T cell derived IL-10 is dispensable for tolerance induction in a murine model of allergic airway inflammation

Regulatory mechanisms initiated by allergen specific immunotherapy are mainly attributed to T cell-derived IL-10. However, it has not been shown that T cell-derived IL-10 is required for successful tolerance induction. Here, we analyze cellular sources and the functional relevance of cell type specific IL-10 during tolerance induction in a murine model of allergic airway inflammation. While tolerance induction was effective in IL-10 competent mice, neutralizing IL-10 prior to tolerogenic treatment completely abrogated the beneficial effects. Cellular sources of IL-10 during tolerance induction were identified by using transcriptional reporter mice as T cells, B cells and to a lesser extent DCs. Interestingly, tolerance induction was still effective in mice with T cell-, B cell-, B and T cell- or DC-specific IL-10 deficiency. In contrast, tolerance induction was not possible in mice lacking IL-10 in all hematopoetic cells, while it was effective in bone marrow chimera that lacked IL-10 only in non-hematopoetic cells. Taken together, allergen specific tolerance depends on IL-10 from hematopoetic sources. The beneficial effects of allergen specific immunotherapy cannot solely be attributed to IL-10 from T cells, B cells or even DCs, suggesting a high degree of cellular redundancy in IL-10 mediated tolerance

Loss of Trex1 in Dendritic Cells Is Sufficient To Trigger Systemic Autoimmunity

Defects of the intracellular enzyme 3' repair exonuclease 1 (Trex1) cause the rare autoimmune condition Aicardi-Goutières syndrome and are associated with systemic lupus erythematosus. Trex1(-/-) mice develop type I IFN-driven autoimmunity, resulting from activation of the cytoplasmic DNA sensor cyclic GMP-AMP synthase by a nucleic acid substrate of Trex1 that remains unknown. To identify cell types responsible for initiation of autoimmunity, we generated conditional Trex1 knockout mice. Loss of Trex1 in dendritic cells was sufficient to cause IFN release and autoimmunity, whereas Trex1-deficient keratinocytes and microglia produced IFN but did not induce inflammation. In contrast, B cells, cardiomyocytes, neurons, and astrocytes did not show any detectable response to the inactivation of Trex1. Thus, individual cell types differentially respond to the loss of Trex1, and Trex1 expression in dendritic cells is essential to prevent breakdown of self-tolerance ensuing from aberrant detection of endogenous DNA

Immunogene und immunsuppressive Eigenschaften des transmembranen Hüllproteins gp41 von HIV

