Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: A lesson learned from flow cytometry

Abstract

Forkhead box P3 (Foxp3) is well known for its highly restricted expression in T regulatory cells (Tregs). A recent study suggested the existence of a Foxp3 positive macrophage subpopulation in mouse bone marrow, spleen, liver, lymph nodes, and thymus that exhibited immune regulatory effect similar to Tregs. Before this report was retracted, we attempted to study the function of this macrophage subpopulation in a mouse model of hyperlipidemia. Bone marrow and spleen cells isolated from C57BL/6 apo E−/− mice were stained with anti-CD11b, anti-F4/80 and anti-Foxp3 and analyzed by flow cytometry. Our results showed that 3.06–8.08% of CD11b+F4/80+ macrophages from bone marrow cells and 0.24–2.21% from splenic were Foxp3-positive. Unexpectedly, unstained or isotype stained controls also showed strong autofluorescence and similar percentages of these cells fell within the same FL1 channel that counted the anti-Foxp3 stained population. Back gating of the autofluorescent population onto a SSC/FSC plot showed that this population of cells had a higher side scatter. The peritoneal macrophages (PMø) exhibited similar autofluorescence. We used qPCR to further evaluate the expression of Foxp3 mRNA in PMø that were treated with M-CSF, M-CSF+IL-4, M-CSF+TGFβ1 or in BMDM treated with TGFβ1 in the presence of anti-CD3 and CD28 antibody co-stimulators. No expression of Foxp3 mRNA was detected in either cell culture systems, whereas robust Foxp3 gene expression was induced in naïve CD4+ cells stimulated with TGFβ1. Consistent with these findings, fluorescence microscopy showed no Foxp3 protein expression in PMø, however Foxp3 expression was easily detected in induced Tregs. We conclude that the reported expression of Foxp3 in macrophages is likely an artifact and that a stringent multimodality approach is critical to demonstrate candidate gene expression in any cell type.

Macrophages Are Phenotypically and Functionally Diverse across Tissues in Simian Immunodeficiency Virus-Infected and Uninfected Asian Macaques

“…The characterization of NHP macrophages by flow cytometry has been restricted by the availability of cross-reactive reagents and is complicated both by the autofluorescence inherent to macrophages as well as the nonspecific capture of antibodies resulting from their high Fc receptor expression (25,26). In order to identify macrophages from NHPs, we first incubated single-cell suspensions of RM tissue homogenates with fluorescent microspheres to demarcate specifically phagocytic cells (Fig.…”

Far‐Red Fluorescent Lipid‐Polymer Probes for an Efficient Labeling of Enveloped Viruses

“…Using our far‐red fluorescent lipid‐polymer probes, we could achieve an efficient tracking of virions inside hepatocytic cells, with no optical contamination due to their autofluorescence up to 700 nm (Figure B and Figure ). Several cell types are autofluorescent, which could lead to false‐positive staining and erroneous conclusions …”

Decreased regulatory function of CD4+CD25+CD45RA+ T cells and impaired IL‐2 signalling pathway in patients with type 2 diabetes mellitus

“…CD4 + CD25 + Treg cells, as a subset of CD4 + T cells with unique immune system suppressive activity, are important determinants of immune and/or metabolic responses . CD4 + CD25 + Treg cells are discriminated by the expression of intracellular forkhead box P3 (FOXP3), a master transcription factor for Treg cells . CD25 or a subunit of IL‐2 receptor (IL‐2Rα), is markedly expressed at a high level on the Tregs surface.…”