Leucocyte-enriched buffy coats (transfusion centre, Mainz, Germany) were obtained from non-allergic, non-atopic, tetanus-immunized healthy blood donors. The study was approved by the local ethics committee. Informed consent was obtained from all donors before participation in the study. Peripheral blood mononuclear cells

(PBMC) were isolated from heparinized blood by Ficoll-Paque 1·077 g/ml (PAA Laboratories GmbH, Cölbe, Germany) density centrifugation. To enrich CD14+ monocytes, 1 × 107 PBMC per well were incubated for 45 min in a six-well plate (Greiner, Frickenhausen, Germany) in Iscove’s modified Dulbecco’s medium containing l-glutamine and 25 mm Hepes (IMDM; selleck kinase inhibitor PAA Laboratories GmbH) supplemented with an antibiotic-antimycotic solution containing 100 μg/mL streptomycin, 100 U/mL penicillin, and 250 ng/ml amphotericin B (PAA) and 3% autologous plasma at 37°. After washing of the non-adherent cells with pre-warmed PBS, the remaining monocytes (purity > 90%) were incubated in 3 ml/well Sunitinib cost IMDM supplemented with 1% heat-inactivated autologous plasma, 1000 U/ml IL-4 (Strathmann Biotech GmbH, Hannover, Germany) and 200 U/ml granulocyte–macrophage colony-stimulating factor (GM-CSF) (Leukine®; Immunex Corp., Seattle, WA). On day 6, the resulting

immature DCs were pulsed with different amounts of OVA or AGE-OVA, as indicated in the figures, in the presence or absence of 10 μg/ml polymyxin B sulphate (Sigma-Aldrich) or 1 μg/ml tetanus toxoid (Behring-Werke, Marburg, Germany), and further stimulated with 1000 U/ml TNF-α, 2000 U/ml IL-1β (Strathmann Biotech GmbH) and 1 μg/ml PGE2 (Cayman Chemical, Ann Arbor, MI) to induce their full maturation. Forty-eight hours after stimulation, the supernatant of mature DCs was collected for determination of IL-6 and IL-12p40. The cells were then harvested, washed twice and used in T-cell stimulation assays. Mature DCs expressed high levels (> 90%) of CD80, CD83, CD86 and MHC class II molecules as determined by flow cytometry. Autologous CD4+ T cells were obtained from PBMC using antibody-coated paramagnetic MicroBeads (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) according

to the protocol of the manufacturer. Separation below was controlled by flow cytometry (purity > 98%). For proliferation assays, 1 × 105 CD4+ T cells were co-cultured in 96-well plates (Greiner) in triplicate with 1 × 104 autologous allergen-pulsed DCs in 200 μl of IMDM supplemented with 5% heat-inactivated autologous plasma. After 5 days, the cells were pulsed with 37 kBq/well of [3H]TdR ([methyl-3H]thymidine; ICN, Irvine, CA) for 6 hr, and [3H]TdR incorporation was evaluated in a beta counter (1205 Betaplate; LKB Wallac, Turku, Finland). For cytokine production assays, 5 × 105 CD4+ T cells were cultured in 48-well plates with 5 × 104 autologous allergen-pulsed DCs in 1 ml of IMDM supplemented with 5% heat-inactivated autologous plasma.

Soluble RAGE (sRAGE), a truncated form of the receptor, is composed of only the extracellular ligand-binding domain lacking the cytosolic and transmembrane domains. sRAGE is produced either by alternative splicing of RAGE mRNA or by carboxyterminal truncation of RAGE through metalloproteinase [25, 26]. sRAGE has the same ligand-binding selleck chemical specificity as RAGE and may function as a ‘decoy’ by binding

pro-inflammatory ligands including HMGB1 and preventing them from accessing cell surface RAGE [27]. In addition, Zong et al. [28] demonstrated that RAGE forms homodimers at the plasma membrane and dimerization is an important step in RAGE signalling. sRAGE can also bind RAGE and incubation of cells with sRAGE inhibits RAGE dimerization and subsequent activation of NF-κB pathways. Therefore, decreased sRAGE levels may indicate activation of RAGE signalling and enhanced inflammation. Up to now, decreased serum level of sRAGE has been detected in multiple sclerosis,

