Role of the Akt signalling pathway in CD8 T cell immune response

Abstract

Understanding the signalling pathways controlling activation and differentiation of CD8 T cells will facilitate their exploitation for anti-cancer therapy. The Akt/PKB signalling pathway influences CD8 T cell function and differentiation but published data are discrepant. Utilising a PDKK465E knock-in mutant mouse model, in which T cells exhibit impaired Akt activity, data presented here show that sub-optimal Akt activity limits CD8 T cell IFN-γ and IL-2 production, granzyme B expression and cellular cytotoxicity. Additional chemical inhibition of Akt further hindered cytotoxicity and granzyme B expression. Microarray and confirmatory qPCR showed reduced expression of transcripts encoding the membrane trafficking protein BAIAP3 in PDKK465E cells which may be responsible for some of these effects.
Previous data from the group showed that PDKK465E CD8 T cells have a skewed memory population. Here single cell RNA sequencing and unsupervised clustering was used to define 7 cell subsets at the peak of Listeria Monocytogenes infection and in which a memory precursor effector cell-like phenotype was favoured by PDKK465E cells. Additionally, mRNA encoding ribosomal proteins was enriched in PDKK465E compared with WT CD8 T cells, and also in more memory-like cells across both samples. PDKK465E cells also exhibited enrichment in OXPHOS-related transcripts, a feature previously correlated with a memory phenotype.
Use of PDKK465E mutant cells, pharmacological inhibition (Akti) and a constitutive form of Akt (Myr-Akt), allowed further examination of effects of altered Akt activity on oxidative phosphorylation. These data showed no significant improvement in spare respiratory capacity (SRC) in Akt-inhibited WT CD8 T cells cultured in IL-2 although Akt inhibition in PDKK465E CD8 T cells resulted in a significant improvement in SRC. Enforced Akt activity in CD8 T cells cultured in IL-15 reduced SRC. However, SRC remained higher than IL-2-treated cells, indicating that whilst increased Akt activity impairs oxidative phosphorylation, there are aspects of IL-15 signalling that cannot be overcome by constitutive Akt activity. Despite a beneficial metabolic and cytotoxic profile, Myr-Akt transduced IL-15 treated CD8 T cells were only marginally superior to IL-15 treated controls cells at conferring tumour therapy.
SGK1, a kinase sharing homology with Akt, was also examined for its effects in CD8 T cells. Under stimulation with normal or restrictive concentrations of IL-2, pharmacological inhibition of SGK1 had negative effects on cellular proliferation. Further, stimulation of SGK1 through increased NaCl concentrations in culture media caused increased survival of CD8 T cells in both cytokine replete and limiting conditions.
Together the data presented in this thesis show that reduced Akt signalling causes a defect in the cytotoxic capacity of CD8 T cells in vitro and alters the transcriptome of CD8 T cells at the peak of an immune response, inducing a more MPEC phenotype. In addition, new subsets of CD8 T cells were identified through Drop-Seq. Mitochondrial oxidative phosphorylation was shown to be negatively regulated by Akt activity. Lastly, SGK1 was shown to increase survival and proliferation of CD8 T cells. These data have implications for CD8 T cell differentiation and treatments aimed at manipulating Akt for therapeutic benefit.

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Identifiers

ORCID for Aymen Al-Shamkhani: orcid.org/0000-0003-0727-4189

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