Moreover, purified DNA was able to activate a TLR9- and IRF1-dependent pathway leading to IL-12p70 induction. In summary, our data suggest that TLR7 and TLR9 collaborate in a fungal recognition mechanism that targets nucleic acids (RNA and DNA, respectively) and activates a common, MyD88- and IRF1-dependent,

pathway. Activation of this pathway was absolutely dependent on phagocytosis and phagosomal acidification, both of which are known requirements for TLR9- and TLR7-mediated recognition. An additional feature of the TLR7/9-dependent responses described here is their cell-type specificity. Indeed, BMDC, but not BMDM, mounted robust cytokine responses to yeast nucleic acids. The reasons for these differences are presently unclear, but they may relate to differential

BIBW2992 TLR or IRF1 expression or to differential STAT1 phosphorylation in response to nucleic acid stimulation [51]. Our data are only apparently in contrast with previous reports indicating that TLR9-defective mice display similar [28, 38] or even increased [14] resistance to C. albicans. Differences between our data and those of others were unequivocally linked, in the present study, to the different doses used for challenge. In fact, increased susceptibility GS-1101 datasheet to C. albicans infection in the absence of TLR7 or TLR9 was observed only using a low challenge dose. When we challenged mice with the high doses used in the studies cited above, no effect of TLR7 or TLR9 deficiency was observed. Our data are in agreement with the notion that lack of specific host factors has different and even opposite effects on the outcome of experimental infection depending on the challenge dose, the associated

severity of infection, and risk of death [19, 52, 53]. Thus, it appears that the Arachidonate 15-lipoxygenase contribution of TLR7 or TLR9 to host defenses against C. albicans can be evidenced only under experimental conditions associated with mild, sublethal infection. The use of low rather than high challenge doses seems logical, since under most natural circumstances, the immune system is exposed to low numbers of microbial cells in the initial stages of infection. Moreover, overwhelming infection is often associated with the deleterious release of pathophysiological mediators by the host and/or of immunosuppressive products by the pathogen, both of which may obscure the contribution of individual immune factors [19, 52-54]. Collectively, our data indicate the presence of at least two different cellular mechanisms underlying fungal recognition that lead to the production of two different sets of defense factors. The first mechanism, underlying the production of IL-23 and TNF-α, relies predominantly on the detection of cell-wall structures by receptors located on the host cell surface, such as dectin-1. This mechanism does not necessarily require phagocytosis and is largely independent from TLR or TRL adaptors.

The CD277 molecule is expressed in both T and NK cells 1, 13 (Supporting Information Fig. 1 and 2). CD277 has three

isoforms BTN3A1, BTN3A2 and BTN3A3, with (BTN3A1 and BTN3A3) or without (BTN3A2) the B30.2 domain in their cytoplasmic part 5 (Fig. 5A). The used mAb (clone 20.1) does not discriminate between the Ig domains of the three BTN3A isoforms, which share a very high level of identity (>95%). Moreover, the CD277 mAb recognizes in a similar manner all the different isoforms expressed in an ectopic cellular Selleckchem VX 809 model (Fig. 5B). Quantitative PCRs were performed to determine the different relative levels of mRNA expression for each isoform in T and NK cells isolated from human PBMCs (Fig. 5C). Both BTN3A1 and BTN3A2 represented the main forms expressed by CD4+ and CD8+ T-cell subsets whereas the decoy form, BTN3A2 was the unique form strongly expressed by NK cells (Fig. 5C and D). BTB3A3 is poorly expressed in these immune cells. These results are further confirmed using available data from GEO omnibus (data not shown). Rapamycin cost To identify a role for these two major CD277 isoforms (Fig. 5D), the KGHYG-1 NK cell line was nucleofected with constructs encoding for FLAG epitope tagged BTN3A1 or BTN3A2. This cell line expresses the natural cytotoxicity receptor, NKp30 and stimulation of this receptor by specific antibodies is able to induce IFN-γ production in this NK cell line (data not

shown). The overexpression of the BTN3 isoforms is monitored by anti-FLAG mAb cell surface staining (Fig. 6A). The transiently transfected NK cells were stimulated by anti-NKp30 and/or anti-FLAG mAbs, and the IFN-γ production assessed by FACS analysis (Fig. 6B). The NKp30 stimulation, but not BTN3A1 or BTN3A2 triggering alone, induces IFN-γ production.

