To our knowledge, this is the first study to determine CD8+ Tregs in HCV-infected patients and in HIV/HCV co-infected patients. The elevated frequencies of CD4+ Tregs and CD8+ Tregs in HCV-infected and HIV/HCV co-infected patients might illustrate the necessity for the immune system to limit a vigorous immune response against the chronic viral infection, while favouring persistent viral infection. Whether the increased

frequencies of CD4+ Tregs and CD8+ Tregs as well as chronic immune activation (CD38+ HLA-DR+) in co-infected patients compared with HCV-infected patients have any relation to the increased risk of fibrosis progression MK-8669 in patients with HIV co-infection is uncertain, keeping in mind that we found no differences in CD4+ Tregs, CD8+ Tregs or T cell activation between HCV-infected patients with or without fibrosis. Microbial translocation is known to be a key contributor to

the elevated chronic immune activation found in HIV-infected Selleck SAHA HDAC patients [22]. Furthermore, microbial translocation has been found to be associated with progression of fibrosis in HCV patients [23]. Further studies assessing the impact of microbial translocation on the increased risk of fibrosis progression in HIV/HCV co-infection are warranted. The function of Tregs in HCV-infected and HIV/HCV co-infected patients has not been described. Recently, it was demonstrated that co-expression of CD45RA and Foxp3 can be used to further characterize CD4+ Tregs into three functionally distinct subpopulation, that is, resting Tregs (CD45RA+ Foxp3low), activated Tregs (CD45RA− Foxp3high) and non-suppressive Tregs (CD45RA− Foxp3low) [31]. Resting and activated Tregs represent two stages of differentiation and both have active Foxp3 gene transcription and suppressive activity. In contrast, the non-suppressive Tregs are characterized by an unstable Foxp3 expression, high production of IL-2 and IFN-γ, and no suppressive Protirelin activity. Thus, the non-suppressive Tregs may illustrate activated cells transiently expressing Foxp3. In our cohort, lower frequencies of resting Tregs as well as higher frequencies

of activated Tregs were found in HCV-infected and HIV/HCV co-infected patients compared with healthy controls. Probably due to the limited study population, significant differences of activated Tregs were only observed between HCV infected without fibrosis and healthy controls. Thus, CD4+ Tregs in patients with chronic HCV infection and especially in patients with HIV/HCV co-infection seem to be functionally more activated. However, the frequency of non-suppressive Tregs was also higher in HCV infected with fibrosis indicating that a considerable fraction of CD4+ Tregs in this patient group may in fact be activated cells with no suppressive capacity. Furthermore, to evaluate whether elevated frequency of Tregs resulted in altered cytokine production, production of the cytokine IL-10 was measured in PBMC.

Analysis of blood cells from injected mice showed that GA associated with a mononuclear CD11bhi cell population (Fig. 1A, left panels). This association was specific for GA, because Alexa488-OVA

did not Atezolizumab manufacturer bind to these cells. Alexa488 staining on CD11bhi cells was also observed when GA-Alexa488 was injected into MHC class II–deficient mice (Fig. 1A, right panels), showing that MHC class II was not necessary for targeting of GA to these cells in vivo. Further characterization of the cell surface markers on GA+ cells from both wild-type and MHC class II–deficient mice identified them as F4/80lo/Ly6G−, consistent with a monocyte phenotype (Fig. 1B and data not shown). GA-Alexa488+ monocytes were observed within 20 min of GA administration, and >95% monocytes were GA+ after 3–6 h (Fig. 1C). Taken together, our findings showed that GA rapidly and specifically targets blood monocytes after intravenous administration. Previous work in our group has shown that naïve blood CD11bhi F4/80lo Ly6G− cells exhibit the capacity to suppress T cell proliferation in vitro [15]. In this study,

co-culture with blood monocytes from naïve mice also suppressed T cells stimulated with anti-CD3/anti-CD28-coated Selleck VX809 beads, and this effect was enhanced in monocytes isolated from mice that had been treated with GA (Fig. 2A). GA-treated monocytes also exhibited enhanced suppression of antigen-specific proliferation of CD4 T cells click here (Fig. 2B). To determine whether intravenous GA treatment could suppress T cell proliferation in vivo, CFSE-labelled, MOG-specific TCR transgenic CD4 T cells were adoptively transferred into

