Inhibited IFN-gamma Production Research Articles

Mechanism of interferon-gamma production by monocytes stimulated with myeloperoxidase and neutrophil extracellular traps

Neutrophil extracellular traps (NETs) have an important role in antimicrobial innate immunity and release substances that may modulate the immune response. We investigated the effects of soluble factors from NETs and neutrophil granule proteins on human monocyte function by using the Transwell system to prevent cell-cell contact. NET formation was induced by exposing human neutrophils to phorbol myristate acetate (PMA). When monocytes were incubated with PMA alone, expression of interleukin (IL)-4, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha mRNA was upregulated, but IL-10, IL-12, and interferon (IFN)-gamma mRNA were not detected. Incubation of monocytes with NETs enhanced the expression of IL-10 and IFN-gamma mRNA, but not IL-12 mRNA. Myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. Both a nuclear factor-kappaB inhibitor (PDTC) and an intracellular calcium antagonist (TMB-8) prevented upregulation of IFN-gamma production. Neither a combined p38alpha and p38beta inhibitor (SB203580) nor an extracellular signal-regulated kinase inhibitor (PD98059) suppressed IFN-gamma production. Interestingly, a combined p38gamma and p38delta inhibitor (BIRB796) significantly decreased IFN-gamma production. These findings suggest that myeloperoxidase induces IFN-gamma production by monocytes via p38gamma/delta mitogen-activated protein kinase.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Utilize Activin-A to Suppress Interferon-γ Production by Natural Killer Cells.

Following allogeneic hematopoietic stem cell transplantation (HSCT), interferon (IFN)-γ levels in the recipient’s body can strongly influence the clinical outcome. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are lucrative as biological tolerance-inducers in HSCT settings. Hence, we studied the molecular mechanism of how UC-MSCs influence natural killer (NK) cell-mediated IFN-γ production. Allogeneic NK cells were cultured in direct contact with UC-MSCs or cell-free supernatants from mesenchymal stem cell (MSC) cultures (MSC-conditioned media). We found that soluble factors secreted by UC-MSCs strongly suppressed interleukin (IL)-12/IL-18-induced IFN-γ production by NK cells by reducing phosphorylation of STAT4, NF-κB, as well as T-bet activity. UC-MSCs secreted considerable amounts of activin-A, which could suppress IFN-γ production by NK cells. Neutralization of activin-A in MSC-conditioned media significantly abrogated their suppressive abilities. Till date, multiple groups have reported that prostaglandin (PG)-E2 produced by MSCs can suppress NK cell functions. Indeed, we found that inhibition of PGE2 production by MSCs could also significantly restore IFN-γ production. However, the effects of activin-A and PGE2 were not cumulative. To the best of our knowledge, we are first to report the role of activin-A in MSC-mediated suppression of IFN-γ production by NK cells.

Bisphenol A suppresses interferon-gamma production and related biochemical pathways in human PBMC