However, co-engagement of NKp30 with a CD277 isoform, modulates the NKp30-induced IFN-γ production. BTN3A1 stimulation seems to increase this cytokine production, whereas BTN3A2 stimulation decreases the NKp30-induced IFN-γ production. These results suggest a differential functional role of these two CD277 isoforms in NK cells. In this study, we describe differential effects of the CD277 molecule as a co-regulator of the immune signal in T cells Olopatadine but not in NK cells (Fig. 1). There is no effect noted on NK cells consistent with the selective expression of the BTN3A2 isoform that lacks much of the cytosolic domain (Fig. 5). However, in the context where only the BTN3A2 isoform is co-engaged, this molecule could induce some negative signals in NK cells (Fig. 6). CD277 cross-linking elicits a robust co-stimulation of T-cell proliferation, cytokine production and CD25 expression. We showed that the stimulation of BTN3/CD277 proteins with a home-made mAb (clone 20.1, 1) increases, in a dose-dependent manner, the rates of early and late T-cell activation events induced by a combination of CD3+/−CD28 mAbs (Figs. 3 and 4).

3). Furthermore, the same treatment regimen reduced significantly the levels of IFN-γ, IL-1β, IL-2, IL-17 and TNF-α both in the spleen and pancreatic lymph nodes compared to control mice (Fig. 4a,b). The same differences, with the exception of TNF-α being undetectable, were also observed in murine sera in the same experimental conditions (Fig. 4c). Finally, prolonged treatment with apoTf did not change significantly the proportion of splenic CD4+ regulatory T cells (Treg) (CD4+/CD25+/FoxP3+) cells compared to control mice (Fig. 5). ApoTf plasma levels were significantly lower in patients with Opaganib clinical trial ND-type 1 diabetes compared to matched

controls, while this difference was not observed comparing patients with CR or LS disease (Fig. 6).When biochemical and clinical features of ND-type 1

diabetes were correlated with apoTf levels we found a significant association with HbA1c determined at disease onset using both laboratory methods (r = −0·452, P = 0·045 with RID; r = −0·564, P = 0·01 with nephelometry) but not with basal or stimulated C peptide levels, RAD001 order GADA and IA2 antibodies, weight loss prior to diagnosis or symptom duration (data not shown). No correlation with any of the analysed clinical and biochemical features was encountered in patients with LS or CR type 1 diabetes (data not shown). The data presented herein were obtained from different murine and cellular models as well as human samples to demonstrate for the first time that recombinant human apoTf or human-derived apoTf acts to inhibit significantly the inflammatory ADP ribosylation factor pathways leading to diabetes. The affected pathways included cytokine-induced beta cell death in

vitro and disease onset in well-established models. In particular, apoTf was associated with milder signs of insulitis and profound modulation of cytokine secretory profile in NOD mice. Several findings may prove significant in our understanding of type 1 diabetes pathogenesis and the role of apoTf. First, the prolonged ex-vivo treatment with apoTf leads to down-modulation of the destructive Th1 and Th17 autoimmune responses [17,21,22] that produce IL-1β, IL-2, TNF-α, IFN-γ, IL-17 and IL-18 [23], which are crucial to diabetes development in the NOD mouse. Th1, Th17 and Treg are thought to be regulated reciprocally and, therefore, changes in Treg could be expected in the immune modulating activity we observed during apoTf treatment in NOD mice [24]. Nevertheless, we could not observe significant changes in the prevalence of Treg (CD4+/CD25+/FoxP3+) cells in the spleen of animals treated for 12 weeks. Further studies are being carried out to demonstrate whether ApoTf exerts its anti-diabetogenic effect by up-regulating Treg function without modifying their numbers or whether it acts via Treg-independent pathways.

