Additional file 8: of Efficient identification of neoantigen-specific T-cell responses in advanced human ovarian cancer

Figure S8. Characterization of JAK1 neoepitope-specific CD4+ T-cells. (a) Peptide reactivity of a JAK1 neoepitope-specific CD4+ T-cell line. IFN-γ and GM-CSF production on CD4+ T-cells against JAK1 mutated (IEILRNLYHEIIV) or wild-type (IEILRNLYHENIV) peptide-pulsed autologous EBV-B-cells were determined by intracellular cytokine staining. (b) TCR usage of JAK1 neoepitope-specific CD4+ T-cell line. T-cells were stained with TCR Vβ subtype-specific antibodies and analyzed by flow cytometry. (c) Purity of Vβ13.6+ cells after magnetic-beads sorting. (d) Avidity of JAK1 neoepitope-specific T-cell clone. CD4+ T-cell clones were stimulated with autologous EBV-B-cells pulsed with the indicated concentration of mutated or wild-type peptide for 6 h in the presence of Golgi stop. IFN-γ production from Vβ13.6+ cells were determined by flow cytometry. The data represents mean ± s.d. of duplicate wells. (e) Recognition of autologous tumor-derived cells by Vβ13.6+ T-cell clone. PBMC or AMC were co-cultured with Vβ13.6+ JAK1 neoepitope-specific CD4+ T clones or without T-cells (−) for 24 h. AMC: ascites-derived mononuclear cells. IFN-γ production was measured by ELISA. The data represent mean + s.d. of duplicate wells. *p