Technical support issues arising from supporting information (oth

Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Figure S1. Effect VX-809 research buy of various TLRs ligands on reporter gene activity in HT-29 (A) and Caco-2 (B) cells. Reporter gene activity was measured after 24 h stimulation.

Different letters indicate statistically different results with a p value <0.05; Student's T test. Figure S2. Response of TSLP reporter clone (Caco-2) to butyrate (2 mM) and different concentration of Trichostatin (TSA) tested alone or in combination with IL-1 (10 ng/ml) or PMA (1ìM). Reporter gene activity was measured after 24 h stimulation. Results are mean ± SD of triplicate measurements of a representative of three independent experiments. ** = p < 0.01, *** = p < 0.001. Figure

S3. TSLP promoter-driven luciferase activity measured using a 3 kb-long promoter construct. Different letters indicate statistically different results with a p value <0.05; Student's T test. Figure S4. Effect of Flagellin on NF1 or/and NF2 mutations of the TSLP promoter-driven luciferase activity. Different letters indicate statistically different results with a p value <0.05; Student's T test. Figure S5. Characterization of NF1 and Belinostat NF2 binding site by promoter deletion and site directed mutagenesis on various epithelial cell lines. HeLa (a), HEK 293 (b), A549 (c). Morin Hydrate Results are mean ± standard deviation (SD) of triplicate measurements of a representative of three independent experiments. Different letters indicate statistically different results with a p value <0.05; Student's T test. Figure S6. Effect of PMA (a), Butyrate (b) and combination of both (c) on TSLP

promoter-driven luciferase activity. Various 5′deletions of the TSLP promoter were cloned in pCDNA3.1-Luc and transiently transfected in Caco-2 cells. Length of each construct is indicated. Cells were stimulated 24 h after transfection and reporter gene activity was assayed 24 h after stimulation. Results are mean ± standard deviation (SD) of triplicate measurements of a representative of three independent experiments. Different letters indicate statistically different results with a p value <0.05; Student's T test. "
“Recent studies have suggested Fas-mediated elimination of antigen-presenting cells as an important mechanism down-regulating the induction of autoimmune responses. It remains unknown whether this mechanism restricts the magnitude of immune responses to non-self antigens. We used a mouse model of a cutaneous CD8+ T-cell-mediated immune response (contact hypersensitivity, CHS) to test if CD4+CD25+ T cells expressing FasL regulate hapten-specific effector CD8+ T cell expansion through the elimination of Fas-expressing hapten-presenting DC.

As pDCs are the principal secretors of IFN-I, the prevailing hypo

As pDCs are the principal secretors of IFN-I, the prevailing hypothesis for IFN-I impairment is centred on pDCs [5, 21, 47]. pDCs that have been induced to produce large amounts of IFN-I in a primary antiviral response are either depleted, through mechanisms such as NK cell-mediated cytotoxicity [48, 49], or are induced to mature and have to be replaced by haematopoesis, or they acquire a transient state of unresponsiveness and paralysis such as Nivolumab that reported in experiments using in vitro stimulation after in vivo viral infections [50]. Although, in our mouse model using avirulent

SFV, we did not observe quantitative reduction in pDCs [16], others have reported significant decrease in numbers of pDCs soon after acute or during persistent viral infections [21, 51]. Consistent with the above animal data, human patients infected with hepatitis B virus (HBV), hepatitis

C virus (HCV) or HIV have decreased numbers of circulating pDCs [52-55]. In addition, patients with HCV infection receiving IFN-Iα therapy exhibit decreased numbers of pDCs in blood compared with untreated controls [56]. Thus, a strong negative correlation exists between the quantity of the IFN-I response and pDC numbers. Recent study by Swiecki et al.[51] has shown that pDC depletion during systemic viral infection occurs in an IFN-I-dependent manner through upregulation of pro-apoptotic expressions of Bid, Bim, Noxa and Bax and downregulation of anti-apoptotic Bcl-xl and Bcl-2. Besides quantitative changes, qualitative differences in pDCs have also Tyrosine Kinase Inhibitor Library been documented. pDCs isolated from mice undergoing IFN-I exhaustion are unable to produce IFN-I in response to CpG,