Although it is unclear why the MicroScan results for clindamycin were often above the range within ± 2 log2 dilutions as revealed by the reference method, it may be associated with clindamycin acting bacteriostatically and the

MicroScan panel being read visually. Bacillus cereus BSIs were reported to be found in immunosuppressed patients, patients receiving continuous intravenous therapy, patients with underlying malignancy, and neonates (Drobniewski, 1993; Gaur et al., 2001). In this study, use of antimicrobials for more than 3 days during the 3-month period before isolation of B. cereus was significantly larger in the BSI group compared with the see more contaminated blood culture group. In conclusion, our results suggest that the virulence gene profiles may be indistinguishable between BSI isolates and isolates from contaminated

blood cultures. In each group, there was wide diversity in the patterns of the virulence genes examined. Compared with the reference MICs, some isolates showed discrepant MIC values determined by the MicroScan or the Etest method for some antimicrobials. We consider that antimicrobial susceptibility data are essential when selecting the treatment regimen for B. cereus infections, because of the existence of isolates showing higher MICs for antimicrobials such as β-lactams and quinolones as shown in this study. Therefore, it is important to characterize the clinical utility and the performance limitations of antimicrobial susceptibility testing methods routinely used for Inhibitor Library research buy clinical B. cereus isolates. Our results also suggest that Silibinin prior antimicrobial therapy may be a risk factor for BSIs due to B. cereus. To prevent BSIs caused

by B. cereus, therefore, clinicians should make efforts to improve the quality of antimicrobial therapy. T.H. was partially supported by a Grant-in-Aid for Scientific Research (20790413) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. No conflict of interest to declare. “
“Enteropathogenic Escherichia coli (EPEC) strains produce a bundle-forming pilus (BFP) that mediates localized adherence (LA) to intestinal epithelial cells. The major structural subunit of the BFP is bundlin, which is encoded by the bfpA gene located on a large EAF plasmid. The perA gene has been shown to activate genes within the bfp operon. We analyzed perA gene polymorphism among typical (eae- and bfpA- positive) EPEC strains isolated from healthy and diarrheal persons in Japan (n= 27) and Thailand (n= 26) during the period 1995 to 2007 and compared this with virulence and phenotypic characteristics. Eight genotypes of perA were identified by heteroduplex mobility assay (HMA). The strains isolated in Thailand showed strong autoaggregation and had an intact perA, while most of those isolated in Japan showed weak or no autoaggregation, and had a truncated perA due to frameshift mutation.

For this reason, methods of abrogating the activity of Treg cells might be critical for the successful immunotherapeutic treatment of cancer. Studies showed that Treg and Th17

cells co-existed in the microenvironment of different types of tumour, and the development of Th17 cells was described to be linked to that of Treg in a reciprocal fashion; however, information on human bladder cancer Th17/Treg development and differentiation is limited. Our data revealed that Th17 cells were correlated inversely with Treg cells and correlated positively with IFN-γ+ CD4+ T cells in the same tumour microenvironment. It has shown that recombinant IL-2 is a promising agent for the activation of immune response against tumour selleck and plays a central role in balancing Treg cells and IL-17+ T cells in multiple diseases. Kryczek et al. reported that IL-2 regulated MK0683 solubility dmso the balance between tumour Treg and Th17 cells by stimulating the differentiation of Treg and inhibiting that of Th17 cells [35]. However, Leveque et al. revealed that under some stimulated conditions, IL-2 rapidly converted epithelial ovarian cancer (EOC) Treg into Th17 cells, down-regulated

FoxP3 expression, and lost their suppressive capacity [17]. Due to the above conflicting data, we sought to determine whether IL-2 would also play a role in balancing Treg cells and IL-17+ T cells in bladder cancers. Our results indicated that tumour-infiltrating Treg cells cultured in the presence of the autologous irradiated CD3– fraction and IL-2 could be converted into Th17 cells, which might be involved in the mechanism that instillations of IL-2 into the urinary bladder is effective in the treatment of superficial bladder cancer. In conclusion, the present data suggest that Th17 cells, together with Treg cells, might contribute to the immunopathogenesis of bladder cancer, and inhibition of Th17 cell development might be a novel immune evasion mechanism. We further identified MycoClean Mycoplasma Removal Kit that IL-2 played a role

in balancing Treg cells and IL-17+ T cells by converting bladder cancer Treg into Th17 cells, our results encouraged a deep in vivo exploration of its effects on in situ immune responses. Further studies are still needed to identify the mechanisms of underlying regulation and dynamic interaction among Th17 cells and Treg and Th1 cells in human pathological conditions such as bladder cancer. The authors have no financial conflict of interest. This study was supported by Heilongjiang Province Science Foundation for Youths (project number: QC2009C05), China Postdoctoral Science Foundation, Innovation of science and technology of Harbin youth (project number: 2008RFQXS008) and Foundation of Heilongjiang Educational Committee (project number: 11531160).

Interestingly, one genotype, −2849AA, is thought to be associated with a threefold reduced risk toward acquisition of pre-eclampsia.61 Recurrent spontaneous abortion has been linked to an increase in CD56+ cells as well as an increase in TNF-α.62,63 However, the balance of this inflammatory cytokine may be skewed as a result of a lack of IL-10 production.

PBMCs from women with RSA show increased cytotoxicity because of high levels of TNF-α, but levels of IL-10 production are significantly lower than control PBMCs.64,65 Similarly, PBMCs from women with RSA show lower production of IL-10 upon stimulation with trophoblastic antigen when compared to normal pregnancy controls.66 We have previously demonstrated that decidual and placental tissue from spontaneous abortions showed reduced presence of IL-10 with no effect on IFN-γ compared to Selleckchem Saracatinib tissue from elective terminations.17 Thus, poor IL-10 production coupled with increased production of inflammatory molecules may be a trigger for early pregnancy loss or preterm birth. Furthermore,

placental explants obtained from women undergoing preterm labor showed poor IL-10 production coupled to elevated prostaglandin release when compared to normal pregnancy control samples.67 Based on these observations, we established mouse models for fetal resorption and preterm birth using IL-10−/− mice. As was aforementioned, our data are significant in that low doses of inflammatory triggers cause Tanespimycin fetal loss or preterm birth depending on the gestational age–dependent exposure to the trigger.19,34,35 These pregnancy complications are strongly linked with immune programming in the form of cytotoxic activation of uterine NK cells, macrophages, or T cells and TNF-α production depending on the nature of the inflammatory trigger. These results provide impetus for further investigation

into the nature of infection/inflammation and the ensuing immune responses in both mouse models and humans. It is well accepted now that IL-10 influences immune responses in a variety MycoClean Mycoplasma Removal Kit of ways. In the context of pregnancy, we propose that IL-10 exerts profound effects on linking immunity, angiogenesis, and maintenance of expression of molecules regulating fluid volume across the placenta. Our work in IL-10−/− mice for the first time provides important clues to the pathogenesis of fetal loss, preterm birth, and pre-eclampsia. These observations have given rise to the hope that IL-10-based therapy may some day become a reality for enigmatic pregnancy maladies. We would like to thank Tania Nevers for insightful critique and reading of the manuscript. This work was supported in part by grants from NIH and NIEHS, P20RR018728 and Superfund Basic Research Program Award (P42ES013660). This work was also supported in part by the Rhode Island Research Alliance Collaborative Research Award 2009-28.