Die Entwicklung eines effektiven HIV-Impfstoffes ist bis heute nicht gelungen.Konventionelle Immunisierungsstrategien mit rekombinant hergestellten Hüllproteinen des Virus in verschiedensten Formen induzierten keine subtypenübergreifende, protektive Immunantwort gegen HIV. Die Gewinnung und Charakterisierung der gp41-spezifischen breit neutralisierenden monoklonalen Antikörper 2F5 und 4E10 bildete die Grundlage einer Reihe neuer epitopgerichteter Ansätze für die HIV-Impfstoffentwicklung. Bisherige Immunisierungsstudien basierten auf der Verwendung des linearen Hauptepitopes (E2) der beiden Antikörper aus dem C-terminalen Teil der Ektodomäne von gp41. Nach neueren Erkenntnissen, reicht für eine effektive Neutralisation durch 2F5 oder 4E10 die Bindung dieser Antikörper an ihr lineares Epitop in der membran proximalen externen Region (MPER) von gp41 allein nicht aus. Vielmehr wurde die Beteiligung einer N-terminalen Domäne (E1) von gp41 an der neutralisationsaktiven Bindung von 2F5 bzw. 4E10 postuliert. In dieser Arbeit wurden die beiden 2F5 und 4E10 spezifischen Epitopbereiche E1 und E2 des gp41 erstmals in das strukturell verwandte transmembrane Hüllprotein des Koala Retrovirus (KoRV) eingebracht. Die Applikation der hergestellten Antigene erfolgte sowohl in Form der codierenden DNA mittels ballistischer Immunisierung (GeneGun®) als auch durch bakteriell exprimierte Proteine. Mit beiden Strategien konnten für drei Hybridproteine in den ersten Studien eine HIV-1 gp41 spezifische, breit neutralisierende humorale Immunantwort induziert werden. Diese Ergebnisse konnten jedoch in späteren Studien nicht reproduziert werden. Die Analyse der induzierten Immunantworten zeigte eine Verlagerung der Hauptimmunantwort als deren Ursache eine bakterielle Fremdinfektion der Versuchtiere diskutiert wurde. Zur Evaluierung der Immunisierungsstudien wurde ein neuartiger real time PCR basierter in vitro Neutralisationstest um Kontrollen zur Virusspezifität und Cytotoxizität erweitert.The development of an effective HIV vaccine is considered the to play a key role in controlling the HIV pandemic. Conventional immunisation strategies using recombinant envelope proteins of the virus did not lead to the induction of a broad range protective immunity. A new target sequence for the induction of a broadly neutralising humoral immune response has been discovered through the characterization of the gp41 specific broadly neutralising monoclonal antibodies 2F5 and 4E10. Until now all attempts to induce 2F5/4E10 like neutralising antibodies failed. So far only the linear main epitope (E2) of 2F5 and 4E10, located in the C-terminal part of the gp41 ectodomain was used as the target sequence. However, it was recently shown that an N-terminal domain (E1) of gp41 increases the avidity of 2F5 to its epitope. The E1 domain may therefore be involved in the mediation of a neutralisation active binding. For the first time immunisation strategies have been developed that target both previously identified domains (E1 and E2) of gp41. The sequences corresponding to E1 and E2 have been introduced at homologous positions in the structurally related transmembrane envelope protein p15E of the Koala Retrovirus (KoRV). These generated hybrid antigens have been used for immunisation of wistar rats. They were applied as recombinant proteins expressed in E.coli and as DNA using a ballistic immunisation (GeneGun®) approach. Although in first trials neutralising antibodies specific for gp41 of HIV-1 were induced, these results could not be reproduced. Analysis of the induced antibodies showed a shift of their binding specifity. A bacterial infection of the used animals was identified as the cause of the unexpected shift in the antigen specific humoral immune response. For evaluation of the immunisation studies a new neutralisation assay based on the measurement of provirus integration by duplex real time PCR has been extended for controls of virus specifity and cytotoxicity

Induction of Antibodies Specific for Gp41 of HIV-1 by Gene Gun DNA Vaccination

All attempts to induce broadly neutralising antibodies such as mAb 2F5 and mAb 4E10 targeting conserved epitopes in the membrane proximal external region (MPER) of the transmembrane envelope (TM) protein gp41 of HIV-1 failed so far. In contrast, in previous studies, immunising with the ectodomain of the TM protein p15E of different gammaretroviruses, we successfully induced neutralising antibodies. These antibodies recognised epitopes located in the fusion peptide proximal region (FPPR) and in the MPER of p15E. The epitope in the MPER of p15E corresponds to that of the mAb 4E10 in gp41 in terms of location within the protein and partial sequence homology. In order to present the MPER of gp41 (containing the 2F5 and 4E10 epitopes) membrane-associated, rats were immunised with DNA constructs corresponding (i) to the entire gp41, (ii) to the C-terminal helix of gp41 and (iii) to hybrid proteins composed of a backbone derived from p15E of a gammaretrovirus with inserted FPPR and MPER from gp41 of HIV-1. After transfection in vitro these proteins were found expressed at the cell surface and the accessibility of the 2F5 epitope was demonstrated by flow cytometry. However, DNA vaccination in rats resulted only in low titres of antibodies specific for the MPER of HIV-1, and none of the sera was neutralising