Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide.

To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN-promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6-20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles.

Tumour islet Foxp3+ T-cell infiltration predicts poor outcome in nonsmall cell lung cancer

The impact of host immunity on outcome in nonsmall cell lung cancer (NSCLC) is controversial. We examined the relationship between lymphoid infiltration patterns in NSCLC and prognosis. Tumour- and stroma-infiltrating CD3+, CD8+ and forkhead box P3 (Foxp3)+ T-lymphocytes were identified using immunohistochemistry and a novel image analysis algorithm to assess total, cytotoxic and regulatory T-lymphocyte counts, respectively, in 196 NSCLC cases. The median cell count was selected as a cut-point to define patient subgroups and the ratio of the corresponding tumour islet:stroma (TI/S) counts was determined. There was a positive association between overall survival and increased CD8+ TI/S ratio (hazard ratio (HR) for death 0.44, p<0.001) but an inverse relationship between Foxp3+ TI/S ratio and overall survival (HR 4.86, p<0.001). Patients with high CD8+ islet (HR 0.48, p<0.001) and Foxp3+ stromal (HR 0.23, p<0.001) counts had better survival, whereas high CD3+ and CD8+ stromal counts and high Foxp3+ islet infiltration conferred a worse survival (HR 1.55, 2.19 and 3.14, respectively). By multivariate analysis, a high CD8+ TI/S ratio conferred an improved survival (HR 0.48, p=0.002) but a high Foxp3+ TI/S ratio was associated with worse survival (HR 3.91, p<0.001). Microlocalisation of infiltrating T-lymphocytes is a powerful predictor of outcome in resected NSCLC.

Développement d’immunothérapies spécifiques pour le traitement de l’hépatite autoimmune de type 2 chez un modèle murin

L’hépatite autoimmune (HAI) est une maladie grave affectant le foie et présentant un haut taux de mortalité lorsque non traitée. Les traitements disponibles sont efficaces, mais de graves effets secondaires leur sont associés. Ces effets secondaires sont généralement le résultat d'une forte immunosuppression et d’autres sont spécifiques à chaque médicament. Aucune immunothérapie spécifique n’est présentement disponible pour le traitement de l’HAI. Récemment, un modèle murin d’HAI a été développé dans notre laboratoire par xénoimmunisation des souris C57BL/6 avec les antigènes humains de l'HAI de type 2. Ce modèle présente la plupart des caractéristiques biochimiques et cliniques retrouvées chez les patients atteints d'HAI de type 2. Dans cette étude, nous avons évaluée l’efficacité de deux types de traitement pour l’HAI de type 2 à l’aide de notre modèle murin. Dans un premier temps, l’anticorps anti-CD3ε a été étudié en prophylaxie et en traitement. Nous avons montré qu’une posologie de 5µg d’anti-CD3 i.v. par jour pendant 5 jours consécutifs induit une rémission chez les souris avec HAI de type 2 établie (traitement). Cette rémission est caractérisée par une normalisation des niveaux d’alanine aminotransférase et une diminution significative de l’inflammation hépatique. Cette rémission semble être associée à une déplétion partielle et transitoire des lymphocytes T CD3+ dans la périphérie et une augmentation des lymphocytes T régulateurs CD4+, CD25+ et Foxp3+ dans le foie. La même posologie lorsqu’elle est appliquée en prophylaxie n’a pas réussi à prévenir l’apparition de l’HAI de type 2. La deuxième voie de traitement consiste en l’administration par voie intranasale d’un forte dose de formiminotransférase cyclodésaminase murin (mFTCD), un autoantigène reconnu dans l’HAI de type 2. Une administration en prophylaxie par voie intranasale de 100µg de mFTCD par jour durant 3 jours consécutifs arrive à prévenir l’HAI de type 2 en diminuant l’inflammation hépatique au bout de deux semaines post-traitement.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Background: Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods: In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results: DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion: The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases. © 2009 Santos et al; licensee BioMed Central Ltd.