Although the mechanism of LAG-3 function remains unclear, a conserved KIEELE motif in the cytoplasmic domain of LAG-3 is essential 2. In contrast to CD4, LAG-3 is only expressed on the cell surface of activated T cells 1, 7–10. LAG-3 surface expression is further regulated by two metalloproteases, ADAM10 and ADAM17, which cleave surface LAG-3, a proportion of which is both constitutive and TCR-ligation induced 11. Importantly, prevention of LAG-3 cleavage blocks T-cell proliferation

and cytokine secretion 11 suggesting that LAG-3 surface expression is under tight regulatory control. This observation raised the question of whether other mechanisms are used to control the expression and distribution of LAG-3. Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), which is another inhibitory molecule for T-cell activation, Trichostatin A chemical structure is mainly stored in Rucaparib price intracellular compartments such as the trans-Golgi network, endosomes and lysosomes 12–17. Surface expression is tightly regulated by controlled internalization and trafficking to the plasma membrane. This raised the possibility that LAG-3 surface expression might also be regulated by modulating its intracellular storage and trafficking. In this study, we addressed the following questions.

First, what is the extent of intracellular storage and localization of LAG-3 versus its relative CD4? Second, what is the sub-cellular localization of LAG-3 and CD4 in activated T cells? Third, what is the fate of intracellular LAG-3? In order to determine cellular distribution of CD4 and

LAG-3, we performed intracellular staining for CD4 or LAG-3 using flow cytometry. Freshly isolated naïve CD4+ T cells do not express LAG-3 10; so naïve T cells were first stimulated with plate-bound anti-CD3 and anti-CD28 for 72 h and then treated with pronase to remove cell surface CD4 and LAG-3 from activated CD4+ T cells. Pronase treatment removed most of the surface CD4 and LAG-3 on activated T cells (Fig. 1A). While intracellular staining revealed that a relatively small amount (23%) of CD4 is present inside cells, in Venetoclax order contrast a greater amount (49%) of LAG-3 appears to be retained intracellularly (Fig. 1A and B). One might speculate that the slightly lower LAG-3 surface expression compared with CD4 following T-cell activation and the increased percentage of intracellular LAG-3 versus CD4 is due to its continuous cleavage by the metalloproteases ADAM10 and ADAM17 that limits surface LAG-3 expression 11, 18. However, when T cells were treated with the metalloproteinase inhibitor TAPI (Calbiochem), cell surface LAG-3 expression was only slightly increased (data not shown). While prevention of LAG-3 cleavage by TAPI slightly changed the ratio of surface and intracellular LAG-3, the effect was small and not sufficient to account for the differences observed between LAG-3 and CD4. The extent of intracellular LAG-3 storage was also examined by Western blot analysis.

The disease activity of SLE was assessed clinically by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)17 on the day of kidney biopsy. Baseline serum creatinine, urine protein, complement levels (C3 Decitabine datasheet and C4) and anti-double strand (ds) DNA antibody titre were also measured. Glomerular filtration rate (GFR) was estimated by a standard equation.18 Kidney biopsy specimen was evaluated according to the International Society of Nephrology (ISN) classification of lupus nephritis.19 The activity index (AI) and chronicity index (CI) of each biopsy specimen were scored by standard methods.19 The method of laser micro-dissection has

been described in our previous studies.16,20,21 Briefly, cryosections of 10 µm thickness were prepared on a cryostat (Leica Microsystems, Wetzlar, Germany) using disposable Nutlin 3 microtome blades (Leica Microsystem) in RNase-free conditions and were mounted on MembraneSlide 0.17 PEN slides (Carl Zeiss PALM Microlaser Technologies, Bernried, Germany). Immediately after taking the slides out of the cryostat, the sections were fixed in 70% ethanol and dehydrated in 100% ethanol. Sections were air-dried at room temperature. Laser micro-dissection of the snap-frozen kidney biopsy specimens was performed using the PALM Microlaser System

