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.
CD83 is one of the best-known surface markers for fully mature dendritic cells (mature DCs), and its cell-type-and maturationspecific regulation makes the CD83 promoter an interesting tool for the genetic modulation of DCs. To determine the mechanisms regulating this DC-and maturation-specific CD83 expression, chromatin immunoprecipitation (ChIP)-on-chip microarray, biocomputational, reporter, electrophoretic mobility shift assay (EMSA), and ChIP analyses were performed. These studies led to the identification of a ternary transcriptional activation complex composed of an upstream regulatory element, a minimal promoter, and an enhancer, which have not been reported in this arrangement for any other gene so far. Notably, these DNA regions contain a complex framework of interferon regulatory factor (IRF)-and NF-B transcription factor-binding sites mediating their arrangement. Mutation of any of the IRF-binding sites resulted in a significant loss of promoter activity, whereas overexpression of NF-B transcription factors clearly enhanced transcription. We identified IRF-1, IRF-2, IRF-5, p50, p65, and cRel to be involved in regulating maturation-specific CD83 expression in DCs. Therefore, the characterization of this promoter complex not only contributes to the knowledge of DC-specific gene regulation but also suggests the involvement of a transcriptional module with binding sites separated into distinct regions in transcriptional activation as well as cell-type-and maturation-specific transcriptional targeting of DCs.
Obesity has become a major health problem in the industrialized world. Immune regulation plays an important role in adipose tissue homeostasis; however, the initial events that shift the balance from a noninflammatory homeostatic environment toward inflammation leading to obesity are poorly understood. Here, we report a role for the costimulatory molecule programmed death-ligand 1 (PD-L1) in the limitation of diet-induced obesity. Functional ablation of PD-L1 on dendritic cells (DCs) using conditional knockout mice increased weight gain and metabolic syndrome during diet-induced obesity, whereas PD-L1 expression on type 2 innate lymphoid cells (ILC2s), T cells, and macrophages was dispensable for obesity control. Using in vitro cocultures, DCs interacted with T cells and ILC2s via the PD-L1:PD-1 axis to inhibit T helper type 1 proliferation and promote type 2 polarization, respectively. A role for PD-L1 in adipose tissue regulation was also shown in humans, with a positive correlation between PD-L1 expression in visceral fat of people with obesity and elevated body weight. Thus, we define a mechanism of adipose tissue homeostasis controlled by the expression of PD-L1 by DCs, which may be a clinically relevant finding with regard to immune-related adverse events during immune checkpoint inhibitor therapy.
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 inhibits 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-hCD83 tg 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 .
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