RNA-seq of degranulating and non-degranulating NK cells under inhibitory and stimulatory treatment

Cytotoxic cells (T CD8+ cells and NK cells) are the primary executors of tumour cell killing. However, not all cells are equally prone to kill. Within circulating lymphocytes, certain gene expression patterns may better prepare immune cells to attack.Upon co-culture with tumour cells, cytotoxic cells will undergo a process known as degranulation, which is based on the release of lysosomal-like structures, cytotoxic granules, that contain proteins specialized on disturbing tumour cell membrane integrity. This results in the killing of tumour cells. Cytolytic granules are decorated with lysosomal proteins such as the lysosome-associated membrane proteins (LAMP) family members, which in basal conditions are restricted to lysosomal compartments. Upon fusion of cytolytic granules with the cell membrane, such proteins become transitorily enriched and available to antibodies. This can be used as a tool to distinguish and sort out cell populations that have been recently activated.In our project, we use LAMP1 as a marker of NK cell activation upon challenge with tumour cells. We make use of this strategy to select which cells are better equipped to get activated, and what are the underlying transcriptional signatures involved in this phenotype. In this project we aim to identify intrinsic modulators of cytotoxic cell activation. To this end, we have the following objectives:- Definition of the NK cell activation priming signature in basal conditions.- Comparison of transcriptional signatures following treatment with stimulatory/inhibitory cytokines and the combination of both.- Uncovering new potential targets for immune therapy. - Analysis of the resulting data from the RNAseq to define the transcriptional signature of activated cells, perform gene set enrichment analysis, identification of enriched pathways and getting insight on the ups-tream regulators of the gene signatures.- Confirmation of hits in primary cells and generation of genetically modified NK cell lines/inhibitors/activators to challenge protein function.- Mechanistic study of protein hits.- TCGA study for relevance in cancer indications.- (If established) Parallel NGS study with T CD8+ cells.- Writing a manuscript containing main findings for the NK cells hits, and potentially for the T CD8+ cell study. Natural killer cells isolated from 6 different healthy donors were each co-cultured with tumor cells to induce degranulation either or not in the prescence of TGFbeta, IL2, or both. Subsequently, NK cells were sorted based on NK cell (CD56, CD3e CD45) and degranulation (LAMP1) markers and processed for RNA sequencing. The total number of samples equals 6 donors * 4 treatments * 2 LAMP1 statuses = 48. *************************************************************** Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access. ***************************************************************