PBMCs were isolated by standard Ficoll density gradient centrifugation using Leucosep® tubes (Greiner, Bio-one, Alphen aan den Rijn, The Netherlands). PBMCs were collected and stored in liquid nitrogen until use. Recombinant proteins were produced as described previously 56. In short, PCR was used to amplify the selected Mtb H37Rv genes from genomic H37Rv DNA. The PCR products were cloned using Gateway Technology (Invitrogen, San Diego, CA, USA) and were subsequently sequenced. Escherichia coli this website strain BL21 (DE3) was used

to over-express Mtb proteins. Recombinant proteins were further purified as described previously 56. All recombinant proteins were tested in quality control assays including, size and purity check, determination of residual endotoxin levels as well as non-specific T-cell stimulation and cellular toxicity in lymphocyte stimulation assays 55. PPD (batch RT49) was purchased from Statens Serum Institute (Copenhagen, Denmark). Synthetic peptides were synthesized as previously described 57. Peptides from Mtb DosR antigens Rv1733c, Rv2029c, Rv2031c and control antigen Ag85B were 20-mers peptides with 10 aa overlap, except peptides 20–22 of Ag85B which were 15-mers with 10 aa overlap (Table S1A–D). The 20-mer peptides of Rv1733c and Rv2029c were elongated with two lysine (K) residues at the C-terminal to improve solubility. The HLA-A*0201-restricted,

HIV-1 p17 Gag77–85 epitope (SLYNTVATL) Selleck PI3K Inhibitor Library was used as control peptide 58. T-cell phenotype analysis was performed as previously described 59. In brief, PBMCs were stimulated for 16 h with protein (10 μg/mL) or peptide pools (5 μg/mL) in the presence of co-stimulatory antibodies anti-CD28/anti-CD49d (Sanquin, The Netherlands and BD Biosciences respectively). After Sclareol 4–6 h, Brefeldin A (3 μg/mL; Sigma) was added to the culture. Cell surface staining was performed for the following

markers; CD3-PB, CD4-PercP/Cy5.5, CD8-AmCyan, CD45RA-PE/Cy5, CD25-APC/Cy7 and CCR7-PE/Cy7. Subsequently, intracellular markers were stained with IFN-γ-Alexa700, TNF-α-APC, IL-2-PE and CD69-FITC (BD Biosciences) using Intrastain kit (Dako Cytomation, Denmark). Samples were acquired on an LSRII. CD4+ and CD8+ populations of ≥2×105 events were analyzed using FlowJo (Treestar, Ashland, OR, USA) and SPICE software (provided by Dr. Mario Roederer, Vaccine Research Center, NIAID, NIH, USA). Boolean gate analysis was used to study the different single, double and polyfunctional CD4+ and CD8+ T cells. Proliferation was measured using carboxy-fluorescein diacetate, succinimidyl ester (CFSE) dilution and flow cytometry. PBMCs from study subjects were thawed, washed and labeled with CFSE (Molecular Probes, Leiden, The Netherlands) at a final concentration of 5 μM for 10 min at 37°C. Washed, counted and viable cells were seeded in triplicates in 96-well round-bottom plates at a concentration of 1.

In contrast, the entire nephrogenic mesenchyme of Six2 mutants commits to nephron formation at the onset of kidney development, prematurely terminating the nephrogenic programme with only a small number of renal vesicles in place.[7, 8] Thus, Six2 has a unique regulatory activity among these factors: promoting the self-renewal of the nephron progenitor population. Self-renewal of nephron progenitors is normally opposed by Wnt signalling from

the adjacent branching tips of the ureteric epithelium. Here, Wnt9b is expressed in a graded fashion with GSK-3 beta pathway higher levels beneath the tips where induced mesenchyme cells first aggregate then epithelialize to generate renal vesicles, and at lower levels above the tip where the ureteric epithelium directly contacts the main body of the nephron progenitor pool.[9] Wnt9b-directed canonical Wnt signalling mediated by a β-catenin containing transcriptional complex induces

