BackgroundRecent studies suggest that the chemotactic G-protein-coupled-receptor (GPCR) formyl-peptide-receptor-like-1 (FPRL1) and the receptor-for-advanced-glycation-end-products (RAGE) play an important role in the inflammatory response involved in neurodegenerative disorders such as Alzheimer’s disease (AD).Therefore, the expression and co-localisation of mouse formyl peptide receptor (mFPR) 1 and 2 as well as RAGE in an APP/PS1 transgenic mouse model using immunofluorescence and real-time RT-PCR were analysed. The involvement of rat or human FPR1/FPRL1 (corresponds to mFPR1/2) and RAGE in amyloid-β 1–42 (Aβ1-42)-induced signalling were investigated by extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, the cAMP level in primary rat glial cells (microglia and astrocytes) and transfected HEK 293 cells was measured. Formyl peptide receptors and RAGE were inhibited by a small synthetic antagonist WRW4 and an inactive receptor variant delta-RAGE, lacking the intracytoplasmatic domains.ResultsWe demonstrated a strong increase of mFPR1/2 and RAGE expression in the cortex and hippocampus of APP/PS1 transgenic mice co-localised to the glial cells. In addition, the Aβ1-42-induced signal transduction is dependant on FPRL1, but also on FPR1. For the first time, we have shown a functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition a possible physical interaction between FPRL1 as well as FPR1 and RAGE was shown with co-immunoprecipitation and fluorescence microscopy.ConclusionsThe results suggest that both formyl peptide receptors play an essential role in Aβ1-42-induced signal transduction in glial cells. The interaction with RAGE could explain the broad ligand spectrum of formyl peptide receptors and their important role for inflammation and the host defence against infections.

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T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer characterized by skewed epigenetic patterns, raising the possibility of therapeutically targeting epigenetic factors in this disease. Here we report that among different cancer types, epigenetic factor TET1 is highly expressed in TALL and is crucial for human TALL cell growth in vivo. Tet1 knockout mice and knockdown in human T-cells did not perturb normal T-cell proliferation, indicating that TET1 expression is dispensable for normal T-cell growth. The promotion of leukemic growth by TET1 was depending on its catalytic property to maintain global 5hydroxymethylcytosine (5hmC) marks, thereby regulating cell cycle, DNA repair genes and TALL associated oncogenes. Furthermore, overexpression of the Tet1 catalytic domain was sufficient to augment global 5hmC levels and leukemic growth of TALL cells in vivo. We demonstrate that PARP enzymes, which are highly expressed in TALL patients, participate in establishing H3K4me3 marks at the TET1 promoter and that PARP1 interacts with the TET1 protein. Importantly, the growth related role of TET1 in TALL could be antagonized by the clinically approved PARP inhibitor Olaparib, which abrogated TET1 expression, induced loss of 5hmC marks and antagonized leukemic growth of TALL cells, opening a therapeutic avenue for this disease. 10 transplantation (Fig. S2H). Moreover, in a published microarray analysis of TALL cell lines transplanted into xenografts, a similar increase in TET1 expression was observed (Fig. S2I), suggesting that high TET1 expression is associated with leukemic growth in vivo. Indeed, forced expression of Tet1-CD in TALL cell lines significantly augmented leukemic growth in vitro and in vivo (Fig. 3D-E). These data clearly suggest that the growth-promoting role of TET1 in TALL is at least partly dependent on its enzymatic activity. TET1 depletion induces loss of 5hmC marks at promoters and gene bodies of genes involved in cell cycle, DNA repair and NOTCH pathway Next we analyzed 5hmC levels in TALL cells via Intracellular fluorescence (IF) in our knockdown, rescue and overexpression experiments. IF-flow cytometry for 5hmC marks in TET1 depleted JURKAT cells, primary TALL patients and TET1 knockout bulk TALL cell lines revealed a significant decrease in global 5hmC levels (Fig. 4A-B and Fig. S3A-B) and increase in 5mC levels (Fig. S3C). Conversely, overexpression of Tet1-CD in TET1 depleted cells or wild type TALL cells induced a global increase in 5hmC levels (Fig. 4C-D). Furthermore, we performed hydroxymethylated DNA immunoprecipitation (hMeDIP)-seq in TET1 depleted JURKAT cells. In hMeDIP-seq, TET1 depletion resulted in more than a 59% reduction of global 5hmC enrichment at the promoter (-5kbTSS), gene body (GB) and intergenic regions compared to scrambled control (referred to as TET1 dependent 5hmC or T1-5hmC regions from here) (Fig. 4E-F). In detail, a total of 2,404 and 6,115 5hmC enriched promoters and GB were observed in the scrambled arm, respectively, out of which more th...

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High epitope-specific sensitivity of CD8 1 T cells is required for optimal immune protection against intracellular pathogens as well as certain malignancies. The quality of antigen recognition of CD8 1 T cells is usually described as "avidity" to its cognate peptide MHCI complex. T cell avidity is mainly dependent on the structural qualities of the T cell receptor (TCR), as convincingly demonstrated by recombinant TCR reexpression experiments. Based on reversible MHCI multimer staining and k off -rate measurements of monomeric peptide MHCI complexes, we recently established a microscopic assay for determining the structural avidity of individual CD8 1 T cells. Here we demonstrate that this assay can be adapted for rapid flow-cytometric avidity screening of epitope-specific T cell populations. Furthermore, we show that-in combination with conventional nonreversible MHCI multimer staining-even very small epitope-specific CD8 1 T cell populations can be analyzed directly ex vivo without the need for previous TCR cloning or T cell sorting. This simplified approach provides highly accurate mean TCR-ligand k off -rate values for poly-or oligoclonal T cell populations and is ideally suited for high-throughput applications in basic research as well as clinical settings. V C 2016 International Society for Advancement of Cytometry Key terms TCR-ligand-k off -rate; structural TCR avidity; dissociation kinetics; direct avidity screening; T cell functionality; T cell therapy; online injection; uninterrupted acquisition; temperature controlled analysis; MHC multimer double staining THE importance of cellular adaptive immunity in the control of viral and some bacterial infections has been convincingly demonstrated by depletion or adoptive transfer experiments, as well as in genetic mouse models. Thereby, antigen-specific CD8 1 T cells seem to play a dominant role for protection against intracellular pathogens (1). Also in certain cancers, the presence of functional CD8 1 T cells could be associated with the induction of tumor regression (2,3). Different immunotherapeutic approaches therefore aim at the reconstitution of a functional CD8 1 T cell response in patients. If antigen-specific T cells are still present but silenced by the micromilieu, application of "checkpoint inhibitors" can serve to re-activate the efficacy of existing T cells (4). However, if endogenous antigen-specific T cells are lacking or existing cells carry T cell receptors (TCRs) with weak antigen recognition, adoptive T cell transfer of more potent T cells might represent a promising approach to restore a protective antigen-specific CD8 1 T cell response. Research over the last decades has indicated that not only the quantity but also the quality of prevalent or adoptively transferred T cells correlates with the success of T cell-based immunotherapies (5-7).