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. 2018 Oct 4;3(19):e122467. doi: 10.1172/jci.insight.122467

Figure 2. Sorting strategy, functional screen assays, and identification of neoantigen-reactive TCRs in Pt.4078.

Figure 2

(A) Gating strategy used in flow cytometry–based sort for CD3+PD-1+ and/or CD134+ TILs. (B–D) ELISPOT assays measuring IFN-γ secretion of microwell cultures upon coculture with target cells. (B) Following expansion, pools of 2 cultures were tested against autologous DCs pulsed with 2 peptide pools (PP), indicated by symbols. (C) Cultures from the reactive pools were tested separately against autologous DCs pulsed with all suspected peptide pools. (D) IFN-γ ELISPOT and CD137 flow cytometry analysis showing reactivity of the TIL cultures following coculture with autologous DCs pulsed with single 25mer peptides from each peptide pool. (E) Allogeneic T cells retrovirally transduced with neoantigen-reactive TCRs cocultured with autologous DCs pulsed with serially diluted mutated and WT 25mer peptides. ‘>’ denotes greater than 500 spots. All data are representative of at least 3 independent experiments except in A.