Stimulatory Conditions Research Articles

Mucosa of human detrusor impairs contraction and β‐adrenoceptor‐mediated relaxation

To elucidate the impact of the mucosa on detrusor muscle function by investigating force of contraction under various stimulatory conditions and during subsequent relaxation using catecholamines. Detrusor tissue was obtained from patients who had undergone cystectomy for bladder cancer and strips of intact or mucosa-denuded muscle were set up for force measurement. Preparations were precontracted with KCl, carbachol or electric-field stimulation (EFS). Precontracted strips were relaxed using increasing concentrations of catecholamines in the absence and presence of the subtype-selective β-adrenoceptor (AR) blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L-748,337 (β3-ARs). Force development in response to KCl (40 mM), carbachol (1 μM) or EFS was larger in the absence of mucosa than in intact muscle strips. The force of contraction of mucosa-denuded strips with detached urothelium incubated in the same chamber was as low as in intact strips. Noradrenaline relaxed precontracted detrusor strips to a significantly larger extent and at lower concentrations in denuded than in intact strips. CGP 20712A did not affect noradrenaline-induced relaxation of denuded and intact strips, and ICI 118,551 did not influence noradrenaline-induced relaxation in denuded strips, but abolished the difference between denuded and intact strips. The antagonism of the relaxant effects of noradrenaline by L-748,337 was slightly smaller in intact than denuded strips. The mucosa of human detrusor strips impairs force development when stimulated with KCl, carbachol or EFS. The mucosa also blunts the relaxing effects of catecholamines. The latter effect does not involve the activation of β1-ARs but only of β2-ARs, whereas β3-ARs mediate the relaxation of human detrusor.

Macrophage M1/M2 Polarization Dynamically Adapts to Changes in Cytokine Microenvironments in Cryptococcus neoformans Infection

ABSTRACTThe outcome of cryptococcal pneumonia correlates with local macrophage polarization status, as M1 and M2 polarization marks protective and nonprotective responses, respectively. Overall, pulmonary macrophage polarization status changes over time during a cryptococcal infection. This could have been caused by repolarization of individual macrophages or by a replacement of M2-polarized cells by new M1-polarized cells. To explore the ability of macrophages to change between polarization states, we conducted a series of experiments using in vitro macrophages. Coculture of macrophages with Cryptococcus neoformans resulted in development of a weak M1-like phenotype, with modestly increased inducible nitric oxide synthase (iNOS) but lacking interleukin 6 (IL-6) induction. The C. neoformans-induced M1-like polarization state was plastic, as macrophages stimulated first with C. neoformans and then with gamma interferon (IFN-γ) or IL-4 expressed mRNA polarization patterns similar to those stimulated with cytokines alone. To further evaluate macrophage polarization plasticity, cytokine stimulatory conditions were established which fully polarized macrophages. IFN-γ and IL-4 stimulation differentially induced complete M1 and M2 polarization, defined by differential expression of marker mRNA panels, surface marker expression, and tumor necrosis factor alpha (TNF-α) protein production. Switching IFN-γ- to IL-4-stimulating conditions, and vice versa, resulted in uniform changes in profiles of polarization marker genes consistent with the most recent cytokine environment. Furthermore, the ability of sequentially stimulated macrophages to inhibit C. neoformans reflected the most recent polarizing condition, independent of previous polarization. Collectively, these data indicate that M1/M2 macrophage polarization phenotypes are highly plastic to external signals, and interventions which therapeutically repolarize macrophages could be beneficial for treatment of cryptococcosis.