a TLR-9 agonist, after treatment ex vivo [21]. Interestingly, the functional defect of pDCs is limited to IFN-I production because synthesis and secretion of other cytokines such as TNF-α, IL-12 and MCP-1 are not impaired [21]. Collectively, it is likely that the inability of the host to mount an IFN-I response during the refractory period against a secondary Celecoxib challenge is due to both a pDC intrinsic defect in IFN-I production and an overall reduction in pDC numbers, the consequence being a vastly reduced IFN-I output, which may render the host less susceptible to secondary bacterial infections. Research into viral/bacterial co-infections has in recent years become much more fashionable due to its potential clinical significance. Most studies have focused on understanding how viral infections cause heightened susceptibility to subsequent bacterial infections. Much less attention has been directed on understanding how the host has evolved mechanisms to enhance resistance against such secondary bacterial infections. The evidence presented above supports our hypothesis that inhibition of IFN-I production is a mechanism by the host to reduce susceptibility to bacterial infections during recovery from primary virus infections.

0 cm radius of the image A behavior was considered to have ended

0 cm radius of the image. A behavior was considered to have ended when an infant looked away, initiated a different type of manual behavior, changed hands, or removed the hand (or hands). Uninterrupted repetitions of a given gesture type were counted as one instance of that categorical type of behavior. Thus, several uninterrupted repetitions of the same manual action were conservatively scored as a single behavior. We evaluated GDC-0068 the qualitative (“categorical”) types of manual exploration behaviors as well as the total number of behavior changes initiated in

sequence (“sequential”) for each display. In the Categorical level of analysis, infants’ manual gestures were classified as one of five gross categories of reaching behavior (e.g., touching, grasping, rubbing,

scratching, or patting). These qualitatively different types of reaching behaviors were recorded and tallied for each display. At the categorical level, infants could potentially receive a score between 0 and 5 representing the number of qualitatively different types of manual gestures initiated toward each display. In the Sequential level of analysis, a finer grain assessment of successive actions was reviewed. The total quantity of gesture changes that occurred in sequence were recorded and tallied for each display. Olaparib order For example, if an infant was observed rubbing a picture display with one hand followed by tapping with both hands, followed by rubbing with one hand, then those manual behaviors would be recorded as two categorical gestures and three MRIP sequential gestures. For both measures of manual exploration, an impossible preference score was calculated for each infant by computing the total number of behaviors initiated toward the impossible cube divided by the sum of gestures

initiated to both the possible and impossible cube displays. Preference scores were then compared with 50/50 chance. We also documented the frequency of social referencing, vocalizations, and mouthing behaviors as independent and complementary measures of infants’ differential responses toward each type of display. Social referencing was defined as an occurrence of the infant looking to the parent or the experimenter only after the child had initially visually inspected the display at least once. Instances of social referencing were logged each time the child referred back to the parent/experimenter after viewing and/or touching the stimulus display. Social referencing behavior has been a useful indicator of infants’ perceptual judgments and impending actions during an ambiguous, uncertain situation involving novel or unusual stimuli (Klinnert, Emde, Butterfield, & Campos, 1986; Walden & Kim, 2005).

After washing three times with TBST and once with TBS, TMB) was a

After washing three times with TBST and once with TBS, TMB) was added and the membranes were let stand for 3 mins while the color developed. The reaction was stopped by rinsing the membranes with distilled water. The antigen-blotted membranes were prepared as described above and incubated for 1 hr in Block Ace at room temperature. Before incubating with membranes, 15 mL of urine samples were preincubated with 7 μL of E. coli lysate with shaking to block nonspecific binding for 1 hr at room temperature. Next, the membranes were incubated with the primary antibody for 1 hr at room temperature with shaking. Other procedures were the

same as for the serum assay. Statistically significant differences ZD1839 clinical trial was determined by the Mann-Whitney’s U-test. Differences with P < 0.05 were considered significant. Affinity purification detected MPB64 as a His-Tag fusion Pexidartinib protein, mostly in the insoluble fraction, with a molecular mass of about 30 kDa. We speculate that recombinant MPB64 is sequestered into inclusion bodies by E. coli and thus rendered insoluble (Fig. 1). We examined the reactivity of MPB64 protein by western blotting using pooled serum from five patients with active TB and serum from healthy individuals as a control. All of the fractions of pooled patient serum that were tested, including the sonicated soluble

fraction, the soluble fraction after freezing and thawing, and the sonicated insoluble fraction, showed a specific band at about 30 kDa (Fig. 2a and b). In contrast, serum from healthy individuals showed no such bands (Fig. 2c). These findings confirmed that MPB64 is specifically present in the serum of patients with active TB and is detected by an MPB64-specific IgG antibody. In order to determine the amounts of antigen, we blotted several amounts (300 ng to 18.3 pg/dot, each ¼ dilutions) of proteins to membranes in duplicate. The results of these dot-blot assays are shown in Figure 3a: we detected signals from 300 ng to 4.7 ng Protein tyrosine phosphatase of antigen in the patients’ pooled serum. However, we did not detect signals from 18.8 ng in healthy subjects. Based on these results, we decided that the optimal amount of purified