CD45.1+ congenic mice. T cells were transferred in the presence of either MOG35–55 alone or MOG35–55 and GA, and 2–4 days later, in vivo T cell proliferation was measured by flow cytometry. As shown in Fig. 2C, in vivo T cell proliferation was reduced in GA-treated mice in comparison with mice injected with MOG35–55 alone. Taken together, these findings showed that intravenous GA treatment greatly delayed T cell proliferation in vivo, which is likely due to the enhanced capability of blood monocytes to suppress antigen-specific T cell proliferation. Subcutaneous administration of GA is commonly used for MS treatment and has been shown to suppress EAE [7]. To address the question of whether suppression of pathogenic T cell proliferation by monocytes was also contributing to the efficacy of subcutaneous GA treatment, we adopted a co-immunization model of EAE treatment modified from Gilgun-Sherki et al. [22]. Mice were injected subcutaneously with a CFA emulsion containing combinations of the disease-causing MOG35–55 peptide and GA. To investigate antigen-specific T cell expansion, CFSE-labelled MOG-specific TCR transgenic cells were adoptively transferred into congenic mice, and the recipients immunized with CFA+MOG35–55 peptide with or without GA. As shown in Fig.

In G93A mSOD1 mice [75], degeneration of the anterior

horn neurones was noted early on in the disease process [110]. Ultrastructural studies showed membrane bound vacuoles originating from the degenerating mitochondria, via distension of the outer mitochondrial membrane, expansion of the IMS, preceding disintegration of the IMM [56]. The notion of a causal role of this mitochondrial dysmorphology in the pathogenesis of ALS has arisen, due to the observations that these defects occur at a presymptomatic stage in G37R and G93A mSOD1 mice [56]. Furthermore, at the onset of disease symptoms, the dominant pathological event in the ventral horn is a rapid increase in the number of vacuolated mitochondria, Vismodegib cost correlating with decline in muscle strength and preceding motor neuronal cell death [56,74,111,112]. It is postulated that this death is due to apoptosis, with the relative density of cytochrome c immunoreactivity noticeably reduced in the swollen mitochondria, suggestive of its pro-apoptotic release into the cytosol [56]. However, over-expression of wild-type SOD1 may also lead to vacuolation of mitochondria [113], and as mitochondrial vacuolation is not seen in all mSOD1 mouse models, it is important to consider whether more subtle disruption of mitochondrial morphology occurs. The initial cause of this mitochondrial

dysmorphology is unclear, although mSOD1 has been implicated in the process, with vacuolation of mitochondria correlating with accumulation of mSOD1 in the mitochondrial IMS of transgenic Selleck MAPK Inhibitor Library mice [113]. Furthermore, mSOD1 Progesterone has been found to be present in only mildly swollen mitochondria, suggesting that the translocation of mSOD1 into the IMS may trigger vacuolation

of the mitochondria, possibly via dysfunctional interaction with mitochondrial chaperones, eliciting structural damage [56,114]. A fragmented network of motor neuronal mitochondria in the anterior horn of SALS patients is suggestive of defective fusion, or an increase in the levels of fission [49]. This is supported by investigation of cultured motor neurones derived from G93A mSOD1 transgenic mice; mitochondria were found to have a lower aspect ratio, suggestive of ‘rounding up’ of individual mitochondria [115]. Furthermore, investigation of a mSOD1 expressing NSC-34 cell line revealed fragmentation of the mitochondrial network alongside remodelling of the mitochondrial cristae [12,116]. Recent analysis of mitochondrial morphology in differentiated NSC-34 cells transfected with IMS-targeted mSOD1 revealed a significant decrease in mitochondrial length, indicative of fragmentation of the mitochondrial network in the presence of mSOD1 [109]. Thus, loss of mitochondrial fusion or an increase in mitochondrial fission may be a component of the pathogenic process in ALS.

IL-5 and GM-CSF were determined in supernatants using specific ELISA

Kit assays (eBiosciences). The results are expressed as the mean±SD. Data were analyzed using Student’s t-test (Prism selleck chemicals GraphPad Software, San Diego, CA, USA). This work was supported by grants from the Italian Ministry of Health, Associazione Italiana Ricerca sul Cancro, Ministero dell’Istruzione, Università e Ricerca (PRIN 2005), Fondazione Cariplo, Agenzia Spaziale Italiana (Progetto OSMA), LR.26 del Friuli Venezia Giulia. The authors thank Silvia Piconese and Mario Colombo (Istituto Tumori, Milan, Italy) for providing OX40-deficient Tregs. They are grateful to Francesco Vitrani for helpful suggestions. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance anti-CTLA-4 antibody to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted

by the authors. “
“Interleukin-19 (IL-19) plays an important role in asthma by stimulating T helper type 2 (Th2) cytokine production. Interestingly, IL-4, a key Th2 cytokine, in turn up-regulates IL-19 expression in bronchial epithelial cells, so forming a positive feedback loop. In atopic dermatitis (AD), another Th2 disease closely related to asthma, IL-19 is up-regulated in the skin. We propose to use IL-4 transgenic (Tg) mice and human keratinocyte culture to delineate the molecular mechanisms involved in the up-regulation

of IL-19 in AD. IL-19 is similarly up-regulated in the skin of IL-4 Tg mice as in human AD. O-methylated flavonoid Next we show that IL-4 up-regulates IL-19 expression in keratinocytes. Interestingly, the up-regulation was suppressed by a pan-Janus kinase (Jak) inhibitor, suggesting that the Jak–signal transducer and activator of transcription (Jak-STAT) pathway may be involved. Dominant negative studies further indicate that STAT6, but not other STATs, mediates the up-regulation. Serial 5′ deletion of the IL-19 promoter and mutagenesis studies demonstrate that IL-4 up-regulation of IL-19 in keratinocytes involves two imperfect STAT6 response elements. Finally, chromatin immunoprecipitation assay studies indicate that IL-4 increases the binding of STAT6 to its response elements in the IL-19 promoter. Taken together, we delineate the detailed molecular pathway for IL-4 up-regulation of IL-19 in keratinocytes, which may play an important role in AD pathogenesis. “
“The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains.

Intracellular staining was performed with the Foxp3 staining buffer kit, according to the manufacturer’s protocol (eBioscience or BD Biosciences). CD4 microbeads were purchased from Miltenyi Biotec (Auburn, CA). Flow cytometry analysis was performed using FlowJo software. Peripheral LNs and spleens were harvested from 8-week-old female mice. CD4+ T cells where enriched by Automacs using CD4 microbeads, labeled with anti-CD4 PE-Cy5, anti-CD25 PE, and CD45RB FITC or anti-CD4 selleck kinase inhibitor PE-Cy5 and anti-CD45RB PE and purified by cell sorting. The purity of CD4+CD25−CD45RBhi, CD4+CD25+, CD4+GFP−CD45RBhi, CD4+GFP+ cells was >98%. RAG KO mice

were injected i.v. with sorted CD4+ T-cell subpopulations in PBS. Mice received 5 × 105 CD4+CD45RBhigh from WT GITR or GITR KO mice alone or in combination with 2 × 105 CD4+ GFP+ GITR WT, CD4+ CD25+ GITR WT, or CD4+ CD25+ GITR KO cells; one group of mice received 2 × 105 CD4+ GFP+ GITR WT alone. Fc-GITR-L (200 μg) was injected i.v. one day after T-cell reconstitution, and then once weekly until the study was terminated. Mice were weighed weekly basis. CD4+CD25−T cells and CD4+CD25+ T cells were purified by cell sorting; postsort purity was >98%. Suppression assays were performed as previously described [3]. Statistical studies were compared using Mann–Whitney U test, and differences were considered statistically significant with p < 0.05. These studies Ivacaftor price were supported by funds

from the Intramural Program of the National Institute of Allergy and Infectious Diseases. The authors declare no Unoprostone financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized 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 S1. Fc-GITR-L expands the absolute numbers of Treg and Tconv cells, has no effect on their suppressive function in vitro. C57BL/6J mice were injected with human IgG1 (solid circle)

or Fc-GITR-L i.p. (open circle). Sixty-four hours after treatment, mice were injected with BrdU and 8 hours later total LN and spleen where harvested and BrdU incorporation determined by flow cytometry. (A) Percentage of Foxp3+ and Foxp3- T cells that incorporated BrdU. Data are derived from 4 mice per group. (B) Cell sorted CD4+CD25+ T cells from IgG1 or Fc-GITR-L injected mice were cultured at the indicated ratio with CD4+CD25- T cells and the mixture was activated with anti-CD3 monoclonal antibody and irradiated APC. (C) C57BL/6J mice were injected with human IgG1 (solid circle) or Fc-GITR-L i.p. (open circle), mice where harvested on day 3, 6 and 9 post Fc-GITR-L treatment. (∗∗∗, P <0.0001). The data represents the mean ± SEM, derived from four mice per group and representative of 3 independent experiments. Figure S2.

