The molecular mechanisms of cytokine-mediated activation of human CD8+ T cells

Thesis

Background: CD8+ T cells can be activated through both TCR-dependent and TCR-independent pathways, the former response was shown to be largely regulated by mTORC2 over mTORC1. This raises the question of whether TCR-independent CD8+ T cell activation also involves the mTOR pathway and if so, which complex dominates. Additionally, because mTORC1 has been shown to regulate IRE1-α during ER stress, this study aims to explore whether the mTORC1-IRE1α pathway contributes to cytokine-induced CD8⁺ T cell activation, especially in NKG2A⁺ T cells and MAIT cells, known to be hyperresponsive to IL-12 and IL-18.

Methods: Human primary peripheral blood mononuclear cells (PBMCs), purified CD8+ T cells and total T cells were used in this study. Small guide RNA and Cas9 protein or chemical inhibitors were used to study the function of proteins including mTORC1, mTORC2 and IRE1α. The knockout efficiency was assessed by western blotting. The activity of mTOR complexes was measured by phospho-flow cytometry (Phosflow). The expression of IFN-γ in CD8+ T cells or total T cells were measured by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. The phenotype and the activation of CD8+ or CD3+ subsets were assessed by flow cytometry.

Results: Chemical inhibition of mTORC1 had a more pronounced downregulation effect on cytokine-induced IFN-γ production than mTORC2 in total CD8+ T cells. Both chemical inhibition and genetic knockout of IRE1α potently supressed XBP1s expression and reduced the production of cytokine-induced IFN-γ in total CD8+ T cells. Regulation of cytokine-mediated activation by mTOR and IRE1α was further confirmed in CD8+NKG2A+CD161+ T and MAIT cells. Despite the shared feature of hyperresponsiveness to IL-12 and IL-18 by CD8+NKG2A+CD161+ T and MAIT cells, the former are less sensitive to glycolysis inhibition.

Conclusions: In contrast to TCR-dependent activation, cytokine-induced CD8+ T cell activation is dependent more on mTORC1 than mTORC2 signalling. The regulation of cytokine-mediated activation by mTOR was partially dependent on IREα-XBP1s pathway. Additionally, NKG2A+CD161+ T and MAIT cells exhibit similar functional characteristics in response to cytokine stimulation but differed in energy utilisation.

Authors: Cheng, L

• DOI: 10.5287/ora-4rpbdyjy9
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English