Elisabeth Zinser

Prevention and Treatment of Experimental Autoimmune Encephalomyelitis by Soluble CD83

CD83 is up-regulated on the surface of dendritic cells (DCs) during maturation and has been widely used as a marker for mature DCs. Recently, we reported the recombinant expression of the extracellular immunoglobulin domain of human CD83 (hCD83ext). Using this soluble form of CD83, allogeneic as well as specific cytotoxic T lymphocyte proliferation could be blocked in vitro. Here we report the functional analysis of soluble CD83 in vivo, using murine experimental autoimmune encephalomyelitis (EAE) as a model. Strikingly, only three injections of soluble CD83 prevented the paralysis associated with EAE almost completely. In addition, even when the EAE was induced a second time, CD83-treated mice were protected, indicating a long-lasting suppressive effect. Furthermore, soluble CD83 strongly reduced the paralysis in different therapeutic settings. Most important, even when the treatment was delayed until the disease symptoms were fully established, soluble CD83 clearly reduced the paralyses. In addition, also when EAE was induced a second time, soluble CD83-treated animals showed reduced disease symptoms. Finally, hCD83ext treatment almost completely reduced leukocyte infiltration in the brain and in the spinal cord. In summary, this work strongly supports an immunosuppressive role of soluble CD83, thereby indicating its therapeutic potential in the regulation of immune disorders in vivo.

Role of CD83 in the immunomodulation of dendritic cells

Glycoprotein CD83 is one of the best-known maturation markers for human dendritic cells (DCs). The fact that CD83 is strongly upregulated together with co-stimulatory molecules such as CD80 and CD86 during DC maturation suggests it plays an important role in the induction of immune responses. Infection studies with herpes simplex virus type 1 (HSV-1) and the inhibition of the CD83 mRNA specific transport from the nucleus to the cytoplasm suggested a possible functional role for CD83. The first clear proof that CD83 is indeed important for DC biology came from recently performed studies using a soluble form of the extracellular CD83 domain. DC-mediated T cell proliferation could be completely inhibited using this recombinant molecule. Additional studies elucidated immunostimulatory as well as regulatory effects of the CD83 molecule. Furthermore, CD83–/– knockout mice revealed a block in CD4+ T cell generation, a new possible immunomodulatory function of CD83.

Immunosuppression Involving Soluble CD83 Induces Tolerogenic Dendritic Cells That Prevent Cardiac Allograft Rejection

Background. Dendritic cells (DCs) are crucial regulators of immunity and important in inducing and maintaining tolerance. Here, we investigated the potential of a novel DC-immunomodulating agent, soluble CD83 (sCD83), in inducing transplant tolerance. Methods. We used the C3H-to-C57BL/6 mouse cardiac transplantation model that exhibits a combination of severe cell-mediated rejection and moderate antibody-mediated rejection and investigated whether sCD83 could augment a combination therapy consisting of Rapamycin (Rapa) and anti-CD45RB monoclonal antibody (&agr;-CD45) to prolong allograft survival. Results. Monotherapies consisting of Rapa and &agr;-CD45 were incapable of preventing rejection. However, all treatments involving sCD83 were capable of (1) down-modulating expression of various DC surface molecules, such as major histocompatibility complex class II and costimulatory molecules, (2) reducing the allogeneic stimulatory capacity of the DCs, and (3) significantly inhibiting antidonor antibody responses. Most striking results were observed in the triple therapy-treated group, sCD83+Rapa+&agr;-CD45, where cell-mediated rejection and antibody-mediated rejection were abrogated for over 100 days. Donor-specific tolerance was achieved in long-term surviving recipients, because donor skin transplants were readily accepted for an additional 100 days, whereas third-party skin grafts were rejected. Success of triple therapy treatment was accompanied by enhancement of tolerogenic-DCs that conferred antigen-specific protection on adoptive transfer to recipients of an allogeneic heart graft. Conclusions. Our study revealed that sCD83 is capable of attenuating DC maturation and function, and inducing donor-specific allograft tolerance, in the absence of toxicity. Thus, sCD83 seems to be a safe and valuable counterpart to current DC-modulating agents.

Denileukin diftitox (ONTAK) induces a tolerogenic phenotype in dendritic cells and stimulates survival of resting Treg

Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.

