Effect of SLAMF6 on T cell calcium signalling in response to antigen stimulation

Sammanfattning

SLAMF6 is an immunoregulatory molecule studied in cancer research as a possible target for immunotherapy in acute myeloid leukaemia (AML). The absence of SLAMF6 has been shown to elevate T cell activation and improve anti-tumour responses (1). The regulatory mechanism of SLAMF6 is not fully understood. The aim of this thesis was to investigate the effect of SLAMF6 on T cell signalling using fluorescence microscopy. The experimental system was developed stepwise. First a mobile SLB containing relevant proteins (including CD58, SLAMF6, and peptide loaded major histocompatibility complex (pMHC)) was created. Cells were then added to the SLB to analyse binding patterns. The T cells were subsequently activated by pMHC binding T cell preceptors (TCRs) on the T cells, and a dose-response curve of the number of activated cells vs pMHC density was generated. Finally, SLAMF6 was added to the system to investigate how it affects T cell signalling. Fluorescence recovery after photobleaching (FRAP) analysis confirmed the formation of a mobile SLB with proteins successfully incorporated in the bilayer. T cells adhered to the SLB through CD58 mediated interactions and showed accumulation of SLAMF6, UCHT1-Fab and pMHC in the cell-SLB contact at the cell surface. T cell activation was observed through calcium signalling, measured using the calcium sensitive dye Fluo-4am. The fraction of signalling cells increased with increasing concentration pMHC, enabling the creation of a dose-response curve. Lastly the addition of SLAMF6 reduced the fraction of signalling cells, suggesting that SLAMF6 regulates T cell activation in this system.