primary Sjögren’s syndrome and RA [29–31]. Moreover, it has been demonstrated in a number of experimental animal models in which administration of sRAGE was used as the therapeutic treatment [32, 33]. All these investigations indicate Selleck Poziotinib that sRAGE may represent a future therapeutic target in chronic inflammatory diseases. Only one report published recently investigated the role of sRAGE in the pathogenesis of SLE and showed that serum levels of sRAGE were increased in patients with SLE [34]. However, these results are preliminary because of the low case number (n = 10). Further investigation with a larger cohort of patients with SLE should be valuable to determine the potential role of sRAGE in the pathogenesis of SLE. In this study, we investigated plasma levels of sRAGE in 105 patients with SLE (including 75 patients receiving antilupus treatment and 30 untreated patients) and 43 age- and sex-matched healthy controls to assess Farnesyltransferase whether there was an association between sRAGE levels and disease characteristics. Subjects.  A total of 105 patients (100 women, five men;

age of 32.4 ± 11.3) from Department of Rheumatology, Provincial Hospital affiliated to Shandong University were included in this study. All patients conformed to the American College of Rheumatology classification criteria for the diagnosis of SLE [35]. The SLE disease activity index (SLEDAI) was used to estimate global disease activity and active disease was defined as SLEDAI >4. A total of 74 patients had active SLE, while 31 patients had inactive SLE. Among these 105 cases, 30 patients were newly diagnosed SLE and did not receive any treatment in the past 3 months. Thirty-three patients received monotherapy with corticosteroids, 11 patients received corticosteroids in combination with antimalarials and 31 patients received corticosteroids in combination with immunosuppressors.

Defects in CD44-deficient macrophages migration to the click here lung were previously described following intranasal infection with Mycobacterium tuberculosis 28 and exposure to inhaled lipopolysaccharides 27. Taken together, recruitment of macrophages to the lung is, in part, dependent on CD44. Although most blood cells are CD44+, only small numbers use it to recognize HA. We recently reported that HA-binding activity of CD44 is regulated

by sialidase Neu1. In accordance with this finding, antigen-activated Th2 cells that more effectively bound HA expressed higher levels of Neu1 as compared with Th1 cells. In addition, the CD44KO mice used in this study could express truncated CD44 molecule. However, they were generated by deletion of exon 2 and exon 3 containing

possible HA-binding site 29, 30, suggesting that the ability of the truncated CD44 potentially expressed in those mice to bind HA is gone. Therefore, our presented findings using CD44KO mice and Th1-/Th2-transferred mice strongly suggest that not only the expression, but also the HA-binding ability of CD44, is important for the accumulation of Th2 cells in the lung. In conclusion, our findings indicate that CD44 expressed on Th2 cells plays a critical role in the accumulation of Th2 cells in the lung and the resulting airway inflammation including Gefitinib ic50 the development of AHR induced by antigen challenge. Our observation suggests that CD44 could be a target molecule for the treatment of Th2-mediated airway inflammation Urease including allergic asthma. Further investigations are required to clarify the role of CD44 in chronic airway inflammation. BALB/c and C57BL/6 mice (female, 8–12 wk old) were obtained from Charles River Laboratory (Yokohama, Japan). DO11.10 transgenic mice (BALB/c background) were from Jackson Laboratory (Bar Harbor, ME). CD44-deficient mice on a C57BL/6 background were generated at Amgen Institute (Toronto, Canada; generously provided by Dr. Tak W. Mak from the University Health Network in Toronto, Canada) and were characterized previously 29, 31. We used female mice, 8–12 wk old, bred in the experimental

animal center of Kagawa University and Kawasaki Medical School. Mice were sensitized by intraperitoneal injections of 500 μg Derf allergen (GREER Laboratories, Lenoir, NC) with 2 mg alum on day 0 and day 14. The mice were then challenged by intranasal administration of 800 μg Derf solution on day 29. Negative control animal was injected with phosphate-buffered saline (PBS) plus alum and exposed to PBS in a similar manner. All experiments in this study were approved by the institutional animal care and use committee of Kagawa University and Kawasaki Medical School. Bronchoalveolar lavage was obtained by washing the lungs with 4×1 mL of PBS and centrifuged. The supernatant of the first wash was stored at −80°C until use. Cell pellets of all washes were collected and re-suspended in 1 mL of PBS.

