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.

Memory CD8+ T cells are an essential component of anti-tumor 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 tumors and infections are not well known. Activation of costimulatory receptors, including CD28 and NKG2D, activate PI3K and subsequently may activate the mTOR pathway in CD8+ T cells. Therefore this study compared the activation of the mTOR signaling pathway after costimulation through CD28 or NKG2D receptors in murine effector CD8+ T cells. Compared to CD28 costimulation, activation through CD3 and NKG2D receptors had weaker activation of mTORc1 as shown by decreased phosphorylation of mTORc1 targets S6K1, ribosomal protein S6, and 4EBP-1. NKG2D costimulation also showed increased gene expression of TSC2, a negative regulator of mTORc1, whereas CD28 costimulation increased gene expression of Rheb, an activator of mTORc1, and HIF-1α and VEGFα, pro-angiogenic factors downstream of mTORc1. Strong mTORc1 activation in CD28-costimulated cells also increased expression of transcription factors that support effector cell differentiation, namely T-bet, and BLIMP1, whereas low levels of mTORc1 activation allowed for the expression of Eomes and BCL6 during NKG2D stimulation, and increased expression of central memory markers CD62L and CD127. These data show that compared to CD28, costimulation through the NKG2D receptor leads to the differential activation of the mTOR signaling pathway and potentially supports memory CD8+ T cell differentiation. Citation Format: Bryan McQueen, Kelsey Trace, Emily Whitman, Amorette Barber. NKG2D and CD28 receptors differentially activate mTOR to alter murine effector CD8+ T cell differentiation. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A05.

To induce strong immune responses, naïve CD8+ T cells require concurrent 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. However, the effect of stimulating costimulatory receptors on effector T cells is still unclear. One costimulation receptor that is likely to be engaged in the tumor microenvironment is NKG2D since the ligands for this receptor are expressed on over 80% of tumors. To determine how activation of costimulatory receptors in the tumor microenvironment alters CD8+ T cell functions, this study investigated the differential activation of signaling pathways by two costimulatory receptors, CD28 and NKG2D. To obtain an in-depth view of how stimulation through the NKG2D receptor alters T cell function, the transcriptome of NKG2D- and CD28- costimulated murine CD8+ T cells was analyzed using RNA-sequencing. These analyses showed that NKG2D costimulation of effector CD8+ T cells altered expression of over 5200 genes compared to CD3 stimulation alone. Some genes that were increased in NKG2D-stimulated cells include IFNγ, and granzymes A and C, suggesting the NKG2D stimulation enhances the anti-tumor functions of CD8+ T cells. When comparing NKG2D and CD28 costimulation, many genes were differentially regulated, including genes in the mTOR pathway. To determine if the differential activation of mTORc1 and mTORc2 signaling pathways altered T function, murine effector CD8+ T cells were activated through CD3 in combination with CD28 or NKG2D and treated with no, low, or high amounts of rapamycin to deactivate either mTORc1 or both mTORc1 and mTORc2. NKG2D-stimulated T cells increased expression of Eomes, a transcription factor that is involved in the differentiation of central memory T cells, while CD28 stimulated cells increased expression of TCF1. Rapamycin inhibition studies showed that stronger activation of mTORc1 in CD28 stimulated T cells inhibited Eomes expression and also increased TCF1 expression compared to NKG2D-stimulated T cells. In addition, NKG2D stimulation lead to differential expression of memory markers including CD127, CCR7, CD62L, and KLRG1, with NKG2D stimulation inducing a central memory phenotype compared to CD28 stimulation which caused an effector memory phenotype. Together, these data suggest that differential activation of mTORc1 by the NKG2D receptor alters CD8+ T cell differentiation to become central memory T cells. Thus, NKG2D stimulation likely enhances CD8+ T cell anti-tumor effector functions. Citation Format: Kelsey Trace, Sara Jacobson, Amorette E. Barber. NKG2D receptor stimulation of effector CD8+ T cells alters the transcriptome and activation of mTOR to enhance anti-tumor immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5009. doi:10.1158/1538-7445.AM2015-5009