Dendritic cell (DC)-based immunotherapy has been shown to be a promising strategy for anti-cancer therapy. Nevertheless, only a low overall clinical response rate has been observed in vaccinated patients with advanced cancer and therefore methods to improve DC immuno-stimulatory functions are currently under intense investigation. In this respect, we exposed human monocyte-derived DCs to a physiological temperature stress of 40°C for up to 24 h followed by analysis for (i) expression of different heat shock proteins, (ii) survival, (iii) cell surface maturation markers, (iv) cytokine secretion, and (v) migratory capacity. Furthermore, we examined the ability of heat-shocked DCs to prime naïve CD8⁺ T cells after loading with MelanA peptide, by transfection with MelanA RNA, or by transduction with MelanA by an adenovirus vector. The results clearly indicate that in comparison to control DCs, which remained at 37°C, heat-treated cells revealed no differences concerning the survival rate or their migratory capacity. However, DCs exposed to thermal stress showed a time-dependent enhanced expression of the immune-chaperone heat shock protein 70A and both an up-regulation of co-stimulatory molecules such as CD80, CD83, and CD86 and of the inflammatory cytokine TNF-α. Moreover, these cells had a markedly improved capacity to prime autologous naïve CD8⁺ T cells in vitro in an antigen-specific manner, independent of the method of antigen-loading. Thus, our strategy of heat treatment of DCs offers a promising means to improve DC functions during immune activation which, as a physical method, facilitates straight-forward applications in clinical DC vaccination protocols.

Foxp3-positive regulatory T cells (Tregs) are crucial for the maintenance of immune homeostasis and keep immune responses in check. Upon activation, Tregs are transferred into an effector state expressing transcripts essential for their suppressive activity, migration, and survival. However, it is not completely understood how different intrinsic and environmental factors control differentiation. Here, we present for the first time to our knowledge data suggesting that Treg-intrinsic expression of CD83 is essential for Treg differentiation upon activation. Interestingly, mice with Treg-intrinsic CD83 deficiency are characterized by a proinflammatory phenotype. Furthermore, the loss of CD83 expression by Tregs leads to the downregulation of Treg-specific differentiation markers and the induction of an inflammatory profile. In addition, Treg-specific conditional knockout mice showed aggravated autoimmunity and an impaired resolution of inflammation. Altogether, our results show that CD83 expression in Tregs is an essential factor for the development and function of effector Tregs upon activation. Since Tregs play a crucial role in the maintenance of immune tolerance and thus prevention of autoimmune disorders, our findings are also clinically relevant.

The aryl hydrocarbon receptor (AhR) is an environmental sensor and ligand-activated transcription factor that is critically involved in the regulation of inflammatory responses and the induction of tolerance by modulating immune cells. As dendritic cells (DCs) express high AhR levels, they are efficient to induce immunomodulatory effects after being exposed to AhR-activating compounds derived from the environment or diet. To gain new insights into the molecular targets following AhR-activation in human monocyte-derived (mo)DCs, we investigated whether the natural AhR ligand quercetin or the synthetic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) modulates the function of human moDCs regarding their capability to prime naïve T cells or to migrate. As only quercetin, but not TCDD, impaired T cell activation and migration of LPS-matured DCs (LPS-DCs), we analyzed the mode of action of quercetin on moDCs in more detail. Here, we found a specific down-regulation of the immunomodulatory molecule CD83 through the direct binding of the activated AhR to the CD83 promoter. Furthermore, treatment of LPS-DCs with quercetin resulted in a reduced production of the pro-inflammatory cytokine IL-12p70 and in an increased expression of the immunoregulatory molecules disabled adaptor protein (Dab) 2, immunoglobulin-like transcript (ILT)-3, ILT4, ILT5 as well as ectonucleotidases CD39 and CD73, thereby inducing a tolerogenic phenotype in quercetin-treated maturing DCs. Overall, these data demonstrate that quercetin represents a potent immunomodulatory agent to alter human DC phenotype and function, shifting the immune balance from inflammation to resolution.