Event Abstract Back to Event Bisphenol A suppresses interferon-gamma production and related biochemical pathways in human PBMC Emanuel Raggl1, Simon Geisler1, Johanna M. Gostner1, Dietmar Fuchs1* and Harald Schennach1 1 Innsbruck Medical University, Austria Bisphenol A (BPA) is one of the most widely distributed chemicals. Humans are exposed to it mainly via polycarbonate plastics that are used for food packaging and plastic bottles and contain BPA as a softener. There is accumulating evidence of a carcinogenic and allergy-promoting effect of BPA. In vitro we investigated the impact of BPA on freshly isolated human peripheral blood mononuclear cells (PBMC) from healthy donors. PBMC were incubated with increasing doses of BPA, and after 30 min either left unstimulated or stimulated with the mitogen phytohemagglutinin (PHA) and cell viability was controlled. After 48 h, formation of interferon-gamma (IFN-gamma), macrophage product neopterin as well as tryptophan breakdown by indoleamine 2,3-dioxygenase (IDO), expressed as the kynurenine to tryptophan ratio (Kyn/Trp), were monitored. All these immunobiochemical pathways are characteristic for cell-mediated (= Th1-type) immune response. Upon PHA stimulation, neopterin production and tryptophan breakdown increased significantly compared to unstimulated cells. BPA exerted a dose-dependent suppressive effect in unstimulated and in PHA-stimulated PBMC at 12.5 - 200 µM concentrations. Due to the cross-regulation of Th1- and Th2-type immunity, the suppressive effects of BPA on Th1-type immunity could relate to the allergy-promoting properties of the compound. Moreover, the inhibition of IFN-gamma production and its related antiproliferative effects could be of relevance for the tumor-promoting effect of this chemical. On the other side, the antioxidant compound could also have beneficial effects, possibly delaying or even preventing Th1-type immunopathologies. References Zaknun D, Schroecksnadel S, Kurz K, Fuchs D. Potential role of antioxidant food supplements, preservatives and colorants in the pathogenesis of allergy and asthma. Int Arch Allergy Immunol 2012;157:113-124 Gostner JM, Becker K, Fuchs D, Sucher R. Redox regulation of the immune response. Redox Report 2013;18: 88-94 Gostner J, Ciardi C, Becker K, Fuchs D, Sucher R. Immunoregulatory impact of food antioxidants. Curr Pharm Des (in press) Keywords: bisphenol A, Interferon-gamma, Neopterin, indoleaminedioxygenase, PBMC Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Raggl E, Geisler S, Gostner JM, Fuchs D and Schennach H (2013). Bisphenol A suppresses interferon-gamma production and related biochemical pathways in human PBMC. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00665 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Dietmar Fuchs, Innsbruck Medical University, Innsbruck, Austria, dietmar.Fuchs@i-med.ac.at Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Emanuel Raggl Simon Geisler Johanna M Gostner Dietmar Fuchs Harald Schennach Google Emanuel Raggl Simon Geisler Johanna M Gostner Dietmar Fuchs Harald Schennach Google Scholar Emanuel Raggl Simon Geisler Johanna M Gostner Dietmar Fuchs Harald Schennach PubMed Emanuel Raggl Simon Geisler Johanna M Gostner Dietmar Fuchs Harald Schennach Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Functional distinction of rat liver natural killer cells from spleen natural killer cells under normal and acidic conditions in vitro

The microenvironment within solid tumors has often been shown to exhibit an acidic extracellular pH. Although the morphologic and functional differences in natural killer (NK) cells of the liver and spleen have been reported previously under physiological conditions, the difference under acidic conditions is still unclear. This study was to investigate the differences in the morphological and functional characteristics between rat liver and spleen NK cells under normal and acidic conditions in vitro. Liver and spleen NK cells were isolated and purified from Sprague-Dawley rats by density gradient centrifugation and the Dynabeads(®) FlowComp(TM) Flexi system, and stimulated for 4 days with or without IL-2 or treated with low pH or control for different times. Morphology was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cell death and proliferation assays were performed by flow cytometry, IFN-gamma production was tested by ELISA, and cytotoxic activity was evaluated by lactate dehydrogenase (LDH) release assay. Liver NK cells had significantly higher levels of cytotoxic activity than spleen NK cells under normal and acidic conditions, and the maximum difference was observed at pH 5.6. Further analysis revealed that the cytotoxic activity of NK cells was correlated with morphology, cell death, proliferative activity and IFN-gamma production. By TEM, liver NK cells contained a greater number of electron-dense granules per cell at pH 5.6. Moreover, a modest elevation of cell death and reduction of proliferation of liver NK cells occurred within a range of 5.6-7.2. Interestingly, an acidic extracellular pH only marginally, and not significantly, suppressed IFN-gamma production by liver NK cells. The sharp morphological and functional differences shown by the two types of NK cells in vitro indicate that liver NK cells are unexpectedly resistant to pH shock.

HTLV-1 bZIP factor perturbs immune response to the pathogens in vivo by inhibiting IFN-gamma production

HTLV-1 carriers and the patients with adult T-cell leukemia (ATL) frequently suffer from the opportunistic infections. Although it has been known that HTLV-1 infection evokes cell-mediated immune deficiency, its mechanism remains unknown. HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1, which is constitutively expressed and involved in the proliferation of HTLV-1-infected cells. In this study, we investigated the role of HBZ in immunodeficiency in a mice model. HBZ transgenic (HBZ-Tg) mice were infected with HSV-2 and Listeria monocytogenes and evaluated for cellular immunity to those pathogens. The clearance of both pathogens was impaired in HBZ-Tg mice compared with the non-transgenic littermate. In the both infection, production of IFN-gamma in CD4+ T cells was significantly reduced in HBZ-Tg mice. In addition, ectopic expression of HBZ in primary human CD4+ T cells impaired IFN-gamma production in vitro. As the molecular mechanisms, we found that HBZ suppressed transcription of IFNG promoter. Our results suggest that HBZ plays a crucial role for cellular immunodeficiency in HTLV-1-infected subjects.