renal vesicle formation.[10] Together, these genetic-based data highlight a complex regulatory network underpinning Dabrafenib specification, maintenance, and commitment of nephron progenitors. What is not clear is how the transcriptional pathways direct these events. The majority of functional studies have examined gene knockouts to infer function rather than directly addressing the transcriptional networks at play. A combination of in vivo and in vitro analysis has defined regulatory modules, uncovering some of the basic networks underpinning Six2 regulation.[11] However, a broader insight requires unbiased genome-scale methodology, integrating a full complement of the regulatory factors to take our understanding

to a deeper, systems level. Combining advances in next generation sequencing with chromatin immunoprecipitation-mediated GNA12 enrichment of transcriptional components at their target sites (ChIP-seq) has resulted in exciting new insights into critical control mechanisms regulating complex biological processes. Similarly, integrating ChIP-seq analysis with gene expression data in nephron progenitors is expected to lead to a new level of insight into transcriptional targets and modules of regulation, and to generate a clearer picture of how nephron progenitor status is programmed, maintained then lost on progenitor commitment to nephron fates. We have recently taken advantage of such experimental analyses to identify the gene regulatory networks engaged by Six2 and canonical Wnt-directed transcriptional complexes. Six2+ nephron progenitors were isolated from embryonic mouse kidneys and subjected to ChIP-seq either immediately (Six2 ChIP) or after treatment with a Wnt pathway agonist to induce β-catenin transcriptional engagement and epithelial commitment (β-catenin ChIP). While each factor was bound to an independent set of regulatory elements, a subset of genomic regions was directly engaged by both factors suggestive of overlapping regulatory functions.

2B). Importantly, when titrating the amount of antigen used in these antigen-presentation experiments, we observed https://www.selleckchem.com/JAK.html that low concentrations (30 μg/mL) of the neo-glycoconjugates were already sufficient

to result in potent T-cell proliferation compared to native OVA (i.e. 500 μg/mL; 14, 15), herewith illustrating the strong potential of the neo-glycoconjugates in the activation of T cells. Proliferation of CD4+ T cells activated by DCs pulsed with OVA-3-sulfo-LeA and OVA-tri-GlcNAc was slightly increased compared to T cells primed by native OVA-loaded DCs, despite the presence of mannose on native OVA (Fig. 3A). A much stronger effect of the neo-glycoconjugates was observed on CD8+ T-cell proliferation. OVA-3-sulfo-LeA and OVA-tri-GlcNAc were significantly enhanced cross-presented compared to native OVA, as shown by a tenfold increased Inhibitor Library price proliferation of OVA-specific CD8+ T cells (Fig. 3B). Similar results were obtained when BMDCs were used (Supporting Information Fig. 3). Controls in experiments also included DCs loaded with non-glycan-modified OVA and maltohexaose-modified OVA, which yielded responses that were not significantly different from

those generated with native OVA (proliferation measured at highest concentration of antigen was 6.75×104±749 and 8.55×104±1093 respectively, for CD8+ T cells and 2.14×104±632 and 3.33×104±1093 respectively, for CD4+ T cells (data not shown). Experiments performed with BMDCs derived from MR−/− revealed that the uptake and processing route of the neo-glycoconjugates was MR-dependent as the proliferation of OVA-specific CD4+ and CD8+ T cells was significantly decreased compared to their response using WT BMDCs (Fig. 3C and D). Although the cross-presentation was greatly reduced Alanine-glyoxylate transaminase using the MR−/− BMDCs, there was still some background presentation of OVA-3-sulfo-LeA and OVA-tri-GlcNAc. As our neo-glycoconjugate

preparations did not contain endotoxin above detection level, we conclude that the observed enhanced cross-presentation of OVA-3-sulfo-LeA and OVA-tri-GlcNAc is glycan-mediated and distinct from the previously reported TLR-dependent cross-presentation of native OVA 15. This was confirmed using MyD88-TRIFF−/− BMDCs; similar to using WT BMDCs, cross-presentation of the neo-glycoconjugates was enhanced in MyD88-TRIFF−/− BMDCs compared to native OVA, indicating that the cross-presentation induced by 3-sulfo-LeA and tri-GlcNAc is independent of TLR-signaling (Fig. 3E). Indeed, addition of LPS improved cross-presentation of native OVA. However, when LPS was mixed with the neo-glycoconjugates, mostly cross-presentation of the lowest antigen doses (e.g. 10 and 3 μg/mL) was affected (Fig. 3F). Together these data indicate that both OVA-neo-glycoconjugates target the MR and upon uptake are potently cross-presented to CD8+ T cells. The entered cross-presentation pathway is different from native OVA, as the observed cross-presentation occurs independent of TLR-signaling.

