Cytotoxic T Lymphocyte Generation and SILAC Labeling

Cytotoxic T Lymphocytes were generated as previously described (Hukelmann et al., 2016 (link), Navarro et al., 2011 (link), Navarro et al., 2014 (link)) and expanded in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS (Life Technologies), 50 units/mL penicillin-G, 50 μg/mL streptomycin, and 50 μM β-mercaptoethanol and in the presence of 20 ng/mL IL-2 alone (Proleukin, Novartis). For SILAC labeling, CTLs were cultured for 5 days in SILAC RPMI 1640 medium (Life Technologies), supplemented with 200 mg/L L-proline, 84 mg/L L-arginine, 300 mg/L L-glutamate, 10% dialyzed FBS with a 10 kDa cutoff (Thermo Scientific), 50 units/mL penicillin-G, 50 μg/mL streptomycin, 50 μM β-mercaptoethanol, and 20 ng/mL IL-2. The “light” SILAC media contained L-[12C6, 14N4]arginine (R0) and L-[12C6, 14N2]lysine (K0). The “heavy” media contained L-[13C6, 15N4]arginine (R10) and L-[13C6, 15N2]lysine (K8).
For the IL-2 stimulation of CTLs, cells were “IL-2 quiesced” by the removal of IL-2 for 24 hr, but they were supplemented with 20 ng/ml IL-12 (R&D Systems) to sustain cell viability (at ∼90%) and the expression of the IL-2Rα chain (CD25). For the Tofacitinib and PP2 studies, CTLs were maintained only in the presence of IL-2, and no IL-12 was added to the culture.