX86 treatment (Supporting Information Fig. 4). We hypothesized that, in the immunosuppressive tumor microenvironment, Tem cells were inhibited in their ability to license DCs via CD40L, and that OX40 triggering might provide the right signal for Tem cells to supply an effective CD40/CD40L mediated co-stimulation. Although the intratumoral injection of OX86 did not change the percentage of Tem cells (Fig. 4C), it significantly upregulated CD40L expression on Tem cells (Fig. 4D). Such CD40L up-modulation was specific for Tem cells, as no other T-cell subsets and especially CD44lowCD62Llow recently activated T (Tact) cells responded accordingly (Fig. 4E).