Figure 5.

Isolation of a Mutated SIN3A-Specific TCR

(A) TIL4171F6 T cells were screened against a library of 25-mer long-peptide pools (PPs) encoding mutations. The reactivity of T cells against mutation was measured by IFN-γ ELISPOT assay. (B and C) TIL 4171 F6 T cells were co-cultured with PP-3-pulsed 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 of each single cell is shown in dot plots (B) and the scatter plot (C). (D) 4171TCR was transduced into donor T cells, and then transduced T cells were co-cultured with PP-pulsed DCs. Error bars represent SD. (E) Individual mutated 25-mer peptides corresponding to PP-3 were pulsed on autologous DCs for 24 hr, and peptide-pulsed DCs were co-cultured with 4171TCR-transduced T cells. (F) Purified 25-mer WT or mutated SIN3A peptide (LGKFPELFNWFKIFLGYKESVHLET, N > I) 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.