Cells were stained with TMRE (Sigma-Aldrich, St Louis, MO, USA) in PBS to a final concentration of 125 nM, and incubated for 30 min at 37°C with 5% CO2 to assess mitochondrial membrane potential (ΔΨm). Total mitochondrial mass and membrane potential were also determined using mitotracker green and red dyes (Invitrogen), respectively, according to manufacturers’ instructions. For in vitro culture experiments, CD8+ T cells were purified >90% by magnetic-activated cell sorting (MACS) using anti-CD8α microbeads PD0332991 clinical trial and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) following manufacturer’s instructions. For microarray and Western blot analysis,

CD8+ T cells were purified >98% using the Easysep PE selection kit (StemCell Technologies) using PE-CD8α (eBioscience). Primary naïve CD8+ T cells were cultured in 24-well plates at 1×106 cells/mL at 37°C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich) supplemented with glutamine, 2-mercaptoethanol and antibiotics (all Sigma-Aldrich). Where used, IL-7 (Peprotech, Rocky Hill, NJ, USA) was supplemented at 50 ng/mL. CD8+ T cells were sorted, check details and total RNA was prepared using the RNEasy mini kit (Qiagen). RNA was quality and quantity controlled for degradation on a BioAnalyzer

2100 (Agilent). Since the starting quantity of RNA for each sample did not exceed  μg, two cycle amplification was performed, as recommended by the manufacturer (Affymetrix). The GC-RMA (Robust Multiarray Analysis) algorithm was applied to the probe level data (CEL files). Quality control and data processing was performed Teicoplanin at the Bloomsbury Centre for Bioinformatics, University College London, using the limma,

gcrma, simpleaffy, annotate, annaffy and affycoretools R packages in Bioconductor. Multiple testing correction was applied for the data using the Benjamini and Hochberg False Discovery Rate. Annotation of each probe set was derived using the NetAffx site (Affymetrix). Microarray data were deposited in ArrayExpress (accession number E-TABM-991). Cell pellets containing 1×106 cells were lysed at 4°C in 1 mL 1% NP40 lysis buffer. Protein lysates were run on 12% SDS-PAGE acrylamide gels and protein content analysed on nitrocellulose membrane with the following antibodies: Mcl1 (Rockland), Bcl2, BclXL, Bok, Bax, total Bad, Bim, Bid, Bak, Puma, pBad (Ser112) (all from Cell Signaling Technology), and Actin (Santa Cruz). Densitometry calculations of proteins were calculated in the ImageJ v1.43 (NIH, Public Domain). The authors thank Biological Services for animal husbandry and technical support, Hugh Brady for providing Bad transgenic mice. This work was supported by the Medical Research Council UK under programme code U117573801. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset.