Summary Memory CD8+ T cells are an essential component of anti-tumour and anti-viral immunity. Activation of the mammalian/mechanistic target of rapamycin (mTOR) pathway has been implicated in regulating the differentiation of effector and memory T cells. However, the mechanisms that control mTOR activity during immunity to tumours and infections are not well known. Activation of co-stimulatory receptors, including CD28 and natural killer group 2D (NKG2D), activate phosphatidylinositol-3 kinase and subsequently may activate the mTOR pathway in CD8 + T cells. This study compared the activation of the mTOR signalling pathway after co-stimulation through CD28 or NKG2D receptors in murine effector CD8 + T cells. Compared with CD28 co-stimulation, activation through CD3 and NKG2D receptors had weaker activation of mTORc1, as shown by decreased phosphorylation of mTORc1 targets S6K1, ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1. NKG2D co-stimulation also showed increased gene expression of tuberous sclerosis protein 2, a negative regulator of mTORc1, whereas CD28 co-stimulation increased gene expression of Ras homologue enriched in brain, an activator of mTORc1, and hypoxia-inducible factor-1a and vascular endothelial growth factor-a, pro-angiogenic factors downstream of mTORc1. Strong mTORc1 activation in CD28-co-stimulated cells also increased expression of transcription factors that support effector cell differentiation, namely T-bet, B lymphocyte-induced maturation protein (BLIMP-1), interferon regulatory factor 4, and inhibitor of DNA binding 2, whereas low levels of mTORc1 activation allowed for the expression of Eomes, B-cell lymphoma 6 (BCL6), and inhibitor of DNA binding 3 during NKG2D stimulation, and increased expression of memory markers CD62 ligand and CD127. These data show that compared with CD28, co-stimulation through the NKG2D receptor leads to the differential activation of the mTOR signalling pathway and potentially supports memory CD8 + T-cell differentiation.

To induce strong anti-tumor immune responses, CD8+ T cells require stimulation through the TCR and costimulatory receptors. Each costimulatory molecule can have a unique effect on the function of T cells due to differential activation of downstream signaling pathways and gene expression. One costimulation molecule that is likely to be engaged in the tumor microenvironment is NKG2D since the ligands for this receptor are expressed in over 80% of tumors. In order to determine how activation of costimulatory receptors in the tumor microenvironment affects CD8+ T cell functions, previous studies have compared the activation of CD8+ T cells through CD3 in combination with either CD28 or NKG2D and showed that CD3 and NKG2D stimulated cells secreted less anti-inflammatory cytokines. Both costimulatory receptors activated AKT, however the altered cytokine secretion was due to the unique activation of β-catenin in NKG2D stimulated cells. This study aimed to further investigate the differences in the signaling pathways downstream of AKT that are activated by CD3 /CD28 and CD3/NKG2D. Specifically, this study focused on the activation of mammalian target of rapamycin complex 1 and 2 (mTORC1 and mTORC2) along with nuclear factor kappa B (NFκB), all of which have been shown to have important roles in the CD8+ T cell anti-tumor functions. Activation of the mTORC1, mTORC2 and NFκB pathways were compared in murine CD8+ effector T cells stimulated through CD3 alone, CD3 and CD28 or CD3 and NKG2D. The activation of mTORC1was measured through phosphorylation of its downstream effectors including 4-EBP1, P70SK6 and HIF-1, while mTORC2 measured activation of AKT, SGK1 and NFkβ. Activation of CD8+ T cells through CD3 and NKG2D decreased activation of mTORC1 while increasing activation of mTORC2. Together this shows that activation through NKG2D leads to the differential activation of signaling pathways, thus likely altering the anti-tumor effector functions of the CD8+ T cells. Citation Format: Amorette E. Barber, Taylor Bedsworth, Jasmine Spence, Emily Whitman. Activation of the NKG2D receptor differentially activates mTOR and NFκB signaling pathways in CD8+ T cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1269. doi:10.1158/1538-7445.AM2013-1269