An important study by Belli et al. (76) demonstrated that antibodies to E. maxima Gam56 and Gam82 recognized proteins in the WFB’s of macrogametocytes and oocysts of E. tenella and E. acervulina. Homologous genes encoding for Gam56 and Gam82 were also identified in these species and, when the three sequences were aligned, they were found to be highly homologous around the epitopes of these proteins, hence explaining the cross-species protection afforded by the vaccine. It is not yet understood,

however, how the protective IgG antibodies gain access selleck compound to what is an essentially intracellular parasite. Despite the obvious success of CoxAbic® as the first and, currently, only subunit vaccine for the control of coccidiosis within the poultry industry and now registered in many countries worldwide, a drawback is the expense associated with production. This is because production of the vaccine relies on affinity purification of native gametocyte selleck antigens from parasites. As it is not possible to reliably culture sexual stages of Eimeria in an in vitro culture system, parasites are passaged and isolated from the intestines of chickens raised under strict specific pathogen free (SPF) conditions, which is not only expensive, but also time consuming and laborious

(83). Therefore, recent work has been aimed at determining whether recombinant forms of the gametocyte proteins in APGA could maintain antigenic and immunogenic

properties analogous to the native antigens and, therefore, perhaps replace them. A study by Belli et al. (83) examined bacterially expressed recombinants of Gam56 and Gam82 to determine if they could maintain antigenic determinants recognized by protective antibodies to their native protein counterparts. Antibodies to the native proteins appeared to recognize the same epitopes of the recombinant Gam56 and Gam82, suggesting the epitope sites had been maintained (83). Moreover, immunization of chickens with these recombinant proteins Protein kinase N1 induced a strong antibody response, and sera from these birds recognized the native proteins (83), further indicating that these recombinant proteins mimicked the antibody response elicited by immunization with the native antigens. In Australia, despite the work by our own group, only live vaccines such as EIMERIAVAX 4m, the first Australian produced vaccine, are currently included in the program for coccidiosis control in the poultry industry. There remains a heavy reliance on anticoccidial chemotherapeutics, predominantly ionophore drugs. With increasing drug resistance and the negativity associated with drug residues in poultry meat and eggs, the dependency on chemotherapeutics will inevitably be altered in the near future and, thus, the prospect of vaccines becoming the future of coccidiosis control is likely.

Although numerous studies have described that CT [17, 43, 44] and the heat-labile toxin of E. coli (LT) [45] are potent inducers of Th2-type immune responses to coadministered soluble protein antigens [27, 37, 46, 47], other studies have demonstrated the capacity of CT to augment CTL responses after intranasal immunization [48–50]. Similarly, a non-toxic mutant of LT was found to enhance Th1 responses AZD6244 concentration to coadministered antigens [41]. Therefore, it is likely that CT and LT can enhance the immune responses in both Th1 and Th2-like manners. Considering this, it has been suggested that targeting of the toxins to different immunological sites, their

binding to distinct receptors or their activation/inhibition of distinct G proteins, and the dose administered may all influence the adjuvant effect for Th1 and Th2 cells [41]. We speculate that it might also be possible to shift a mixed Th1/Th2 response to the

predominantly Th2 nasal response elicited with Cry1Ac protoxin by modifying either the dose, route or even by using Cry1A toxins instead of protoxins or by modifying some motif within the protein. Indeed, we have previously attained mixed Th1/Th2 serum antibody responses following immunization with various Cry1A toxins. Moreover, we observed that an eight hydrophobic amino acid motif substitution in Domain I of Cry1A toxins is able to modulate the ratio of IgG subclasses, IgG1/IgG2a induced in serum [51]. Although further studies are still required to elucidate the precise mechanisms

by which Cry1Ac protoxin exerts its immunomodulatory effects, the results presented here contribute to explaining Opaganib chemical structure the high immunogenicity of this protein via the i.n. route. In addition, our data suggest that this protein can be used as a tool to better characterize the compartmentalization of nasal immune responses. The study was funded STK38 by the following grants: CONACyT 43102-M, and 080920; UNAM DGAPA PAPIIT IN221807, PAPIME PE203607 and PAPCA 2009-2010 (project 14). “
“Natural killer T (NK T) cells play a central role as intermediates between innate and adaptive immune responses important to induce anti-tumour reactivity in cancer patients. In two of 14 renal cell carcinoma (RCC) patients, treated with interferon (IFN)-α, we detected significantly enhanced numbers of circulating NK T cells which were typed phenotypically and analysed for anti-tumour reactivity. These NK T cells were T cell receptor (TCR) Vα24/Vβ11+, 6B11+ and bound CD1d tetramers. No correlation was observed between NK T frequencies and regulatory T cells (Tregs), which were also enhanced. NK T cells expressed CD56, CD161, CD45RO and CD69 and were predominantly CD8+, in contrast to the circulating T cell pool that contained both CD4+ and CD8+ T cells, as is found in healthy individuals. It is unlikely that IFN-α triggered the high NK T frequency, as all other patients expressed low to normal NK T numbers.

