ADAM17-mediated CD16 shedding drives monocyte subset misclassification in ex vivo immunological assays
- 1Department of Bio-Medical Science, GAIHST, Gachon University
- 2Department of Microbiology, Gachon University College of Medicine
- 3Division of Gastroenterology and Hepatology, Department of Internal Medicine, Gachon University Gil Medical Center, Gachon University College of Medicine
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.
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BibTeX
@article{kwon2026adam17mediated,
title = {{ADAM17-mediated CD16 shedding drives monocyte subset misclassification in ex vivo immunological assays}},
author = {Soyeon Kwon and Seung Kak Shin and Seungwon Ryu},
journal = {Frontiers in Immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1886931},
url = {https://www.frontiersin.org/articles/10.3389/fimmu.2026.1886931/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - ADAM17-mediated CD16 shedding drives monocyte subset misclassification in ex vivo immunological assays
AU - Soyeon Kwon
AU - Seung Kak Shin
AU - Seungwon Ryu
PY - 2026
DA - 2026/10/13
JO - Frontiers in Immunology
DO - 10.3389/fimmu.2026.1886931
UR - https://www.frontiersin.org/articles/10.3389/fimmu.2026.1886931/full
AB - 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.
SN - 1664-3224
ER -