MPB64 protein for detecting the specific reaction for this assay is 18.8 ng. When we examined serum and urine samples for M. tuberculosis by dot-blot assay using purified MPB64 antigen, we rated the reaction as “2 (++)” if we observed a strong signal, “negative” for no signal, and “1 (+)” for a weak signal. Figure 3 shows examples of each type of assay result. Relevant clinical data and the results of dot-blot assay using MPB64 antigen for a representative patient are shown in Figure 4. The patient had many bacterial cells on culture and an increased ESR on admission. After 2 months of hospitalization, when their TB was considered to be in the active phase, the number of colonies had increased about twofold and the ESR from 50 to 100 mm.

C57BL/6J(B6) mice were obtained

from Joint Venture Sipper

C57BL/6J(B6) mice were obtained

from Joint Venture Sipper BK Experimental Animal (Shanghai, China). MRL/lpr, B6/lpr, B6/OVA323–339-specific TCR-transgenic, B6/CD45.1-transgenic and B6/FcγRIIb−/− mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were bred in specific pathogen-free conditions. All experimental manipulations were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Second Military Medical University (Shanghai). The full-length cDNA of mouse FcγRIIb was cloned by RT-PCR from bone marrow-derived DC. Recombinant adenoviral vectors encoding FcγRIIb, GFP Proteases inhibitor or LacZ were constructed using pAdeasy1 system according to the manufacturers’ instruction (Stratagene Biotechnologies). These recombinant adenoviruses Erastin chemical structure were amplified in HEK 293 cells, purified by CsCl gradient centrifugation, dialyzed and stored at −80°C until use. The titers of Ad-FcγRIIb, Ad-GFP and Ad-LacZ were 1011, 1012 and 1012 respectively. Soluble IC were prepared as previously described 27. Briefly, OVA stock (Sigma-Aldrich) was prepared using PBS to 10 mg/mL, and then 50 μg of OVA was incubated with 500 μg mouse-derived anti-OVA mAb (IgG1, Sigma-Aldrich) for 1 h at 37°C to obtain anti-OVA IC. In some experiments, anti-CD3 mAb (IgG1) as irrelevant mAb and monomeric or aggregated IC/Ig

were used. Monomeric Ig was isolated by a centrifugation of anti-OVA mAb at 100 000×g for 1 h, and then supernatants were collected. Aggregated Ig was prepared

by heating anti-OVA mAb for 1 h of at 63°C. MRL/WT and MRL/lpr mice (10-wk-old) were sacrificed to collect sera for isolation of Ig or IC. Sera were passed through a 0.45-μm filter, and then Ig/IC were isolated using protein G-agarose selleck kinase inhibitor column (Invitrogen). The eluted preparation was dialyzed and concentrated using an Amicon centrifugal filter device with a 30 or 300 kD cutoff to be as Ig or IC, respectively. The purity of Ig or IC was verified using Coomassie staining, and the purity was routinely over 95%. Purified lpr mice-derived IC had been confirmed to be strong positive reactions in assays of both ANA and anti-dsDNA staining, whereas MRL Ig did not (Supporting Information Fig. 6). BM cells from B6/WT or MRL/WT mice were cultured at a density of 2×106 cells/mL in RPMI1640 medium supplemented with 10% FBS (both from PAA Laboratories), recombinant mouse GM-CSF (10ng/mL) and recombinant mouse IL-4 (1 ng/mL) (both from PeproTec). Nonadherent cells were gently washed out on d3, the remaining loosely adherent clusters were cultured for another 3 days, and then CD11c+ immature DCs were obtained by magnetic microbeads (Miltenyi Biotec). DCs were incubated with Ig (anti-OVA 100 μg/mL), IC (10 μg OVA plus 100 μg anti-OVA/mL), MRL-Ig or MRL/lpr-IC (100 μg/mL) for 24 h before stimulation with LPS (100 ng/mL) or CpG (0.3 μM) for another 24 h.