Methods: Thoracic aortas removed from 10-week-old male Sprague-Dawley rats were cut into 3- to 5-mm rings and were cultured for 10 days. Phosphate concentration was titrated in the medium to induce vascular calcification. Ferric citrate was applied as an iron donor with different concentrations. To study the preventive effects, 0.1 mM iron was introduced since 2 days before, on the same time and on the 3th or 6th day in

high phosphate treated aorta until 10th day. Calcification was assessed by Alizarin red staining and the calcium content of aorta was determined by the o-cresolphthalein complex-one method. Results: Vascular calcification was observed in rat aortas which were cultured in a high-phosphate medium. Calcium deposition was dramatically decreased by co-incubation with elevated PFT�� manufacturer iron (0.1 mM) compared with normal iron in medium (2.00 ± 2.32 vs 40.73 ± 17.25 mg/g, p < 0.01). Calcification buy PF-6463922 was significantly prevented if high iron level was introduced before (0.53 ± 0.39 mg/g, p < 0.01) or on the same time (7.38 ± 8.62 mg/g, p < 0.05) when high phosphate level was achieved. The inhibitory effect of iron was not significant after 3 or 6 days exposure to high phosphate concentration. Conclusions: Iron significantly reduced and prevented high phosphate-induced calcification in rat aorta. The inhibitory effect was no longer exit if aortic calcification

had already established. The mechanism(s) for the effects of iron on vascular calcification needed to be explored. FUJII HIDEKI, NAKAI KENTARO, GOTO SHUNSUKE, NISHI SHINICHI Division of Nephrology and Kidney Center, Kobe University Graduate School of Medicine Introduction: Clinical features at hemodialysis initiation affect the prognosis during the subsequent dialysis period, while they were not fully elucidated in very elderly patients. The purpose of this study was to clarify clinical features associated with chronic kidney disease- mineral bone disorder (CKD-MBD) and cardiovascular disease (CVD) at hemodialysis initiation in these

patients. Methods: Twenty consecutive elderly patients with end stage renal disease Glutamate dehydrogenase (ESRD) (≧80 years; VE group) and 35 consecutive control patients with ESRD Results: Diastolic blood pressure and pulse pressure were significantly higher in the VE group than in the control group. Though cardiac function was comparable between the two groups, left ventricular mass index tended to be greater in the control group. Though serum creatinine levels were significantly lower in the VE group, estimated glomerular filtration rate was comparable between the two groups. In addition, despite lower serum phosphate levels and calcium-phosphate products, TAC, AVC and MAC were more severe in the VE group compared to the control group. In the VE group, 12 patients had been followed up by nephrologists (F group) and 8 had not (NF group).

In our previous study 15 we went on to demonstrate for the first time that the net increase in Treg-cell-mediated suppressor potential in asymptomatic HIV+ subjects was due to increased sensitivity of effector cells to be suppressed, rather than an increase in the potency of their Treg cells to mediate suppression, emphasising the importance of assessing Treg-cell function in the context of both the Treg and effector cell simultaneously. This study extends these observations and probes Treg cell quality in HIV+ progressors prior to and after Highly

Active selleck compound Anti-Retroviral Therapy (HAART) initiation. In addition to impacting quality, HIV infection is known to alter Treg cell quantity. Several studies, including ours, report a decline in absolute Treg-cell number

in chronic HIV infection 8, 11, 15. Some studies show Treg-cell frequency to be elevated in HIV infection 16, 17, but this discrepancy may reflect CD4+ T-cell count disparity in HIV+ subjects. A systematic longitudinal analysis of Treg-cell absolute number in HIV+ progressors prior to and after HAART initiation is therefore warranted. Furthermore, the importance of examining Treg-cell quantity in the context of the Treg-cell Metabolism inhibitor counter-regulatory cytokine, IL-17 18, 19, is increasingly being recognised. Studies in nonhuman primate models of lentiviral infection and in HIV-infected human

