Skip to main content
. 2024 Mar 19;12(3):e008402. doi: 10.1136/jitc-2023-008402

Figure 5.

Figure 5

Immunogenicity of shortlisted potential circular RNA neoantigens predicted from patients with CRC. (A) Schematics depicting antitumor effects of trained antigen-specific HLA-A*11 CD8+ T cells on patient-derived organoids. Created with BioRender.com. (B) Enzyme-linked immunospot characterization of IFN-γ secretion by circRNA neoantigens specific T cells after stimulation with HLA-A*11:01 expressing artificial antigen-presenting cells, in the absence (first row) or presence (second row) of CircNeo peptides (MYH9_2 & MYH9_3 pool), as well as individual peptides encoding MYH9_2 (third row) or MYH9_3 (fourth) neoepitopes. Images were taken and spots were counted using the CTL ImmunoSpot analyzer. (C) Quantification of the number of IFN-γ spots per 20,000 CD8+ T cells in. n=3 biological samples per group, each experiment was performed using independently trained T cells from three different healthy donors. One-way ANOVA was used for multiple experimental groups with p value adjustment. Data is shown as mean±SEM. *P<0.0001. (D) Tetramer staining showing the percentage of the control T cells specific to CircNeo peptides (first panel) or the MYH9_2 peptide (fourth panel), CircNeo trained T cells specific to the irrelevant EBV peptide (second panel) or CircNeo peptides (third panel), as well as MYH9_2 trained T cells specific to the irrelevant EBV peptide (fifth panel) and MYH9_2 peptide (last panel). (E) Quantification of tetramer percentages from CircNeo and MYH9_2 trained T cells in (D). n=3 biological samples per group, each experiment was performed using independently trained T cells from three different healthy donors. One-way ANOVA was used for multiple experimental groups with p value adjustment. Data is shown as mean±SEM. **P<0.01. (F) Representative images of organoid-neoantigen specific T cells killing assay showing normal organoid co-cultured with MYH9_2 specific T cells (first row), paired tumor organoid co-cultured with MYH9_2 specific T cells (second row) and tumor organoid co-cultured with MYH9_2 specific T cells in the presence of HLA blockade (third row). Scale bar, 100 µm. (G–I) Dose-dependent killing of tumor organoids by MYH9_2 or RAPGEF5_2 trained CD8+ T cells. Tumor organoids were dissociated into single cells or small clusters prior to co-culture with CD8+ T cells at the effector: target ratio 5:1 and 10:1, and analyzed by flow cytometry. The representative flow cytometry plots from circMYH9_2 T cells were shown in (G). n=2. The two-sided Student’s t-test was used to compare between each two groups. Data is shown as mean±SEM. *P<0.05; **p<0.01; ***p<0.001. ANOVA, analysis of variance; APC, antigen presenting cell; circRNA, circular RNA; EBV, Epstein-Barr virus; HLA, human leukocyte antigen; IFN, interferon; PI, propidium iodide; SFU, spot-forming units; FITC, fluorescein isothiocyanate; BF, bright field.