Conclusion: Renal hL-FABP ameliorated the tubulointerstitial damage in

Aldo-induced renal injury via ROS and suppressing activation of the intrarenal RAS (Figure). KISHIDA MASATSUGU1,2, NISHIYAMA AKIRA3, HAMADA MASAHIRO2, SHIBATA MIKIKO2, KITABAYASHI CHIZUKO2, MORIKAWA TAKASHI2, KONISHI YOSHIO2, ARAI YOSHIE4, ICHIHARA ATSUHIRO4, KOBORI HIROYUKI3, selleck products IMANISHI MASAHITO2 1Department of Hypertension and Nephrology, National Cerebral and Cardiovascular Center, Osaka, Japan; 2Department of Nephrology and Hypertension, Osaka City General Hospital, Osaka, Japan; 3Department of Pharmacology, Kagawa University, Kagawa, Japan; 4Department of Endocrinology and Hypertension, Tokyo Women’s Medical University, Tokyo, Japan Introduction: In a patient with renovascular hypertension, we examined the effect of a direct renin inhibitor (DRI) on blood pressure (BP) and circulating renin-angiotensin system (RAS). Methods: DRI

(aliskiren, 150 or 300 mg/day) was administered to the patient (76 years-old woman) with unilateral renovascular hypertension caused by aortitis. BP and plasma RAS parameters, including Vemurafenib renin activity (PRA), renin concentration (PRC), angiotensinogen concentration (AGT), and soluble form of the (pro)renin receptor concentration (s(P)RR), were measured continuously before and during DRI treatment. Results: Before and 1, 3 hours after the first administration of aliskiren (150 mg), BP was 180/80, 142/64, and 132/68 mmHg, respectively. However, the BP was increased 3-hours after treatment, and returned to 170/70 mmHg at 24 hours. Before and after 1, 3, 24 hours treatment with aliskiren, PRA and PRC levels were 5.7, 1.2, 4.6, 6.7 ng/ml/h (PRA) and 19.2, 619, 755, 608 pg/ml (PRC), respectively. Aliskiren significantly decreased plasma AGT,

but not s(P)RR levels. Higher dose of aliskiren (300 mg/day) did not show apparent BP reduction, although PRA levels were continuously decreased. On the other hand, PRC was increased by approximately 100-fold MRIP after treatment with aliskiren (300 mg/day). Conclusion: In a patient with typical renovascular hypertension, antihypertensive effect of aliskiren was not apparent. Unexpected less antihypertensive efficacy of aliskiren was associated with markedly increases in PRC levels. KIM YANG GYUN, IHM CHUN-GYOO, LEE TAE WON, LEE SANG HO, JEONG KYUNG HWAN, MOON JU YOUNG, LEE YU HO, KIM SE YUN Division of Nephrology Department of Internal medicine Kyung Hee University College of Medicine Introduction: The intrarenal renin-angiotensin system(RAS) contributes not only the generation but also the maintenance of hypertension in the 2-kidney 1-clip(2K1C) Goldblatt hypertensive rats. It is supposed to be regulated differently depending on parts of kidney(cortex or medulla) in 2K1C rats, but there has been sporadic infomration.

