Expansion and isolation of NY-ESO-1–specific T cell clones.

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Expansion and isolation of NY-ESO-1–specific T cell clones. Expansion and isolation of NY-ESO-1–specific T cell clones. PBMCs were obtained from patients with metastatic melanoma. T cell cloning strategy for a representative HLA-A2+, HLA-Cw3+ donor is shown. (A) Schematic outlining the expansion and testing strategy to identify NY-ESO-1–reactive T cell clones. PBMCs were incubated with 28 NY-ESO-1 18-mer peptides (overlapping by 12 aa) and then expanded for 10 d before restimulation with individual peptides in the presence of BFA. Epitopes presented by patient MHC alleles are colored red, blue, and green in those peptides containing the full epitope sequence. (B) Representative flow cytometry measurement of intracellular staining for IFN-γ in expanded PBMCs restimulated with individual NY-ESO-1–derived 18-mer peptides. (C) Schematic outlining the reexpansion strategy using individual 9–10-mer peptides verified to elicit a T cell response. (D) Representative flow cytometry data showing an NY-ESO-1–reactive subpopulation of CD3+CD8+ T cells before sorting. Sorted cells were expanded in the presence of IL-2 and irradiated autologous PBMCs. (E) Representative flow cytometry data showing an NY-ESO-1–reactive subpopulation of CD3+CD8+ T cells following sorting. Michael T. Bethune et al. PNAS 2018;115:45:E10702-E10711 ©2018 by National Academy of Sciences