Activated protein C targets CD8+ dendritic cells to reduce the mortality of endotoxemia in mice

Activated protein C (aPC) therapy reduces mortality in adult patients with severe sepsis. In mouse endotoxemia and sepsis models, mortality reduction requires the cell signaling function of aPC, mediated through protease-activated receptor-1 (PAR1) and endothelial protein C receptor (EPCR; also known as Procr). Candidate cellular targets of aPC include vascular endothelial cells and leukocytes. Here, we show that expression of EPCR and PAR1 on hematopoietic cells is required in mice for an aPC variant that mediates full cell signaling activity but only minimal anticoagulant function (5A-aPC) to reduce the mortality of endotoxemia. Expression of EPCR in mature murine immune cells was limited to a subset of CD8+ conventional dendritic cells. Adoptive transfer of splenic CD11chiPDCA-1- dendritic cells from wild-type mice into animals with hematopoietic EPCR deficiency restored the therapeutic efficacy of aPC, whereas transfer of EPCR-deficient CD11chi dendritic cells or wild-type CD11chi dendritic cells depleted of EPCR+ cells did not. In addition, 5A-aPC inhibited the inflammatory response of conventional dendritic cells independent of EPCR and suppressed IFN-gamma production by natural killer-like dendritic cells. These data reveal an essential role for EPCR and PAR1 on hematopoietic cells, identify EPCR-expressing dendritic immune cells as a critical target of aPC therapy, and document EPCR-independent antiinflammatory effects of aPC on innate immune cells.

700 INFLAMMASOME IS MODULATED BY PEGYLATED INTERFERON MONOTHERAPY IN CHRONIC HEPATITIS C PATIENTS

p=0.008). MC displayed a highly enriched Vdelta1 pool compared to HC and CHC (2.9% vs 1.0% and 1.1% of circulating CD3, p = 0.008 for both) that determined the inversion of the Vdelta1/Vdelta2 ratio (5.9 [MC] vs 0.5 [HC] and 0.6 [CHC], p = 0.003 for both). This ratio inversely correlated to cryocrit percentage (R = 0.39, p = 0.05). Conclusion: The reduced ability of MC T-regs to inhibit IFN-gamma production by target cells and the correlation between CD4+CD25− proliferation in co-cultures and cryocrit percentage suggest a role for T-reg impairment in perpetuating mixed cryoglobulinemia, whereas a predominance of Vdelta1 among gamma-delta T cell subsets may be important to limit cryocrit formation.

The A2aR adenosine receptor controls cytokine production in iNKT cells

The purine nucleoside adenosine is an important anti-inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor-mediated mechanisms. Invariant NKT (iNKT) cells recognize glycolipids presented on CD1d molecules and produce vigorous amounts of cytokines upon activation, hence regulating immune reactions. The mechanisms polarizing their cytokine pattern are elusive. Previous studies demonstrated that adenosine can suppress IFN-gamma production by iNKT cells. We describe the expression of all four known adenosine receptors A1R, A2aR, A2bR and A3R on mouse iNKT cells. We show that IL-4 production in primary mouse iNKT cells and a human iNKT line is efficiently inhibited by A2aR blockade with an inverse relation to IL-4. These data are supported by A2aR-deficient mice, which exhibit largely decreased levels of IL-4, IL-10 and TGF-beta concomitantly with an increase of IFN-gamma upon alpha-galactosylceramide administration in vivo. While A2aR inhibits other lymphocyte populations, A2aR is required for the secretion of IL-4 and IL-10 by iNKT cells. These data suggest adenosine:A2aR-mediated mechanisms can control the cytokine secretion pattern of iNKT cells.