Persistent inflammation in the CNS during chronic EAE despite local absence of IL-17 production

Experimental autoimmune encephalomyelitis (EAE) is an artificially induced demyelination of the central nervous system (CNS) that resembles multiple sclerosis in its clinical, histopathological, and immunological features. Activated Th1 and Th17 cells are thought to be the main immunological players during EAE development. This study was designed to evaluate peripheral and local contribution of IL-17 to acute and chronic EAE stages. C57BL/6 mice were immunized with MOG plus complete Freund's adjuvant followed by pertussis toxin. Mice presented an initial acute phase characterized by accentuated weight loss and high clinical score, followed by a partial recovery when the animals reached normal body weight and smaller clinical scores. Spleen cells stimulated with MOG produced significantly higher levels of IFN-γ during the acute period whereas similar IL-17 levels were produced during both disease stages. CNS-infiltrating cells stimulated with MOG produced similar amounts of IFN-γ but, IL-17 was produced only at the acute phase of EAE. The percentage of Foxp3+ Treg cells, at the spleen and CNS, was elevated during both phases. The degree of inflammation was similar at both disease stages. Partial clinical recovery observed during chronic EAE was associated with no IL-17 production and presence of Foxp3+ Treg cells in the CNS. © 2013 Sofia Fernanda Gonçalves Zorzella-Pezavento et al.

Bacille Calmette-Guérin/DNAhsp65 prime-boost is protective against diabetes in non-obese diabetic mice but not in the streptozotocin model of type 1 diabetes

Type I diabetes is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this study was to evaluate the prophylactic potential of a prime-boost strategy involving bacille Calmette-Guérin (BCG) and the pVAXhsp65 vaccine (BCG/DNAhsp65) in diabetes induced by streptozotocin (STZ) in C57BL/6 mice and also in spontaneous type 1 diabetes in non-obese diabetic (NOD) mice. BCG/DNAhsp65 vaccination in NOD mice determined weight gain, protection against hyperglycaemia, decreased islet inflammation, higher levels of cytokine production by the spleen and a reduced number of regulatory T cells in the spleen compared with non-immunized NOD mice. In the STZ model, however, there was no significant difference in the clinical parameters. Although this vaccination strategy did not protect mice in the STZ model, it was very effective in NOD mice. This is the first report demonstrating that a prime-boost strategy could be explored as an immunomodulatory procedure in autoimmune diseases. © 2013 British Society for Immunology.

Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5′ or 3′ untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.

Anti-CTLA-4 treatment induces IL-10-producing ICOS+ regulatory T cells displaying IDO-dependent anti-inflammatory properties in a mouse model of colitis.

BACKGROUND AND AIMS: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to act as a negative regulator of T cell function and has been implicated in the regulation of T helper 1 (Th1)/Th2 development and the function of regulatory T cells. Tests were carried out to determine whether anti-CTLA-4 treatment would alter the polarisation of naive T cells in vivo. METHODS: Mice were treated with anti-CTLA-4 monoclonal antibody (mAb) (UC10-4F10) at the time of immunisation or colonic instillation of trinitrobenzene sulfonic acid (TNBS). The cytokines produced by lymph node cells after in vitro antigenic stimulation and the role of indoleamine 2,3 dioxygenase (IDO) and of interleukin-10 (IL-10) were tested, and the survival of mice was monitored. RESULTS: Injection of anti-CTLA-4 mAb in mice during priming induced the development of adaptive CD4(+) regulatory T cells which expressed high levels of ICOS (inducible co-stimulator), secreted IL-4 and IL-10. This treatment inhibited Th1 memory responses in vivo and repressed experimental intestinal inflammation. The anti-CTLA-4-induced amelioration of disease correlated with IDO expression and infiltration of ICOS(high) Foxp3(+) T cells in the intestine, suggesting that anti-CTLA-4 acted indirectly through the development of regulatory T cells producing IL-10 and inducing IDO. CONCLUSIONS: These observations emphasise the synergy between IL-10 and IDO as anti-inflammatory agents and highlight anti-CTLA-4 treatment as a potential novel immunotherapeutic approach for inducing adaptive regulatory T cells.

Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice.

Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated ("desensitized") by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various beta-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of beta-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3+ T and B220+ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the beta-arrestin2- and GRK6-deficient animals. Surprisingly, however, both T and B cells from beta-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that beta-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.

Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets.

Stimulated CD4(+) T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation promoting Teff and Treg, respectively, Teff were selectively increased in Glut1 transgenic mice and reliant on glucose metabolism, whereas Treg had activated AMP-activated protein kinase and were dependent on lipid oxidation. Importantly, AMP-activated protein kinase stimulation was sufficient to decrease Glut1 and increase Treg generation in an asthma model. These data demonstrate that CD4(+) T cell subsets require distinct metabolic programs that can be manipulated in vivo to control Treg and Teff development in inflammatory diseases.

In vitro expansion of CD4+CD25highFOXP3+CD127low/- regulatory T cells from peripheral blood lymphocytes of healthy Mycobacterium tuberculosis-infected humans.

CD4+CD25highFOXP3+ regulatory T (Treg) cells have recently been found at elevated levels in the peripheral blood of tuberculosis patients, compared to Mycobacterium tuberculosis latently infected (LTBI) healthy individuals and non-infected controls. Here, we show that CD4+CD25highFOXP3+ T lymphocytes can be expanded in vitro from peripheral blood mononuclear cells (PBMC) of LTBI individuals, but not of uninfected controls by incubating them with BCG in the presence of TGF-beta. These expanded cells from the PBMC of LTBI subjects expressed CTLA-4, GITR and OX-40, but were CD127low/- and have therefore the phenotype of Treg cells. In addition, they inhibited in a dose-dependant manner the proliferation of freshly isolated mononuclear cells in response to polyclonal stimulation, indicating that they are functional Treg lymphocytes. In contrast, incubation of the PBMC with BCG alone preferentially induced activated CD4+ T cells, expressing CD25 and/or CD69 and secreting IFN-gamma. These results show that CD4+CD25highFOXP3+ Treg cells can be expanded or induced in the peripheral blood of LTBI individuals in conditions known to predispose to progression towards active tuberculosis and may therefore play an important role in the pathogenesis of the disease.

Regulatory T cells depress immune responses to protective antigens in active tuberculosis.

RATIONALE: Tuberculosis (TB) remains a leading cause of death, and the role of T-cell responses to control Mycobacterium tuberculosis infections is well recognized. Patients with latent TB infection develop strong IFN-gamma responses to the protective antigen heparin-binding hemagglutinin (HBHA), whereas patients with active TB do not. OBJECTIVES: We investigated the mechanism of this difference and evaluated the possible involvement of regulatory T (Treg) cells and/or cytokines in the low HBHA T-cell responses of patients with active TB. METHODS: The impact of anti-transforming growth factor (TGF)-beta and anti-IL-10 antibodies and of Treg cell depletion on the HBHA-induced IFN-gamma secretion was analyzed, and the Treg cell phenotype was characterized by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Although the addition of anti-TGF-beta or anti-IL-10 antibodies had no effect on the HBHA-induced IFN-gamma secretion in patients with active TB, depletion of CD4(+)CD25(high)FOXP3(+) T lymphocytes resulted in the induction by HBHA of IFN-gamma concentrations that reached levels similar to those obtained for latent TB infection. No effect was noted on the early-secreted antigen target-6 or candidin T-cell responses. CONCLUSIONS: Specific CD4(+)CD25(high)FOXP3(+) T cells depress the T-cell-mediated immune responses to the protective mycobacterial antigen HBHA during active TB in humans.

