Susanne Rößner

Repetitive Injections of Dendritic Cells Matured with Tumor Necrosis Factor α 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)-α (TNF/DCs) induce antigen-specific protection from experimental autoimmune encephalomyelitis (EAE) in mice. Maturation by TNF-α 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-α results in incompletely matured DCs (semi-mature DCs) which induce peptide-specific IL-10–producing T cells in vivo and prevent EAE.

Myeloid‐derived suppressor cell activation by combined LPS and IFN‐γ treatment impairs DC development

Myeloid‐derived suppressor cells (MDSC) and DC are major controllers of immune responses against tumors or infections. However, it remains unclear how DC development and MDSC suppressor activity both generated from myeloid precursor cells are regulated. Here, we show that the combined treatment of BM‐derived MDSC with LPS plus IFN‐γ inhibited the DC development but enhanced MDSC functions, such as NO release and T‐cell suppression. This was not observed by the single treatments in vitro. In the spleens of healthy mice, we identified two Gr‐1lowCD11bhighLy‐6ChighSSClowMo‐MDSC and Gr‐1highCD11blowPMN‐MDSC populations with suppressive potential, whereas Gr‐1highCD11bhigh neutrophils and Gr‐1lowCD11bhighSSClow eosinophils were not suppressive. Injections of LPS plus IFN‐γ expanded these populations within the spleen but not LN leading to the block of the proliferation of CD8+ T cells. At the same time, their capacity to develop into DC was impaired. Together, our data suggest that spleens of healthy mice contain two subsets of MDSC with suppressive potential. A two‐signal‐program through combined LPS and IFN‐γ treatment expands and fully activates MDSC in vitro and in vivo.

Myeloid dendritic cell precursors generated from bone marrow suppress T cell responses via cell contact and nitric oxide production in vitro

Tolerogenic activity of myeloid dendritic cells (DC) has so far been attributed mostly to immature or semi‐mature differentiation stages but never to their precursor cells. Although myeloid suppressor cells (MSC) have been isolated ex vivo, their developmental relationship to DC and their precise phenotype remained elusive. Here, we describe the generation of MSC as myeloid DC precursors with potent suppressive activity on allogeneic and OVA‐specific CD4+ and CD8+ T cell responses in vitro. These MSC appear transiently in DC cultures of bone marrow (BM) cells after 8–10 days under low GM‐CSF conditions or after 3–4 days under high GM‐CSF conditions. They represent CD11c– myeloid precursor cells with ring‐shaped nuclei and are Gr‐1low (i.e. Ly‐6C+, Ly‐6Glow), CD11b+, CD31+, ER‐MP58+, asialoGM1+ and F4/80+. Sorted MSC develop into CD11c+ DC within 6 days. Their suppressor activity partially depends on IFN‐γ stimulation. Suppression is mediated through mechanisms requiring cell contact and nitric oxide but is independent of TNF, CD1d and TGF‐β. Together, our data describe the generation of MSC with distinct suppressor mechanisms in vitro preceding their development into immature DC.

Culture of bone marrow cells in GM-CSF plus high doses of lipopolysaccharide generates exclusively immature dendritic cells which induce alloantigen-specific CD4 T cell anergy in vitro

Dendritic cells (DC) can be generated from mouse bone marrow (BM) in the presence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Bacterial stimuli such as endotoxin / lipopolysaccharide (LPS) can induce their final maturation. When BM‐DC cultures were treated at day 6 or later with LPS, this final maturation was induced in vitro within 24 h. Such mature DC exhibited high levels of surface MHC II molecules and potent T cell sensitizing, but reduced endocytosis capacity. In contrast, immature DC express only few MHC II molecules and are weak T cell stimulators but highly endocytic. When BM‐DC cultures in GM‐CSF were treated with 1 μg / ml LPS at day 0 of the culture or throughout the culture, only immature DC developed as defined by phenotype (MHC II low) and function (high endocytosis, weak primary mixed lymphocyte reaction). Those early LPS‐treated immature DC induced alloantigen‐specific anergy of CD4+ T cells in vitro. These findings might contribute to the understanding of reduced T cell immunity in the course of septic shock and find application in DC‐mediated tolerogenic immunotherapy strategies.

Inhibition of the proteasome influences murine and human dendritic cell development in vitro and in vivo.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) known today and are designated as natures adjuvant since they are the only antigen-presenting cell type capable of inducing naïve T cell responses in vivo. In order to become potent T cell stimulators DC have to mature. This mature DC phenotype is characterized amongst other characteristics by the up-regulation of co-stimulatory molecules such as CD40, CD80, CD86 and the cell surface expression of CD83. Inhibition of their expression blocks the immune responses in vitro and in vivo, and thus represents an interesting strategy to control undesired and/or over-activated immune responses such as in autoimmune disorders, transplant rejections and allergies. Here we investigated the in vitro and in vivo effects of the proteasome inhibitor Velcade in respect to DC phenotype and DC functions in murine and human DC. Interestingly, in vitro, DC maturation as well as DC-mediated T cell stimulation and cytokine production was impaired. Furthermore, administration of the inhibitor in vivo resulted in a reduced mature phenotype of ex vivo generated murine DC. Thus, inhibition of the proteasome interferes with DC maturation and subsequently with DC-mediated T cell stimulation events.

Modulation of murine bone marrow-derived dendritic cells and B-cells by MCS-18 a natural product isolated from Helleborus purpurascens.

MCS-18, a natural product isolated from Helleborus purpurascens has been shown to have several beneficial effects in inflammatory and autoimmune disorders. However, very little is known regarding the immuno-modulatory capacity of MCS-18 in respect to murine bone marrow-derived dendritic cells (BM-DC) and B-cells. Thus, in the present study we examined the effect of MCS-18 on murine BM-DC and B-cells. Interestingly MCS-18 inhibited the expression of important DC-specific molecules and lead to an impaired T-cell stimulation capacity. In addition, MCS-18 also reduced B-cell proliferation and immunoglobulin production.