epidermidis stain harboring PQG56 (spx antisense knock-down plasmid) is increased substantially, in accordance with the phenotype in the homologous spx mutant strain of S. aureus (Pamp et al., 2006). This observation further supports that spx is an important regulator mediating the biofilm formation of S. epidermidis. Biofilm formation by S. epidermidis

is generally considered as a two-step process, including primary attachment and biofilm accumulation. To investigate selleck kinase inhibitor which step is affected by Spx, we first compared the attachment ability of the Spx-overexpressing strain (harboring pQG55) and the vector control strain (harboring pQG53). In primary attachment assays, the Spx-overexpressing strain showed decreased attachment ability (about 34-fold) to polystyrene compared with the WT strain, whereas the strains carrying either pQG53 or pQG54 showed no difference in primary attachment (Fig. 3a and b). To investigate whether the transcription of atlE was affected see more by Spx, quantitative RT-PCR was performed. The result indicates that the transcriptional level of atlE in the Spx-overexpressing strain carrying pQG55 shows no difference compared with the other three strains (Fig. 3c). This indicates that Spx does not affect the attachment ability by regulating

atlE. We then compared the primary attachment on 96-well polyethylene plates between WT and ica-negative strains isolated from our previous work (Li et al., 2005), and no significant difference was found (data not shown). PIA is a key factor in the biofilm accumulation of S. epidermidis (Rupp et al., 1999). To investigate whether the production of PIA was affected by Spx, immuno-dot blot Amino acid assays were performed. The Spx-overexpressing strain was found to produce significantly less PIA compared with the vector control strain (Fig. 4a). The transcription of the icaADBC operon and its repressor icaR among different strains was further examined by quantitative RT-PCR. Decreased

icaADBC, but comparable icaR transcriptional levels were found in the Spx-overexpressing strain compared with the vector control strain (Fig. 4b and c). This result indicates that Spx affects PIA production by regulating the transcription of icaADBC in an icaR-independent manner. In B. subtilis and S. aureus, Spx plays an important role in the oxidative-stress adaptation. The B. subtilis and S. aureus spx mutant strains were hypersensitive to diamide, a thiol-specific oxidant (Nakano et al., 2003a; Pamp et al., 2006). To study whether the overexpression of Spx affects S. epidermidis in the adaptation to diamide, the diamide sensitivity of the Spx-overexpressing strain (harboring pQG55) and the control strain (harboring pQG53) was compared using disk diffusion tests.

The lowest dose regimen from Study B, 5 μg (3×/72 hr), was repeated, and two lower dose regimens, 2 μg (4×/72 hr) and 1 μg (4×/72 hr), were added. The 5 μg

(3×/72 hr) and 2 μg (4×/72 hr) dose regimens had remission rates of 63% and 53%, respectively, similar to the higher dose regimens in Study B. Again, there was no statistically significant difference in remission rates between the 5 μg (3×/72 hr) and 2 μg (4×/72 hr) dose regimens in Study C, or the various dose regimens in Study B. As in the higher dose regimens in Study B, these mice entered remission 1–2 weeks after treatment and the remission was long-lasting, up to 24 weeks of follow-up. However, at the 1 μg (4×/72 hr) dose p38 MAPK apoptosis regimen, the remission rate dropped to 16% and this reduction was significantly different compared with the 2 μg (4×/72 hr) dose regimen (P < 0·05). Yet, for mice that did enter remission, the remission was long-term (up to 24 weeks). Thus, the minimum effective dose of monoclonal anti-CD3 F(ab′)2 for the 4×/72 hr dose regimen is ≥ 1 μg. In both Studies B and C, partial remission was observed in one or two mice within each dose regimen, such Cell Cycle inhibitor that normal glycaemia was detected in these mice for a transient period ranging from 3 to 11 weeks post-treatment. Thereafter, the blood glucose levels rose quickly and were sustained at

levels of ≥ 250 mg/dl. There was no correlation between dose and the numbers of mice exhibiting partial

