Andersen, M. N. | bioRxiv Science

bioRxiv2026

A Circulating TLR2pos CD14neg CD16neg '' Unclassified Subset '' is Decreased in Multiple Myeloma Patients and May Comprise CD163pos Dendritic Cells.

BackgroundStrategies 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 TLR2posCD14dim/negCD16neg population, which we termed the unclassified subset (UCS). MethodsPeripheral 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 TLR2pos gating. PBMCs from additional healthy donors were analyzed to characterize the UCS population, including the impact of using either TLR2pos or a negative selection-based gating strategy. ResultsThe TLR2pos CD14dim/neg CD16neg 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<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 CD163pos dendritic cells (DCs). Importantly, gating strategies influenced the composition of the UCS: negative selection-based gating captured all DC subsets, whereas TLR2pos gating primarily included CD1cpos DCs that were highly CD163pos. ConclusionsThese findings demonstrate that circulating CD163pos CD1cpos 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 TLR2pos CD14dim/neg CD16neg CD163pos cells in MM patients may represent decreased levels of circulating DCs that may contribute to the immune dysregulation in this disease.

immunology↗

bioRxiv2025

Targeted delivery of RNA-based therapeutics enables functional analysis of macrophage subpopulations

Macrophages infiltrate all human tissues where they play key roles in innate immunity, homeostasis, and tissue function. However, extensive clinical and experimental evidence indicates that macrophages also contribute significantly to the progression of several diseases such as cancer, cardiometabolic disorders, and inflammatory and neurodegenerative conditions. Advances in single-cell omics have revealed diverse macrophage populations in both healthy and diseased tissues. However, studying their functions is challenging due to limitations in tools for targeting specific populations. The Cre-lox system, involving Cre recombinase expression driven by macrophage-specific promoters, is widely used for gene manipulation. Despite its utility, this method has drawbacks like leaky expression, variable efficiency, and potential toxicity. Moreover, genetic models are costly and can have unintended effects on immune cells, hindering comprehensive studies on macrophage function. To address this challenge, we developed an advanced lipid nanoparticle-based system for precise RNA therapeutic delivery to macrophages, either broadly or via antibody-mediated targeting of specific subsets. This versatile platform enables the administration of various RNA molecules, such as mRNA, siRNA, and sgRNA for CRISPR/Cas9 applications, in both in vitro and in vivo settings. It allows for targeted cell depletion or gene knockout, facilitating detailed functional analysis. Furthermore, the systems flexibility and precision are enhanced by its compatibility with Cre-specific Cas9 expression, enabling comprehensive genomic and proteomic targeting of specific macrophage subsets.

immunology↗