individuals highlight pathogenic infection to be associated with loss of Th17 cells 19–23. IL-17 serves to maintain the integrity of the mucosal barrier. Loss of Th17 cells may permit microbial translocation across the gastrointestinal mucosa and thereby promote immune activation driven by bacterial lipopolyscaacharide, which is associated with disease progression 20, 24, 25. In this manuscript we provide novel insight into both qualitative and quantitative aspects of Treg cells in chronic HIV infection. We demonstrate that increased sensitivity of effector cells to Treg-cell mediated suppression is a feature of asymptomatic HIV-1 infected patients, but not patients who have progressed onto therapy; BIBF-1120 that this function is not inextricably linked to reduced expression of the counter-regulatory IL-17 cytokine and that reduced Treg and IL-17 numbers is a feature of chronic HIV infection that is not restored by up to 12 months of antiviral therapy. Assessing Treg-cell function is contingent on robust proliferation and cytokine expression by effector cells following TCR ligation. This function is known to be compromised in HIV-1-infected individuals 26, 27. Longitudinal analysis of effector cell proliferative capacity from chronically HIV-1-infected progressor patients prior to the initiation of HAART (Prog.

Similar to the Helicobacter model, IL-23 was responsible for inducing IL-17 production and colon-specific RGFP966 nmr tissue inflammation, and depletion of the Sca-1+ ILCs prevented development of colitis [3]. The idea that IL-17 production by ILCs can contribute to autoimmune disease has also been explored in humans. IL-17-producing cells are increased in the intestine of patients with ulcerative colitis and Crohn’s disease [8]. CD3− cells contributed significantly to the production of IL-17, both IL-17a and IL-17f mRNA

transcripts were increased in CD3− cells isolated from the intestines of patients with IBD as compared with transcripts in healthy controls [8]. In addition, there is an increased frequency of ILCs in the colon and ileum of patients Enzalutamide purchase with Crohn’s disease but not ulcerative

colitis [8]. However, since the absolute numbers of IL-17-producing ILCs in the inflamed intestine are very small, it is still unclear whether these cells play a direct role in driving IBD. Therefore, further studies are needed to determine their exact role. There have been a small number of reports showing that NK cells produce IL-17. Since human NKR-LTi cells have been shown to secrete IL-17 [82], careful analysis and interpretation of the results are essential to avoid confusion between IL-17 production by NKR-LTi cells and that by classical NK cells. In the steady state, NK cells in the spleen do not express RORγt [5]; however, upon infection with Toxoplasma gondii, splenic NK cells have enhanced

RORγt expression and secrete IL-17 [4]. A recent report has also shown that CD56+CCR4+ human peripheral blood NK cells produce both IL-17 and IFN-γ and express the transcription factors RORγt and Tbet [98]. These cells are not NKR-LTi cells, since the NK cells in this study did not express IL-7R (CD127), nor IL-23R, and since NKR-LTi cells are not thought to exist in human peripheral blood [82, 89]. iNKT cells are a subset of T cells that express a semi-invariant TCR that recognizes glycolipids presented by CD1d molecules expressed on APCs. There have been a number of recent reports demonstrating that iNKT selleck compound cells play a role in host protection against infection via the production of IL-17. Expression of RORγt in developing iNKT precursor cells is associated with the development of a preprogrammed IL-17-producing subset that does not express NK1.1 [99]. The signals that induce RORγt expression in iNKT precursors and lineage commitment have not yet been defined. These NK1.1− iNKT cells are capable of secreting IL-17 not only in response to stimulation with the synthetic ligand α-galactosylceramide or its analogue PBS-57, but also following stimulation with natural ligands, including LPS or glycolipids derived from Sphingomonas wittichii and Borrelia burgdorferi [100]. This IL-17-producing NK1.1− subset is present at high frequency in the lung, comprising up to 40% of pulmonary iNKT cells in naïve mice.