High Expression of CD244 and SAP Regulated CD8+ T Cell Responses of Patients with HTLV-I Associated Neurologic Disease

HTLV-I-specific CD8+ T cells have been characterized with high frequencies in peripheral blood and cerebrospinal fluid and production of proinflammatory cytokines, which contribute to central nervous system inflammation in HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, little is known about the differences in CD8+ T cell activation status between asymptomatic carrier (ACs) and patients with HAM/TSP. The expression of CD244, a signaling lymphocyte activation molecule (SLAM) family receptor, was significantly higher on CD8+ T cells in HTLV-I-infected patients, both ACs and patients with HAM/TSP, than those on healthy normal donors (NDs). Blockade of CD244 inhibited degranulation and IFN-γ production in CD8+ T cells of patients with HAM/TSP, suggesting that CD244 is associated with effector functions of CD8+ T cells in patients with HAM/TSP. Moreover, SLAM-associated protein (SAP) was overexpressed in patients with HAM/TSP compared to ACs and NDs. SAP expression in Tax-specific CTLs was correlated in the HTLV-I proviral DNA loads and the frequency of the cells in HTLV-I-infected patients. SAP knockdown by siRNA also inhibited IFN-γ production in CD8+ T cells of patients with HAM/TSP. Thus, the CD244/SAP pathway was involved in the active regulation of CD8+ T cells of patients with HAM/TSP, and may play roles in promoting inflammatory neurological disease.

Suppression of Bystander T Helper 1 Cells by Iris Pigment Epithelium-Inducing Regulatory T Cells via Negative Costimulatory Signals

To determine whether iris pigment epithelium (IPE)-induced T regulatory (Treg) cells can suppress the activation of bystander T cells with cell contact via costimulatory interactions. CD8(+) T cells were cocultured with IPE, x-irradiated, and then used as regulators (IPE-induced Treg cells). The target CD4(+) T cells from wild-type control or knockout donors were used for the assay. T-cell activation was assessed for proliferation by examining both [(3)H]-thymidine incorporation and cytokine production. Expression of costimulatory molecules on IPE-induced Treg cells was evaluated using RT-PCR, immunostaining, and flow cytometry. Expression of costimulatory receptors on target T cells or Treg cells was evaluated by flow cytometry. Neutralizing antibodies were then used to abolish regulatory function. CD8(+) IPE-induced Treg cells significantly suppressed the activation of effector target T cells, e.g., T-cell proliferation and cytokine production such as Th1, Th2, and Th17 cytokines. Although IPE-induced Treg cells expressed various costimulatory molecules, including programmed cell death 1 ligand 1 (PD-L1), only PD-L1 on the Treg cells was actually delivered to target Th1 cells using cell-to-cell interaction (T-T interaction). If neutralizing antibodies for PD-L1 were cocultured with Treg cells, Th1 suppression was impaired. Moreover, Treg cells failed to suppress IFNgamma production by target CD4(+) T cells from programmed cell death 1 (PD-1) knockout donors. Th1-specific inhibition was exclusively achieved with direct cell contact. T cells exposed to IPE in the eye that acquires full regulatory capacity express negative costimulators and suppress bystander Th1-type effector cells.

Sequence flexibility of the immunodominant HLA A*0201 restricted ppUL83 CD8 T‐cell epitope of human cytomegalovirus

The cytomegalovirus ppUL83 protein contains an immunodominant A*0201 restricted epitope between residues 495 and 503. We investigated the tolerance of this epitope to sequence variation in the context of peptide binding to HLA A*0201 and the ability to induce an Interferon gamma (IFNgamma) response through engagement with the T-cell receptor (TCR). The majority of mutations investigated resulted in a decrease in the production of IFNgamma indicating that if such variants occurred in vivo they would not be recognized by CD8 T-cell clones specific for the wild-type epitope. The mechanistic basis for the majority of the mutant peptides was their failure to bind and stabilize class I HLA cell surface expression. However, one peptide with a mutation at the P5 position (methionine to cysteine) resulted in a significant enhanced binding to HLA A*0201 and also an increase in cell surface expression over the wild-type peptide but was unable to engage with the CD8 TCR and trigger IFNgamma production. This peptide acted as a competitive inhibitor of the wild-type peptide but could not fully inhibit IFNgamma production by the latter. We subsequently investigated whether mutations of the HLA A*0201 epitope were evident in immunocompromized patients experiencing either rapid exponential or persistent cytomegalovirus replication.

