Fig. 1: Q148G is an IL-1β mutant with strongly reduced biological activity that can be completely reactivated upon targeting using a CD8α sdAb.
From: Specific targeting of IL-1β activity to CD8+ T cells allows for safe use as a vaccine adjuvant
a Model of IL-1R1 (red, domains D1–D3) and IL-1RAP (orange) binding to IL-1β (purple), showing a detail of the Q148G mutation. b NF-κB-driven luciferase reporter gene expression in HEK-Blue-IL1R cells. NF-κB activity is normalized to background and expressed as fold induction. Data points represent the mean of four independent experiments ± s.e.m. c CD8α ALN-1 design: IL-1β Q148G is coupled to an anti-CD8α sdAb by a 20xGGS linker. The fusion to the BcII10 sdAb is used as an untargeted control. d Representative SDS-PAGE protein gel demonstrating purity after recombinant production in HEK-293-F cells. Dotted lines indicate nonadjacent lanes from the original gel. e NF-κB-driven luciferase reporter gene expression in HEK-Blue-IL1R cells, transiently transfected with CD8α or irrelevant target DNA. NF-κB activity is expressed as absolute luminescence units (LU). Data points represent the mean of at least five independent experiments ± s.e.m. f Confocal microscopy showing nuclear translocation of NF-κB p65 in HEK-Blue-IL1R cells (1:1 mix of cells transfected with CD8α or irrelevant target DNA). Stimulation with vehicle, WT IL-1β, CD8α ALN-1, or untargeted ALN-1 (0.5 nM). The scale bar (white) indicates 50 μm. Asterisk (*) indicates cells of particular interest. g RT-qPCR analysis showing expression of NF-κB-, p38 MAPK-, and AP-1-driven target genes in human 132 1N1 astrocytes, transfected with CD8α or irrelevant target DNA. Stimulation with WT IL-1β, CD8α ALN-1, or untargeted ALN-1 (0.5 nM). Data are normalized to gene expression upon WT IL-1β stimulation. Bars represent mean ± s.e.m. of two independent experiments. See also Supplementary Fig. 1.