The Ex Vivo Induction of Human CD103+CD25hi Foxp3+CD4+ and CD8+ Tregs is IL‐2 and TGF‐β1 Dependent

The expression of the integrin αE (CD103), may enhance the retention of regulatory T cells to peripheral inflammatory sites and possibly contribute to their suppressive potential. The aim of this study was to define the regulatory role of IL-2 and TGF-β1 on the CD103 expression and the optimal in vitro conditions for the induction/expansion of human CD4(+) and CD8(+) Tregs. Cord blood mononuclear cells (CBMC) were stimulated under various culture conditions, including anti-CD3, anti-CD28, IL-2 and TGF-β1. TGF-β1 and IL-2 were both required for optimal expression of CD103. In addition, TGF-β1 and IL-2 synergistically induced CD103 expression on CD8(+) T cells, whereas, only additive induced expression was noted on CD4(+) T cells. Surprisingly, CD103 expression was not dependent upon CD28 costimulation. IL-2 also played a central role in CD103 expression by CD25(hi) Foxp3+ Tregs. IL-2, TGF-β1 and anti-CD3 defined the optimal stimulatory conditions favouring the induction/expansion of both CD4(+) and CD8(+) human Tregs from naive CBMC. Thus, this study provides new insights into the regulatory role of IL-2 upon CD103 expression by human cord blood CD4(+) and CD8(+) T cells. Furthermore, it identifies the in vitro culture conditions driving the differentiation of the novel phenotype CD4(+) and CD8(+) CD103(+) CD25(hi) Foxp3+ Tregs from human CBMC.

Afferent lymphatic cannulation as a model system to study innate immune responses to infection and vaccination

The afferent lymphatics consist of the cells and immunomodulatory signals that are involved in the early response to peripheral stimuli. Examination of this compartment in both homeostatic and stimulatory conditions permits the analysis of the innate biological pathways responsible for the generation of an adaptive immune response in the lymph node. Afferent lymphatic cannulation is therefore an ideal model system to study cellular migration and antigen dispersal kinetics during infection and vaccination. Utilisation of these lymphatic cannulation models has demonstrated the ability to both increase current understanding of infectious diseases, vaccine delivery systems and has the potential to target effector cells and molecules that may be used as novel therapeutic or vaccine targets.

Striatal cholinergic interneurons display activity-related phosphorylation of ribosomal protein S6.

Cholinergic interneurons (CINs) provide the main source of acetylcholine to all striatal regions, and strongly modulate dopaminergic actions through complex regulation of pre- and post-synaptic acetylcholine receptors. Although striatal CINs have a well-defined electrophysiological profile, their biochemical properties are poorly understood, likely due to their low proportion within the striatum (2–3%). We report a strong and sustained phosphorylation of ribosomal protein S6 on its serine 240 and 244 residues (p-Ser240–244-S6rp), a protein integrant of the ribosomal machinery related to the mammalian target of the rapamycin complex 1 (mTORC1) pathway, which we found to be principally expressed in striatal CINs in basal conditions. We explored the functional relevance of this cellular event by pharmacologically inducing various sustained physiological activity states in CINs and assessing the effect on the levels of S6rp phosphorylation. Cell-attached electrophysiological recordings from CINs in a striatal slice preparation showed an inhibitory effect of tetrodotoxin (TTX) on action potential firing paralleled by a decrease in the p-Ser240–244-S6rp signal as detected by immunofluorescence after prolonged incubation. On the other hand, elevation in extracellular potassium concentration and the addition of apamin generated an increased firing rate and a burst-firing activity in CINs, respectively, and both stimulatory conditions significantly increased Ser240–244-S6rp phosphorylation above basal levels when incubated for one hour. Apamin generated a particularly large increase in phosphorylation that was sensitive to rapamycin. Taken together, our results demonstrate for the first time a link between the state of neuronal activity and a biochemical signaling event in striatal CINs, and suggest that immunofluorescence can be used to estimate the cellular activity of CINs under different pharmacological and/or behavioral conditions.

Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines

To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.