Hepatitis C virions subvert natural killer cell activation to generate a cytokine environment permissive for infection

Hepatitis C virus (HCV) is remarkably successful in establishing persistent infections due to its ability to evade host immune responses through a combination of mechanisms including modulation of interferon (IFN) signalling in infected cells, interference with effector cell function of the immune system and continual viral genetic variation. We have previously demonstrated that natural killer (NK) cells can be inhibited in vitro by recombinant HCV glycoprotein E2 via cross-linking of CD81, a cellular co-receptor for the virus. Taking advantage of the recently established tissue-culture system for HCV, we have studied the effects of CD81 engagement by the HCV envelope glycoprotein E2 when the protein is part of complete, infectious viral particles. Specifically, we asked whether exposure to HCV viral particles (HCVcc) affects activation of NK cells and whether altered NK cell activation, in turn, impacts on HCV infectivity. We found that immobilized HCVcc, unlike soluble HCVcc, inhibited IFN-gamma production by interleukin (IL)-12 activated NK cells, and that this effect was mediated by engagement of cellular CD81 by HCV-virion displayed E2. In contrast, NK-production of IL-8 was increased in the presence of HCV. The cytokines produced by IL-12 activated NK cells strongly reduced the establishment of productive HCV infection. Importantly, NK-cell derived cytokines secreted in the presence of HCVcc showed a diminished antiviral effect that correlated with IFN-gamma reduction, while IL-8 concentrations had no impact on HCV infectivity. Exposure to HCVcc modulates the pattern of cytokines produced by NK cells, leading to reduced antiviral activity.

Minimal influence of tocilizumab on IFN-γ synthesis by tuberculosis antigens

Interferon gamma (IFN-gamma) production is a critical step of antituberculosis (anti-TB) immune response. The purpose of this study was to determine the influences of biologics, including the interleukin (IL)-6 receptor-inhibitor tocilizumab (TCZ), and tumor necrosis factor (TNF) antagonists infliximab (INF) and etanercept (ETA), on Mycobacterium tuberculosis (MTB) antigen-induced IFN-gamma production. MTB antigen (ESAT-6 and CFP-10)-induced IFN-gamma-releasing assay was performed with or without addition of biologics (TCZ, ETA, and INF) using whole blood from patients with active TB. ETA and INF inhibited IFN-gamma production in a dose-dependent manner. In whole blood from TB patients, ESAT-6 stimulated significant production of IFN-gamma (1.30 +/- 1.95 IU/ml), and TCZ did not inhibit IFN-gamma production (1.56 +/- 1.88 IU/ml). IFN-gamma production by ESAT-6 was inhibited by ETA and INF (0.98 +/- 1.74, 0.75 +/- 1.66 IU/ml, respectively). CFP-10 stimulated significant production of IFN-gamma (1.46 +/- 1.60 IU/ml), and TCZ did not inhibit IFN-gamma production (1.51 +/- 1.77 IU/ml). IFN-gamma production by CFP-10 was inhibited by ETA and INF (0.91 +/- 0.99, 0.72 +/- 0.88 IU/ml, respectively). TCD did not inhibit MTB antigen-induced IFN-gamma production. As IFN-gamma production is important in antimycobacterial host defenses, the minimal influence of TCZ on IFN-gamma-releasing assay suggests a low risk of latent TB infection reactivation during tocilizumab therapy.

Low-flux hemodialysis suppresses interferon-gamma production: the possible role of beta2-microglobulin

