Ova 257 264 siinfekl peptide

CD8+ T cells were purified from the spleen of the WT C57/BL6 mice, through the use of a Mojo CD8 T cell Negative Selection kit. Naïve T cells were then labeled with 5 μM Alexa Fluor 488 conjugated Carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) dye for 10 minutes at 37°C. The purified CD8 T cells were then activated by plate-coated or Dynabeads-coated CD3/CD28 Ab. In the meantime, CD80 (+) or CD80(−) PDVC57 SCC cells were pre-treated with mitomycin c (4 μg/ml) for 2 hours to arrest their proliferation, and then added on Day 2 post T cell activation and added to T cells at 1:1 ratio in T cell medium.
T cell proliferation, a sign of successful activation, was measured by flow cytometry quantification of CFSE retention in T cells after 3 days co-culture. For tumor/OT-I T cell co-cultures, splenocytes from OT-I mice were activated with OVA 257–264 (SIINFEKL) peptide (2 μg/ml) (InvivoGen) for 2 days, and then the CD8+ T cells were purified, labeled with CFSE, and added to CD80 (+) or CD80 (−) SCC cells at 1:1 ratio, together with 10 μg/ml CD28 agonist Ab (clone 37.51 or D665), 10 μg/ml CD28 antagonist Ab (mPEG PV1-Fab’)(Papotto et al., 2017 (link)), 25 μg/ml CTLA4 blocking Ab (clone UC10–4F10–11), or 25 μg/ml PDL1 blocking Ab (clone 10F.9G2) and co-culture in T cell medium for 3 days