ADAM17-mediated CD16 shedding drives monocyte subset misclassification in ex vivo immunological assays

Human monocytes are classified as classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) subsets, but CD16 stability during ex vivo assays remains uncertain. Here, we examined whether standard PBMC handling alters CD16-based subset identity and whether ADAM17 inhibition can prevent this artifact. In isolated PBMCs, intermediate and non-classical monocytes rapidly lost surface CD16 during culture at 37 °C, with or without LPS stimulation, whereas CD16 expression remained stable at 4 °C and in unseparated whole blood. Pre- versus post-incubation labeling and sorted-cell experiments indicated that CD16 loss was not primarily due to receptor internalization or cell death, but instead produced a pseudo-classical phenotype. Public scRNA-seq analyses showed enriched ADAM17 expression in monocyte/macrophage populations across blood, liver, and kidney datasets. Pharmacological ADAM17 inhibition with TMI-005 or TAPI-1 preserved CD16 expression during LPS stimulation without broadly impairing monocyte cytokine production. We therefore propose an ADAM17 inhibitor-based protocol that combines surface staining, stimulation, and intracellular cytokine profiling to preserve monocyte subset identity during functional assays. These findings identify a practical strategy to reduce CD16-dependent monocyte subset misclassification in ex vivo immunological studies.