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Generation and detection of patient neoantigen-specific T cells

NT Nicholas L. Truex
RH Rebecca L. Holden
BW Bin-You Wang
PC Pu-Guang Chen
SH Stephanie Hanna
ZH Zhuting Hu
KS Keerthi Shetty
OO Oriol Olive
DN Donna Neuberg
NH Nir Hacohen
DK Derin B. Keskin
PO Patrick A. Ott
CW Catherine J. Wu
BP Bradley L. Pentelute
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These experiments were performed as described in a previous publication13. PBMCs were cultured in RPMI-1640 medium supplemented with L-glutamine, nonessential amino acids, HEPES, β-mercaptoethanol, sodium pyruvate, penicillin/streptomycin (Gibco), and 10% AB-positive heat-inactivated human serum (Gemini Bioproduct). For in vitro expansion of antigen-specific T cells, PBMCs were stimulated in 24-well cell culture plates at 5 × 106 cells per well with individual peptides (each at 2 μg/mL) in the presence of IL-7 (20 ng/mL; R&D Systems). On day 3, low-dose IL-2 (20 U/mL; Amgen) was added. Half-medium change and supplementation of cytokines were performed every 3 days. After 14 days, T-cell specificity was tested against the peptide by interferon (IFN)-γ ELISPOT.

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