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

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Dendritic cells (DCs) are key regulators of the immune system. To gain more knowledge on human primary DCs, we recently performed transcriptome and surfactome analyses . We could demonstrate that DC subsets isolated from the lymphohematopoietic system displayed a strong ontogenetic relationship. Interestingly, we further found that the microenvironment in non-lymphoid tissues exhibited a stronger influence on the overall DC transcriptome. As our previous results were obtained from the steady-state, we have now started to investigate the DC subpopulations under inflammatory conditions. Our preliminary data demonstrate that, although the DC subsets isolated from blood, thymic, and splenic tissues have a similar DC subset specific TLR expression profile, thymic DC subsets were diminished in their cytokine secretion upon stimulation. This was in contrast to blood and splenic DCs, which displayed the full activation spectrum, including the expression of costimulatory molecules and pro-inflammatory cyto-/chemokines, such as IL-12 and RANTES. 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 here hypothesize that this effect might derive from a strong tolerogenic imprinting as consequence of the thymus environment. The observed semi-mature phenotype of thymic DCs might resemble the phenotype of tumor DCs. Therefore, we further hypothesize that the tumor microenvironment induces similar epigenetic and/or transcriptomic changes in infiltrating tumor DCs compared to DCs localized in the thymus. In order to test our hypotheses, we here want to i) determine functional differences between human blood and thymic DCs, ii) evaluate similarities and differences between human thymic and tumor DC subsets, and iii) analyze the mechanisms for tolerogenic predisposition of thymic and tumor DC subsets in experimental murine systems. Thus, to investigate transcriptional changes upon TLR ligand stimulation of sorted DC subpopulations isolated from thymi, blood, and tumors, we will perform RNAseq analyses, while the epigenetic status of these cells will be determined by ChIPseq analyses. Furthermore, we will functionally analyze candidate regulatory mechanisms, such as differences in intracellular signaling pathways and transcription factor activation. The measurement of DC and T cell responses will include multi-color flow cytometry, CBA, and PhosFlow assays. We expect that the identification of the key players involved in the tolerogenic predisposition of thymic and tumor DC subsets, allows for the development and testing of intervention strategies, e.g. to promote tumor therapies.