8 From a conceptual standpoint, these side-effects might be anticipated because many cytokines function physiologically in a paracrine fashion, over short distances between cells.6 An important challenge check details is to develop methods to deliver cytokines to tumour sites where they might enhance immune responses without producing undesirable systemic effects. Experimentally this has been achieved in a variety of ways including direct local injections of cytokines,10–12 injection of tumours with viruses encoding cytokine genes,13–15 or by transplanting genetically modified viable tumours into animals.16,17

These approaches have greatly contributed to our understanding of the effects of the local production of cytokines on the number and function of immune cells within the tumour microenvironment and also illustrated the considerable potential of cytokines to enhance anti-tumour immune responses. However, because metastatic lesions are often numerous and not easily accessible, translating these advances into a clinical setting remains a challenge. Hence, there remains a critical

need to develop ways in which the cytokine milieu in the tumour microenvironment can be altered. In our current work, Y-27632 supplier we set out to develop a general strategy to construct cytokines that are biologically inactive but could be activated by proteases. Ultimately this approach could be used to deliver inactive cytokines systemically but have them activated locally by tumour-site-expressed proteases. In principle, this should reduce systemic side-effects but retain the enhancement of anti-tumour immune responses. The strategy we are developing uses a fusion protein approach that takes advantage of proteases that are secreted by tumours. As an initial test of this general strategy, we have used different proteases, prostate-specific Baf-A1 antigen (PSA), matrix metalloproteinase 2 (MMP2) or MMP9. The expression of the protease PSA is highly restricted to prostate epithelial cells; PSA is produced by prostate tumours, and as such, is an excellent target protease

for activating the cytokine fusion protein.18 The MMPs have been known to have critical and varied roles in tumour development and progression and are preferentially expressed in a variety of tumours.19 We have used IL-2 as the test cytokine in the fusion protein because it is a potent factor for T-cell and natural killer (NK) cell development20,21 and the local production of IL-2 within tumours has demonstrated anti-tumour immunological effects in animal models.16,17 Moreover, an IL-2-containing fusion protein might be able to be more easily translated to the treatment of human cancers because IL-2 is already Food and Drug Administration approved for the treatment of certain tumours.7–9 In this report, we examine several strategies of blocking the biological activity of IL-2, yet allowing it to be functionally activated by PSA or MMP proteases.

Moreover, while TREG cells from either Lgals3−/− or WT mice suppressed IFN-γ and IL-4 production by CD4+CD25− T cells PI3K Inhibitor Library (TEFF), inhibition of cytokine production was much more pronounced when TEFF cells were co-cultured with Lgals3−/− TREG cells (Fig. 3C and D). Because the immunosuppressive activity of TREG cells is in part mediated by IL-10 and TGF-β, we examined production of these cytokines in draining LNs from WT- and Lgals3−/−-infected mice. Nonpurified LN cells (Fig. 3E) or purified TREG cells (Fig. 3F) from L. major infected Lgals3−/− mice

restimulated ex vivo with L. major antigen showing enhanced IL-10 mRNA expression as compared with cells obtained from WT mice. Furthermore, increased amounts of TGF-β transcripts were also detected in purified TREG cells from Lgals3−/− compared with WT mice (Fig. 3G). Thus, endogenous

galectin-3 not only controls TREG-cell frequency Opaganib purchase in LN and infection sites, but also limits the immunosuppressive function of these cells during the course of parasitic protozoa infection. To better characterize TREG cells from Lgals3−/− mice, we next evaluated the expression of CD25, CTLA4, CD103, and CD62L in CD4+Foxp3+ T cells from uninfected WT and Lgals3−/− mice. Despite the higher percentage of CD4+Foxp3+CD25+ TREG cells found in uninfected Lgals3−/− mice, the expression of CD62L, CD103, and CTLA4 did not differ significantly between WT and Lgals3−/− animals (Fig. 4A). However, in vitro stimulated TREG cells purified from Lgals3−/− mice synthesized considerably higher

amounts of IL-10 compared with in vitro stimulated WT TREG cells (Fig. 4B). Thus, endogenous galectin-3 controls IL-10 production by TREG cells either in the absence or presence of L. major infection. Previous studies showed that TREG cells preferentially express the Notch ligand Jagged-1, which confers an immunosuppressive phenotype to these cells [19-21]. We check analyzed expression of Jagged-1 on TREG and TEFF cells purified from uninfected WT and Lgals3−/− mice. Remarkably, TREG cells from Lgals3−/− mice showed higher Jagged-1 expression even in the absence of stimulation when compared with WT TREG cells (mean fluorescence intensity 139.50 ± 3.21 versus 96.68 ± 0.84, respectively; Fig. 5A). In contrast, TEFF from Lgals3−/− mice display higher Jagged-1 expression only after in vitro stimulation, in comparison with TEFF cells isolated from WT mice (mean fluorescence intensity 115.48 ± 4.87 versus 81.31 ± 2.05, respectively; Fig. 5A). It has been reported that Notch signaling plays an important role during development, expansion, and function of both TEFF and TREG cells [22]. We analyzed the expression of Notch receptors on TEFF and TREG cells isolated from uninfected WT and Lgals3−/− mice. We found that resting TEFF cells from Lgals3−/− mice displayed enhanced expression of Notch-1, Notch-3, and the Notch target gene Hes-1 (Fig. 5B).

