Patients have mosaic and germline variants in TLR8 with normal expression of TLR8 protein and enhanced responsiveness to TLR8 stimulation. (A) Abdominal computed tomography scan from P1 showing marked splenomegaly. (B) H&E staining of bone marrow biopsy specimen (P3) showing hypocellularity for age with lymphohistiocytic aggregate (arrow). (C) Leder staining demonstrating myeloid hypoplasia (P3). (D) Family pedigrees of the 6 patients with variants in TLR8. Symbols with dots indicate mosaicism, and the solid black box indicates a germline variant in P6 (ND, not done). (E) ddPCR of patient DNA showing droplets with TLR8 variant p.P432L (upper left), WT TLR8 (lower right), both templates (upper right), or no TLR8 template (lower left) for P1 and the mother. Monos, monocytes; NK, natural killer; WB, whole blood, NT, no TLR8 template. (F) Percentage of droplets with variant or WT sequence in DNA or cDNA from whole blood or PBMCs, saliva, fibroblast lines, and/or sorted cell populations. (G) Intracellular TLR8 expression by flow cytometry in cells from age-matched healthy controls (solid red lines) and patients (P1, P3, and P5, solid blue lines). Similar expression of TLR8 was observed in patient monocytes and neutrophils. CD3⁺ T cells did not express TLR8. Red dashed line and blue dashed line indicates unstained control in healthy controls and patients respectively. (H) Expression of phosphorylated p65 (NF-κB) in monocytes (CD14⁺) from patients (P1 and P3) and healthy age-matched male controls stimulated with indicated doses of TLR8 agonist TL8-506. A small percentage (5% to 6%) of patient monocytes responded to the lower dose (100 ng/mL) of the stimulant. Healthy cells responded at the highest dose of TLR8 stimulation. There was no statistical difference between the patient cells and healthy cells with respect to their response at the highest dose of stimulation.