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. 2022 Mar 9;603(7902):721–727. doi: 10.1038/s41586-022-04499-2

Fig. 4. Substitutants are presented at the cell surface and activate T cells.

Fig. 4

a, A model predicting the effect of IFNγ on the presentation and recognition of SIINFEKL and SIINwEKL by anti-H2-Kb-bound SIINFEKL antibodies. b, Dot plot depicting the APC median fluorescence intensity (MFI) of H2-Kb-bound SIINFEKL peptides in MD55A3 cells expressing H2-Kb (MD55A3 H2-Kb) in combination with the V5–ATF41–63–tGFP–SIINxxKL reporters. Each dot represents an independent biological replicate (n = 3). ***P < 0.001, ordinary one-way ANOVA using Sidak’s multiple comparison test. c, HT29 H2-Kb control and SIINwEKL-expressing cells were pre-treated with IFNγ, IDOi and tryptophan depletion as indicated and used in co-cultures with OT-I-derived T cells for 4 h. T cell activation was assessed by flow cytometry analysis of intracellular IFNγ positivity. Dots represent values obtained from independent experiments. Lines represent mean ± s.d. of three independent experiments. ***P < 0.001, ordinary one-way ANOVA using Sidak’s multiple comparison test. d, Tumour-killing efficacy of OT-I T cells in co-cultures with control HT29 H2-Kb or HT29-H2-Kb-SIINwEKL cells. Dots represent the mean relative cell number of the indicated co-cultures plus s.d. of three independent experiments. ***P < 0.001 by ordinary one-way ANOVA using Sidak’s multiple comparison test. e, Immunopeptidomics analysis of mock and IFNγ-treated RA glioblastoma cells. Heat map depicting the number of various substitutions detected in immunopeptidomics analysis of mock (Ctrl) or IFNγ-treated RA glioblastoma cells. Only the peptides identified in two biological replicates were counted. f, Flow cytometric analysis of CD8+ T cells following co-culture of naive CD8+ T cells and autologous monocyte-derived dendritic cells pulsed with peptide or DMSO vehicle. Plots show T cells reactive to streptavidin–PE and streptavidin–PE-CF594-labelled pMHC multimers complexed with the KLHL4 substitutant peptide YFDPHTNKF (well 1). g, A model depicting the induction of W>F substitutants following tryptophan depletion associated to IFNγ treatment and T cell activation.