Generally, IgG is infused via the intravenous (IVIG) or subcutaneous (SCIG) route. For IVIG infusion, published data demonstrate that higher IgG doses and trough levels

provide patients with improved protection from infection. The same conclusions are not yet accepted for SCIG; data from two recent Phase III studies and a recent post-hoc analysis, however, suggest the same correlation between higher SCIG dose and serum IgG concentration and decreased incidence of infection seen with IVIG. Other measures learn more of clinical efficacy have not been considered similarly. Thus, combined analyses of these and other published SCIG studies were performed; a full comparison of the 13 studies was, however, limited by non-standardized definitions SB203580 in vitro and reporting. Despite these limitations, our analyses

indicate that certain clinical outcomes improve at higher SCIG doses and associated higher serum IgG concentrations, and suggest that there might be opportunity to improve patient outcomes via SCIG dose adjustment. “
“Klebsiella pneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is becoming increasingly multidrug resistant. However, the underlying molecular pathogenesis of this bacterium remains elusive, limiting the therapeutic options. Understanding the mechanism of its pathogenesis may facilitate the development of anti-bacterial therapeutics. Here, we show that Lyn, a pleiotropic Src tyrosine kinase, is involved in host defense against Kp by regulating phagocytosis process and simultaneously Morin Hydrate downregulating inflammatory responses. Using acute infection mouse models, we observed that lyn−/− mice were more susceptible to Kp with increased mortality and severe lung injury compared with WT mice. Kp infected-lyn−/− mice exhibited elevated inflammatory cytokines (IL-6 and TNF-α), and increased superoxide in the lung and other organs. In addition, the phosphorylation of p38 and NF-κB p65 subunit increased markedly in response to Kp infection in lyn−/− mice. We also demonstrated

that the translocation of p65 from cytoplasm to nuclei increased in cultured murine lung epithelial cells by Lyn siRNA knockdown. Furthermore, lipid rafts clustered with activated Lyn and accumulated in the site of Kp invasion. Taken together, these findings revealed that Lyn may participate in host defense against Kp infection through the negative modulation of inflammatory cytokines. “
“Accumulating evidence suggests that Th17 cells and Tregs may exhibit development plasticity and that CD4+ Tregs can differentiate into IL-17-producing T cells; however, whether Th17 cells can reciprocally convert into Tregs has not been described. In this study, we generated Th17 clones from tumor-infiltrating T lymphocytes (TILs).

Thus the blockade in differentiation of maturing T and B

cells in the Snai3-expressing HSC occurs between the c-Kit+Sca− stage and the more mature common lymphoid progenitor population. The data presented in this report indicate that the expression of Snai3 in bone GDC-0973 nmr marrow progenitors alters neither the maintenance of the stem cells nor the early stages of stem-cell differentiation but does dramatically skew the production of cells committed to the lymphoid or myeloid lineages. The Snai3 protein could alter these maturation profiles either through the repressor function of the SNAG domain of the protein, or by competing with endogenous transcriptional regulators for binding to E box sites. The identification of genes whose expression is influenced by the presence of Snai3 in these precursor populations may provide key insight into the regulation of differentiation of myeloid- and lymphoid-precursor cells. Animals were housed in the Animal Resource Center (University of Utah Health Science Center, Salt Lake City, UT) according to the guidelines of the National Institutes PS-341 chemical structure of Health. C57BL/6 and B6.SJL-Ptprc Pepc/BoyJ were obtained from The Jackson Laboratories. C57BL/6CrSlc-Tg(ACTb-EGFP)OsbC14-Y01-FM131 mice ubiquitously expressing GFP were utilized [[27]]. The pBMN-1-GFP retrovirus was obtained from Addgene (plasmid 1736). The coding sequence of Snai3

