A Novel, Five-Marker Alternative to CD16–CD14 Gating to Identify the Three Human Monocyte Subsets

Abstract

Human primary monocytes are heterogeneous in terms of phenotype and function, but are sub-divided only based on CD16 and CD14 expression. CD16 expression distinguishes a subset of monocytes with highly pro-inflammatory properties from non-CD16 expressing “classical” monocytes. CD14 expression further subdivides the CD16 + monocytes into non-classical CD14 low and intermediate CD14 high subsets. This long-standing CD16–CD14 classification system, however, has limitations as CD14 is expressed in a continuum, leading to subjectivity in delineating the non-classical and intermediate subsets; in addition, CD16 expression is unstable, making identification of the subsets impossible after in vitro culture or during inflammatory conditions in vivo . Hence, we aimed to identify the three monocyte subsets using an alternative combination of markers. Additionally, we wanted to address whether the monocyte subset perturbations observed during infection is real or an artifact of differential CD16 and/or CD14 regulation. Using cytometry by time-of-flight (CyTOF), we studied the simultaneous expression of 34 monocyte markers on total monocytes, and derived a combination of five markers (CD33, CD86, CD64, HLA-DR, and CCR2), that could objectively delineate the three subsets. Using these markers, we could also distinguish CD16 + monocytes from CD16 − monocytes after in vitro stimulation. Finally, we found that the observed expansion of intermediate (CD14 high ) monocytes in dengue virus-infected patients was due to up-regulated CD16 expression on classical monocytes. With our new combination of markers, we can now identify monocyte subsets without CD16 and CD14, and accurately re-examine monocyte subset perturbations in diseases.

The Peripheral Immune System and Amyotrophic Lateral Sclerosis

“…HLA DR is expressed in CD16 + monocytes, while CD14 + monocytes reduce HLADR expression when activated (105). Other markers can also be used to distinguish monocyte subsets (106).…”

Standardization of sampling and sample preparation for analysis of human monocyte subsets in peripheral blood

“…Defined in mice. [94] Classical monocytes Intermediate monocytes Non-classical CD33 high , CD86 +/high , CD64 + , HLA-DR + , CCR2 high CD33 high CD86 +/high CD64 + HLA-DR high CCR2 low CD33 low CD86 high [175] Classical monocytes Intermediate monocytes Non-classical CD300e high CD14 high CD16 negative CD300e hi CD14 hi CD16 + CD300e hi CD14 low/-CD16 + [111] Classical monocytes Intermediate monocytes Non-classical CD14 ++ CD16 − CD14 ++ CD16 + CD14 + CD16 ++ [34,165,[168][169][170][171][172]176]* *There is a great variation in the overall antibody panel and gating strategy applied, but all the studies identified monocyte subsets by CD14 and CD16 expression.…”

Decrease of Non‐Classical and Intermediate Monocyte Subsets in Severe Acute SARS‐CoV‐2 Infection

“…CD14 ++ CD16 dim CL monocytes and CD14 ++ CD16 + INT monocytes account for 90–95% of peripheral blood monocytes and are known to respond to bacterial‐associated signals. Conversely, CD14 dim CD16 + NC monocytes account for the remaining 5–10% and are more responsive to virus‐associated stimuli (2, 3). In this study, patients with acute SARS‐CoV‐2 infection were studied and NC, INT, and CL monocytes subsets were evaluated by flow cytometry at the first clinical presentation.…”