Figure 3.

Identification of neoantigens targeted by circulating CD8⁺PD-1⁺ cells isolated from subjects NCI-3784, NCI-3903 and NCI-3713. (a,b) Reactivity of NCI-3784 TMG3- (top), TMG5- (middle) and TMG8-reactive (bottom) CD3⁺CD8⁺ lymphocytes to autologous DCs pulsed with the individual mutant 25-mers encoded by the indicated TMG (a) or to autologous DCs pulsed with FLNA_R>C, KIF16B_L>P or SON_R>C 25-mers, or their WT counterparts (b). (c) Recognition of B cells pulsed with serial dilutions of the Mut KIF1PB_P>S minimal peptide or its WT counterpart by NCI-3903 TMG9-reactive CD3⁺CD8⁺ lymphocytes. (d) Frequency of the top five TRB clonotypes, as determined by deep-sequencing analysis of TRB from NCI-3903 TMG9-reactive lymphocytes. (e,f) IFN-γ ELISPOT assays (e) and frequency of 4–1BB expression (f) of NCI-3713 pretreatment PBMC that were sorted into CD8⁺, CD8⁺PD-1⁻, CD8⁺PD-1⁺ and CD8⁺PD-1^hi cells, expanded in vitro and cocultured with B cells pulsed with either DMSO as a control or the WT and Mut epitopes from the indicated proteins. Experiments were performed without duplicates. All data are representative of at least two experiments.