During the course of a malaria infection, a wide array of immune effectors are activated. The first acute phase stimulates an inflammatory response with the release of cytotoxic compounds followed by acquired response and antibody production. Previous exposure to the pathogen confers a partial protection to a subsequent infection, a phenomenon coined ‘premunition’ by very early work on avian malaria [51]. Cellier Holzem et al. [52] infected immunologically naive domestic canaries with Plasmodium relictum. Thirty-four days after this primary

infection, when the birds had recovered their initial haematocrit and body mass values, surviving canaries were re-infected with the homologous strain. In agreement with the idea of premunition, re-exposed birds were better able to cope with the infection, keeping parasitaemia at lower levels and managing to maintain constant haematocrit

Palbociclib and body mass. Primary infected canaries produced more haptoglobin, a protein of the acute-phase response, compared with noninfected birds. However, haptoglobin did not differ between primary and secondary infected birds, suggesting that while inflammatory effectors are involved in the control of the initial acute phase of the infection, long-lasting partial immunity relies on memory effectors. Pioneering work conducted on CB-839 in vitro rodent malaria has stressed the importance of host immunity as a component of malaria virulence. Pro-inflammatory cytokines are important immune effectors involved in malaria resistance. Up-regulation of pro-inflammatory cytokines is often associated with a resistance phenotype

prone to immunopathology damage. On the contrary, up-regulation of anti-inflammatory cytokines confers a susceptible phenotype to microparasites and a protection towards immunopathology. Long et al. [53, 54] used phenotypic manipulations of both pro- and anti-inflammatory cytokines in mice infected oxyclozanide with Plasmodium chabaudi. They found that blockade of IL-10 (an anti-inflammatory cytokine) reduced parasitaemia but, nevertheless, exacerbated malaria virulence (i.e. mouse mortality) [53]. Similarly, blocking the TNF-α receptors induced an increase in parasite density while reducing disease severity [54]. Overall, there is strong evidence based on human and rodent studies that malaria virulence has an immune-based component [55, 56]. Building on this previous work, Bichet et al. [57] experimentally infected domestic canaries whose inducible nitric oxide synthase (iNOS) activity was inhibited by a drug (aminoguanidine). Inducible nitric oxide synthase catalyses the production of nitric oxide (NO), a nitrogen reactive species with cytostatic and cytotoxic effect on different Plasmodium species both in vitro and in vivo [58].

2B). Since by using other combinations of inbred mouse strains we previously identified a locus quantitatively controlling thymic Treg-cell development on chromosome 17 [14], we assessed if the same locus was involved in the quantitative regulation of Treg-cell

differentiation in NOD mice. To address this question, we first analyzed the proportion of thymic CD25high CD4SP Treg cells in the congenic mouse strains NOD.B10-H2b and NOD.B6-H2b. These two congenic lines, that carry the B10- or B6-derived H2 locus of H-2b haplotype on an NOD genetic background, respectively, showed a ‘low’ (B6-like) percentage of Treg cells (data not shown). This observation indicated a major influence of an H2-linked locus on buy Lumacaftor the quantitative development of Treg cells. To better define the region of interest, we analyzed other recombinant NOD.B6 congenic

mouse strains [17]. NOD.B6-R76 (R76) mice carry a <20 Mbp B6-derived chromosomal region centromeric to the H2 locus. These mice displayed low (B6-like) proportions of thymic Foxp3+ CD4SP Treg cells. In contrast, thymocytes from the NOD.B6-R156 (R156) strain, carrying a distinct Decitabine order B6-derived region centromeric to H2, had high (NOD-like) proportions and numbers of Foxp3+ CD4SP Treg cells (Fig. 3A and B). Peripheral percentages and numbers of Treg cells were comparable in all the strains analyzed (Supporting Information Fig. 1). In conclusion, a ≤20 Mbp long region centromeric to the H2 complex on mouse chromosome 17 harbors a gene (or multiple genes) that quantitatively controls Treg-cell development. Interestingly, the Trd1 locus contains the diabetes susceptibility locus Idd16. The locus on chromosome 17 controlling Treg-cell development previously reported by us was located telomeric of

H2 and is therefore clearly distinct from the one we report here [14]. It was previously shown that R76 congenic mice develop diabetes with delayed kinetics when compared with those of NOD animals [17]. PAK6 To analyze whether changes in Treg-cell development may somehow be linked to diabetes by influencing Treg-cell function in the periphery, we compared NOD and R76 Treg-cell suppressive activity in vitro. We purified NOD and R76 CD4+CD25high CD127− splenic Treg cells and analyzed their capacity to inhibit proliferation of CD4+CD25−CD127+ splenic Tconv cells induced with plate-bound anti-CD3ε antibody. As shown in Supporting Information Fig. 2, NOD and R76 Treg cells inhibited proliferation of NOD and R76 Tconv cells with similar efficiency. Together, these data show that the intrinsic suppressive function of Treg cells and the sensitivity of Tconv cells to Treg-cell–mediated suppression are similar in NOD and R76 mice.

