Fig. 5: Increased uORF-derived peptide presentation in mitotic cancer cells promotes targeted immune responses.
a Schematic of the uORF reporter system. Two different 5’ UTRs (eIF4G2 and TPX2) were selected and cloned into the pGL3-promoter vector. The sequence of the uORF-derived peptide was replaced with the SIINFEKL sequence, followed by firefly luciferase. b Detection of the p:MHC complex SIINFEKL:H-2Kb by flow cytometry, shown as Median fluorescence intensity (MFI), in TC1 cells transfected with the indicated reporters. Cells were treated with Taxol (1 µM) or DMSO (Control) for 16 h. Data represent mean ± SD from biologically independent experiments (n = 3). P values were calculated using a two-tailed unpaired t-test. c MFI of the SIINFEKL peptide bound to H-2Kb in TC1 cells transfected with wild-type uORF-SIINFEKL reporters or start codon mutant uORF-SIINFEKL reporters. Cells were treated with Taxol (1 µM) for 16 h. Data represent mean ± SD from biologically independent experiments (n = 3). P values were calculated using a two-tailed unpaired t-test. d Schematic illustrating how the presentation of uORF-derived SIINFEKL peptides induced by mitotic arrest leads to OT-I T cell recognition. Proliferating cells expressing uORF-SIINFEKL reporters are not recognized by OT-I cells, whereas enhanced uORF translation in mitotically arrested cells allows for their recognition. e, f Percentage of TC1 cell killing in vitro by activated OT-I cells. TC1 cells were transfected with the indicated reporters and arrested in mitosis with Taxol (1 µM) for 16 hrs. or treated with DMSO for the same period. Data represent mean ± SD from biologically independent experiments (n = 5). P values were calculated using a two-tailed unpaired t-test. NS non-significant. Source data are provided as a Source Data file.