In addition to changes at the mRNA level, master transcription factors drive epigenetic modifications of many Th effector genes that reinforce the dominant phenotype [61, 62]. These epigenetic patterns are passed on to the cell’s progeny, creating a single Th

clone with similar epigenetic imprinting, that is, a Th-cell phenotype. Cytokine production by Th cells typically requires a few days of differentiation following the initial activation [41, 42], but phenotype induction at the transcriptional level already occurs within a few hours [63-65]. Over the last decade, Th-cell feedback mechanisms have been studied extensively using mathematical modelling. Whereas older studies focused on Th1/Th2 differentiation [66-68], more recent studies have included Palbociclib purchase Treg and the novel Th-cell phenotypes [69-73]. Most of these mathematical models incorporate positive feedback and cross-inhibition. These models are typically parameterized in such manner that only single master transcription factors can be expressed, but co-expression can occur with other parameter regimes [71]. Interestingly, models have been formulated both at the inter- and intracellular level, and models at either level are capable of explaining Th differentiation

in response to outside signals, showing that there is redundancy in the system. Some studies have attempted to incorporate feedbacks at the genetic and epigenetic levels into models [56, 73], although only a single feedback loop is sufficient to explain Th-cell phenotypes. Modelling has also illustrated that AZD6244 cell line master regulator heterodimer formation Thiamet G is sufficient for explaining mutual inhibition [71]. In addition to make the inducible phenotypes mathematically tractable as

alternative ‘steady states’ or ‘attractors’ of a dynamical system, these models provide insight into the development of Th-cell phenotypes over time, that is, the time series of changes that these cells undergo. These models show that early skewing leads to progressive differentiation into Th-cell phenotype as seen by experimental studies [43, 65]. In addition to traditional approaches, Th-cell differentiation has been studied intensively using high-throughput techniques. The targets of many important Th transcription factors have been mapped [9, 13, 14, 63, 74], and expression profiling has been performed by a number of groups [8, 63, 65, 75]. We and others have advocated a time series approach to Th-cell differentiation, because the Th-cell transcriptome is very dynamic in time. Indeed, we have shown that the mRNA signature of Th cells changes rapidly after the cognate priming and that genes can be classified into a ‘core’ and ‘turnover’ groups, and these also differ when different phenotypes are induced [65].

Levels of activated JAK and signal transducer and activator of transcription (STAT) proteins were detected by immunoblot analysis. Target-gene expression levels were measured by reverse transcription–polymerase chain reaction (RT–PCR) or real-time PCR. The JAK inhibitors CP-690,550 this website and INCB028050 both suppressed activation of JAK-1/-2/-3 and downstream STAT-1/-3/-5, as well as the expression levels of target proinflammatory genes (MCP-I, SAA1/2) in oncostatin-M (OSM)-stimulated rheumatoid synovial fibroblasts. In contrast, the JAK-3-selective inhibitor, PF-956980, suppressed STAT-1/-5 activation but did not affect

STAT-3 activation in OSM-stimulated rheumatoid synovial fibroblasts. In addition, PF-956980 significantly suppressed MCP-1 gene expression, but did not block SAA1/2 gene expression in OSM-stimulated rheumatoid synovial fibroblasts. These data suggest that

Opaganib research buy JAK-3-selective inhibition alone is insufficient to control STAT-3-dependent signalling in rheumatoid synovial fibroblasts, and inhibition of JAKs, including JAK-1/-2, is needed to control the proinflammatory cascade in RA. The Janus kinase (JAK) family of cytoplasmic tyrosine kinases mediates signalling by association with type 1 and type II cytokine receptors [1]. JAK activation leads to activation of their downstream substrates, the signal transducer and activator of transcription Florfenicol (STAT) proteins, followed by their nuclear translocation and subsequent activation of target genes [2]. Dysfunctional JAK/STAT signalling has been implicated in various haematological and immunological disorders [3] and other pathological inflammatory conditions, such as rheumatoid arthritis (RA) [4]. Because JAKs play an essential role in cellular signalling pathways involved in regulating the immune and inflammatory process [5, 6], targeting of the JAK family members may cause immunosuppression or anti-inflammatory effects [7]. Clarification of the

modification of downstream signalling cascades induced by JAK inhibition is thus important for elucidating the molecular mechanisms whereby JAK inhibitors might exert their beneficial effects against RA. JAK-3 is important in proinflammatory cytokine-mediated signalling [8, 9], which is involved in the pathogenesis of RA. The use of kinase inhibitors with wide-ranging effects on immune/inflammatory mediators may have a more beneficial response than biological agents that target a single cytokine [10, 11]. Small-molecule inhibitors of JAKs are emerging as promising therapies for RA [12]. However, the inhibitory activities responsible for the beneficial effects of these inhibitors against RA are unknown. The JAK-3 inhibitor CP-690,550 has demonstrated efficacy in clinical trials of RA [13-15]. Although CP-690,550 inhibits JAK-3, it also exerts overlapping activities against JAK-1 and JAK-2 [16].