The

control mice were treated with BM and CY only. Donor skin grafts survived longer than 100 days in chimeric mice but were rejected shortly in control CY-treated mice (mean ± SD = 12 ± 3 days, Fig. 1D). Skin grafts from third-party control C3H (H-2k) mice were used to determined if chimeric KU-57788 datasheet mice corroborate donor-specific tolerance. Skin grafts from C3H mice were rejected shortly in chimeric mice (Fig. 1D, mean ± SD = 11 ± 2 days), suggesting that antigen-specific tolerance was established in the animals with mixed chimerism. The major drawback for BM transplantation is donor T cell-mediated GVHD. Previous studies have demonstrated that adoptive transfer of donor DN Treg cells can inhibit CD8+ T cell-mediated autoimmunity and GVHD [[27, 28]]. To determine if adoptive transfer of DN Treg cells play a role in GVHD in the current model, we put it to test by comparison with CD4+ TAM Receptor inhibitor or CD8+ T cells. C57BL/6 CD4+ T cells or CD8+ T cells purified from BM donor C57BL/6 mice were i.v. injected to BALB/c mice (4 × 106/mouse) on day 0. All mice received CY and BM transplantation as the DN Treg-cell treatment described in Fig. 1. As shown in Fig. 2A and B, all mice that received DN

Treg cells survived beyond 100 days without a decrease in body weight or signs of GVHD. Pathology analysis showed that hepatocytes, liver cell cords, and portal and venous structures were normal with no evidence of GVHD (Fig. 2C). In contrast, the mice that received CD4+ or CD8+ T cells developed GVHD with weight loss and mortality (Fig. 2A and B). Infiltrating mononuclear cells, proliferation in bile ducts, and abnormal portal and venous structure, and typical lesions of chronic GVHD were evident (Fig. 2C). Hence, these data indicate that adoptive transfer CD4+ or CD8+ T cells, but not DN Treg cells, induces GVHD in our protocol. T cells play a major role in BM graft rejection [[29, 30]]. Our data indicate that DN Treg cells in combination with immunosuppression can help AZD9291 donor BM transplantation

and establish-mixed chimerism (Fig. 1). We are interested in determining the mechanism of T-cell suppression in our protocol. We tested the effect of adoptive transfer of DN Treg cells on various clones of T cells bearing different T-cell receptors (TCRs). To focus on the effect on T cells, we depleted NK cells in recipients. BALB/c mice (n = 3) were treated by intraperitoneal (i.p.) injection of NK-cell depletion antibody (anti-Asialo, GM1) on day −4 and −1. Recipient BALB/c mice were treated with cyclophosphamide (200 mg/kg, i.p.) on day 0 and 3. Donor C57BL/6 DN Treg cells (107) were injected into BALB/c mice at same day, while mice of control group were treated with PBS. Recipient mice lymph node cells were harvested on day 8, stained with TCR Vβ antibodies, each combined with anti-CD4 antibody, and anti-CD8 antibody before flow cytometry analysis.