Interferon-gamma (IFN-gamma) is required for the acquired immune response involving maturation and activation of antigen-presenting cells (APC); both IFN-gamma production and activation of APC are impaired in ESRD patients on low-flux hemodialysis (HD). High levels of uremic toxins including beta2-microglobulin (beta2M) are thought to play a role in immunosuppression. To test this hypothesis, we conducted an A-B-A crossover study to examine the influence of high-flux HD (A) versus low-flux HD (B) in ESRD patients (n = 14) using biocompatible synthetic dialyzer membranes (Polyamix(R), Gambro, Hechingen, Germany) and ultrafiltered bicarbonate-buffered dialysis fluid. Each study period lasted 6 months and at the end of each period, Kt/V urea, plasma levels of albumin, C-reactive protein (CRP) and beta2M were determined. In addition, production of IFN-gamma induced by heat-killed Staphylococcus epidermidis (Staph epi) was performed in whole blood cultures. Kt/V urea (mean +/- SEM) was 1.45 +/- 0.06 in Period A, 1.36 +/- 0.07* in Period B, and 1.51 +/- 0.07 in Period A' (*p < 0.01). beta2M resting levels increased from 26.6 +/- 1.42 mg/l in Period A to 34.7 +/- 2.30* mg/l in Period B, and decreased to 21.6 +/- 1.07 mg/l at the end of Period A' (*p < 0.0001). Albumin and CRP levels did not differ significantly. Staph epi-induced IFN-gamma production was 1.2 +/- 0.39 ng/1,000 PBMC at the end of Period A, 0.34 +/- 0.10 ng/1,000 PBMC* in Period B, and 2.71 +/- 0.67 ng/1,000 PBMC at the end of Period A' (*p < 0.05). There was an inverse correlation between b2M levels and whole blood IFN-gamma production (R2 = 0.26). High levels of uremic toxins such as beta2M suppress IFN-gamma production in ESRD patients under low-flux HD. High-flux HD reduces beta2M levels by 30% and improves IFN-gamma production suggesting an improved cellular immune response in ESRD patients.

Human CD8+CXCR3+ T cells have the same function as murine CD8+CD122+ Treg

The importance of CD8(+)CD122(+) Treg in the maintenance of immune homeostasis has been previously demonstrated in mice. Because the expression pattern of CD8 and CD122 in humans is different from that in mice, human CD8(+) Treg that correspond to the murine CD8(+)CD122(+) Treg have not been identified. In this study, we performed DNA microarray analyses to compare the gene expression profiles of CD8(+)CD122(+) cells and CD8(+)CD122(-) cells in mice and found that CXCR3 was preferentially expressed in CD8(+)CD122(+) cells. When we analyzed the expression of CD122 and CXCR3 in murine CD8(+) cells, we observed a definite population of CD122(+)CXCR3(+) cells. CD8(+)CXCR3(+) cells in mice showed similar regulatory activities to CD8(+)CD122(+) cells by in vivo and in vitro assays. While CD8(+)CD122(+)CXCR3(+) cells are present in mice, CD8(+)CXCR3(+) cells, but not CD8(+)CD122(+) cells, are present in humans. In the in vitro assay, human CD8(+)CXCR3(+) cells showed the regulatory activity of producing IL-10 and suppressing IFN-gamma production from CD8(+)CXCR3(-) cells. These results suggest that human CD8(+)CXCR3(+) T cells are the counterparts of murine CD8(+)CD122(+) Treg.

T-box 21 transcription factor is responsible for distorted TH2 differentiation in human peripheral CD4+ T cells

Regardless of T(H)1/T(H)2 theory, CD4(+) T cells of patients with allergic asthma, a typical T(H)2 disease, and those of healthy subjects expressed equivalent levels of IFN-gamma, even though T(H)2 cytokines were significantly upregulated in asthmatic patients. The mechanisms underlying distorted T(H)2 cell polarization in human T cells were elucidated. Cytokine-producing activity and the expression of T(H)1/T(H)2-specific transcription factors in naïve, T(H)1/T(H)2, or both CD4(+) T cells derived from human peripheral and cord blood were comparatively analyzed. The mechanisms of the differential expression of T-box 21 transcription factor (T-bet) in the cells were assessed by determining the chromatin accessibility at the TBX21 gene. The functional roles of T-bet and other transcription factors in human T(H)1/T(H)2 differentiation were further investigated. T(H)2 cells derived from naive CD4(+) T cells in peripheral blood but not in cord blood produced IFN-gamma. T-bet was expressed in peripheral, but not cord blood, resting naive T cells. Consistently, the accessibility at the proximal TBX21 gene promoter in peripheral naive T cells was higher than that in cord blood naive T cells. IFN-gamma-producing activity was induced in T(H)2-differentiated cord blood T cells by means of ectopic expression of T-bet. In addition, a reduction of T-bet in peripheral T cells suppressed IFN-gamma production. T-bet not only upregulated IFN-gamma but also downregulated IL-4 and IL-13 gene transcription, independently of the modification of T(H)1/T(H)2 balance. The expression of T-bet at a naive stage is crucial for the development of IFN-gamma-producing T cells in human peripheral blood, even in T(H)2-related diseases.