remission. Overall, all of the mice that entered remission did so within 1–2 weeks after treatment, consistent with previous studies,10 and the majority of remissions observed were durable for at least the 12-week observation period. In addition to modulation of the CD3–TCR complex, the PD parameters routinely assessed Janus kinase (JAK) in clinical studies of otelixizumab include changes in various immune-cell subsets such as CD4+, CD8+ and CD4+ FoxP3+ T cells. Because we wanted to mirror the PD parameters routinely collected in clinical situations, we specifically elected to evaluate similar flow-cytometric PD parameters in the peripheral blood of mice from Studies B and C. In Studies B and C, the proportions of CD4+, CD8+ and CD4+ FoxP3+ T cells were assessed before dosing and again within 24 hr of the last dose. We elected to use the CD4+ FoxP3+ phenotype to identify Treg cells in the periphery, given that FoxP3 expression directly correlates with Treg-cell function, regardless of the CD25 expression levels20 and because CD25 is also found on activated CD4+ T cells. In Study B, T-cell subsets were also evaluated at the 12-week end-point. We first compared T-cell subset proportions between two groups: (i) placebo and (ii) all mice that received antibody in Studies B and C.

In experiments 1 and

2, the animals were evaluated every other day for frequency and severity of arthritis. Scoring was performed in a blinded manner without knowledge of the treatment groups and previous scores. Severity was graded as described find more previously [22], scoring 1–3 in each paw (maximum of 12 points per mouse) as follows: (i) swelling or erythema in one joint; (ii) swelling or erythema in two joints; or (iii) severe swelling of the entire paw or ankylosis. At termination of the experiments, mice were anaesthetized for blood withdrawal, and then killed by cervical dislocation. Sera were collected individually and stored at −20°C until used. Successful removal of the ovaries was confirmed by weighing the uteri. For experiment 2, one femur was placed in formaldehyde for analysis of bone mineral density.

The paws (experiments 1 and 2) were placed in formaldehyde, decalcified and embedded in paraffin. Sections were stained with haematoxylin and eosin and encoded before examination. In sections from each animal, the distal and proximal areas of all four paws were graded separately on a scale of 0–4 and the score was then divided R428 by 2, which yielded a maximum histological destruction score of 16 points per mouse, assessed as follows: 1 = synovial hypertrophy; 2 = pannus, discrete erosions of cartilage and bone; 3 = severe erosions of cartilage and bone; and 4 = complete ankylosis. In experiment 3, spleens were collected and frozen individually in liquid nitrogen, and kept at −20°C until use. One femur was subjected to a peripheral quantitative computed tomography (pQCT) scan with a Stratec pQCT XCT Research M, software version 5·4B (Norland, Fort Atkinson, WI, USA)

at a resolution of 70 µm, as described previously [23]. Trabecular BMD was determined with a metaphyseal scan at a point 3% of Cell press the length of the femur from the growth plate. The inner 45% of the area was defined as the trabecular bone compartment. Cortical BMD was determined with a mid-diaphyseal scan. For measurement of bone resorption, serum levels of fragments of type I collagen were assessed using a RatLaps enzyme-linked immunosorbent assay (ELISA) kit (Nordic Bioscience Diagnostics A/S, Herlev, Denmark). Serum levels of osteocalcin, a marker of bone formation, were determined with a mouse osteocalcin immunoradiometric assay (IRMA) kit (Immutopics, Inc., San Clemente, CA, USA). As a marker of cartilage destruction, serum levels of cartilage oligomeric matrix protein (COMP) were determined with an animal COMP® ELISA kit (AnaMar Medical AB, Uppsala, Sweden). By use of a previously described ELISA, serum levels of anti-CII antibodies were determined [24]. A bioassay with cell line B13·29, subclone B9 (which is dependent on IL-6 for growth), was used to measure serum levels of IL-6, as described previously [25,26].

2) while

not altering the frequency of the other cell populations (Supporting Information Fig. 3). With the purpose of analyzing the relevance of MDSCs as key factors for maintaining homeostasis, we analyzed at 21 dpi the parasitemia and survival of treated mice after a dose of 5FU at 10 or 15 dpi, or two doses, at 10 plus 15 dpi, and the results were compared with those of untreated controls. Surprisingly, when 5FU was administered at 10 dpi, the parasitemias were lower compared with those of untreated controls, whereas the parasitemias were significantly higher when the drug was given at 15 dpi (Fig. 6B). In addition, mouse survival was about 50% when 5FU was administered at 10 dpi whereas selleck compound the survival Galunisertib ic50 was approximately 20% in mice treated at 15 dpi, but there was no survival when two doses were administered, 10 plus 15 dpi (Fig. 6C). In parallel, we also analyzed whether MDSCs depletion at 15 dpi was able to restore the Con A proliferative response of infected splenocytes. As expected, a recovery of the splenocytes proliferation was observed (Fig. 7A). Consistent with this result, a significant reduction in the percentage of CD8+TN+ T cells