T‐helper 17 cells: The driving force of psoriasis and psoriatic arthritis

There is growing evidence that two recently recognized, unique subsets of CD4(+) T-cells, T-helper 17 cells (Th17) and CD4(+) CD25(+) regulatory T-cells (Treg), may play important roles in the pathogenesis of psoriasis. This study sought to investigate the relationship between Th17 cells and psoriasis or psoriatic arthritis (PsA). Peripheral blood CD4(+) T-cells were isolated from 47 patients with psoriasis and 47 controls, and were cultured under various stimulatory conditions. The Th17 cells and regulatory Treg cells were detected by flow cytometry. Plasma interleukin (IL)-17 was measured by enzyme-linked immunosorbent assay. The proportion of Th17 and Treg cells from peripheral blood mononuclear cells showed no difference between patients with psoriasis and controls. However, psoriasis and PsA patients demonstrated a higher proportion of induced Th17 cells, which was correlated with psoriasis area severity index score. The level of plasma IL-17A in psoriasis was higher than that in controls. Th17 cells may play important roles in the pathogenesis of psoriasis and psoriatic arthritis.

High glucose concentration impairs ATP outflow and immunoglobulin production by human peripheral B lymphocytes: Involvement of P2X7 receptor

Aims/hypothesisPatients with diabetes are more prone to bacterial infections mostly due to hyperglycemia-induced suppression of immune cells function. B lymphocytes by secreting antibodies inhibit microbial replication, but the impact of high glucose concentration on humoral immune response is not fully resolved. The aim of this work was to investigate the effect of high glucose concentration on B cells response to stimulation with a bacterial antigen and autocrine regulation. MethodsPurified human peripheral blood B cells were cultured at different glucose concentrations and stimulated in vitro with Staphylococus aureus Cowan I (SAC) plus IL-2. B cells proliferation, differentiation and IgM expression were analyzed by flow cytometry. B cell ATP release and involvement of P2 purinergic receptors in regulation of IgM secretion was assessed. ResultsB cells cultured at 25mM glucose in response to SAC stimulation released significantly less (∼55%) IgM comparing to cells maintained in 5mM glucose. Under resting and stimulatory conditions B cells released significant quantities of ATP to the culture media, but ATP level decreased when B cells were maintain in high glucose. SAC-induced B cell IgM release was totally blocked by highly selective antagonist (Az11645373) of P2X7 receptor. IgM secretion increased in the presence of potent P2X7 receptor agonist (BzATP), but this effect was abolished by high glucose concentration. Conclusions/interpretationHigh glucose concentration impairs B cell function by suppression of P2X7 receptor-dependent IgM release in response to in vitro bacterial antigen stimulation. This alteration may greatly contribute to the impaired humoral immune response in diabetics.

TCR‐induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56lck residues

Biochemical studies have demonstrated that phosphorylation of lymphocyte cell kinase (p56(lck) ) is crucial for activation of signaling cascades following T cell receptor (TCR) stimulation. However, whether phosphorylation/dephosphorylation of the activating or inhibitory tyrosine residues occurs upon activation is controversial. Recent advances in intracellular staining of phospho-epitopes and cytometric analysis, requiring few cells, have opened up novel avenues for the field of immunological signaling. Here, we assessed p56(lck) phosphorylation, using a multiparameter flow-cytometric based detection method following T cell stimulation. Fixation and permeabilization in conjunction with zenon labeling technology and/or fluorescently labeled antibodies against total p56(lck) or cognate phospho-tyrosine (pY) residues or surface receptors were used for detection purposes. Our observations showed that activation of Jurkat or primary human T cells using H(2) O(2) or TCR-induced stimulation led to simultaneous phosphorylation of the activating tyrosine residue, Y394 and the inhibitory tyrosine residue, Y505 of p56(lck) . This was followed by downstream calcium flux and expression of T cell activation markers; CD69 and CD40 ligand (CD40L). However, the extent of measurable activation readouts depended on the optimal stimulatory conditions (temperature and/or stimuli combinations). Treatment of cells with a p56(lck) -specific inhibitor, PP2, abolished phosphorylation at either residue in a dose-dependent manner. Taken together, these observations show that TCR-induced stimulation of T cells led to simultaneous phosphorylation of p56(lck) residues. This implies that dephosphorylation of Y505 is not crucial for p56(lck) activity. Also, it is clear that cytometric analysis provides for a rapid, sensitive, and quantitative method to supplement biochemical studies on p56(lck) signaling pathways in T cells at single cell level.