These counts returned to basal levels during the recovery phase. These findings are in accordance with the literature reports that showed increased number of blood eosinophils following helminthic infections (15).

Their subsequent disappearance from the blood has been attributed to migration to the site of the infection where they degranulate, releasing eosinophil secondary granule proteins (16). Production selleck screening library of cytokines by secondary lymphoid organ cultures stimulated with specific antigens and Con A was used to characterize cellular immunity. Considering IFN-γ induction by specific stimuli, a significant production was detected during the acute phase but not at the recovery phase. The opposite happened with IL-10 production, i.e. absence of this cytokine at the acute selleck period and presence of detectable levels during the recovery phase. Analysing these data together with antibody levels (IgG subclasses and IgE), we could suggest that an initial mixed pattern (Th1/Th2) at the acute phase

was followed predominantly by a Th2 polarization during the recovery phase. Production of IFN-γ and IL-10 stimulated by polyclonal activation with Con A showed a similar pattern, i.e. a general decreased production of these mediators by cultures of spleen and lymph nodes. A theoretical explanation for this finding is that T lymphocytes capable of producing these cytokines migrate from lymphoid organs to the places of temporary (lungs) or final (intestine) establishment of the worm. This possibility is supported by recent literature reports (3,8,17). Together these results

show that experimental inoculation of Lewis rats with S. venezuelensis triggers an infection that is similar in terms of kinetics of parasite establishment and immunity to experimental strongyloidiasis in other rodents and also in human S. stercoralis infection. The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) that supported this study with grants. “
“Human Amino acid parvovirus B19 (B19) has been, for decades, the only parvovirus known to be pathogenic in humans. Another pathogenic human parvovirus, human bocavirus (HBoV), was recently identified in respiratory samples from children with acute lower respiratory tract symptoms. Both B19 and HBoV are transmitted by the respiratory route. The vast majority of adults are IgG seropositive for HBoV, whereas the HBoV-specific Th-cell immunity has not much been studied. The aim of this study was to increase our knowledge on HBoV-specific Th-cell immunity by examining HBoV-specific T-cell proliferation, Interferon-gamma (IFN-γ), IL-10 and IL-13 responses in 36 asymptomatic adults. Recombinant HBoV VP2 virus-like particles (VLP) were used as antigen. HBoV-specific responses were compared with those elicited by B19 VP2 VLP.

Again, this adds impetuous to the need for clinical intervention trials with supplement of the circulating

25-OHD pool, which may be less harmful than supplementation with active vitamin D. Currently there is growing interest in the phosphaturic bone-hormone fibroblast growth factor 23 (FGF-23), which acts by binding to a membrane LBH589 in vitro bound α-Klotho-FGF receptor 1c complex in the distal tubules of the kidney, and by an unknown signalling mechanism reduces phosphate reabsorption in the proximal tubules.133 FGF-23 also acts as a negative regulator of PTH secretion by the parathyroid glands, and also directly inhibits 1,25-OHD production in the kidneys by reducing CYP27B1 activity.133 FGF-23 levels are elevated in early kidney disease, Nutlin-3a cell line and in various observational studies have shown association with vascular calcification, increased left ventricular mass in all stages of CKD, and importantly is an independent predictor of mortality in incident dialysis patients.134 It has been suggested that the

early changes in FGF-23 concentrations to maintain a normal serum phosphate in CKD may explain the alteration in vitamin D metabolism observed and could be the underlying causative factor for increased cardiovascular risk, not abnormal vitamin D metabolism per se. However, to date no Klotho protein complex has been isolated in any tissue pertinent to the cardiovascular system outside the kidneys, and in response to the supposition that supraphysiological levels of FGF-23 encountered

could act in a non-receptor driven fashion, it should be noted that in HAS1 non-renal conditions associated with excessive FGF-23 (e.g. X-linked hypophosphataemia or tumour-induced osteomalacia) notable increases in cardiovascular risk are not encountered. This is a growing area of research attention and more data should be available in the near future. Patients with CKD are at significant risk of cardiovascular disease, beyond that of the normal population, and this is not fully explained by the traditional Framingham risk factors. Vitamin D deficiency is increasingly common as CKD progresses, for a variety of reasons. Experimental and clinical studies suggest that vitamin D may improve cardiovascular risk through such diverse mechanisms as improved glycaemic control, anti-inflammatory actions, enhanced endothelial function, decreased atherosclerosis and atherogenesis, suppression of the RAS, reduction of proteinuria, and improved cardiovascular physiology (summarized in Fig. 2).

