Minor role of bystander tolerance to fetal calf serum in a peptide-specific dendritic cell vaccine model against autoimmunity: comparison with serum-free cultures.

Dendritic cells (DCs) are currently considered as promising tools for vaccination against tumors and also autoimmune responses. A major point of concern has been the use of fetal calf serum (FCS) as a source of heterologous antigen in DC cultures. FCS peptides can be presented by the DCs and cause T-cell responses in the recipient. We investigated the role of FCS in an autoimmune model where DC injections can prevent peptide-specifically from experimental autoimmune encephalomyelitis (EAE). We show that murine bone marrow-derived DCs generated in FCS-containing or serum-free media resulting in a similar phenotype, maturation potential, and functions. Peptide-specific protection could be achieved similarly with FCS-DC or serum-free DCs. Although FCS-DC induced strong CD4 T cell proliferation and cytokine production against FCS, these T cells lack antigenic recall during EAE. Even if FCS was reinjected, the effect on EAE resulted only in a 3-day delay of disease onset. Together, our data show that presentation of bystander antigens by peptide-specific DC vaccinations may have little influence on T-cell responses in vivo if the bystander antigen cannot be recalled by specific T cells.

Dendritic cells matured with TNF can be further activated in vitro and after subcutaneous injection in vivo which converts their tolerogenicity into immunogenicity.

Dendritic cell (DC) maturation can occur by different types of stimuli. Previously, we described that murine DC matured with tumor necrosis factor (TNF) up-regulate surface MHC and costimulatory molecules but lack cytokine release, and therefore termed them semi-mature DC. These TNF/DC-induced tolerance after intravenous (i.v.) injection in a model of experimental autoimmune encephalomyelitis (EAE). Here, we show that TNF/DC are not terminally differentiated but can still respond to the microbial stimulus lipopolysaccharide. Subcutaneously injected TNF/DC induce an unpolarized T(H)1/T(H)2 response and are not protective in the experimental autoimmune encephalomyelitis model. Although TNF/DC home to the draining lymph node, they remain negative for intracellular cytokine stainings. However, the nonmigrating, endogenous DC started to produce interleukin (IL)-12p40, TNF and little IL-6, IL-10, and MCP-1 in a bystander fashion. Together, DC matured with the inflammatory stimulus TNF remains responsive to further signals in vitro and in vivo. These signals can be provided by pathogens or the subcutaneous injection route, which can convert them from tolerogenic to immunogenic DC. These findings are important for selecting the appropriate injection route of human DC for tumor immunotherapy.

IL-4 supports the generation of a dendritic cell subset from murine bone marrow with altered endocytosis capacity.

Dendritic cells (DC) of myeloid origin can be generated from mouse bone marrow (BM) using granulocyte macrophage-colony stimulating factor (GM-CSF). Immature major histocompatibility complex (MHC) II(low) DC are known to bear a high endocytosis capacity, in contrast to DC precursors and mature DC. Now we found that a subset of MHC II(low) DC in BM-DC cultures is unable to exert mannose receptor-mediated endocytosis of fluorescein isothiocyanate (FITC)-dextran (DX) and resembles immature Langerhans cells (LC). The FITC-DX endocytosis activity of LC-like cells occurs at an earlier stage of development, where the surface MHC II expression is absent or very weak. This LC-like subset expresses higher levels of E-cadherin but lower amounts of the markers Gr-1, scavenger receptor 2F8, and CD11b, when compared with the highly endocytic DC subset. The latter myeloid DC resemble monocyte-derived DC (MoDC). The sorted LC-like population develops completely and exclusively into mature MHC IIhigh DC, and the MoDC-like cells remain immature MHC II(low) DC or develop into adherent MHC IIneg macrophages or mature into MHC IIhigh DC. The development of LC-like cells is promoted by interleukin-4. Thus, we show here that the simultaneous development of LC-like and MoDC-like DC subsets occurs in standard bulk cultures with GM-CSF, suggesting the existence of two different precursors for LC and MoDC in BM.

Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF.

Little is known about the distinct roles of the two types of IL-4R on DC. Here we report that IL-4 and IL-13 are able to promote DC maturation, as evaluated by up-regulation of MHC class II and costimulatory molecules, when the concentration of GM-CSF is relatively lower than the dose of IL-4 or IL-13. In addition, under these conditions both cytokines enable DC to respond to maturation stimuli such as bacterial products or proinflammatory cytokines. Both IL-4 and IL-13 act synergistically with weak maturation stimuli such as TNF-alpha or CD40. The IL-4R signaling for DC maturation requires the IL-4R alpha-chain and STAT6, but not Janus kinase 3, indicating that IL-4R type II signaling is preferentially responsible for these effects. In contrast, the production of IL-12 p70, but not IL-10 and TNF, induced by microbial products was enhanced only by IL-4, not by IL-13 or Y119D, a selective type II IL-4R agonist, in vitro and in vivo. This enhancement was dependent on the presence of Janus kinase 3, indicating that this function is exclusively mediated by the type I IL-4R. In short, we discerned the individual roles of the two IL-4R types on DC function, showing that IL-4R type I promotes IL-12 secretion independently of GM-CSF concentration, while IL-4R type II promotes the up-regulation of MHC class II and costimulatory surface markers in a GM-CSF concentration-dependent manner.

Repetitive injections of dendritic cells matured with tumor necrosis factor alpha induce antigen-specific protection of mice from autoimmunity.

Mature dendritic cells (DCs) are believed to induce T cell immunity, whereas immature DCs induce T cell tolerance. Here we describe that injections of DCs matured with tumor necrosis factor (TNF)-alpha (TNF/DCs) induce antigen-specific protection from experimental autoimmune encephalomyelitis (EAE) in mice. Maturation by TNF-alpha induced high levels of major histocompatibility complex class II and costimulatory molecules on DCs, but they remained weak producers of proinflammatory cytokines. One injection of such TNF/DCs pulsed with auto-antigenic peptide ameliorated the disease score of EAE. This could not be observed with immature DCs or DCs matured with lipopolysaccharide (LPS) plus anti-CD40. Three consecutive injections of peptide-pulsed TNF/DCs derived from wild-type led to the induction of peptide-specific predominantly interleukin (IL)-10-producing CD4(+) T cells and complete protection from EAE. Blocking of IL-10 in vivo could only partially restore the susceptibility to EAE, suggesting an important but not exclusive role of IL-10 for EAE prevention. Notably, the protection was peptide specific, as TNF/DCs pulsed with unrelated peptide could not prevent EAE. In conclusion, this study describes that stimulation by TNF-alpha results in incompletely matured DCs (semi-mature DCs) which induce peptide-specific IL-10-producing T cells in vivo and prevent EAE.