(base pair (bp) 79–942 of NM_013914.2) was cloned into the Bam HI and XhoI sites of the vector. The Snai3 encoding cDNA was obtained by RT-PCR amplification of mouse thymus cDNA using 5′-CGGATCCATGCCGCGCTCCTTCCTGGTGA and 5′-GCTCGAGCTAGGGGCCAGGACAGCAGC oligonucleotides. PCR amplification was performed using Platinum pfx (Invitrogen, Grand Island, NY, USA). PCR amplification was 55°C annealing

http://www.selleck.co.jp/products/BafilomycinA1.html (30 s), 68°C extension (2 min) and 95°C denaturing (30 s) for 40 cycles. After subcloning the sequence was confirmed to match that of the reference sequence of NM_013914.2. Plat E cells were grown in stem cell media (SCM): Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 15% FCS, P/S, 1 μg/mL puromycin (Sigma, St. Louis, MO, USA), and 10 μg/mL blasticidin (Sigma) except during virus production when antibiotics were subtracted [[28]]. Retroviral vectors were transfected into the Plat E packaging cell line using Fugene HD reagent (Roche, Pleasanton, CA, USA) at a 6:1 ratio. Cells were incubated at 37°C for 24 h then switched to 32°C for virus production in fresh media. Supernatant was collected and filtered through a 0.45 μm filter prior to use in transduction. B6.SJL were injected with 300 μL 10 mg/mL 5′fluorouracil (Sigma) in PBS [[29]]. Four days later their BM was collected and cultured with RBC in SCM with 100 ng/mL SCF (Sigma), 20 ng/mL IL-6 (Sigma), and 10 ng/mL IL-3 (Sigma) at 5–6 × 106 cell/mL for 2 days at 37°C. Stem cell cultures were collected and red blood cell (RBC) lysed with ammonium chloride potassium (ACK). Remaining cells were resuspended in 7.

As shown in Fig. 3A, the expression levels of FOXP3 and IFN-γ in expanded E3-Th17 cells were not significantly altered even after culture for 9 days. However, the number of IL-17-producing cells significantly decreased during the culture, from above 60% to approximately 40%. Recent studies have shown that the stable expression of FOXP3 in

naturally occurring Tregs involves epigenetic regulations, including DNA methylation and histone modification 41, 43. Furthermore, these studies demonstrated that human FOXP3 contains several highly conserved demethylation regions that are exclusive for Tregs. Thus, we next investigated whether expanded Th17 cells expressing FOXP3 exhibited FOXP3 DNA demethylation. We designed the human FOXP3 methylation-specific primers based on the Treg-specific demethylated region (TSDR) within the OSI-906 mouse FOXP3 CpG island 43–45, and then compared the FOXP3 methylation levels in expanded Th17 cells, CD4+CD25+ naturally occurring Tregs and OKT3-activated naïve CD4+ T cells. As expected, the TSDR within FOXP3 of CD4+CD25+ Tregs was almost completely demethylated

compared with that of CD4+CD25– T cells (Fig. 3B). In contrast to CD4+CD25+ Tregs, FOXP3 methylation levels of two OKT3-activated naïve T cells were similar to levels in CD4+CD25– T cells (100% methylation), although approximately 15% of these activated cells expressed FOXP3. However, Th17 clones derived from different rounds of expansion displayed partial methylation in GSI-IX manufacturer TSDR within FOXP3, and this decreased significantly with increasing stimulation and expansion cycles. In addition, demethylation

levels of FOXP3 in Th17 clones at different expansion cycles were correlated positively with FOXP3 expression (Fig. 3B). These results indicate that epigenetic modification of FOXP3 occurred in Th17 cells following multiple cycles of in vitro TCR stimulation, resulting in increased Interleukin-3 receptor and stable expression of FOXP3 in expanded Th17 cells. It is well known that TCR–ligand interactions are critical for T-cell lineage commitment, including FOXP3 induction and Treg lineage differentiation 3, 16. Given that Th17 clones differentiate into IFN-γ-producing and FOXP3+ T cells after in vitro expansion, we next investigated whether TCR stimulation is the primary determinant for this process. E1-Th17 clones were expanded in vitro with allogeneic PBMCs in the presence or absence of OKT3 and then evaluated for the IL-17, IFN-γ, and FOXP3 expression. As shown in Fig. 4A, the proportions of IL-17-producing cell populations in Th17 clones were significantly decreased after in vitro expansion, regardless of whether the system included OKT3 or not. Notably, the Th17 clones contained higher percentages of IL-17-producing cells when cultures included both PBMCs and OKT3 than those in the absence of OKT3.