Myeloid differentiated cells derived from patient iPSCs and gene-edited CD34s with the TLR8 variants demonstrated increased responsiveness to TLR8 stimulation. (A) Cytospin analysis of cells derived from neutrophil differentiation of WT or p.P432L iPSC clones from patient (P2) and WT or p.F494L iPSC clones from patient (P3) and their upregulation of phosphorylated p65 (NF-κB) in response to stimulation with the indicated doses of the TLR8-ligand TL8-506. (B) H&E stain of cytospin from cells derived from neutrophil differentiation of WT and gene-edited (p.P432L) healthy donor human CD34⁺ HPCs and their upregulation of phosphorylated p65 (NF-κB) in response to the TL8-506. Cells in flow plots are gated on CD45⁺CD66b⁺ neutrophils. Data are representative of 2 independent experiments. (C) Cytospin analysis of cells derived from macrophage differentiation of WT or p.P432L induced pluripotent stem cell (iPSC) clones from patient (P2) and phosphorylated p65 (NF-κB) in response to stimulation with the indicated doses of the TLR8-ligand TL8-506. Cells are gated on CD45⁺CD14⁺ macrophages. (D-E) iPSC-derived macrophages with WT or p.P432L TLR8 from patient (P2) were cultured overnight with the indicated doses of TL8-506 (TLR8 agonist), CL264 (TLR7 agonist), or LPS (TLR4 agonist). (D) Cytokines were measured in the cell culture supernatant and demonstrate that macrophages with variant TLR8 produced significantly more IL-6 and TNF-α with low-dose TLR8 stimulation (TL8-506, 25ng/mL) compared with cells with WT TLR8. There was no difference in the cytokine response to TLR7 stimulation (CL264), and doses of CL264 <100 ng/mL did not result in cellular activation (data not shown). (E) WT and p.P432L macrophages had a similar response to the TLR4 ligand LPS with respect to production of TNF-α and IL-6. NS, no stimulation. Data are presented as mean ± SEM and includes data from 3 independent experiments, analyzed by 2-way ANOVA. Findings that are statistically significant are denoted by an asterisk (*P ≤ .05).