Abstract
Mucosal associated invariant T (MAIT), invariant natural killer T (iNKT) and V𝛾9+V𝛿2+ T cells are all subsets of an important type of immune cell known as the innate-like T (ILT) cells. ILT’s are known to help bridge the gap between the innate and adaptive immune response by providing a specific yet rapid response to foreign antigens. ILT’s have a complex and tightly regulated transcriptional program that facilitates their development, differentiation and effector functions, but it is their expression of promyelocytic leukaemia zinc finger (PLZF) that defines their status as an innate-like T cell. While it is well understood the role of PLZF in the context of myeloid leukaemia, the exact purpose for its expression in ILT’s is not yet sufficiently studied. Additionally, while the transcriptional program of conventional T cells has been thoroughly established, the nuances of the ILT transcriptional program are yet to be determined. Furthermore, evidence has shown that ILT’s can be broken up into additional sub-categories based on their expression level of different markers, however very few of these sub-categories have been identified. Due to the importance of these cells in normal immune function, as well as their implication in numerous pathological conditions, this project was undertaken with the goal of producing a spectral flow cytometry panel that allows for a better understanding of the molecular biology of ILT’s. Spectral flow cytometry is a rapidly advancing immunological technique that uses light emission capturing technology to characterize and measure both molecular and physical properties of individual cells. This thesis focuses on detailing the complex design process that resulted in the production of a 20 colour immunophenotyping spectral flow cytometry panel. The panel includes a number of different cell surface markers, transcription factors and cytokines with the goal of using it to explore the transcriptional program that controls ILT’s and for the phenotyping of a number of different cell sub-types, to further our overall knowledge of the ILT bracket of immune cells.