(PALM Microlaser Technologies), which is equipped with a pulsed high-quality laser beam, computer-controlled microscope stage and micromanipulator. Under direct

visual control, areas of interest in the histological specimens were selected through the PALM RoboSoftware (PALM Microlaser Technologies) by moving the computer mouse and micro-dissected by cutting the contour of the selected areas with the adjusted laser beam. The isolated tissue was then laser-catapulted into a microcentrifuge tube filled with guanidine thiocyanate containing lysis buffer for the subsequent RNA isolation. Approximately 20–30 glomerulus and 20 randomly selected tubulointerstitial areas were isolated from each specimen. The tissue lysate of glomerulus and tubulointerstitium were kept Sorafenib cost at −80°C until RNA extraction was performed with the RNAqueous-Micro Kit (Applied Biosystems, Foster City, CA, USA), following manufacturer’s instruction. The RNAqueous-Micro Kit (Applied Biosystems) was used for the extraction of total RNA. TaqMan microRNA Reverse Transcription kit (Applied Biosystems) and High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) were used for reverse transcription. Intrarenal expression of miR-146a, miR-155, miR-198 miR-638 and miR-663 were quantified by reverse transcription-quantitative polymerase chain reaction (RT-QPCR) with the ABI Prism 7900 Sequence Detection System (Applied Biosystems). These targets were selected because previous studies on PBMC or urine showed that they were differentially expressed between lupus nephritis patients and normal controls.

mirabilis. Orf9 belongs to the group 1 family of glycosyltransferases (Pfam00534, E value = 9 × e−28) and shares 33% identity to glycosyltransferase of Herpetosiphon aurantiacus. Therefore, orf7, orf9, and orf12 were proposed to encode the three glycosyltransferases and were named wpaA, wpaB, and wpaD, respectively. Among four known pathways for synthesis and translocation LY2835219 concentration of O-antigen (Hug et al., 2010; Valvano, 2011), the Wzx/Wzy-depending pathway occurs in the synthesis of the majority of O-antigens, especially heteropolymeric O-antigens. Both Wzx (flippase)

and Wzy (O-antigen polymerase) are highly hydrophobic inner membrane proteins, usually sharing little sequence identities with their homologues. In the O40-antigen gene cluster, orf6 and orf8 are the only two genes encoding predicted membrane proteins. Orf6 has 12 predicted transmembrane segments, which is a typical topology for Wzx proteins, and shares 46% identity or 63% similarity with putative flippase of E. coli O91. It was proposed that orf6 encodes the O-antigen flippase and was named wzx. Orf8 exhibited no sequence identity to any protein in GenBank. However, the transmembrane region search indicated that it had 10 predicted transmembrane segments with a large www.selleckchem.com/products/Rapamycin.html periplasmic loop of 34 amino acid residues. One or two such loops have

been reported for a number of O-antigen polymerases (Islam et al., 2010; Islam et al., 2011; Daniels et al., 1998) and seemed to be important in the recognition of the O-unit or/and for the catalytic activity (Valvano,

2011). Therefore, orf8 was proposed to encode O-antigen polymerase and, accordingly, was designated wzy. These findings suggested that the biosynthesis of the P. alcalifaciens O40-antigen is mediated by the Wzx/Wzy-dependent process. orf15, orf16, and orf17 are homologues of wza, wzb, and wzc genes required for the biosynthesis and export of group 1 and Thalidomide 4 capsular polysaccharides (CPS) (Whitfield, 2006). In particular, tyrosine–protein kinase Wzc and its cognate tyrosine phosphatase Wzb are essential for maintaining polymerization process, and Wza is involved in forming an outer membrane pore through which the CPS is translocated (Collins et al., 2007). Together with a nonessential gene named wzi, the wza, wzb, and wzc genes comprise a conserved locus within group 1 CPS biosynthesis clusters of E. coli (Whitfield, 2006). In contrast, in E. coli group 4 capsular producers, the wza, wzb, and wzc genes are accompanied by the ymcABCD genes and located outside the CPS gene cluster. Both group 1 and 4 capsules can be anchored to the cell surface by means of core-lipid A giving rise to the so-called KLPS. Some strains coexpress KLPS with a “normal” LPS, whereas others produce KLPS as the only serotype-specific polysaccharide (Whitfield, 2006). The latter seems to be the case of P.

