Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell-mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigen-specific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4(+)CD25(-) T cells could be expanded 8-fold into alloantigen-specific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO(+)CCR7(-) memory cells, and had a CD4(high), CD25(+), Foxp3(+), and CD62L (L-selectin)(+) phenotype. Although these CD4(high)CD25(+)Foxp3(+) alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.

HIV-1 tropism and CD4 T lymphocyte recovery in a prospective cohort of patients initiating HAART in Ribeir��o Preto, Brazil

While human immunodeficiency virus (HIV)-1 chemokine co-receptors 5 tropism and the GWGR motif in the envelope third variable region (V3 loop) have been associated with a slower disease progression, their influence on antiretroviral response remains unclear. The impact of baseline V3 characteristics on treatment response was evaluated in a randomised, double blind, prospective cohort study with patients initiating highly active antiretroviral therapy with lopinavir or efavirenz plus azithothymidine/3TC (1:1) over 48 weeks. Similar virological and immunological responses were observed for both treatment regimens. The 43 individuals had a mean baseline CD4 T cell count of 119 cells/mm�� [standard deviation (SD) = 99] and a mean viral load of 5.09 log10 copies/mL (SD = 0.49). The GWGR motif was not associated with a CD4 T cell response, but predicted R5 tropism by the geno2pheno[clinical20%] algorithm correlated with higher CD4 T cell levels at all monitoring points (p < 0.05). Moreover, higher false-positive rates (FPR) values from this analysis revealed a strong correlation with CD4 T cell recovery (p < 0.0001). Transmitted drug resistance mutations, documented in 3/41 (7.3%) cases, were unrelated to the assigned antiretroviral regimen and had no impact on patient outcomes. In conclusion, na��ve HIV-1 R5 infected patients exhibited higher CD4 T cell counts at baseline; this difference was sustained throughout therapy. The geno2pheno[clinical] option FPR positively correlated with CD4 T cell gain and may be useful in predicting CD4 T cell recovery.

Soissons, [abbaye] St-Jean-des-Vignes, le triforium [d��g��ts dus �� la guerre] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55108

Soissons, [abbaye] St-Jean-des-Vignes, clo��tre, porte 18e s. [d��g��ts dus �� la guerre] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55107

Soissons, fa��ade de St-Jean-des-Vignes [abbaye, d��g��ts dus �� la guerre] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55103

Cambrai, place Thiers, salle des concerts [d��g��ts dus �� la guerre] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55506

The roles of PKC&#948; and Src kinases in the glucocorticoid induction and the TGF-&#946; superinduction of the mouse mammary tumor virus transcription in B lymphocytes

R��sum�� : Le virus tumoral de la glande mammaire de la souris (MMTV) est un r��trovirus provoquant le d��veloppement de tumeurs dans les glandes mammaires des souris susceptibles femelles. Au cours de son ��volution, le virus s'est adapt�� et s'exprime dans des cellules sp��cialis��es. Les lymphocytes B sont les premi��res cellules infect��es et elles sont essentielles pour la propagation de l'infection aux glandes mammaires. Dans notre ��tude, le virus MMTV a ��t�� utilis�� afin d'examiner les voies de signalisation induites par les glucocortico��des (dexam��thasone (dex), une hormone st��ro��dienne) et le transforming growth factor-f3 (TGF-P, une cytokine), deux mol��cules impliqu��es dans l'activation de la transcription �� partir du promoteur du MMTV dans les cellules B. Le TGF-P seul n'influence pas l'activit�� du promoteur du MMTV. Par contre, en synergie avec dex, le TGF-P provoque une super-induction de l'expression du promoteur par rapport �� une stimulation par le glucocortico��de seul. Cette super-induction est r��gul��e par une famille de prot��ines, les Smads. Ainsi, dans les lymphocytes B, l'utilisation du MMTV a permis de mettre en ��vidence une nouvelle synergie entre les glueocortieo��des et le TGF-p. pans ce travail, l'utilisation d'inhibiteurs pharmacologiques et de mutants �� dominant-n��gatifs �� nous a pet mis de d��montrer qu'une Prot��ine Kinase C delta (PKC5) active est impliqu��e dans la transduction du signal lors de la r��ponse au dex ainsi que celle au TGF-P. N��anmoins, la PKC5 est r��gul��e diff��remment dans chaque voie sp��cifique : la voie du TGF-p n��cessitait l'activation du PKC5 par diacylglycerol (DAG) et la phosphorylation de tyrosines sp��cifiques, alors que la voie impliquant les glucocortico��des ne le n��cessitait pas. Nous avons aussi d��montr�� qu'une tyrosine kinase de la famille Src est responsable de la phosphorylation des tyrosines sur la PKC5. Les essais de kinase in vitro nous ont permis de d��couvrir que plusieurs Src kinases peuvent phosphoryler la PKC6 dans les cellules B et qu'elles ��taient constitutivement actives. Enfin, nous avons montr�� qu'il existe une interaction prot��ine - prot��ine induite par dex, entre le r��cepteur aux glucocortico��des (GR) et la PKC5 dans les cellules B, une association qui n'a pas ��t�� d��montr��e auparavant. Par ailleurs, nous avons analys�� les domaines d'interactions entre PKC5 et GR en utilisant les essais de ��GST pull-down��. Nos r��sultats montrent que le domaine r��gulateur de la PKC5 et celui qui interagit avec l'ADN du GR sont impliqu��s. En r��sum��, nous avons trouv�� que dans une lign��e lymphocytaire B, le virus MMTV utilise des m��canismes pour r��guler �� la fois la transcription et la voie de signalisation qui sont diff��rents de ceux utilis��s dans les cellules mammaires ��pith��liales et les fibroblastes. Nos d��couvertes pourraient ��tre utilis��es comme mod��les pour l'��tude de g��nes cellulaires impliqu��s dans des processus tels qu'inflammation, immunit�� ou canc��rog��n��se. Summary: Mouse Mammary Tumor Virus (MMTV) is a retrovirus that causes tumors in the mammary glands of susceptible female mice and has adapted evolutionarily to be expressed in specialized cells. The B lymphocytes are the first cells to be infected by the MMTV and are essential for the spread of infection to the mammary glands. Here, we used the MMTV as a model system to investigate the signalling cascade induced by giucocorticoids (dexamethasone, "dex", a steroid hormone), and by Transforming Growth Factor-beta (TGF-P, a cytokine) leading to its transcriptional activation in B lymphocytes. By itself, TGF-I3 does not affect the basal activity of the MMTV promoter. However, TGF-13 significantly increases glucocorticoid-induced expression, through its effectors, the Smad factors. Thus, MMTV in B cells demonstrates a novel synergism between glucocorticoids and TGF-16. In this thesis project, we present evidence, based on the use of pharmacological inhibitors and of dominant-negative mutants, that an active Protein Kinase C delta (PKC6) is required as a signal transducer for the dex response and for the TGF-P superinduction as well. The PKC6 is differentially regulated in each specific pathway: whereas the TGF-13 superinduction required PKC6 to be activated by diacylglycerol (DAG) and to be phosphorylated at specific tyrosine residues, the glueocorticoid-induced pathway did not. We also showed that a protein tyrosine kinase of the Src family is responsible for the phosphorylation of tyrosines on PKC6. By performing in vitro kinase assays, we found that several Src kinases of B cells were able to phosphorylate PKC6 and that they were constitutively active. Finally, we demonstrate a dex-dependent functional protein-protein interaction between the glucocorticoid receptor (GR) and PKC6 in B cells, an association that has not been previously described. We further analysed the interacting domains of PKG6 and GR using in vitro GST pull-down assays, whereby the regulatory domain of PKC6 and the extended DNA-binding domain of the GR were involved. In summary, we found that in B-lymphoid cell lines, MMTV uses novel mechanisms of transcriptional control and signal transduction that are different from those at work in mammary epithelial or fibroblastic cells. These findings will be used as model for cellular genes involved in cellular processes such as immune functions, inflammation, or oncogenic transformation that may have a similar pattern of regulation.

