Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.

T cells are crucial effectors of glioma rejection induced by local IL-12 application and CTLA-4 blockade.

Dendritic cells (DCs) play a central role in shaping immunogenic as well as tolerogenic adaptive immune responses and thereby dictate the outcome of adaptive immunity. Here, we report the generation of a CD8α+ DC line constitutively secreting the tolerogenic cytokine interleukin (IL)-35. IL-35 secretion led to impaired CD4+ and CD8+ T lymphocyte proliferation and interfered with their function in vitro and also in vivo. IL-35 was furthermore found to induce a tolerogenic phenotype on CD8α+ DCs, characterized by the upregulation of CD11b, downregulation of MHC class II, a reduced costimulatory potential as well as production of the immunomodulatory molecule IL-10. Vaccination of mice with IL-35-expressing DCs promoted tumor growth and reduced the severity of autoimmune encephalitis not only in a preventive but also after induction of encephalitogenic T cells. The reduction in experimental autoimmune encephalitis severity was significantly more pronounced when antigen-pulsed IL-35+ DCs were used. These findings suggest a new, indirect effector mechanism by which IL-35-responding antigen-presenting cells contribute to immune tolerance. Furthermore, IL-35-transfected DCs may be a promising approach for immunotherapy in the context of autoimmune diseases.

Because of their unique capacity to cross-present Ags to CD8+ T cells, mouse lymphoid tissue–resident CD8+ dendritic cells (DCs) and their migratory counterparts are critical for priming antiviral T cell responses. High expression of the dsRNA sensor TLR3 is a distinctive feature of these cross-presenting DC subsets. TLR3 engagement in CD8+ DCs promotes cross-presentation and the acquisition of effector functions required for driving antiviral T cell responses. In this study, we performed a comprehensive analysis of the TLR3-induced antiviral program and cell-autonomous immunity in CD8+ DC lines and primary CD8+ DCs. We found that TLR3-ligand polyinosinic-polycytidylic acid and human rhinovirus infection induced a potent antiviral protection against Sendai and vesicular stomatitis virus in a TLR3 and type I IFN receptor–dependent manner. Polyinosinic-polycytidylic acid–induced antiviral genes were identified by mass spectrometry–based proteomics and transcriptomics in the CD8+ DC line. Nanostring nCounter experiments confirmed that these antiviral genes were induced by TLR3 engagement in primary CD8+ DCs, and indicated that many are secondary TLR3-response genes requiring autocrine IFN-β stimulation. TLR3-activation thus establishes a type I IFN–dependent antiviral program in a DC subtype playing crucial roles in priming adaptive antiviral immune responses. This mechanism is likely to shield the priming of antiviral responses against inhibition or abrogation by the viral infection. It could be particularly relevant for viruses detected mainly by TLR3, which may not trigger type I IFN production by DCs that lack TLR3, such as plasmacytoid DCs or CD8− DCs.

Experimental autoimmune myocarditis (EAM) is a CD4 + T-cell-mediated model of human inflammatory dilated cardiomyopathies.Heart-specific CD4 + T-cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation-induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV−/− K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α-myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV−/− K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4 + T cells and in expression of IFN-γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN-γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN-γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis. Keywords: CD4 + T cells Experimental autoimmune myocarditis (EAM) Endothelial antigen presentation Interferon-γ MHC class IIAdditional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Prof. Hans Acha-Orbea e-mail: hans.acha-orbea@unil.ch C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2016. 46: 656-664 Immunomodulation 657 IntroductionInflammatory dilated cardiomyopathy is a common cause of heart failure in young patients and often results from myocarditis in predisposed individuals [1]. CD4 + T-cell-mediated experimental autoimmune myocarditis (EAM) is a model for inflammatory heart disease and is induced in mice either by immunization with α-myosin heavy chain (α-MyHC) derived peptides emulsified in CFA [2][3][4] or by vaccination with α-MyHC-loaded activated dendritic cells (DCs) [5]. Typically, both Th1 and Th17 cells are induced during the course of EAM. It is assumed that both of these subsets mediate myocarditis as mice deficient in either interleukin 17 (IL-17) or interferon γ (IFN-γ) develop cardiac pathology [6,7]. Remarkably, heart-specific autoimmune inflammation has been shown to be a consequence of impaired central tolerance induction to α-MyHC during T-cell development in mice and in humans [8]. CD4 + T-cell-orchestrated immune responses are driven by MHC class II (MHCII) mediated antigen presentation. MHCII is primarily expressed on professional APCs. Cardiac APCs were suggested to present cardiac myosin before the onset of cardiac pathology [9], and DCs, the major APC subset able to initiate primary immune responses, were shown to be critical for ...