Lentiviral-Mediated Transcriptional Targeting of Dendritic Cells for Induction of T Cell Tolerance In Vivo

Abstract Dendritic cells (DCs) are important APCs able to induce both tolerance and immunity. Therefore, DCs are attractive targets for immune intervention. However, the ex vivo generation and manipulation of DCs at sufficient numbers and without changing their original phenotypic and functional characteristics are major obstacles. To manipulate DCs in vivo, we developed a novel DC-specific self-inactivating lentiviral vector system using the 5′ untranslated region from the DC-STAMP gene as a putative promoter region. We show that a gene therapy approach with these DC-STAMP-lentiviral vectors yields long-term and cell-selective transgene expression in vivo. Furthermore, transcriptionally targeted DCs induced functional, Ag-specific CD4 and CD8 T cell tolerance in vivo, which could not be broken by viral immunization. Tolerized CTL were unable to induce autoimmune diabetes in a murine autoimmune model system. Therefore, delivering transgenes specifically to DCs by using viral vectors might be a promising tool in gene therapy

Immunocytochemical localization of the alpha-1B adrenergic receptor and the contribution of this and the other subtypes to vascular smooth muscle contraction: analysis with selective ligands and antisense

ABSTRACT The contribution of the alpha-1B adrenergic receptor (AR) to vascular smooth muscle contraction has been assessed using a combination of immunological, molecular biological and pharmacological approaches. A subtype-selective antibody detected alpha-1B immunoreactivity in the medial layer of the aorta, caudal, femoral, iliac, mesenteric resistance, renal and superior mesenteric arteries. Receptor protection assays and antisense oligonucleotides were used to assess the contribution of the alpha-1B AR to contraction. The alpha-1B AR was implicated in mediating the phenylephrine-induced contraction of the mesenteric resistance artery. The alpha-1D AR was implicated in mediating the contraction of the aorta, femoral, iliac and superior mesenteric arteries. Similarly, the alpha-1A AR was implicated in mediating contraction of the caudal and renal arteries. In vivo application of antisense oligonucleotides targeted to the translational start site of the alpha-1B AR had no effect on the phenylephrine-induced contraction of the femoral or renal arteries. In contrast, antisense oligonucleotides directed against the alpha-1D AR significantly inhibited the phenylephrine response in the femoral artery but had no effect on the renal artery. Application of alpha-1A AR antisense oligonucleotides inhibited the contraction of the renal artery without effect on the femoral artery. These data show that (1) alpha-1B AR immunoreactivity is widely distributed in the same peripheral arteries in which previous studies detected its mRNA, and (2) despite this distribution, receptor protection and antisense oligonucleotide studies indicate that the alpha-1B AR mediates the contraction of only the mesenteric resistance artery. The alpha-1 AR family is a member of the G proteincoupled superfamily of receptors. In analogy to bacteriorhodopsin, these receptors have the now very familiar proposed structure of seven transmembrane spanning domains connected by hydrophilic loops alternately exposed to the extracellular and intracellular environment. The structure of G protein-coupled receptors in general and alpha-1 ARs specifically have been the subject of several reviews (Bylund et a

Roquin Paralogs 1 and 2 Redundantly Repress the Icos and Ox40 Costimulator mRNAs and Control Follicular Helper T Cell Differentiation

SummaryThe Roquin-1 protein binds to messenger RNAs (mRNAs) and regulates gene expression posttranscriptionally. A single point mutation in Roquin-1, but not gene ablation, increases follicular helper T (Tfh) cell numbers and causes lupus-like autoimmune disease in mice. In T cells, we did not identify a unique role for the much lower expressed paralog Roquin-2. However, combined ablation of both genes induced accumulation of T cells with an effector and follicular helper phenotype. We showed that Roquin-1 and Roquin-2 proteins redundantly repressed the mRNA of inducible costimulator (Icos) and identified the Ox40 costimulatory receptor as another shared mRNA target. Combined acute deletion increased Ox40 signaling, as well as Irf4 expression, and imposed Tfh differentiation on CD4+ T cells. These data imply that both proteins maintain tolerance by preventing inappropriate T cell activation and Tfh cell differentiation, and that Roquin-2 compensates in the absence of Roquin-1, but not in the presence of its mutated form

Lentiviral-mediated transcriptional targeting of dendritic cells for induction of T cell tolerance in vivo

Dendritic cells (DCs) are important APCs able to induce both tolerance and immunity. Therefore, DCs are attractive targets for immune intervention. However, the ex vivo generation and manipulation of DCs at sufficient numbers and without changing their original phenotypic and functional characteristics are major obstacles. To manipulate DCs in vivo, we developed a novel DCspecific self-inactivating lentiviral vector system using the 5 untranslated region from the DC-STAMP gene as a putative promoter region. We show that a gene therapy approach with these DC-STAMP-lentiviral vectors yields long-term and cell-selective transgene expression in vivo. Furthermore, transcriptionally targeted DCs induced functional, Ag-specific CD4 and CD8 T cell tolerance in vivo, which could not be broken by viral immunization. Tolerized CTL were unable to induce autoimmune diabetes in a murine autoimmune model system. Therefore, delivering transgenes specifically to DCs by using viral vectors might be a promising tool in gene therapy

Cleavage of roquin and regnase-1 by the paracaspase MALT1 releases their cooperatively repressed targets to promote TH17 differentiation

Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (TFH cells) is linked to mutation of the gene encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and accumulated as cells of the TH17 subset of helper T cells in the lungs. Roquin inhibited TH17 cell differentiation and acted together with the endoribonuclease regnase-1 to repress target mRNA encoding the TH17 cell-promoting factors IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ. This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase MALT1. Thus, this pathway acts as a 'rheostat' by translating TCR signal strength via graded inactivation of post-transcriptional repressors and differential derepression of targets to enhance TH17 differentiation