Inflammasome-derived IL-1β regulates the production of GM-CSF by CD4+ T cells and γδ T cells (171.24)

Abstract Recent findings have demonstrated an indispensable role for granulocyte-macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). However the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1 signaling centrally regulates GM-CSF production during EAE. IL-1R1 is required to induce GM-CSF production following polyclonal T cell stimulation under both TH17 (i.e. IL-23) and TH1 (i.e. IL-12) stimulatory conditions. Furthermore, we demonstrate that γδ T cells are also potent producers of GM-CSF during the inflammatory response and that their secretion of GM-CSF is similarly dependent on IL-1 signaling. Interestingly, we find that the expression of GM-CSF by myelin-reactive T cells during EAE is not restricted to discrete TH1 and TH17 cell lineages. Rather GM-CSF can be co-expressed with both IFNγ and IL-17. Inflammasome-derived IL-1β was identified to be an upstream amplifier of GM-CSF producing T cells during the priming phase of EAE. Finally, we show a central role for MYD88 in the control of GM-CSF production by both CD4+ T cells and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1β and the IL-1R1/MYD88 signaling axis in the regulation of GM-CSF production.

Germinal centers

Germinal centers

Osteogenic stimulatory conditions enhance growth and maturation of endothelial cell microvascular networks in culture with mesenchymal stem cells

To optimize culture conditions for in vitro prevascularization of tissue-engineered bone constructs, the development of organotypic blood vessels under osteogenic stimulatory conditions (OM) was investigated. Coculture of endothelial cells and mesenchymal stem cells was used to assess proangiogenic effects of mesenchymal stem cells on endothelial cells. Four different culture conditions were evaluated for their effect on development of microvascular endothelial cell networks. Mineralization, deposition of extracellular matrix, and perivascular gene expression were studied in OM. After 3 days, endothelial cells established elongated capillary-like networks, and upregulated expression of vascular markers was seen. After 15 days, all parameters evaluated were significantly increased for cultures in OM. Mature networks developed in OM presented lumens enveloped by basement membrane-like collagen IV, with obvious mineralization and upregulated perivascular gene expression from mesenchymal stem cells. Our results suggest osteogenic stimulatory conditions to be appropriate for in vitro development of vascularized bone implants for tissue engineering.

Inflammasome-Derived IL-1β Regulates the Production of GM-CSF by CD4+ T Cells and γδ T Cells

Recent findings have demonstrated an indispensable role for GM-CSF in the pathogenesis of experimental autoimmune encephalomyelitis. However, the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1R is required for GM-CSF production after both TCR- and cytokine-induced stimulation of immune cells in vitro. Conventional αβ and γδ T cells were both identified to be potent producers of GM-CSF. Moreover, secretion of GM-CSF was dependent on IL-1R under both IL-12- and IL-23-induced stimulatory conditions. Deficiency in IL-1R conferred significant protection from experimental autoimmune encephalomyelitis, and this correlated with reduced production of GM-CSF and attenuated infiltration of inflammatory cells into the CNS. We also find that GM-CSF production in vivo is not restricted to a defined CD4(+) T cell lineage but is rather heterogeneously expressed in the effector CD4(+) T cell population. In addition, inflammasome-derived IL-1β upstream of IL-1R is a critical regulator of GM-CSF production by T cells during priming, and the adapter protein, MyD88, promotes GM-CSF production in both αβ and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1β and the IL-1R/MyD88 signaling axis in the regulation of GM-CSF production.

