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Cytotoxic CD8+T lymphocytes (CTL) eliminate infected cells or transformed tumour cells by releasing perforin-containing cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca2+-influx through store operated Ca2+channels, formed by STIM-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca2+-dependent target cell killing. Here, we isolated total RNA from natural killer (NK) cells, non-stimulated CD8+T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8+T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca2+-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8+T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca2+dependent cytotoxicity, candidates were also analyzed under Ca2+-limiting conditions. Overall, this strategy led to the identification of KCNN4, RCAN3, CCR5 and BCL2 as potential candidates to regulate the efficiency of Ca2+-dependent target cell killing.

Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL.

A systematic understanding of different factors influencing cell type specific microRNA profiles is essential for state-of-the art biomarker research. We carried out a comprehensive analysis of the biological variability and changes in cell type pattern over time for different cell types and different isolation approaches in technical replicates. All combinations of the parameters mentioned above have been measured, resulting in 108 miRNA profiles that were evaluated by next-generation-sequencing. The largest miRNA variability was due to inter-individual differences (34 %), followed by the cell types (23.4 %) and the isolation technique (17.2 %). The change over time in cell miRNA composition was moderate (