Fig. 5: Antigen anchoring to F-actin in dead cells potentiates DNGR-1-dependent XP by cDC1s.
a–e, Stimulation of pre-activated OT-I CD8+ T cells after overnight co-culture with Mutu DCs or Flt3L cDC1 and necrotic HeLa cells expressing the indicated fusion proteins. Graphs show concentration of IFN-γ in the supernatant of cultures. a, Comparison of the ability of LA-OVA- and mutLA-OVA-expressing HeLa cells to stimulate OT-I T cells following presentation of dead cell-associated antigen by Mutu DC. b, XP of cells as in a in the presence of anti-DNGR-1 (1F6) or isotype-matched control antibody (MAC49). c, Comparison of DNGR-1-deficient (KO) Mutu DC complemented with WT DNGR-1 (C9A/KO) or double tryptophan mutant (2 W/KO) DNGR-1 for ability to cross-present dead LA-OVA or mutLA-OVA cells. d, Comparison of F-actin-specific affimer-OVA (Aff-OVA) and control affimer OVA (CtrlAff-OVA) dead cells with LA-OVA dead cells. e, XP of (mut)LA-OVA cells by Flt3L cDC1. Plotted data represent mean of technical duplicates (a–c) or mean ± s.d. of technical triplicates (d,e). Data are representative of three (a–c) and two (d,e) independent experiments, respectively. f,g, Induction of OVA-specific CD8+ T cell responses following immunization of WT or sGSNKO mice with dead LA-OVA or mutLA-OVA cells mixed with poly I:C (25 µg per mouse). Scatter plots show data pooled from three (f) or two (g) independent experiments, and the number of biological replicates in each group ranges from n = 4 to 13. P values shown for pairwise comparison of groups in each graph were analyzed using ordinary one-way ANOVA and Tukey’s multiple comparison test. f, Percentage (left) of H-2Kb/OVA pentamer+ CD44+ double-positive cells of total CD8+ T cells and their absolute number (right). g, Percentage (left) of IFN-γ+ CD44+ double-positive cells of total CD8+ T cells and their absolute number (right). Where pairwise comparisons are statistically significant, exact P values have been annotated on the graph, and where indicated, ns, not significant.