(Fig. 7B) was associated with an increase in the percentage of activated CD107a+CD8+ T cell (Fig. 7C). CD107a has been previously shown to be a marker for cytotoxic CD8+ T-cell activity [29]. Interestingly, we also detected a higher level of IL-6 and IFN-γ inflammatory cytokines in plasma from 5FU-treated mice compared with untreated ones, as well as an elevated concentration of TNF-α in both untreated and treated groups

(Fig. 7D). Finally, the 5FU treatment increased the number of Th1 (CD4+IFN-γ+) and Th17 (CD4+IL-17A+) cells (Fig. 7E) at 19 IKBKE dpi. It is clear that there is a complex interplay between host and parasite that influences the outcome of an infection. Recently, we demonstrated that during acute T. cruzi infection, BALB/c mice showed a reduced inflammatory response, and an improved survival and tissue repair compared with B6 mice, the latter developed a severe inflammation and liver/cardiac pathology [23]. In the present study, our data clearly indicate that there was a higher number of MDSCs infiltrating the liver and spleen of infected BALB/c mice than in B6 mice. An analysis of MDSCs subsets in the liver and spleen revealed that the number of G-MDSCs was higher in infected BALB/c with respect to B6 mice, suggesting a protective role for G-MDSCs in the resolution of inflammation. In agreement with this concept, an increased accumulation of G-MDSCs has been correlated with reduced tissue injury in various experimental models of inflammation [30-32]. In cancer, the frequency of each MDSCs subset appears to be influenced by the type of tumor [2]. The study of the suppressor mechanisms exerted by splenic MDSCs from infected BALB/c mice revealed that the suppression of lymphocyte proliferative response was mediated by ROS and NO production but not by arginase activity.

Plates were then washed four times with PBS containing 0.05% Tween-20. Serum sample were diluted 1:300 in PBS and a threefold dilution series www.selleckchem.com/products/PLX-4032.html was performed. A total of 100 μL per well of the serum dilution was transferred to the LCMV-coated plates. After 1 hour of incubation at room temperature, plates were washed four times, followed by incubation with 100 μL per well of HRP-conjugated goat-anti-mouse IgG (Jackson ImmunoResearch) diluted 1:30 000 in PBS, followed by 1 hour incubation. Thereafter, plates were again washed four times and 50 μL per well of the peroxidase substrate OPD (SIGMA) were applied

and the color reaction stopped after 10 min by adding 100 μL per well of 2 M sulfuric acid. OD was determined at a wavelength of 492 nm. LCMV-specific Ab titers were determined by an endpoint titer 0.1 OD over background. To determine the viral antigen specificity of these Abs, cell lysates of LCMV-infected and noninfected B16 melanoma cells find more were immunoprecipitated with IgG from LCMV immune serum that were bound to protein G-coupled sepharose (GE Healthcare). Samples were separated by 4–12% gradient SDS-PAGE (SERVA) and visualized with rabbit anti-LCMV serum

(1:5000), followed by HRP-conjugated donkey anti-rabbit IgG (Dianova). The ECL plus detection system (GE Healthcare) was applied for visualization. Single-cell suspensions of splenocytes were obtained by mechanical disruption. IFN-γ production of CD8+ T cells was determined by intracellular IFN-γ staining (anti-IFN-γ; clone XMG 1.2, ebioscience) after restimulation of 106 splenocytes with 10−7M LCMV GP33 peptide or LCMV NP396 peptide in the presence of 10 μg/mL Brefeldin A (SIGMA). CTL- and NK-cell activity was determined in a 51Cr-release assay. Target cells were loaded with 51Cr for 2 hours at 37°C and then incubated for 5 hours at 37°C with splenocytes that were previously titrated in a threefold

dilution series. Duplicate wells were assayed Amoxicillin for each effector-to-target ratio and percentages of specific lysis were calculated. Data were analyzed using SigmaPlot Version 9.0 software. Significant differences were evaluated with Mann–Whitney U-test using InStat3 software (GraphPad). The authors thank Maike Hofmann for helpful discussions and critical comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft DFG (Pi295/6-1 to H.P. and SFB490 to A.W.). The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized 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.