A Circulating TLR2 ^pos CD14 ^neg CD16 ^neg ″ Unclassified Subset ″ is Decreased in Multiple Myeloma Patients and May Comprise CD163 ^pos Dendritic Cells.

<jats:p> <jats:bold>Background</jats:bold> : Strategies to define human monocytes by flow cytometry vary considerably across studies. Recently, toll-like receptor 2 (TLR2) has been proposed as a marker to identify ″ all monocytes ″in human peripheral blood. However, the TLR2-defined monocytes also contained a previously ignored TLR2 <jats:sup>pos</jats:sup> CD14 <jats:sup>dim/neg</jats:sup> CD16 <jats:sup>neg</jats:sup> population, which we termed the unclassified subset (UCS). <jats:bold>Methods</jats:bold> : Peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS) were analyzed by multiparameter flow cytometry using TLR2 <jats:sup>pos</jats:sup> gating. PBMCs from additional healthy donors were analyzed to characterize the UCS population, including the impact of using either TLR2 <jats:sup>pos</jats:sup> or a negative selection-based gating strategy. <jats:bold>Results</jats:bold> : The TLR2 <jats:sup>pos</jats:sup> CD14 <jats:sup>dim/neg</jats:sup> CD16 <jats:sup>neg</jats:sup> UCS population was present in healthy controls, MGUS, and MM patients. The UCS expressed the monocyte-macrophage scavenger receptor CD163 and was significantly reduced in MM patients compared to healthy donors (P&lt;0.002). Further phenotypic characterization in healthy blood donors revealed that approximately 80% of UCS cells expressed CD163 at levels comparable to classical monocytes, yet phenotypically resembled CD163 <jats:sup>pos</jats:sup> dendritic cells (DCs). Importantly, gating strategies influenced the composition of the UCS: negative selection-based gating captured all DC subsets, whereas TLR2 <jats:sup>pos</jats:sup> gating primarily included CD1 <jats:sup>cpos</jats:sup> DCs that were highly CD163 <jats:sup>pos</jats:sup> . <jats:bold>Conclusions</jats:bold> : These findings demonstrate that circulating CD163 <jats:sup>pos</jats:sup> CD1 <jats:sup>cpos</jats:sup> DCs are included in the TLR2pos cell population previously described as exclusively monocytes, highlighting the impact of gating strategy on monocyte subset identification. Further, the lower level of TLR2 <jats:sup>pos</jats:sup> CD14 <jats:sup>dim/neg</jats:sup> CD16 <jats:sup>neg</jats:sup> CD163 <jats:sup>pos</jats:sup> cells in MM patients may represent decreased levels of circulating DCs that may contribute to the immune dysregulation in this disease. </jats:p>