Figure 3.

Isolation of highly enriched, mutation-reactive T cell lines. For each panel, examples of T cell mini-lines 1F3 and 4C5 are shown. A. Mini-lines identified in the screen were REP expanded and stimulated in duplicate wells with peptides encompassing each tumor-specific mutation (N = 37 mutations) in ELISPOT assays. Bars represent the number of IFN-γ spot forming cells in each well. X-axis represents each individual mutation peptide pool with the reactive peptide indicated. B. FACS sorting plots for T cell line that proved reactive to a single mutation. CD8 (X-axis) and 4-1BB (Y-axis) double positive cells were FACS sorted. C. IFN-γ ELISPOT assay of T cell lines responding to individual minimal peptides contained within the mutation specific peptide pool. Bars represent the number of spot forming cells per well. Amino acids sequences of each peptide are shown on the X-axis. D. Flow cytometry plots of T cell lines stained with antibodies towards Vβ regions (X-axis) and 4-1BB (Y-axis). T cell lines were stained with the full complement of Vβ antibodies, and only the positive Vβ stains are shown (Vβ 13.1 for well 1F3; and Vβ 1 for well 4C5). E. IFN-γ ELISPOT assay of T cell cross-reactivity to WT versions of minimal epitopes. Lines represent the number of IFN-γ spot forming cells responding to 10-fold titrations of cognate mutated minimal epitope (squares) and the corresponding wild type peptide (triangles).