1/5 2-specific antibody We found that the amount of peptide requ

1/5.2-specific antibody. We found that the amount of peptide required for detectable TCR internalization was reduced in the high (−9MCTL) compared with the low (−5MCTL) avidity cells (Fig. 2a). This result suggested the possibility that TCR signalling differed in the high versus low avidity cells

at a given level of pMHC. To further address the response of the lines to TCR engagement we analysed the production of IFN-γ following stimulation with immobilized anti-CD3 antibody (Fig. 2b). We found that the high and low avidity lines exhibited significant differences in the amount of anti-CD3 required to produce IFN-γ. Hence, high and low avidity cells differ in their requirement for pMHC and in their sensitivity to activation by anti-CD3 antibody. These data suggest that the sensitivity to peptide antigen may be the result of differences in the signalling that results from TCR engagement. Following initiation of TCR signalling, the AG-014699 ic50 Selleckchem BTK inhibitor cascade bifurcates,

with distinct pathways leading to increases in cytoplasmic calcium levels and phosphorylation of ERK.34,35 Both of these signals have been shown to be critical for TCR activation.35,36 We first determined whether high versus low avidity cells differed in their ability to signal for calcium uptake when cells were stimulated with titrated amounts of peptide antigen. The CTL were loaded with the calcium-sensitive dye Fluo3 AM and basal readings were obtained for 60 seconds. Antigen-presenting cells pulsed with 10−6, 10−9, or 10−12 m peptide were then added. Calcium levels were measured for an additional 240 seconds to allow CTL–APC interaction, followed by addition of extracellular Ca2+ to assess uptake from the extracellular environment. As shown in Fig. 3, high avidity CTL had detectable increases in Fluo3 at all the peptide concentrations assessed, with the levels increasing in a dose-dependent fashion. In contrast, low-avidity CTL exhibited only a minimal increase in

Fluo3 fluorescence at 10−9 m. Stimulation with APC bearing 10−6 m peptide was required to achieve calcium levels similar to those observed when high avidity cells were activated with 10−12 m peptide. EL4 cells alone failed to induce any calcium response (data not shown). However, of note, when the optimal activating peptide concentration for Sitaxentan each line was used (as defined by the lowest concentration that resulted in maximum IFN-γ levels) the two lines exhibited similar levels of calcium flux. We next assessed the kinetics and magnitude of MAPK-ERK1/2 phosphorylation over time following stimulation with a range of peptide concentrations. At the early time-point of 10 min, increases in phospho-ERK1/2 were apparent only in high avidity CTL (Fig. 4). Phosphorylation in this population was detectable at this time with all peptide concentrations, although there was a clear dose-dependent increase. Low avidity CTL exhibited a detectable increase in phospho-ERK1/2 at 30 min (Fig. 4).

5 mg/kg) were asymptomatic Both motor activity and acoustic star

5 mg/kg) were asymptomatic. Both motor activity and acoustic startle response are sensitive measures for assessing selleck chemical the effects of pyrethroids at doses far below those producing convulsions.

Infection challenge with C. albicans either pre- or post-exposure to deltamethrin caused increase in the CFU both in liver and spleen. The ability of a pathogen to cause infection depends on a successful invasion of the host, which, in turn, requires that the various host defence mechanisms are not working to their full potential. For the host, an infectious process is a highly complex situation to deal with, even in situations where the immune system is not disturbed by toxic chemicals or in the deficiency find more of essential nutrients [27–29]. Evidence on modulation of white blood cells and lymphocyte populations by deltamethrin is limited and seems to be dependent on age, sex and species of the animals [19]. Our study shows that deltamethrin has a potential

to compromise the immunity and impair host resistance to C. albicans infection in mice. In a recent report by Suwanchaichinda et al. [30], mice exposed to deltamethrin in soyabean oil by gavage at doses of 5 and 10 mg/kg showed immunosuppression and enhanced susceptibility to malaria infection (Plasmodium berghei). Deltamethrin induced thymus atrophy by interfering with the cell signalling cascades in male BALB/c mice injected i.p. with a single dose of 25 mg/kg [31]. The immunosuppressive properties

