Abstract
Mucosal-associated invariant T (MAIT) cells are an innate-like T cell population which are abundantly present in blood, mucosal surfaces, such as the gut, and in the liver in humans. MAIT cells express a semi-invariant T cell receptor (TCR), Vα7.2-Jα33/12/20, which is restricted to bacteria derived metabolites from riboflavin synthesis pathway presented by MHC class-I related (MR1) protein expressed on many antigen presenting cells (APCs). Recent studies have identified a protective role for MAIT cells against bacterial and viral infection which is mediated by expansion of MAIT cells in infected tissues. MAIT cells in peripheral blood can be easily detected with flow cytometry via their Vα7.2 chain of the MAIT cell TCR, combined with other MAIT cell markers such as CD3, CD8, CD26, CD161, and IL-18Rα. Present methods to identify MAIT cells in tissue during disease are imperfect due to the lack of negative control tissue which has impeded method development. In this study, modified Jurkat.MAIT TCR1/2/3 cell lines expressing the MAIT cell TCR, Vα7.2-Jα33, along with wild-type Jurkat.76 and empty vector Jurkat.EV cell lines were used to test the reliability of immunohistochemistry (IHC) and BaseScope for the detection of the MAIT cell TCR. Real-time PCR and flow cytometry were first used to assess the expression of the MAIT cell TCR in the Jurkat cell lines. Jurkat cell lines were then fixed in formalin and made into paraffin embedded cell clots. The formalin fixed paraffin embedded (FFPE) Jurkat cell clots expressing Vα7.2-Jα33 were utilised as positive controls and the Jurkat.76 and Jurkat.EV cell clots negative for Vα7.2-Jα33 were used as negative controls for the development of IHC and BaseScope assays. Chromogenic and fluorescent immunohistochemistry were attempted in both FFPE Jurkat cell clots and liver sections. Chromogenic IHC successfully detected Vα7.2+ expression in FFPE Jurkat cell clots. However, only fluorescent IHC was able to visualise Vα7.2+ expression in both FFPE Jurkat cell clots and liver sections. Additionally, co-stained Vα7.2+CD3+ cells were also detected with fluorescent IHC in FFPE liver sections confirming the presence of Vα7.2+ T cells which are mostly MAIT cells. FFPE Jurkat cell clots and liver sections were also successfully probed for MAIT cell TCR, Vα7.2-Jα33/12/20, by BaseScope assay. This thesis explored two different methods of MAIT cell identification in FFPE tissue. Both IHC and BaseScope methods were able to detect the MAIT cell TCR in FFPE Jurkat cell clots and liver sections, but further optimisation is suggested to allow for accurate identification of absolute cell counts. Once optimised, these methods can be reliably used to accurately identify MAIT cells in tissues and thus can be utilised for future research examining
the role of tissue associated MAIT cells in disease.