Background: PARP inhibitors niraparib and talazoparib are FDA approved for special cases of breast cancer. PARP is an interesting repair protein which is frequently affected in cancer cells. We studied the combined action of talazoparib or niraparib with ionizing radiation in melanoma cells and healthy fibroblasts. Methods: Homologous recombination (HR) status in six different melanoma cell lines and healthy fibroblasts was assessed. Cell cultures were treated with PARP inhibitors talazoparib or niraparib and ionizing radiation (IR). Apoptosis, necrosis and cell cycle distribution was analyzed via flow cytometry. Cell migration was studied by scratch assays. Results: Studied melanoma cell cultures are HR deficient. Studied healthy fibroblasts are HR proficient. Talazoparib and niraparib have congruent effects within the same cell cultures. In all cell cultures, combined treatment increases cell death and G2/M arrest compared to IR. Combined treatment in melanoma cells distinctly increases G2/M arrest. Healthy fibroblasts are less affected by G2/M arrest. Treatment predominantly decelerates or does not modify migration. In two cell cultures migration is enhanced under the inhibitors. Conclusions: Although the two PARP inhibitors talazoparib and niraparib appear to be suitable for a combination treatment with ionizing radiation in our in vitro studies, a combination treatment cannot generally be recommended. There are clear interindividual differences in the effect of the inhibitors on different melanoma cells. Therefore, the effect on the cancer cells should be studied prior to a combination therapy. Since melanoma cells increase more strongly than fibroblasts in G2/M arrest, the fractional application of combined treatment should be further investigated.

The aryl hydrocarbon receptor (AhR) is a decisive regulatory ligand-dependent transcription factor. It binds highly diverse ligands, which can be categorized as either endogenous or exogenous. Ligand binding activates AhR, which can adjust inflammatory responses by modulating immune cells such as dendritic cells (DCs). However, how different AhR ligand classes impact the phenotype and function of human monocyte-derived DCs (hMoDCs) has not been extensively studied in a comparative manner. We, therefore, tested the effect of the representative compounds Benzo(a)pyrene (BP), 6-formylindolo[3,2-b]carbazole (FICZ), and Indoxyl 3-sulfate (I3S) on DC biology. Thereby, we reveal that BP significantly induces a tolerogenic response in lipopolysaccharide-matured DCs, which is not apparent to the same extent when using FICZ or I3S. While all three ligand classes activate AhR-dependent pathways, BP especially induces the expression of negative immune regulators, and subsequently strongly subverts the T cell stimulatory capacity of DCs. Using the CRISPR/Cas9 strategy we also prove that the regulatory effect of BP is strictly AhR-dependent. These findings imply that AhR ligands contribute differently to DC responses and incite further studies to uncover the mechanisms and molecules which are involved in the induction of different phenotypes and functions in DCs upon AhR activation.

The CD83 is a type I membrane protein and part of the immunoglobulin superfamily of receptors. CD83 is involved in the regulation of antigen presentation and dendritic cell dependent allogeneic T cell proliferation. A soluble form of CD83 inhibit dendritic cell maturation and function. Furthermore CD83 is expressed on activated B cells, T cells and in particular on regulatory T cells. Previous studies on murine CD83 demonstrated this molecule to be involved in several immune-regulatory processes, comprising that CD83 plays a key role in the development und function of different immune cells. In order to get further insights into the function of the human CD83 and to provide preclinical tools to guide the function of CD83/sCD83 for therapeutic purposes we generated Bacterial Artificial Chromosomes (BAC) transgenic mice. BACs are excellent tools for manipulating large DNA fragments and are utilized to engineer transgenic mice by pronuclear injection. Two different founders of BAC transgenic mice expressing human CD83 (BAC-hCD83tg mice) were generated and were examined for the hCD83 expression on different immune cells as well as both the in vitro and in vivo role of human CD83 (hCD83) in health and disease. Here, we found the hCD83 molecule to be present on activated DCs, B cells and subtypes of CD4+ T cells. CD8+ T cells, on the other hand, showed almost no hCD83 expression. To address the function of hCD83, we performed in vitro mixed lymphocyte reactions (MLR) as well as suppression assays and we used the in vivo model of experimental autoimmune encephalomyelitis (EAE) comparing wild-type and hCD83-BAC mice. Results herein showed a clearly diminished capacity of hCD83-BAC-derived T cells to proliferate accompanied by an enhanced activation and suppressive activity of hCD83-BAC-derived Tregs. Furthermore, hCD83-BAC mice were found to recover faster from EAE-associated symptoms than wild-type mice, encouraging the relevance also of the hCD83 as a key molecule for the regulatory phenotype of Tregs in vitro and in vivo.