Figure 7.

Primary T Cell Expressing Neoantigen-Specific TCR Recognizes Tumor

(A) TCRdist analysis of TCRB repertoire sequences showing the tree diagram of TCRB CDR3 homology. Clonality was not reflected. Red arrows indicate neoantigen peptide reactive CDR3 sequence. As a negative control, rearranged CDR3-derived di-amino acids (red rectangle) were mutated into AlaAla.

(B and C) TCRαβ derived from CTL responding to Dpagt1pep included TCR36, and TCRαβ derived from CTL responding to Cry1pep is termed as TCR8 and TCR10. Reactive TCRα and β chains were selected, and they were tandemly linked with Furin-SGSG-P2A sequence and cloned into pMXs-IRES-GFP. Selected combination of TCRα and TCRβ were transfected to TG40 CD8A/B (TCR36, B; TCR8, TCR10, C). These TG40 cells were cocultured with each peptide-pulsed EL4 for 24 h. The percentage of CD69 upregulation by TCR36 or TCR8 and TCR10 and their AA mutation transduction in GFP⁺ cells for MutDapgt1 peptide or MutCry1 peptide is shown. Data were pooled from six or three independent experiments, and the values represent mean ± SD. Each black circle shows independent values.

(D) TCR36-transduced or TCR8- and TCR10-transduced GFP⁺CD8⁺ T cells were cocultured with MC38 in the presence or absence of neoantigens peptide for 48 h. Subsequently, supernatants were measured for IFN-γ by ELISA. Data were pooled from seven to ten independent experiments, and the values represent mean ± SD. Data were analyzed by unpaired Student's t test, ∗p < 0.05, ∗∗p < 0.01.