Topical Application of Soluble CD83 Induces IDO-Mediated Immune Modulation, Increases Foxp3+ T Cells, and Prolongs Allogeneic Corneal Graft Survival

Modulation of immune responses is one of the main research aims in transplant immunology. In this study, we investigate the local immunomodulatory properties of soluble CD83 (sCD83) at the graft-host interface using the high-risk corneal transplantation model. In this model, which mimics the inflammatory status and the preexisting vascularization of high-risk patients undergoing corneal transplantation, allogeneic donor corneas are transplanted onto sCD83-treated recipient animals. This model allows the direct and precise application of the immune modulator at the transplantation side. Interestingly, sCD83 was able to prolong graft survival after systemic application as well as after topical application, which is therapeutically more relevant. The therapeutic effect was accompanied by an increase in the frequency of regulatory T cells and was mediated by the immune-regulatory enzyme IDO and TGF-β. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-β, a cytokine previously shown to be an essential mediator of IDO-dependent, long-term tolerance. These findings open new treatment avenues for local immune modulation after organ and tissue transplantation.

12/15-Lipoxygenase-mediated enzymatic lipid oxidation regulates DC maturation and function.

DCs are able to undergo rapid maturation, which subsequently allows them to initiate and orchestrate T cell-driven immune responses. DC maturation must be tightly controlled in order to avoid random T cell activation and development of autoimmunity. Here, we determined that 12/15-lipoxygenase-meditated (12/15-LO-mediated) enzymatic lipid oxidation regulates DC activation and fine-tunes consecutive T cell responses. Specifically, 12/15-LO activity determined the DC activation threshold via generation of phospholipid oxidation products that induced an antioxidative response dependent on the transcription factor NRF2. Deletion of the 12/15-LO-encoding gene or pharmacologic inhibition of 12/15-LO in murine or human DCs accelerated maturation and shifted the cytokine profile, thereby favoring the differentiation of Th17 cells. Exposure of 12/15-LO-deficient DCs to 12/15-LO-derived oxidized phospholipids attenuated both DC activation and the development of Th17 cells. Analysis of lymphatic tissues from 12/15-LO-deficient mice confirmed enhanced maturation of DCs as well as an increased differentiation of Th17 cells. Moreover, experimental autoimmune encephalomyelitis in mice lacking 12/15-LO resulted in an exacerbated Th17-driven autoimmune disease. Together, our data reveal that 12/15-LO controls maturation of DCs and implicate enzymatic lipid oxidation in shaping the adaptive immune response.

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.

Leukoreduction system chambers are an efficient, valid, and economic source of functional monocyte-derived dendritic cells and lymphocytes

The demand for human monocyte-derived dendritic cells (moDCs), as well as for primary human B and T lymphocytes for immunological research purposes has been increased in recent years. Classically, these monocytes are isolated from blood, leukapheresis products or buffy coats of healthy donors by plastic adherence of peripheral blood mononuclear cells (PBMCs), followed by stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, while lymphocytes are usually isolated from the non-adherent fraction (NAF) by magnetic cell sorting. However, donor-blood is a limited resource and not every blood bank offers leukapheresis products or buffy coats for laboratory use. Additionally, a leukapheresis is very expensive and also the generation/isolation of cells is time- and cost-intensive. To overcome some of these obstacles, we evaluated if low-cost leukoreduction system chambers (LRSCs), which arise after routine donor plateletpheresis procedures, and are usually discarded, would be an alternative and appropriate source of PBMCs to generate moDCs and to isolate lymphocytes. By analyzing the number and phenotype of immature and mature dendritic cells (DCs), as well as of B and T lymphocytes derived from LRSCs, we found all cells to be of high quantity and quality. Further investigations on DCs comprising transwell migration assays, allogeneic mixed lymphocyte reactions (MLR), cytokine secretion assays, and cytotoxic T cell induction assays revealed high migratory, as well as stimulatory capacity of these cells. In addition, DCs and T cells were efficiently electroporated with mRNA and showed characteristic cytokine production after co-culture, demonstrating LRSCs as an efficient, valid, and economic source for generation of moDCs and lymphocytes for research purposes.

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.

Teaching Basic Science and Clinical Medicine at a Distance: An Evaluation of Satellite Communication

The evaluation plan arid preliminary results are reported for the ATS‐6 Satellite interactive telecommunication program for teaching at sites remote from an established medical school. The technical aspects of the system were seen as quite adequate and did not appear to substantially inhibit interaction. In the Fairbanks broadcasts analyzed, the average number of interactions ranged from 1.49 to 2.60 per minute. Ninety percent of the interaction flowed across the Satellite during Omak transmissions. Participants tended to initiate communication when they were on camera rather than when viewing their counterparts. Omak participants directed a higher percentage of the communication in student/resident programs (70%) than in faculty continuing education programs (59%). Interaction analysis indicated the categories of giving opinion, information, and suggestion were most frequent. Asking for opinion and suggestion occurred infrequently. The most frequent emotional expression was offering solidarity (e.g., rea...