Rôle de la molécule CD47 sur le lymphocyte T dans la régulation de la réponse immunitaire

L’importance respective des lymphocytes T régulateurs naturels générés dans le thymus ou induits en périphérie dans la régulation immunitaire et la résolution de l’inflammation est désormais bien établie. Nous avons contribué à mettre en évidence une nouvelle voie d’induction de lymphocytes T régulateurs périphériques à partir de cellules T humaines CD4+CD25- naïves et mémoires. Nous avons montré que l’engagement de la molécule ubiquitaire transmembranaire CD47 sur la cellule T par un anticorps monoclonal ou par le peptide 4N1K (peptide dérivé du domaine carboxy-terminal de la thrombospondine-1 et spécifique du site de liaison à CD47) induisait des lymphocytes T CD4+ régulateurs exerçant une fonction suppressive sur les lymphocytes T effecteurs. Les propriétés suppressives induites par la thrombospondine-1 confortent les fonctions anti-inflammatoires de cette protéine de la matrice extracellulaire. L’inhibition exercée par les lymphocytes T régulateurs induits dépend du contact intercellulaire entre les cellules T régulatrices et leurs cibles, et est indépendante du TGF-. Nos résultats démontrent également le rôle de CD47 sur le lymphocyte T CD4+ dans la réponse immunitaire spécifique de l’antigène in vivo. En effet, les souris BALB/c déficientes pour CD47 présentent un biais de la sécrétion d’anticorps et de cytokines de type Th1, alors que les souris BALB/c sont décrites comme exprimant un profil de production de cytokines de type Th2. Nos travaux mettent en évidence le rôle de CD47 dans l’inhibition du développement d’une réponse cellulaire et humorale de type Th1 in vivo, confirmant de précédentes études in vitro réalisées avec des cellules T CD4+ humaines. Nous présentons également le rôle inhibiteur de l’engagement de CD28 in vitro sur la différenciation en cellules Th17 des lymphocytes T CD4+ naïfs isolés de souris BALB/c. Le mécanisme proposé est dépendant de la production de l’IL-2 et de l’IFN- et indépendant de la présence de lymphocytes T régulateurs. Notre étude du rôle de deux molécules transmembranaires CD47 et CD28 exprimées sur la cellule T CD4+, contribue à une meilleure connaissance des mécanismes impliqués dans la tolérance immunologique, la résolution de l’inflammation et la différenciation des cellules T "helper" CD4+.

Prevention of experimental diabetes by Uncaria tomentosa extract: Th2 polarization, regulatory T cell preservation or both?

Ethnopharmacological relevance: Uncaria tomentosa (Willd.) DC (Rubiaceae) is a species native to the Amazon rainforest and surrounding tropical areas that is endowed with immunomodulatory properties and widely used around the world. In this study we investigated the immunomodulatory potential of Uncaria tomentosa (UT) aqueous-ethanol extract on the progression of immune-mediated diabetes.Materials and methods: C57BL/6 male mice were injected with MLDS (40 mg/kg) and orally treated with UT at 10-400 mg/kg during 21 days. Control groups received MLDS alone or the respective dilution vehicle. Pancreatic mononuclear infiltrate and beta-cell insulin content were analyzed by HE and immunohistochemical staining, respectively, and measured by digital morphometry. Lymphocyte immunophenotyping and cytokine production were determined by flow cytometry analysis.Results: Treating the animals with 50-400 mg/kg of UT caused a significant reduction in the glycemic levels, as well as in the incidence of diabetes. The morphometric analysis of insulitis revealed a clear protective effect. Animals treated with UT at 400 mg/kg presented a higher number of intact islets and a significant inhibition of destructive insulitis. Furthermore, a significant protection against the loss of insulin-secreting presented beta-cells was achieved, as observed by a careful immunohistochemical evaluation. The phenotypic analysis indicated that the groups treated with higher doses (100-400 mg/kg) presented CD4(+) and CD8(+) T-cell values similar to those observed in healthy animals. These same higher doses also increased the number of CD4(+)CD25(+)Foxp3(+) regulatory T-cells. Moreover, the extract modulated the production of Th1 and Th2, with increased levels of IL-4 and IL-5.Conclusions: The extract was effective to prevent the progression of immune-mediated diabetes by distinct pathways. (C) 2011 Elsevier B.V. All rights reserved.

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: Activation of CD8+ cells, interferon-γ recovery and reduction of lung injury

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-γ and to restrict the growth of bacilli.