Figure 3.
Neoantigen Screening from PD-L1-KO Tumors
(A) Circos plot showing the detected mutations in the MC38 tumor. Tumor tissues were sampled at day 15 after tumor inoculation. Mutations of MC38 were identified by whole-exome sequence and RNA sequencing (RNA-Seq). FPKM value and MHC-I affinity values were also plotted.
(B) Venn diagram of identified missense mutations between MC38 WT and PD-L1-KO tumors. Reactive peptide sequences and their IC50 values as calculated by NetMHCpan ver3.0. H2-Kb-restricted peptides for MC38 neoantigen candidates were synthesized and prepared.
(C) Experimental scheme of neoantigen candidate screening. MC38-PD-L1-KO tumor-bearing mice were treated with anti-PD-1 and anti-CTLA-4 on day 7, 10, and 13. Fifteen days after tumor inoculation, CD8 T cells were purified from pooled splenocytes and the tumor draining lymph node (4 mice per experiment) and cocultured with 30 Gy-irradiated splenocytes in the presence or absence of 10 μg/mL neoantigen candidate peptides for 72 h.
(D) Upper, culture supernatants were measured for IFN-γ production by ELISA. Data are pooled from five independent experiments. Lower, character of neoantigen peptides.