iNOS expression and NO production are known to be dominantly regulated by the transcription factor NF-κB.23,40 Therefore, we first checked whether rRv2626c activates the NF-κB transcription factor in macrophages. RAW 264·7 macrophages were either left untreated or treated with rRv2626c (5 μg). The positive control group received LPS plus IFN-γ. Nuclear extracts were prepared from these

cells and the expression of NF-κB was mounted using an electrophoretic mobility shift assay. It was observed that stimulation with rRv2626c caused an increase in the intensity of the NF-κB complex Cytoskeletal Signaling inhibitor in vitro compared with the untreated group (Fig. 4a; compare lane 4 with lane 2) suggesting induced expression of NF-κB. A similar increase was apparent in cells stimulated with LPS plus IFN-γ (lane 3) as compared with the control (lane 2). The specificity of the DNA–protein interaction was confirmed by homologous and heterologous competition during the binding reaction. In the presence of a 100-fold molar excess of unlabelled wild-type consensus NF-κB oligonucleotides, the complex completely disappeared MS-275 purchase (lane 6) but was unaffected even in the presence of a 100-fold molar excess of unlabelled NF-κB mutant oligonucleotides (lane 7) that carried a mutation in the bases critical for NF-κB binding. To conclusively demonstrate the specific involvement of NF-κB, a nuclear

extract prepared from RAW 264·7 cells treated with PDTC, a specific inhibitor of this transcription factor,41–43 was used in the electrophoretic mobility shift assay. PDTC treatment was found to inhibit rRv2626c-induced NF-κB activity (compare lane 5 with lane 4). The levels of nuclear p50 and p65 subunits of NF-κB present in rRv2626c-stimulated GPX6 macrophages were further confirmed using NF-κB-specific antibody. The immunoblotting results again showed increased nuclear translocation of p50 and p65, indicating

that rRv2626c induces NF-κB activity (Fig. 4b; compare lane 3 with lane 1) in macrophages, and this was almost comparable to that induced by LPS plus IFN-γ (lane 2). Treatment with PDTC, as expected, caused a reduction in nuclear translocation of both p50 and p65 subunits of NF-κB (lane 4). Having shown the direct involvement of NF-κB, we once again assayed for activation of iNOS by western blotting as well as NO production in the presence or absence of PDTC followed by stimulation with rRv2626c. While rRv2626c induced iNOS expression (Fig. 4c; lane 3) comparable to that induced by LPS plus IFN-γ (Fig. 4c; lane 2), treatment with PDTC inhibited rRv2626c-induced iNOS expression (Fig. 4c; compare lane 4 with lane 3). The subsequent production of NO in these experimental groups was measured. Again, it was observed that rRv2626c increased NO production as a function of concentration (Fig. 4d; bars 2, 3 and 4), and NO production was inhibited by PDTC treatment (Fig. 4d; bars 5, 6 and 7) in a concentration-dependent manner.

n.-primed mice. Figure 3B shows data for CD8+

T cells tested 4 and 10 wk after i.m. priming, at 4 wk after a booster immunization of i.m.-primed mice given i.vag. or i.m., and at 1 year after Bcl-2 inhibitor an i.m/i.m. prime-boost regimen. In all experiments, tet−CD8+ T cells from immune mice were also analyzed and their phenotypes mirrored those of naïve mice (data not shown). Four weeks after i.n. immunization with AdC6gag, CD44 was upregulated on Gag-specific CD8+ T cells from spleens, blood, ILN and NALT (Fig. 3A). This increase was less pronounced on tet+CD8+ cells from the GT, presumably reflecting that most T cells from the GT were already antigen-experienced. Most of the tet+CD8+ T cells from the GT expressed comparable levels of CD62L although a small population was CD62Lhi. It should be pointed out that expression of CD62L was also Y-27632 mouse low on most of the genital CD8+ T cells from naïve mice. Expression of α4β7 was low on most cells except for a small population of tet+CD8+ T cells present in spleen and blood. The booster immunization did

not have a pronounced effect on the expression of CD44, CD62L or CD27. α4β7 expression was again increased on some of the tet+CD8+ T cells from spleens and ILN. At 4 wk after i.m. immunization, CD44 expression was upregulated on tet+CD8+ T cells from spleens, ILN and GT (Fig. 3B). We detected a downregulation of CD62L expression on tet+CD8+ T cells from spleens, blood and the GT but not on those from ILN. CD27 expression was decreased on a subpopulation of tet+CD8+ T cells from blood, spleens and GT. At 4 wk after i.n. or i.vag. boost, expression levels of CD44, CD62L, CD27 and α4β7 mirrored those seen at 10 wk after priming, and there were no striking differences among groups that received an AdC6gag i.m. prime followed by a heterologous boost through the i.m. or i.vag. routes. At 1 year after the i.m. prime-boost vaccine regimen, expression of CD44 on tet+CD8+ T cells isolated from the different compartments (NALT was not tested in this experiment) overlapped with those seen on part of CD8+ T cells of

