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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.

CTL and NK cells recognize and eliminate cancer cells. However, immune evasion, down regulation of immune function by the tumor microenvironment, or resistance of cancer cells are a major problem. While CTL and NK cells are both important to eliminate cancer, most studies address them individually. In a new experimental human model, we analysed combined primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, MART-1-specific CTL or NK cells eliminated SK-Mel-5 cells within 24 hours indicating that SK-Mel-5 cells are initially not resistant. However, at lower effector-to-target ratios, which resemble conditions of the immune contexture in human cancer, a significant number of SK-Mel-5 cells survived. Whereas CTL pre-exposure induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, NK cell pre-exposure induced resistance in surviving SK-Mel-5 cells to NK cells but not to MART-1 specific CTL. In contrast, there was even a slight enhancement of CTL cytotoxicity against SK-Mel-5 cells following NK cell pre-exposure. In all other combinations, resistance to subsequent cytotoxicity was higher, if melanoma cells were pre-exposed to larger numbers of CTL or NK cells. Increases in human leukocyte antigen class I expression correlated with resistance to NK cells, while reduction in MART-1 antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. This study quantifies the interdependence of CTL and NK cell cytotoxicity and may guide strategies for efficient CTL-NK cell anti-melanoma therapies.Key points summaryCytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. CTL and NK work in parallel, but most studies address them individually.In a new human experimental model, antigen-specific CTL and NK cell cytotoxicity interdependence against melanoma is shown.Whereas high numbers of antigen-specific CTL and NK cells eliminate all melanoma cells, lower, more physiological numbers induce resistance, in case secondary CTL or NK cell exposure follow initial CTL cell exposure or if secondary NK cell exposure follows initial NK cell exposure; only if secondary CTL exposure follows initial NK cell exposure no resistance of melanoma but even a slight enhancement of cytotoxicity was observed.Alterations in HLA-I expression correlated with resistance to NK cells, while reduction in antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous antigen.The results should help to better understand and optimize immune therapies against cancer.Graphical abstract

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.