Extended Data Fig. 6: circFAM53B-encoded peptide elicits anti-tumour immunity via binding to HLA.

(a) The binding predictions for circFAM53B-encoded peptides to HLA-A*02:01 or HLA-A*11:01 using the IEDB algorithm (Rank, Score¹) and SYFPEITHI (Score²). The circFAM53B-encoded unique amino acid sequences are shown in red. (b) Quantification of the spot count per 5 × 10⁴ T cells, determined by IFNγ ELISpot. (c) Percentages of IFNγ-stained cells in the in vitro primed CD8⁺ T cells are shown, evaluated by flow cytometry. **P = 0.0014 (circFAM53B(181-219)), 0.0011 (MUC1(12-20)). (d) Representative flow cytometric plots of tumour death induced by the in vitro primed CTLs and the quantitation of PI⁺ tumour cells are shown. ***P = 0.0003 (circFAM53B(181-219)), 0.0003 (circFAM53B(192-200)), 0.0004 (MUC1(12-20)). (e) Percentages of CD8⁺ T cells stained for intracellular perforin or GZMB are shown, evaluated by flow cytometry. (f-i) The CTLs primed by circFAM53B(181-219)-pulsed DCs were rechallenged by circFAM53B WT, circFAM53B KD and “rescued” breast tumour cells MCF-7. The “rescued” breast tumour cells were established by transfecting circFAM53B KD cells with liposome only (mock), empty vector (vec), full length of circFAM53B (circFAM53B^fl), truncated circFAM53B^Δ1-117, circFAM53B^Δ60-117 RNAs, and mutated circFAM53B RNAs, respectively. (f) Scheme for circFAM53B mutation and truncation. (g) Immunoblotting for Flag and circFAM53B-219 expression in indicated cells (n = 3 independent experiments). For gel source data, see Supplementary Fig. 6. (h) Percentages of T cells stained for intracellular IFNγ, perforin and GZMB are shown, evaluated by flow cytometry. *P = 0.0141, **P = 0.0056. (i) Percentages of the PI⁺ dead tumour cells induced by in vitro primed CTLs are shown, evaluated by flow cytometry. ***P = 0.0003 (mock), 0.0004 (vec), 0.0004 (circFAM53B^Δ1-117). (j) Quantification of the spot count per 5 × 10⁴ T cells determined by IFNγ ELISpot. (k) Percentages of T cells stained for the indicated intracellular cytokines, evaluated by flow cytometry. (l) Percentages of the PI⁺ dead tumour cells induced by in vitro primed CTLs are shown, evaluated by flow cytometry. (m) MHC peptide binding predictions for circFAM53B-encoded peptides to HLA-DRB1*01:01 using the IEDB algorithm (Rank) and SYFPEITHI (Score²). The circFAM53B-encoded unique amino acid sequences are shown in red. (n) Percentages of T cells stained for the indicated intracellular cytokines, evaluated by flow cytometry. IFNγ: *P = 0.0116 (circFAM53B(191-204)), 0.0105 (circFAM53B(192-205)). TNF: ***P = 0.0002 (circFAM53B(191-204)), 0.0008 (circFAM53B(192-205)). IL-2: *P = 0.0416 (circFAM53B(192-205)). (o) Representative flow cytometric plots and quantitation of circFAM53B(192-200)-pentamer staining in the in vitro primed T cells. **P = 0.0014. (p) Clonotyping comparison between circFAM53B(192-200)-pentamer⁺ versus circFAM53B(192-200)-pentamer⁻ CTLs. The non-overlapping TCR repertoires are shown. Results are mean ± s.d. of n = 3 (b-e, h-l, n, o) independent experiments producing similar results. ****P <  0.0001. P values, compared with T cells primed by unloaded DCs (0 µg/ml (b, c) or (-) (d, o)), untreated T cells (UT) (e, j-l, n), CTLs rechallenged by circFAM53B WT tumour cells (h), circFAM53B WT tumour cells (i), were determined by two-tailed one-way ANOVA with Dunnett’s multiple-comparisons test.

Source Data