Anti-inflammatory activity of p-coumaryl alcohol-gamma-O-methyl ether is mediated through modulation of interferon-gamma production in Th cells.

p-Coumaryl alcohol-gamma-O-methyl ether (CAME) was isolated from Alpinia galanga and shown to contain a phenylpropanoid structure similar to p-coumaryl diacetate (CDA). CDA is known to have antioxidant and anti-inflammatory activity, but the biochemical activities of CAME are unknown. Inflammation is mediated by inflammatory cytokine production, in particular, by CD4+ T helper cells (Th cells), but it is unclear whether phenylpropanoids affect cytokine production in Th cells. In this study, we decided to investigate the functions of CAME and CDA in CD4+ Th cells. Mouse CD4+ Th cells were isolated from C57BL6 mice and stimulated with an antibody against T cell receptors in the presence of phenylpropanoids. Cytokine production was measured by elisa and intracellular cytokine staining. Gene knockout mice and tetracycline-inducible transgenic mice were used to examine the molecular mechanisms of phenylpropanoids on modulation of cytokine production. CAME potently reduced intracellular reactive oxygen species in Th cells, as does CDA. However, although CDA was cytotoxic, CAME selectively and potently suppresses interferon-gamma (IFNgamma) production in CD4+ Th cells, without toxicity. This effect was caused by attenuated expression of the transcription factor, T-box protein expressed in T cells (T-bet), and T-bet was essential for CAME to inhibit IFNgamma production in CD4+ Th cells. CAME selectively and substantially suppresses IFNgamma production in CD4+ Th cells by decreasing T-bet expression. As increased IFNgamma production by CD4+ Th cells can mediate inflammatory immune responses, a selective IFNgamma suppressor, such as CAME may be an effective, naturally occurring, compound for modulating inflammatory immune disorders.

Antigenic stimulation with cytochrome P450 2J expressed in mouse hepatocellular carcinoma cells regulates host anti-tumour immunity

Cytochrome P450 2J subfamily (CYP2J) enzymes expressed in mouse hepatocellular carcinoma (HCC) cells were identified as an antigen recognized by specific CD4(+) T cells and the structure of its T cell epitope was determined by proteomics-based exploration. The major histocompatibility complex (MHC) class II binding peptides were isolated from I-A(k)/peptide complex of dendritic cells (DCs) loaded or unloaded with MIH-2 mouse HCC cells. MHC class II-binding peptides found in MIH-2-loaded DCs but not in unloaded DCs were determined by tandem mass spectrometric analysis. The peptide, consisting of amino acid 276-290 (DFIDAFLKEMTKYPE) of mouse CYP2J enzymes, was identified as an antigenic peptide presented in the context of MHC class II. Preventive treatment of mice with CYP2J peptide stimulated interferon (IFN)-gamma production of splenocytes and suppressed the growth of implanted CYP2J-positive MIH-2 cells but not CYP2J-negative murine bladder tumour cells. However, continuous treatment of MIH-2-bearing mice with CYP2J peptide significantly suppressed IFN-gamma production of splenocytes and accelerated the growth of implanted MIH-2 tumours in vivo. Increased frequencies of CD4(+)forkhead box P3 regulatory T cells and CD11b(+)Gr-1(+) myeloid suppressor cells were observed in splenocytes from the continuously immunized mice. These results indicate that antigenecity of CYP2J isoforms expressed in HCC cells activate host anti-tumour immunity at an initial stage of HCC, but suppress host anti-tumour immunity with excessive antigenic stimulation at an advanced stage.

HSP60 as a Target of Anti-Ergotypic Regulatory T Cells

The 60 kDa heat shock protein (HSP60) has been reported to influence T-cell responses in two ways: as a ligand of toll-like receptor 2 signalling and as an antigen. Here we describe a new mechanism of T-cell immuno-regulation focused on HSP60: HSP60 is up-regulated and presented by activated T cells (HSP60 is an ergotope) to regulatory (anti-ergotypic) T cells. Presentation of HSP60 by activated T cells was found to be MHC-restricted and dependent on accessory molecules - CD28, CD80 and CD86. Anti-ergotypic T cells responded to T-cell HSP60 by proliferation and secreted IFNγ and TGFβ1. In vitro, the anti-ergotypic T cells inhibited IFNγ production by their activated T-cell targets. In vivo, adoptive transfer of an anti-ergotypic HSP60-specific T-cell line led to decreased secretion of IFNγ by arthritogenic T cells and ameliorated adjuvant arthritis (AA). Thus, the presentation of HSP60 by activated T cells turns them into targets for anti-ergotypic regulatory T cells specific for HSP60. However, the direct interaction between the anti-ergotypic T regulators (anti-HSP60) and the activated T cells also down-regulated the regulators. Thus, by functioning as an ergotope, HSP60 can control both the effector T cells and the regulatory HSP60-specific T cells that control them.