Methods of studying field germination and seedling physiology: present potential and drawbacks

Seed germination and seedling growth are equally important processes for population survival. Observation of germinating orchid seeds in a natural substrate is inhibited by their minute size. There are basically two approaches to studying germination in situ: Search for spontaneous seedlings, and field sowing experiments; these may be supplemented with experiments in vitro. Finds of spontaneous seedlings are clearly irregular and fortuitous but may yield valuable, if fragmentary, input to hypothesis building. An understanding of the habitat dynamic may be helpful in the search of such seedlings. The seed package technique for germination in situ and its various adaptations can yield considerable information concerning spatial distribution and timing, identity of participating fun­gi, seedling growth rate and stimulatory environmental conditions. This approach constitutes the backbone to studies of germination ecology and still holds much unexplored potential. However, conditions that are suitable for orchid seed germination in nature are not necessarily identical to those supporting adult plant growth. Experiments in vitro on artificial substrate normally employs either immature or strongly surface sterilized seeds, and thus the results can be misleading re­gar­ding the behavior of natural seeds and seedlings in the field. However, such experiments may be designed for added information on specific questions, provided that growth conditions are manipulated suitably. Experiments in vitro, especially symbiotic and on series of minimal media, have much to offer in sup­por­ting and extending field observations of substrate and site preferences.

Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5

Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.

Targeted Sos1 deletion reveals its critical role in early T-cell development

Activation of the small G protein Ras is required for thymocyte differentiation. In thymocytes, Ras is activated by the Ras guanine exchange factors (RasGEFs) Sos1, Sos2, and RasGRP1. We report the development of a floxed allele of sos1 to assess the role of Sos1 during thymocyte development. Sos1 was required for pre-T-cell receptor (pre-TCR)- but not TCR-stimulated developmental signals. Sos1 deletion led to a partial block at the DN-to-DP transition. Sos1-deficient thymocytes showed reduced pre-TCR-stimulated proliferation, differentiation, and ERK phosphorylation. In contrast, TCR-stimulated positive selection, and negative selection under strong stimulatory conditions, remained intact in Sos1-deficient mice. Comparison of RasGEF expression at different developmental stages showed that relative to Sos2 and RasGRP1, Sos1 is most abundant in DN thymocytes, but least abundant in DP thymocytes. These data reveal that Sos1 is uniquely positioned to affect signal transduction early in thymocyte development.

Bayesian hierarchical modeling for signaling pathway inference from single cell interventional data

Recent technological advances have made it possible to simultaneously measure multiple protein activities at the single cell level. With such data collected under different stimulatory or inhibitory conditions, it is possible to infer the causal relationships among proteins from single cell interventional data. In this article we propose a Bayesian hierarchical modeling framework to infer the signaling pathway based on the posterior distributions of parameters in the model. Under this framework, we consider network sparsity and model the existence of an association between two proteins both at the overall level across all experiments and at each individual experimental level. This allows us to infer the pairs of proteins that are associated with each other and their causal relationships. We also explicitly consider both intrinsic noise and measurement error. Markov chain Monte Carlo is implemented for statistical inference. We demonstrate that this hierarchical modeling can effectively pool information from different interventional experiments through simulation studies and real data analysis.

Differential Glucose-Regulation of MicroRNAs in Pancreatic Islets of Non-Obese Type 2 Diabetes Model Goto-Kakizaki Rat

