Thomas Felzmann

Interferon-γ–triggered indoleamine 2,3-dioxygenase competence in human monocyte-derived dendritic cells induces regulatory activity in allogeneic T cells

The role of the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO) in down-regulating human alloresponses has recently been controversially debated. We here demonstrate that human monocyte-derived dendritic cells (mDCs) can be endowed with sustained IDO competence in vitro by 48-hour activation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). IFN-gamma also amplified proinflammatory cytokine secretion during activation. Yet, on reculture after activation cytokine production ceased, whereas IDO enzymatic activity continued. Manipulation of tryptophan metabolism did not affect proinflammatory cytokine release, suggesting that IFN-gamma triggers IDO activity and proinflammatory cytokine release as distinct cellular programs. IDO-competent DCs down-regulated allogeneic T-cell responses, but this IDO-mediated effect was overcome by slightly modifying cell culture conditions. Nevertheless, the CD4(+)CD25(+) T-cell fraction stimulated by IDO-competent DCs displayed substantial suppressor activity. This suppressive activity (1) required allogeneic stimulation for its induction, (2) affected third-party T cells, and (3) was reduced by the IDO inhibitor methyl-thiohydantoin-tryptophan. It became also manifest when DC/T-cell cocultures were initiated with naive (CD4(+)CD25(-)CD45RA(+)) T cells, indicating the differentiation of adaptive regulatory T cells. Together, these findings suggest that IFN-gamma triggered IDO competence in human mDCs constitutes a critical factor for endowing allogeneic T cells with regulatory activity.

Phase I study of tumor Ag-loaded IL-12 secreting semi-mature DC for the treatment of pediatric cancer

BACKGROUND Cancer vaccines employing DC in their capacity as APC have been tolerated well and have shown some efficacy in clinical studies. IL-12, a cytokine critical for type 1 T-helper (Th1) lymphocyte and cytotoxic T-lymphocyte (CTL) differentiation, when released from a DC-based cancer vaccine, may support the generation of a cellular T-cell response. METHODS We applied tumor cell lysate plus keyhole limpet hemocyanin (KLH)-loaded and 48-h lipopolysaccharide (LPS) plus IFN-gamma-stimulated fully mature DC, which do not release IL-12, subcutaneously to eight patients, and maximally 6-h stimulated semi-mature (sm) DC, which are potent producers of IL-12, subcutaneously (n=6) or intranodally (n=8) as a cancer vaccine to patients suffering from advanced solid pediatric malignancies. RESULTS No serious adverse events were observed following application of IL-12-releasing smDC. Following immunization the majority of patients responded positively to KLH in a delayed-type hypersensitivity (DTH) test. In addition, three of six intranodally treated patients responded to the tumor Ag in the DTH test. DISCUSSION We conclude that treatment with a DC-based cancer vaccine enabled to release the immune regulatory cytokine IL-12 is safe and feasible and has the potential to induce a cellular immune response in pediatric cancer patients.

Monocyte enrichment from leukapharesis products for the generation of DCs by plastic adherence, or by positive or negative selection.

BACKGROUND DCs for use in immunotherapy are frequently generated from peripheral blood monocytes. However, there are different approaches to monocyte enrichment. METHOD Plastic adherence is a widely used method for the enrichment of monocytes collected in a leukapheresis procedure. Alternatively,monocytes may be enriched by positive selection using magnetic beads coupled to CD14 Abs, or by cell depletion using beads coupled to Abs against CD2 and CD19 to remove non-monocytes. RESULTS Positive selection resulted in the highest purity of immature DCs (97 +/- 1%), but in a low yield (8 +/- 3%). In contrast, depletion of non-monocytes gave a good yield (21 +/- 6%), but insufficient purity (42 +/- 10%). Conventional adherence procedures resulted in a good yield (25 +/- 5%) and reasonable purity (72 +/- 4%). All three monocyte enrichment procedures resulted in DCs that underwent maturation upon exposure to a combination of lipopolysaccharide and IFN-gamma. These DCs had a typical immune phenotype, they released similar amounts of IL-12, and had the capacity to support MLR. CONCLUSION Our data provide a basis to choose a monocyte enrichment procedure that favors high purity or a high yield. However, if a manual open system suffices, plastic adherence is a reasonable alternative.

Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines

Manufacturing procedures for cellular therapies are continuously improved with particular emphasis on product safety. We previously developed a dendritic cell (DC) cancer vaccine technology platform that uses clinical grade lipopolysaccharide (LPS) and interferon (IFN)‐y for the maturation of monocyte derived DCs. DCs are frozen after 6 hrs exposure at a semi‐mature stage (smDCs) retaining the capacity to secret interleukin (IL)‐12 and thus support cytolytic T‐cell responses, which is lost at full maturation. We compared closed systems for monocyte enrichment from leucocyte apheresis products from healthy individuals using plastic adherence, CD14 selection, or CD2/19 depletion with magnetic beads, or counter flow centrifugation (elutriation) using a clinical grade in comparison to a research grade culture medium for the following DC generation. We found that elutriation was superior compared to the other methods showing 36 ± 4% recovery, which was approximately 5‐fold higher as the most frequently used adherence protocol (8 ± 1%), and a very good purity (92 ± 5%) of smDCs. Immune phenotype and IL‐12 secretion (adherence: 1.4 ± 0.4; selection: 20 ± 0.6; depletion: 1 ±0.5; elutriation: 3.6 ± 1.5 ng/ml) as well as the potency of all DCs to stimulate T cells in an allogeneic mixed leucocyte reaction did not show statistically significant differences. Research grade and clinical grade DC culture media were equally potent and freezing did not impair the functions of smDCs. Finally, we assessed the functional capacity of DC cancer vaccines manufactured for three patients using this optimized procedure thereby demonstrating the feasibility of manufacturing DC cancer vaccines that secret IL‐12 (9.4 ± 6.4 ng/ml). We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing.

Immune Suppression by γδ T-cells as a Potential Regulatory Mechanism After Cancer Vaccination With IL-12 Secreting Dendritic Cells

In cancer patients undergoing immune therapy with lipopolysaccharides/interferon-γ activated interleukin (IL)-12 secreting dendritic cells (DCs) we observed enhanced proliferative capacity of pyrophosphate-responsive peripheral blood (PB) γδ T-cells. This was not noted before as in other clinical trials DCs were used that were not enabled for IL-12 secretion and mice do not have a corresponding subset of PB γδ T-cells. In vitro examination of IL-12 DC/PB γδ T-cell interactions revealed a potential of PB γδ T-cells to negatively regulate the proliferative capacity of CD4 and CD8 T-cells. We further demonstrate that IL-12 is critical in the activation of PB γδ T-cells. In contrast, the regulatory activity of PB γδ T-cells in immune responses against strong recall antigens or alloantigens did not require activated DCs, but depended on pyrophosphate activation of PB γδ T-cells. Depletion of PB γδ T-cells abrogated the regulatory activity in IL-12 DC/peripheral blood mononuclear cell cocultures; adding back graded numbers of PB γδ T-cells restored it. Our observations revealed a potential PB γδ T cell-mediated negative regulatory feedback mechanism triggered by IL-12 DCs, which may critically impact on the design of DC cancer vaccines.

Immunophenotypic characterization of myelomonocytic cells in patients with myelodysplastic syndrome.

Summary. Infections, an important determinating factor in the clinical course of myelodysplastic syndromes (MDS), result in activation of myelomonocytic cells. In this study we demonstrate activation‐associated immunophenotypic changes of cell surface antigens on monocytes and granulocytes observed in two groups of MDS patients, one with low and another one with high clinical risk, and compared them to healthy individuals. Significantly changed expression of the complement receptors 1 (CD35) and 3 (CD11b), the Fcγ receptor I (CD64), the leucocyte‐homing receptor (CD44) and the activation associated membrane proteins CD67 and M5 were found on monocytes and/or granulocytes of MDS patients. In low‐risk MDS patients we observed activation‐associated phenotypic changes only in monocytes, whereas in high‐risk MDS patients, both monocytes and granulocytes showed such changes. Additionally, we performed respiratory burst experiments and observed an impaired response of monocytes and granulocytes derived from MDS patients. Despite the fact that all patients were free of infection by clinical criteria, cell surface phenotyping as well as the reduced respiratory burst capacity of myelomonocytic cells suggests in vivo preactivation of these cells.