of deltamethrin can be ascribed to its ability eltoprazine to diffuse into cells via its property of liposolubility and inhibitory effect on protein synthesis. These reports show that as a result of exposure to deltamethrin there is a risk of weakened resistance to infection challenge. CFU in liver remained high in deltamethrin treated mice. In spleen and liver, CFU were more than deltamethrin alone treated animals. This shows a high infection rate in spleen and liver. When mice were treated with deltamethrin before and after the challenge by C. albicans, it significantly suppressed the humoral immune response. Lymphocytes play a major role in immune defense against the bacterial infections [32, 33]. For instance, TH1 cells and interferon-γ are required to control the primary peak parasitemia in mice infected with Plasmodium chabaudi and this mechanisms is antibody-independent [34]. CD4 +  TH1 and TH2 cells and antibodies are required after the peak to eliminate the parasites. Depletion of natural killer cells can also cause a rapid increase in fungal or bacterial infection [35]. Studies on the insecticide sulfluramid caused suppression ranging from 70 to 89% (6–57 μmol/kg/day) in mice [36]. Decreases in IgG classes (IgG1, IgG2b, IgG3) were also reported following exposure to perfluoroctanic acid for 10 days [37]. Taken together, our data suggest that humoral immunological function may be a target for pesticides.

Finally, we emphasize the need for creating novel SALS disease mo

Finally, we emphasize the need for creating novel SALS disease models based on the results of omics analysis, especially based on the observation that dynactin-1 gene expression was downregulated in SALS motor neurons. “
“Pathological arterial wall changes have been cited as potential mechanisms of cerebrovascular disease in the HIV population. We hypothesize that dilatation would be present in arterial walls of patients

with HIV compared to controls. Fifty-one intracranial arteries, obtained from autopsies PF 01367338 of five individuals with HIV infection and 13 without, were fixed, embedded, stained, and digitally photographed. Cross-sectional areas of intima, media, adventitia and lumen were measured by preset color thresholding. A measure of arterial remodeling was obtained by calculating the ratio between the lumen diameter and the thickness DNA Damage inhibitor of the arterial wall. Higher numbers indicate arterial dilatation, while lower numbers indicate arterial narrowing. HIV-infected brain donors were more frequently black (80% vs. 15%, P = 0.02)

compared with uninfected donors. Inter and intra-reader agreement measures were excellent. The continuous measure of vascular remodeling was significantly higher in the arteries from HIV donors (β = 2.8, P = 0.02). Adjustments for demographics and clinical covariates strengthen this association (β = 9.3, P = 0.01). We found an association of HIV infection with outward brain arterial remodeling. This association might be mediated by a thinner media layer. The reproduction

of these results and the implications of this proposed pathophysiology merits further study. “
“A. Smallwood, A. Oulhaj, C. Joachim, S. Christie, C. Sloan, A. D. Smith and M. Esiri (2012) Neuropathology and Applied Neurobiology38, 337–343 Cerebral subcortical small vessel disease and its relation to cognition in elderly second subjects: a pathological study in the Oxford Project to Investigate Memory and Ageing (OPTIMA) cohort Background: Subcortical small vessel disease (SVD) is known to contribute to vascular cognitive impairment and vascular dementia, but understanding about the extent of its influence is limited because there is a lack of consensus about how this pathology should be assessed. Methods: In this study we have made use of a simple, novel, image-matching scoring system to assess the extent of SVD in a group of 70 cases from the prospectively assessed Oxford Project to Investigate Memory and Ageing (OPTIMA) cohort. These cases were found at autopsy to have cerebrovascular disease and no other pathology except Braak stage 4 or less tau pathology, and insufficient amyloid plaque pathology to meet Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) criteria for the diagnosis of Alzheimer’s disease.

Additionally, the Flow Coupler resulted in significantly more vas

Additionally, the Flow Coupler resulted in significantly more vascular thrombotic events when compared to the non-flow Coupler. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014. “
“The purpose of this work was to report our initial experience with lymphaticovenular anastomoses (LVA), a controversial technique for lymphedema treatment. Although LVA technique was described many years ago, the procedure is not as widespread as it was supposed to be, taking into account the high impact that lymphedema has in the quality of life of patients. Thus, 12 patients, Hydroxychloroquine nmr 5 with lower limb and

