Flow cytometric definitions of human monocytes vary across studies. A recent approach proposed using TLR2pos expression to define “all monocytes” in peripheral blood. However, application of a TLR2-based gate also identified a previously overlooked population with low or absent CD14 and CD16 expression. The investigators designated this population the unclassified subset (UCS) and sought to determine its phenotype and relevance in monoclonal gammopathies, including multiple myeloma (MM) and MGUS.
The study analyzed peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with MM or MGUS by multiparameter flow cytometry using a TLR2pos gating strategy. Additional healthy-donor PBMCs were examined to more fully characterize the UCS and to compare alternative gating approaches. Specifically, the authors compared TLR2pos gating with a negative selection–based gating strategy to assess how gate choice influenced the cellular composition captured as TLR2pos or as monocyte-equivalent cells.
Using TLR2pos gating, the authors observed a population defined by TLR2pos CD14dim/neg CD16neg phenotypes that did not fit conventional monocyte subset definitions. They termed this population the unclassified subset (UCS). The UCS was present across cohorts: healthy controls, MGUS patients, and MM patients. Quantitative comparisons revealed that the UCS population was significantly reduced in MM patients compared with healthy donors (reported P < 0.002).
Phenotypic analysis demonstrated that UCS cells expressed the monocyte-macrophage scavenger receptor CD163. In healthy donor samples, approximately 80% of UCS cells expressed CD163 at levels comparable to those seen on classical monocytes. Despite CD163 expression, further phenotyping indicated that the UCS more closely resembled a population of CD163pos dendritic cells (DCs) rather than conventional monocytes. The UCS also included cells positive for CD1c, a marker associated with a major circulating DC subset.
The UCS was detected in all groups analyzed: healthy controls, patients with MGUS, and patients with MM. The most notable group-level difference reported was the reduced frequency of TLR2pos CD14dim/neg CD16neg CD163pos cells in MM patients versus healthy donors (P < 0.002). The manuscript presents this reduction as a potential indicator of decreased circulating DCs within the TLR2-defined population in MM.
The authors examined how alternative gating approaches affected the cell populations captured. When a negative selection–based gating strategy was used, the captured population included all dendritic cell subsets. In contrast, the TLR2pos gating strategy preferentially included CD1cpos DCs that were highly CD163pos. Thus, the choice of gating method meaningfully influenced whether DC subsets were classified within the TLR2-defined monocyte population and altered the apparent composition of circulating monocyte/related cells.
The findings demonstrate that a TLR2pos-defined “all monocytes” gate contains a CD163pos CD1cpos DC population that had been previously unrecognized when TLR2 was used as a monocyte marker. Because the UCS—enriched for CD163pos CD1cpos DCs—was decreased in MM patients, the authors suggest this may reflect reduced circulating DC levels in MM. Such a reduction could contribute to the immune dysregulation characteristic of MM. The study highlights how gating strategy can change subset identification and interpretation when profiling circulating myeloid cells.
This report is a preprint posted to bioRxiv and has not been peer reviewed. The source provides no detailed sample sizes, experimental replicates, or extended functional assays within the abstract; those details would need to be consulted in the full preprint PDF or supplementary material. The authors declared no competing interests and reported funding sources in the manuscript.
Note: Specific methodological details, sample size, and additional phenotypic or functional data are reported in the full preprint and supplementary materials but are not detailed in the abstract provided here.