Phenotypic and Functional Effects of Novel Akt Inhibitor Perifosine on Immune System.

Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agent which targets cell membranes and inhibits Akt activation. Perifosine inhibits multiple myeloma (MM) cell growth in vitro and in vivo. Currently perifosine is under phase II clinical evaluation in MM. Although perifosine has shown significant direct antitumor effects, its effect on immune system has not yet been clarified. The objective of this study was to investigate the effects of perifosine on immune system including the activity of antigen presenting cells (APCs), T cells and NK cells. Perifosine was used at a clinically relevant concentrations of 2.5 μM, 5 μM and 10 μM. Monocyte-derived dendritic cells (DCs) from healthy donors were used as the APCs. We observed that the perifosine up to 48 hours had no effect on viability (>90%), as assessed by annexin V and PI staining. Alteration of DC phenotype by perifosine was further examined by flow cytometry. Our results demonstrated that perifosine treatment led to a dose-dependent downregulation of surface antigen expression, associated with costimulation (CD40, CD80 and CD86), antigen presentation (HLA-ABC, HLA-DQ) and maturation (CD83) on immature DCs at 24 and 48 hours. The significant downregulation of CD40, CD83, HLA-ABC and HLA-DQ was also observed on mature DCs. Perifosine also inhibited immature DC uptake of antigens (40-kDa Dextran-FITC, 45-kDa protein A-Alexa Fluor 488 and 20-kDa protein G-Alexa Fluor 488) in a dose-dependent manner. Since DCs play a crucial regulatory role via cytokine production, we next determined IL-12p70 and IL-10 secretion by LPS-induced DCs with and without perifosine treatment. Compared to controls, perifosine treatment at 24 hours significantly inhibited LPS-induced-IL-12p70 production by DCs (trt vs. untrt = 166 pg/ml (2.5μM), 111 pg/ml (5μM) and 45 pg/ml (10μM) vs. 192 pg/ml), as well as inhibited IL-10 production (trt vs. untrt = 371 pg/ml (2.5μM), 306 pg/ml (5μM) and 179 pg/ml (10μM) vs. 472 pg/ml). These data suggest that perifosine is able to affect both immature and mature DCs and could contribute to inhibition of DC-mediated immune responses. Furthermore, we evaluated effect of perifosine on T cells obtained from healthy donor peripheral blood mononuclear cells (PBMCs) by negative selection. Although perifosine treatment, up to 10μM, had no effect on T cell survival, it inhibited IFN-gamma production by T cells stimulated with PMA and ionomycin compared to that of the control (intracellular flow cytometry, trt vs. untrt= 11.7% vs. 21.3%). The IFN-gamma inhibition by perifosine treatment (10μM) was further confirmed by ELISA upon stimulation with anti- CD2/CD3/CD28 activation beads (trt vs. untrt =11340 pg/ml vs. 18150 pg/mL). Similarly, viability of NK cells obtained from PBMCs by negative selection was not significantly affected by perifosine; however, decreased cytotoxicities were observed by NK cells treated with perifosine (10μM) against the U266 myeloma cell line (trt vs. untrt=30% vs. 51% (E:T=10:1) and 17% vs 30% (E:T=5:1) respectively). In addition, the IFN-gamma production by IL-2 and IL-12- induced NK cells was significantly inhibited following the perifosine treatment (trt vs. untrt=286 pg/ml (2.5μM), 210 pg/ml (10μM) vs. 439 pg/ml). These studies demonstrate that perifosine treatment significantly affects phenotype and function of human DCs, T cells and NK cells. Our pre-clinical data therefore indicates the need to monitor immune functions in patients under the Akt inhibitor treatment.