Generating Cytotoxic T Lymphocytes (CTLs)

CTLs were generated as described previously (Waugh et al., 2009 (link)). In brief, lymphocytes isolated from spleens or lymph nodes of P14-LCMV or OTI transgenic mice or nontransgenic mice were activated for 48 h with either 100 ng/ml of soluble LCMV or OTI-specific peptide, gp33-41 and SIINFEKL, respectively, or 0.5 µg/ml anti-CD3 antibody (2c11). Cells were then cultured in 20 ng/ml IL-2 (Proleukin) at 37°C for an additional 6 d. For conditional deletion of PDK1, CTLs were prepared by activating PDK1^flox/flox TamoxCre splenocytes with 2c11 for 2 d followed by culturing in IL-2 for 5 d, with 0.6 µM of 4′OHT (Sigma-Aldrich) being added for the final 72 h of culture. Where indicated, cells were treated with various inhibitors: 1 µM Akti1/2 (EMD Millipore), 20 nM rapamycin (EMD Millipore), 10 µM IC87114 (synthesized in-house), and 10 µM LY294002 (Promega).
For short-term (20 h) TCR stimulations, naive CD8⁺ T cells were purified from lymph nodes of HIF1^flox/flox CD4Cre and HIF1^WT/WT CD4Cre nontransgenic mice or alternatively from P14-LCMV or OTI transgenic mice by magnetic cell sorting (Miltenyi Biotec). P14-LCMV and OTI naive CD8⁺ T cells were activated with gp33-41 plus 3 ng/ml anti-CD28 (clone 37.51; eBioscience) and SIINFEKL, respectively. Nontransgenic naive CD8⁺ T cells were activated with 2c11 plus anti-CD28.