After selection with G418 we performed

PCR screening (PCR: 5-tcaacctacaaacggaaagaa and 5′-ctaaacccaaacacagaccta). As a PCR control we cloned a similar genomic fragment of the IgG1 region in front of IgE. The test-arm fragment was 155 bp longer, as the actual target vector region, in order to avoid PCR contaminations (Supporting Information Fig. 4). The expected PCR size for controls is 1050 bp and for correct integration of the target vector is 895 bp (Supporting Information Fig. 4). Then, we verified positive clones by southern blots using an external genomic probe (Fig. 1A). Three independent positive clones were injected and chimeric offspring was bred to Deleter mouse strain on the 129Sv genetic background [42]. Testing of the Cre-deletion was done using the primers:

5′-atgggagtttctgtgattct selleck kinase inhibitor and 5′-gcccagaaggataagaaaac for the IgE knock-in (PCR-B, 590 bp) (Fig. 1A). After Cre deletion backcrossing for nine generations to C57BL/6 was performed. Some of these mice were then mated to CD23-deficient mice [23] on a C57BL/6 background. All studies with mice were performed in accordance with German animal experimentation law. Immunoglobulin isotype-specific ELISA was done using goat anti-mouse immunoglobulin anti-sera (Southern Biotech, USA) except for IgE detection, which was done with monoclonal anti-IgE antibodies 84.1C and EM95.3 [43, 44] and total murine IgG, which was done by goat anti-mouse IgG (Jackson, USA). For antigen-specific PD0325901 ELISAs, we coated with 10 μg/mL TNP-OVA. We used pooled sera

from immunized mice as a standard and titrated the samples in serial dilutions and gave the titers of specific Igs as relative Units/mL. Anti-CD23 (B3B4, BD Biosciences, USA), anti-CD45RB-B220, anti-IgG1 (Clone A85-1, BD or RMG1-1, Biologend) and anti-IgE (Clone23G3, BD; or EM95.3) FITC and PE-labeled antibodies were used in FACS analysis on cells that have been preincubated with mouse IgG as Fc block (Jackson Immuno Research, USA) on a FACScalibur (BD Biosciences, USA). For the detection of surface IgE and IgG1 after N. brasiliensis infection, mesenteric lymph node cells were prepared by mechanical disruption in 70 μm cell strainers (BD Falcon) and washed with an acid buffer (0.085 M NaCl, 0.005 Atazanavir M KCl, 0.01 M EDTA, and 0.05 M NaAcetate (pH 4)) to remove extrinsic IgE bound to CD23. For the detection of mouse mast cell protease-1 (MMCP-1) in plasma of anaphylactic mice, we used an ELISA kit (eBiosciences, USA) according to the manufacturer. In vitro antibody production was examined in total spleen cells stimulate with 20 μg/mL LPS, with and without IL-4 (Peprotech, USA) for 4–5 days. For antigen-specific immune response 3-month old mice were sensitized by injection with 100 μg TNP-OVA (Biosearch Technologies, USA), precipitated with alum (Serva, Heidelberg, Germany), subcutaneously and i.p. After 14 days mice received a similar booster injection.

c-C3BP or rGAPDH was observed (Figure 3c, d). The H.c-C3BP or rGAPDH interaction with C3 was specific and strong, which was evident from the fact that the column-bound C3 was eluted at high salt wash (0·5 m NaCl) or by lowering the pH to 2·2. To test whether H.c-C3BP or rGAPDH binding to C3 would influence complement function, a simple haemolytic assay was performed where the lysis of sensitized sheep erythrocytes by serum complement proteins was measured. As shown in Figure 3(e, f), a dose-dependent inhibition of erythrocyte lysis by H.c-C3BP and rGAPDH was observed. To rule out that the observed inhibition was not due to suppression of the classical pathway, binding of C1q protein by H.c-C3BP was

measured. No interaction among these proteins was evident in the microtitre plate assay (not shown). To confirm selleck chemicals whether the inhibition of erythrocyte lysis by H.c-C3BP or rGAPDH was due to suppression of C3 activation, the formation of membrane attack complex (MAC) was measured on the LPS-coated surface. A dose-dependent decrease in the formation of MAC was observed in the presence of H.c-C3BP or rGAPDH (Figure 3g, h). The presence of H.c-C3BP (GAPDH) in the ES products of H. contortus suggests that the protein should

