Extended Data Fig. 4. Generation of HEK293T cell lines able to present OVA on H-2K^b.

a, Confocal microscope images of HEK293T cells stably expressing cytosolic GFP, H-2K^b and either C7, C9::C7 or C9(Y7F)::C7 receptors (anti-Dectin-1 staining). Nuclei were stained with DAPI. Images are representative of 3 similar images (scale bars 5 μm). b, As for (a) but using flow cytometry c, IL-2 from B3Z hybridoma cells cultured with the above H-2K^b-expressing HEK293T cells in the presence of the indicated concentrations of SIINFEKL peptide. Data are plotted as mean (± s.d.) of experimental triplicates from one of three independent experiments (n = 3). P values were determined by two-way ANOVA. d, Confocal images of HEK293T cells stably transfected with empty vector or plasmid encoding either C7, C9::C7 or C9(Y7F)::C7 receptors and pulsed with biotinylated zymosan for 1 hr before fixation and labelling of uninternalised zymosan with fluorescent streptavidin (scale bars 10 μm).. The number of internalised zymosan particles (right) was enumerated from the images (left). Data represented as mean (± s.e.m.) and each dot represents an independent experiment. e, RAW264.7 cells expressing H-2K^b and either C9::C7 or C9(Y7F)::C7 receptors were pulsed with zymosan-OVA for 4 hrs before fixation with 0.5 % PFA and incubated with OT-I cells. IFN-γ was assessed by ELISA. f, LLOMe (1 mM) was added during zymosan-OVA pulse of RAW264.7 cells expressing H-2K^b and C9(Y7F)::C7 receptor. All data in (e, f) are plotted as mean (± s.e.m.) of experimental triplicates. n.s., not significant; **P ≤ 0.01; ****P ≤ 0.00001.