Effect of rapamycin and KU-0063794 on CTL gene expression. Mus musculus

Comparison of transcriptional profile of CD8 cytotoxic T lymphocytes terated with the mTORC1 inhibitor rapamycin or the mTOR inhibitor KU-0063794 and comparison with proteomic analysis. Abstract: High resolution mass spectrometry maps the cytotoxic T lymphocyte (CTL) proteome and the impact of mammalian target of rapamycin complex 1 (mTORC1) on CTL. We show that the CTL proteome is dominated by metabolic regulators and granzymes and that mTORC1 selectively represses and promotes expression of a protein subset (~10%) including key CTL effector molecules and signaling proteins. mTORC1 also controlled flux through a subset of metabolic pathways rather than acting as an on/off switch for global CTL metabolism. Proteomic data highlighted the potential for mTORC1 negative control of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production in CTL. Further work revealed that mTORC1 represses PIP3 production and determines the mTORC2 requirement for activation of the serine/threonine kinase AKT. Unbiased proteomic analysis thus provides a comprehensive understanding of CTL identity and mTORC1 control of CTL function. Overall design: Spleens from three biological replicates (control and drug treated samples were generated from the same spleens) from P14 TCR transgenic mice were harvested and activated with the antigenic peptide gp33-41 and IL-2/12 for 48h. After activation, cells were further clonally expanded in the presence of IL-2/12 for a further 96h prior to RNA extraction and hybridization to Affymetrix microarray.