BackgroundThe Goto-Kakizaki (GK) rat is a well-studied non-obese spontaneous type 2 diabetes (T2D) animal model characterized by impaired glucose-stimulated insulin secretion (GSIS) in the pancreatic beta cells. MicroRNAs (miRNAs) are short regulatory RNAs involved in many fundamental biological processes. We aim to identify miRNAs that are differentially-expressed in the pancreatic islets of the GK rats and investigate both their short- and long term glucose-dependence during glucose-stimulatory conditions.Methodology/Principal FindingsGlobal profiling of 348 miRNAs in the islets of GK rats and Wistar controls (females, 60 days, N = 6 for both sets) using locked nucleic acid (LNA)-based microarrays allowed for the clear separation of the two groups. Significant analysis of microarrays (SAM) identified 30 differentially-expressed miRNAs, 24 of which are predominantly upregulated in the GK rat islets. Monitoring of qPCR-validated miRNAs during GSIS experiments on isolated islets showed disparate expression trajectories between GK and controls indicating distinct short- and long-term glucose dependence. We specifically found expression of rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335 to be regulated by hyperglycaemia. The putative targets of upregulated miRNAs in the GK, filtered with glucose-regulated mRNAs, were found to be enriched for insulin-secretion genes known to be downregulated in T2D patients. Finally, the binding of rno-miR-335 to a fragment of the 3′UTR of one of known down-regulated exocytotic genes in GK islets, Stxbp1 was shown by luciferase assay.Conclusions/SignificanceThe perturbed miRNA network found in the GK rat islets is indicative of a system-wide impairment in the regulation of genes important for the normal functions of pancreatic islets, particularly in processes involving insulin secretion during glucose stimulatory conditions. Our findings suggest that the reduced insulin secretion observed in the GK rat may be partly due to upregulated miRNA expression leading to decreased production of key proteins of the insulin exocytotic machinery.

Modulatory role of Ser126 phosphorylation in the regulation of the renal Na‐K‐Cl cotransporter (NKCC2)

Phosphorylation of N-terminal threonine residues is required for NKCC2 activation. An additional residue, Ser126, lying closely downstream, was initially identified as being phosphorylated in vitro by AMP-activated kinase, and recently shown to be phosphorylated in vivo in a regulated fashion, likely mediated by PKA. To determine the relative importance of Ser126 phoosphorylation in NKCC2 regulation, we have used site-directed mutagenesis and heterologous expression in Xenopus oocytes. In hypotonic or isotonic solutions, wt NKCC2 exhibits roughly 50% of the maximal activity observed under stimulatory conditions (hypertonicity, With-No-lysine kinase isoform 3 (WNK3) coexpression). A Ser126A mutant exhibits markedly reduced activity under hypotonic conditions, but its activity under hypertonic conditions was increased compared to that of wild-type NKCC2. Stimulation by WNK3 or hypertonicity was not dependent on the presence of a serine at position 126. However, the phosphorylation level of the regulatory threonines was reduced in the Ser126A mutant, under any stimulatory condition, compared to that observed in the wild-type cotransporter. Thus, we conclude phosphorylation at residue Ser126 might be critical under conditions where the main threonines are not phosphorylated; it also modulates the phosphorylation of the main threonines by the regulatory pathways. MICINN BFU2007/62119

MiR-155, a Modulator of FOXO3a Protein Expression, Is Underexpressed and Cannot Be Upregulated by Stimulation of HOZOT, a Line of Multifunctional Treg

MicroRNAs (miRNAs) play important roles in regulating post-transcriptional gene repression in a variety of immunological processes. In particular, much attention has been focused on their roles in regulatory T (Treg) cells which are crucial for maintaining peripheral tolerance and controlling T cell responses. Recently, we established a novel type of human Treg cell line, termed HOZOT, multifunctional cells exhibiting a CD4+CD8+ phenotype. In this study, we performed miRNA profiling to identify signature miRNAs of HOZOT, and therein identified miR-155. Although miR-155 has also been characterized as a signature miRNA for FOXP3+ natural Treg (nTreg) cells, it was expressed quite differently in HOZOT cells. Under both stimulatory and non-stimulatory conditions, miR-155 expression remained at low levels in HOZOT, while its expression in nTreg and conventional T cells remarkably increased after stimulation. We next searched candidate target genes of miR-155 through bioinformatics, and identified FOXO3a, a negative regulator of Akt signaling, as a miR-155 target gene. Further studies by gain- and loss-of-function experiments supported a role for miR-155 in the regulation of FOXO3a protein expression in conventional T and HOZOT cells.