Functional maturation of dendritic cells by exposure to CD40L transgenic tumor cells, fibroblasts or keratinocytes.

Tumor antigen pulsed dendritic cells (DCs) can induce anti-tumor immunity. We studied strategies for the reliable generation of such a tumor vaccine by functional maturation of DCs via interaction of CD40 with its ligand (CD40L, CD154). Exposure of immature DCs to CD40L transgenic cells, soluble recombinant human CD40L molecules or lipopolysaccharide induced expression of the co-stimulatory molecules, CD80 and CD86, and supported an allogeneic mixed leukocyte reaction. In contrast, the release of IL-12, an important mediator of anti-tumor immunity, and antigen-specific expansion and IFNgamma secretion of lymphocytes, was strongly triggered only by DCs exposed to CD40L transgenic cells.

Toll-Like Receptor 4 Engagement Drives Differentiation of Human and Murine Dendritic Cells from a Pro- into an Anti-Inflammatory Mode

The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs’ initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.

CD40 ligation restores type 1 polarizing capacity in TLR4-activated dendritic cells that have ceased interleukin-12 expression.

Inflammation triggered by microbial lipopolysaccharide (LPS) through Toll‐like receptor (TLR) 4 in the presence of interferon (IFN)‐γ induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation program. This DC differentiation is characterized not only by a dynamic immune activating but also by tolerance‐inducing phenotype associated with down‐modulation of cytokines previously considered to be irreversible. CD40L on activated T cells further modifies DC differentiation. Using DNA micro‐arrays, we showed down‐regulated mRNA levels of TLR signalling molecules, whereas CD40/CD40L signalling molecules were up‐regulated at a time when LPS/IFN‐γ‐activated DCs had ceased cytokine expression. Accordingly, we demonstrated that CD40/CD40L but not TLR4 or TLR3 signalling mediated by LPS or poly (cytidylic‐inosinic) acid (poly I:C) and dsRNA re‐established the capacity for secreting interleukin (IL)‐12 in primarily LPS/IFN‐γ‐activated DCs, which have exhausted their potential for cytokine secretion. The resulting TH1 polarizing DC phenotype – which lacked accompanying secretion of the crucial immune suppressive factor IL‐10 – maintained the potential for activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T‐cell‐mediated stimulus at an exhausted DC state, which prevents an immune tolerant DC phenotype. These findings impact on the rational design of TLR‐activated DC‐based cancer vaccines for the induction of anti‐tumoural CTL responses.

The MAPK-Activated Kinase MK2 Attenuates Dendritic Cell–Mediated Th1 Differentiation and Autoimmune Encephalomyelitis

Dendritic cell (DC)–mediated inflammation induced via TLRs is promoted by MAPK-activated protein kinase (MK)-2, a substrate of p38 MAPK. In this study we show an opposing role of MK2, by which it consolidates immune regulatory functions in DCs through modulation of p38, ERK1/2-MAPK, and STAT3 signaling. During primary TLR/p38 signaling, MK2 mediates the inhibition of p38 activation and positively cross-regulates ERK1/2 activity, leading to a reduction of IL-12 and IL-1α/β secretion. Consequently, MK2 impairs secondary autocrine IL-1α signaling in DCs, which further decreases the IL-1α/p38 but increases the anti-inflammatory IL-10/STAT3 signaling route. Therefore, the blockade of MK2 activity enables human and murine DCs to strengthen proinflammatory effector mechanisms by promoting IL-1α–mediated Th1 effector functions in vitro. Furthermore, MK2-deficient DCs trigger Th1 differentiation and Ag-specific cytotoxicity in vivo. Finally, wild-type mice immunized with LPS in the presence of an MK2 inhibitor strongly accumulate Th1 cells in their lymph nodes. These observations correlate with a severe clinical course in DC-specific MK2 knockout mice compared with wild-type littermates upon induction of experimental autoimmune encephalitis. Our data suggest that MK2 exerts a profound anti-inflammatory effect that prevents DCs from prolonging excessive Th1 effector T cell functions and autoimmunity.