Figure 3. A*01:01/ILDTAGKEEY is immunogenic.

(A) Bulk TILs were able to kill cognate melanoma in a fluorescent cell in vitro–killing assay. The fluorescently labeled melanoma cell line 17T was coincubated with IHW01161 and the cell line 135T was coincubated with IHW01070. Error bars represent the SEM of biological triplicates. (B and C) Bulk TILs were specifically reactive toward the neoantigen. TILs were coincubated with minimal epitope–pulsed A*01:01⁺ B-LCLs. (B) IFN-γ ELISA results. Error bars represent the SEM of biological triplicates; 2-way ANOVA with Tukey’s correction for multiple comparisons. (C) IFN-γ ELISA peptide titration assay with 17TIL (IHW0116) and 135TIL (IHW01070). Plots show the mean with SD of biological triplicates; 2-way ANOVA with Sidak’s correction for multiple comparisons. (D–F) Delineation of the neoantigen-specific subpopulations. (D) IFN-γ secretion assay of the melanoma cells 17TIL (coincubatad with peptide-pulsed IHW01161 cells) and 135TIL (coincubated with peptide-pulsed IHW01070 cells). P < 0.00001, by χ² for both. (E) Differential expansion of 17TILs in response to coincubation with B-LCL IHW01161 and either the WT or mutant peptide. P < 0.00001, by χ² test. (F) CD8⁺ gated TILs double-stained with the Q61K and Q61R tetramers. FSC-A, forward scatter area. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.