Cambrai, rue des Prisons [d��g��ts dus �� la guerre] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55495

Cambrai, rue des Carmes [d��g��ts dus �� la guerre] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55502

Soissons, fa��ade Sud de St-Jean-des-Vignes [abbaye, d��g��ts dus �� la guerre] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55106

Verdun, rue des Rouyers [d��g��ts dus aux bombardements] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55250

Verdun, rue des Hauts Fins [d��g��ts dus aux bombardements] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55244

Verdun, rue des Capucins [d��g��ts dus aux bombardements] : [photographie de presse] / [Agence Rol]

R��f��rence bibliographique : Rol, 55247

Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death.

We have explored in vitro the mechanism by which human immunodeficiency virus, type 1 (HIV-1) induces cell death of primary CD4+ T cells in conditions of productive infection. Although HIV-1 infection primed phytohemagglutinin-activated CD4+ T cells for death induced by anti-CD95 antibody, T cell death was not prevented by a CD95-Fc decoy receptor, nor by decoy receptors of other members of the TNFR family (TNFR1/R2, TRAILR1/R2/OPG, TRAMP) or by various blocking antibodies, suggesting that triggering of death receptors by their cognate ligands is not involved in HIV-induced CD4 T cell death. HIV-1 induced CD4 T cell shrinkage, cell surface exposure of phosphatidylserine, loss of mitochondrial membrane potential (Deltapsim), and mitochondrial release of cytochrome c and apoptosis-inducing factor. A typical apoptotic phenotype (nuclear chromatin condensation and fragmentation) only occurred in around half of the dying cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a broad spectrum caspase inhibitor, prevented nuclear chromatin condensation and fragmentation in HIV-infected CD4+ T cells and in a cell-free system (in which nuclei were incubated with cytoplasmic extracts from the HIV-infected CD4+ T cells). Nevertheless, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not prevent mitochondrial membrane potential loss and cell death, suggesting that caspases are dispensable for HIV-mediated cell death. Our findings suggest a major role of the mitochondria in the process of CD4 T cell death induced by HIV, in which targeting of Bax to the mitochondria may be involved.

Protection from HIV-1 infection of primary CD4 T cells by CCR5 silencing is effective for the full spectrum of CCR5 expression.

Stable gene silencing by RNA interference (RNAi) can be achieved by expression of small hairpin RNAs (shRNAs) from RNA polymerase III promoters. We have tested lentiviral vectors expressing shRNAs targetting CCR5 in primary CD4 T cells from donors representing various CCR5 and CCR2 genetic backgrounds covering the full spectrum of CCR5 expression levels and permissiveness for HIV-1 infection. A linear decrease in CCR5 expression resulted in a logarithmic decrease in cellular infection, giving up to three logs protection from HIV-1 infection in vitro. Protection was maintained at very high multiplicity of infection. This and other recent reports on RNAi should open a debate about the use of RNAi gene therapy for HIV infection.