Functional studies demonstrate gain of function of TLR8. (A) Analysis of residues altered by TLR8 variants show that positions P432 and F494 contact the symmetric positions in the other subunit of the active ligand-bound dimer. G572D in the ligand-binding state is predicted to enter into a hydrophobic pocket and interact with R375. (B-C) NF-κB reporter cells (HEK Blue Null1 cells) that do not express endogenous TLR8 were transfected with WT TLR8, patient TLR8 variants (encoding p.P432L, p.F494L, p.G572D), or a loss-of-function (LOF) TLR8 variant (encoding p.D543A) and stimulated with the indicated doses of the TLR8-specific agonist TL8-506 (B) or the TLR8/TLR7 agonist CLO75 (C) for 24 hours. Mosaic and germline TLR8 variants lead to gain of function in TLR8 activity as measured by NF-κB transcriptional activity. Data are represented as mean ± standard deviation of biological replicates and representative of 8 independent experiments (TL8-506) or 3 independent experiments (CLO75). **P ≤ .01, ***P ≤ .001 by 2-way ANOVA test.