age-matched naïve mice. This may reflect an increase of CD44 expression on the control CD8+ T cells due to immunosenescence 15. Gag-specific CD8+ T cells isolated from the ILN and GT showed an increase in CD62L expression, which was unexpected for the latter compartment. In Inositol monophosphatase 1 blood and spleen, expression of CD62L and CD27 was similar or only slightly increased above those seen on unprimed CD8+ T cells, suggesting that the Gag-specific CD8+ T cells had differentiated into resting memory cells. Additional markers were analyzed on Gag-specific CD8+ T cells isolated from different compartments after an i.m./i.m. heterologous prime-boost regimen (Fig. 4). For the two early time points, i.e. 4 wk after priming or boosting, cells isolated from the vaginal mucosa were treated and analyzed separately from OUC. CD44, CD62L and CD27 were tested and found to mirror those shown in Fig.

In addition to demonstrating strain independence, experiments were performed to show that Treg-cell control of GC responses was also antigen independent. Figure 3 summarizes the effect of anti-GITR mAb treatment on splenic GC responses induced by i.p challenge of BALB/c mice with IAV. Whereas SRBC induce a Th2-biased response,5 IAV invokes a Th1-polarized reaction.56Figure 3(a) shows that mice immunized i.p. with IAV generate Fluorouracil cost a robust splenic GC response which peaks at day 12 (Fig. 3b). Similar to Th2 antigens,5,6 the GC reaction induced by

IAV was characterized by a steady ratio of IgM+ to switched GC B cells (Fig. 3c). Importantly, anti-GITR mAb administration resulted in a higher frequency and total number of splenic GC B cells at several time-points (Fig. 3b), and significantly increased the proportion of switched GC B cells throughout the entire reaction (Fig. 3c). As opposed to GCs induced with SRBC immunization, we observed no significant difference C59 wnt molecular weight in the distribution of IgG isotypes within the switched GC B-cell pool at any time-points after IAV challenge (data not shown). The results generated above demonstrated the role of Treg cells in controlling both the size of SRBC-induced and IAV-induced GC responses, and the ratio of IgM+ to switched B cells within the

GC population. In these experiments, however, total splenic GC B cells were enumerated because the B220+ PNAhi B-cell population induced after SRBC or IAV injection was presumed to be specific for the challenge antigen. (Please note that specific pathogen-free mice do not exhibit splenic GCs in the absence of immunization, Fig. 1.) We therefore sought to confirm the role of Treg cells in governing GC reactions by tracking antigen-binding GC B cells, instead of the entire B220+ PNAhi splenic B-cell pool. To perform these studies, PE was used as the challenge antigen,57–59 and PE-binding GC B cells were analysed in anti-GITR mAb or control rIgG-treated mice. As shown in Fig. 4(a), i.p. immunization with PE precipitated in alum induced splenic B220+ PNAhi GC B cells, a Non-specific serine/threonine protein kinase sub-set of which retained the ability to bind native

PE. In control animals, the PE-binding GC B-cell response peaked at day 12 (Fig. 4b) and like other normal splenic GC responses, displayed a relatively steady ratio of IgM+ to switched B cells (Fig. 4c). As expected, disruption of Treg cells with anti-GITR mAb administration resulted in an increased total PE-binding GC response, and a progressive increase in the proportion and total number of switched PE-binding GC B cells. In Figs 1–4, splenic GC responses were dysregulated when anti-GITR mAb was given before and soon after immunization. To assess whether already established GCs can be altered by late-stage Treg-cell disruption, mice were challenged with SRBC at day 0 and treated with either anti-GITR mAb or control rIgG on days 8 and 12, or days 12 and 16 post-immunization. Splenic GCs from both groups were examined on days 18 and 24.