also be secreted in the host stomach where it is likely to come in contact with the immune effector cells at the injured site leading to antibody production. This assumption was amply supported by the presence of anti-H.c-C3BP/GAPDH antibodies in H. contortus-infected animals. The H.c-C3BP and rGAPDH reacted with the infected animal sera, whereas no reaction was observed with the serum www.selleckchem.com/products/PLX-4032.html from an uninfected animal in Western blot (Figure 4). For H. contortus infection, six healthy 6- to 8-month-old goats were infected with ~10 000 L3-stage larvae orally, and the blood was collected before infection and every week post-infection, serum separated and stored frozen. Dehydrogenase activity in H.c-C3BP and

rGAPDH was routinely measured in fresh samples. The specific activity http://www.selleck.co.jp/products/Gefitinib.html in H.c-C3BP was 0·3 U/mg protein, whereas it was higher in the rGAPDH sample, 1 U/mg protein. Enzyme activity was low in stored rGAPDH probably due to hydrolysis of the protein (Table 1). This study demonstrates the presence of a complement-C3-binding protein (H.c-C3BP) in the ES products of H. contortus. To our knowledge, this is the first demonstration of such an activity. Initially, H.c-C3BP was isolated using C3–Sepharose column, and the protein band had a size of ~14 kDa, which was used for antibody production and mass spectrometry analysis. The mass spectrometry data suggested H.c-C3BP as glyceraldehyde-3-phosphate dehydrogenase. The peptides that matched GAPDH of H. contortus represented different regions and spread throughout the protein structure. The size of H. contortus GAPDH is ~37 kDa [21], whereas the recombinant form is ~43 kDa including the His tag (this study).

5a) or bLNs (data not shown) of OVA-sensitized and challenged WT or CD137−/− mice showed equally enhanced proliferation, while lymphocytes isolated from controls proliferated only slightly. In addition, we determined cytokine production in supernatants of lymphocyte cell cultures by ELISA. Th2 cytokines IL-5 and IL-13 were increased markedly in cell cultures

of both OVA-immunized CD137−/− and WT mice compared to controls (**P ≤ 0·01) (Fig. 5b), but no significant differences were observed between IL-5 and IL-13 production in spleen cell cultures derived from CD137−/−versus WT mice that underwent the allergy protocol. Th2 cytokine IL-4 and IFN-γ, as signs of the Th1 response, were very low (<50 pg/ml) to undetectable (data not shown). As demonstrated above, we observed similar allergic parameters in CD137−/− and WT mice after OVA sensitization and challenge, demonstrating that CD137 is

click here not required for the development of a Th2-dominated allergic phenotype. Furthermore, we were interested in whether CD137 co-stimulation LY294002 clinical trial is involved in respiratory tolerance induction. Hence, mice were tolerized via mucosal application of OVA before sensitization (Fig. 1, tolerance protocol). Consistent with previous studies [28,30], tolerized WT mice (WT TOL) showed reduced signs of allergic airway disease and resembled the control group (WT Alum). CD137−/− mice were equally protected: we did not detect any significant differences ID-8 with regard to total BALF cell count and eosinophilia (Fig. 2b,c) or pulmonary inflammation and mucus production (Fig. 3). Furthermore OVA-specific IgE, IgG1 and IgG2a serum levels (Fig. 4), in vitro proliferation and Th2 cytokine production were equivalent (Fig. 5a,b). To summarize, all measured parameters were comparable

in tolerized wild-type and CD137−/− mice, suggesting that loss of CD137 is not critical for respiratory tolerance induction in our model. We determined T cell subsets via flow cytometry in spleen and lungs from individual WT and CD137−/− mice on day 21 of the immunization protocols (Fig. 1). Similarly, we found significantly elevated percentages and numbers of CD4+ T cells in lung of OVA-immunized WT and CD137−/− mice (Fig. 6b); in parallel, we observed a slight trend towards reduced proportions of splenic CD4+ T cells after sensitization and challenge (Fig. 6a). With regard to CD8+ T cell frequency, we detected no significant differences after immunization. Again, CD137−/− mice had comparable percentages and absolute numbers in spleen and lung to the WT groups independent of the immunization protocol used. Analysis of Treg (CD4+FoxP3+) cells revealed significantly enhanced percentages in lung (Fig. 6b) of both OVA-immunized mice strains, whereas we did not observe this increase in spleen (Fig. 6a).