Recipient mice received 200 μg anti-mouse IL-17A antibody i.p.

on 4 consecutive days followed by an injection every other day until the age of 7 weeks. For the generation of single-cell suspensions from spleen, LN, and thymus, organs were collected in BSS and were mechanically disrupted on a metallic grid. For isolation of heart-infiltrating cells, euthanized animals were perfused with 20 mL BSS and small heart tissue pieces were digested with 170 check details U/mL collagenase type II (Gibco) and 60 U/mL DNAse 1 (ApliChem) in BSS at 37°C under continuous stirring. The tissue suspension was sheered and mononuclear cells were purified by centrifugation (25 min at 800 × g, 4°C) on a 30–70% Percoll gradient (GE Healthcare). For flow cytometric analysis, cells were resuspended in FACS buffer (PBS, 2% FCS, 10 mM EDTA, 0.05% sodium acide) and incubated with the following monoclonal antibodies: anti-Vβ8.1/8.2-FITC (MR5–2), anti-CD45-FITC (30-F11), anti-Vα2-PE (B20.1), anti-IL-2-PE (JES6–5H4), anti-IL-10-PE (JESS-16E3), anti-IL-17A-PE (TC11–18H10.1), anti-I-Ad-PE (AMS-32.1), anti-Ly6G-PE (1A8), anti-IFN-γ-allophycocyanin (XMG1.2),

MG 132 anti-CD4-PerCP (RM4–5), and anti-TNF-α-PE (MP6-XT22) from BD Pharmingen, anti-CD11b-FITC (M1/70), anti-CD8-allophycocyanin (N418), anti-F4/80- allophycocyanin (CI:A3–1), and anti-IL-4-PE (11B11) from BioLegend, anti-CD11c-allophycocyanin (N418) from eBioscience, and anti-CD62L-allophycocyanin (145/15) from Miltenyi Biotech. Intracellular Foxp3 staining was performed with the mouse regulatory T-cell staining kit using anti-FoxP3-PE (FJK-16) or FoxP3-PE-Cy7 (FJK-16s) antibodies (eBioscience). For assessment of ex vivo production of IFN-γ, IL-17, TNF-α, IL-2, IL-10, and IL-4, 106 lymphocytes were incubated for 5 h at 37 many °C in 96-well round-bottom plates in 200 μL of RPMI per 5% FCS supplemented with 10 μg/mL brefeldin A (Sigma). Cells were stimulated with 0.25 μg myhca614–629 peptide, phorbol myristate acetate (50 ng/mL; Sigma)/ionomycin (500 ng/mL; Sigma) (PMA/I) as positive control, or were left untreated. After surface molecule

labeling, cells were permeabilized with Fix&Perm (BD Bioscience) solution and staining for intracellular cytokines was performed in permeabilization buffer (1× PBS, 2% FCS, 0.1% saponin, 0.1% sodium acide, 5 mM EDTA). Samples were measured using a FACS Calibur or FACS Canto flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Inc.) or CellQuest software (BD Biosciences). Spleen cells were labeled with 10 μL 5 mM carboxyfluorescein succinimidyl ester (CSFE, dissolved in DMSO) (Molecular Probes) in 10 mL PBS for 10 min at 37°C. The staining reaction was stopped with 1 mL FCS (Lonza), followed by washing with PBS. A total of 2 × 105 splenocytes/well were seeded in 96-well round-bottom plate and myhca614–629 peptide was added at the indicated concentrations. CSFE dilution was assessed by flow cytometry after 3 days of incubation at 37°C.