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

Date Published: 2026-10-13

Abstract:
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.

Introduction:
Monocytes are a heterogeneous population of immune cells that have been classified into three major subsets on the basis of their surface expression of CD14 (a co-receptor for Toll-like receptor-4) and CD16 (Fc? receptor-III): classical monocytes (CD14++ CD16?), intermediate monocytes (CD14++ CD16+), and non-classical monocytes (CD14+ CD16++) ( 1 , 2 ). Classical monocytes constitute the majority (approximately 85-90%) of circulating monocytes and are characterized by their pronounced recruitment to sites of…

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