Extended Data Fig. 5: Identification of residues within loop 5 of Unc93b1 that mediate interaction with TLR9.

(a) Schematic of the tested loop 5 mutants of Unc93b1 and the relative TLR9 responses indicated in shades of gray: white indicates a response equivalent to WT while black indicates no response. Asterisks show human Unc93b1 SNPs that have been tested in (f). (b) A larger region in loop 5 of Unc93b1 mediates binding to TLR9. Immunoprecipitation of Unc93b1-Flag from macrophage lines expressing the indicated Unc93b1 mutants (spanning amino acids 267–284, and non-functional HR) followed by immunoblot of TLR9-HA. Representative of two independent experiments. (c) Intracellular cytokine staining of TNFα in macrophage lines shown in (b) after stimulation with CpG-B (25 nM), R848 (100 ng/ml), and LPS (10 ng/ml). Shaded histograms show unstimulated controls. Representative of three independent experiments. (d) Schematics showing relative positions and a sequence alignment (bottom) of swapped regions within the TLR9/3 chimeras. Colored regions indicate TLR3 sequences. NF-κB luciferase assay in HEK293T cells transiently transfected with the indicated TLR9 and Unc93b1 mutants and stimulated with CpG-B (200nM) for 16h. Data are normalized to Unc93b1-independent hIL-1b responses and expressed as luciferase fold change over unstimulated controls. Data are mean ± s.d., n=3 biological replicates. p-values determined by two-way ANOVA followed by a Sidak’s posttest comparing each TLR9 allele coexpressed with Unc93b1 WT vs S282A (TLR9^WT: p<0.0001, TLR9^Mut1: p>0.9999, TLR9^Mut2: p<0.0001, TLR9^Mut3: p<0.0001, TLR9^Mut4: p=0.0020, TLR9^Mut5: p=0.0171. Representative experiment of two independent repeats. (e) TLR9 mutants that rescue signaling in the presence of Unc93b1^S282A also show normal binding to Unc93b1^S282A. HA immunoprecipitation of the indicated TLR9 mutants transiently expressed in HEK293T cells that stably express the indicated Unc93b1-Flag alleles, followed by immunoblot of Unc93b1-Flag. Representative of three independent experiments. (f) Human Unc93b1 variants with SNPs in loop 5 show decreased TLR9 signaling. NF-κB luciferase assay in HEK293T cells expressing TLR9 or TLR7 and the indicated human Unc93b1-Flag variants and stimulated with CpG-B (250 nM) or R848 (250 ng/ml) for 16h, respectively. Data are normalized to Renilla expression and expressed as relative luciferase units (RLUs). Data are mean ± s.d., n=3 biological replicates. p-values are determined by one-way ANOVA followed by a Tukey’s posttest. For CpG-B stimulations: p=0.0048 (WT vs G270S), p=0.0113 (WT vs R277Q), p=0.9994 (WT vs G283R). Representative of four independent experiments. For R848 stimulations: p=0.2001 (WT vs G270S), p=0.0002 (WT vs R277Q), p=0.9933 (WT vs G283R). Representative of three independent repeats experiment. (g) Expression levels of the Unc93b1 mutants used in (f). *p< 0.05, **p< 0.01, ***p< 0.001, ns: not significant. WCL: whole cell lysate. FL: full-length.