Figure 2.

Isolation of a Mutated USP8-Specific TCR

(A) TIL4090F7 T cells were screened against a TMG library. The reactivity of T cells against TMG was measured by IFN-γ ELISPOT assay. (B) TIL4090F7 T cells were co-cultured with TMG-5-transfected autologous DCs for 4 hr, and then they were subjected to single-cell RNA-seq analysis. The expression of IFN-γ and IL-2 mRNA in each single cell was obtained by bioinformatics analysis. FPKM, fragments per kilobase of transcript per million mapped reads. (C) 4090TCR was transduced into donor T cells, and then transduced T cells were co-cultured with TMG-transfected autologous DCs. Error bars represent SD. (D) Mutated 25-mer peptides corresponding to each minigene within TMG-5 were pulsed on autologous DCs for 24 hr, and peptide-pulsed DCs were co-cultured with transduced T cells. (E) Purified 25-mer WT or mutated USP8 peptide (WAKFLDPITGTFHYYHSPTNTVHMY, R > H) was pulsed on autologous DCs for 24 hr, and peptide-pulsed DCs were co-cultured with transduced T cells. The secretion of IFN-γ from T cells was determined by ELISA.