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Identification of mechanisms for the tolerogenic predisposition of human thymic and tumor dendritic cells

Subject Area Immunology
Dermatology
Cell Biology
Term from 2019 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 420943261
 
Final Report Year 2024

Final Report Abstract

Dendritic cells (DCs) are key cells of the immune system. They do not only initiate primary immune responses, they also play an important role in the induction and maintenance of tolerance. In mice and humans, DCs can be distinguished into different subpopulations, e.g., plasmacytoid DCs and conventional DC (cDC) subsets. Inflammatory stimuli (e.g., TLR ligands) induce the maturation of DCs, which is accompanied by an upregulation of costimulatory molecules and the secretion of chemokines and cytokines, important for the polarization into effector T cells. In our previous analyses we have focused on characterization of primary steady state DCs in human lymphoid and non-lymphoid tissues by the analyses of transcriptome and surfactome. Our data displayed a strong ontogenetic relationship of the DCs. Interestingly, the microenvironment in non-lymphoid tissues exhibited a stronger influence on the overall DC transcriptome. Next, we started to analyze tissue DCs under inflammatory conditions. Our preliminary data suggested that the DC subsets isolated from blood, thymic, and splenic tissues have a similar DC reaction to TLR ligand exposition in regard to costimulatory molecule expression. However, TLR ligand stimulated thymic DC subsets were strongly diminished in their cytokine secretion. This missing cyto- and chemokine secretion of thymic DCs was accompanied by a complete block of CD4+ T cell polarization into TH1 cells. As this behavior was observed after the isolation of the DCs and their stimulation in vitro, we hypothesized that the effect might derive from a strong tolerogenic imprinting as consequence of the thymic microenvironment. Further, we postulated that DCs in tumors might be hindered by similar mechanisms. In order to test our hypotheses, we performed experiments to determine functional differences between human blood and thymic DCs. When focusing on previously described tolerogenic stimuli, we found that the addition of Wnt5a to TLR ligand stimulated peripheral blood DCs led to the reduction of cytokine secretion such as IL6, TNFα, and IL23, while co-stimulatory molecules were only slightly altered. For a more unbiased analysis and identification of unknown players that induce transcriptional and epigenetic changes, we established a protocol for the enrichment, cell sort, TLR stimulation, and CITEseq labelling of DCs from fresh thymi and blood. We expect to gain an in-depth view of cellular changes upon TLR stimulation between the analyzed tissues. Further, we developed a protocol allowing for the clear separation of mouse DC2 and DC3 from each other as well as a protocol for the distinction from tissue macrophages in thymus and other tissues. Further, we started to analyze the impact of soluble and surface-bound melanoma factors on DC upon coculture with tumor cells and found that cell-bound melanoma factors suppress the priming of CD80 and CD86 which was associated with a loss of CD80 and CD86 dependent costimulatory capacity of DCs.

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