7 with upper limb lymphedema, underwent LVA surgery under local anesthesia. Two patients were excluded from the study due to the lack of follow-up. At 18 months, 8 out 10 patients showed a variable objective reduction of the perimeter of the limbs and 9 patients presented a subjective clinical improvement. These results joined to the outcomes of the most experienced surgeons in this field are encouraging, although there are still many issues that need to be addressed

with research to optimize the efficacy of this technique. © 2012 Wiley Periodicals, Inc. Microsurgery, 2012. “
“Background: Restoration of elbow and finger extension function is still challenging in management of complete brachial plexus avulsion injury, mainly because of fewer available donor nerves for transfer to the radial nerve. Selective neurotization could be a potentially Selleck Palbociclib alternative for overcoming this dilemma. This study was designed to identify the innervation dominance of the extensor Cediranib (AZD2171) digitorum communis muscle (EDCM) and long head of the triceps brachii (LTB) at the level of division of brachial plexus. Methods: From February 2008 to October 2009, 17 patients with complete brachial plexus avulsion injury underwent the procedure of contralateral C7 nerve root transfer. The posterior divisions of brachial

plexus on the healthy donor side were intraoperatively stimulated and the compound muscle action potentials (CMAPs) from the extensor digitorum communis muscle and long head of triceps brachii were recorded by an electrophysiological device. Results: In 13 out of 17 patients (76.5%), the maximal amplitude of CMAP from EDCM was induced by stimulation of the posterior division of lower trunk (PDLT). The mean amplitudes of CMAP from EDCM with stimulation of the posterior division of upper trunk (PDUT), middle trunk (PDMT), and PDLT were 0.64 ± 0.95, 1.64 ± 1.56, and 5.32 ± 4.67 mV (P < 0.05), respectively. The maximal amplitude of CMAP from LTB was induced mainly by stimulation of the PDMT) and PDLT (6 out of 11 and 5 out of 11 patients). The mean amplitudes of CMAP from LTB with stimulation of the PDUT, PDMT, and PDLT were 0.15 ± 0.24, 5.20 ± 4.27, and 7.48 ± 9.90 mV, respectively.

These patterns were observed regardless of treatment protocol (an

These patterns were observed regardless of treatment protocol (anti-GITR mAb and anti-CD25 mAb), strain (BALB/c EX 527 and C57BL/6) and antigen (SRBC, IAV and PE). Importantly, these findings provide a basis to explain the marked increase in serum antibodies, especially switched isotypes, upon in vivo Treg-cell disruption or depletion. These data are also consistent with reports showing the ability of adoptively transferred Treg cells to suppress in vivo B-cell responses,21,30–42 including GC reactions32,41 and the generation of antibody-forming cells.33,34,36

Although it is clear that Treg cells participate in the control of GC reactions, the target and site of Treg-cell action are currently unknown. Two likely targets are Tfh cells and GC B cells. The Tfh cells are critical in the induction and maintenance of GCs because they provide key co-stimulatory signals through inducible T-cell costimulator (ICOS) and CD154, as well as key cytokines, especially IL-21.75 In

addition, it has been shown that the magnitude of the GC response is directly linked to the size of the induced Tfh-cell pool.76 While Treg-cell suppression of CD4+ T-cell activity is well established,11–13 few investigators have focused on whether Treg cells can specifically alter Tfh function. In a recent study by Erikson and co-workers, however, adoptive transfer of antigen-specific Treg cells was found to down-modulate PD0325901 mouse the expression of ICOS on Tfh cells.41 In addition, Weiner and colleagues reported that induction of Treg cells in vivo compromised the ability of Tfh cells to produce optimal levels of IL-21.39 As ICOS expression77 and IL-21 production78–80 by Tfh cells are crucial for optimal B-cell differentiation and switching, influencing these molecules would serve as an effective means by which Treg cells could

control the GC response. In preliminary experiments, Interleukin-3 receptor we tested whether total numbers of splenic Tfh cells were altered by anti-GITR treatment in SRBC-immunized mice. However, when examining days 8 and 12 (the peak of splenic Tfh-cell induction after antigen challenge), no differences were observed (see Supplementary material, Fig. S4). Germinal centre B cells are also a potential target because a number of studies have demonstrated that Treg cells directly suppress activated B cells in vitro.32,40,42–46 In these experiments, Treg–B-cell contact was required and in several reports, Treg cells effected suppression by killing B cells in either a Fas-dependent43 or granzyme B-dependent40,46 manner. Although Treg cells may indeed directly suppress GC B cells, it is uncertain whether they use a cytotoxic mechanism in vivo. Studies in our laboratory found that both Fas-mutated lpr mice and granzyme B-deficient mice generated normal GC responses after SRBC challenge (data not shown).