Abstract
Spleen is known to contain multiple dendritic and myeloid cell subsets, distinguishable on the basis of phenotype, function and anatomical location. As a result of recent intensive flow cytometric analyses, splenic dendritic cell (DC) subsets are now better characterized than other myeloid subsets. In order to identify and fully characterize a novel splenic subset termed “L-DC” in relation to other myeloid cells, it was necessary to investigate myeloid subsets in more detail. In terms of cell surface phenotype, L-DC were initially characterized as a CD11bhiCD11cloMHCII−Ly6C−Ly6G− subset in murine spleen. Their expression of CD43, lack of MHCII, and a low level of CD11c was shown to best differentiate L-DC by phenotype from conventional DC subsets. A complete analysis of all subsets in spleen led to the classification of CD11bhiCD11cloMHCII−Ly6CloLy6G− cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6Clo and Ly6Chi monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11bhiCD11cloMHCII−Ly6C−Ly6G− cells, which are CD43+, Siglec-F− and CD115−. Changes in the prevalence of L-DC compared to other subsets in spleens of mutant mice confirmed the phenotypic distinction between L-DC, cDC and monocyte subsets. L-DC development in vivo was shown to occur independently of the BATF3 transcription factor that regulates cDC development, and also independently of the FLT3L and GM-CSF growth factors which drive cDC and monocyte development, so distinguishing L-DC from these commonly defined cell types.
Highlights
Characterization of dendritic cell (DC) subsets in spleen has progressed rapidly in terms of phenotype and function; other splenic myeloid subsets are less clearly defined
In order to further explore the relationship between L-DC and the three major myeloid subsets described above, T, B, and red blood cell-depleted splenocytes were gated as CD11bhiCD11cloMHCII− cells, and further tested for Ly6C and Ly6G expression
In addressing the issue of myeloid cell subset classification in spleen, a recent paper suggested a unified nomenclature for DC, monocytes, and macrophages based primarily on ontogeny, and secondly by location, function, and phenotype [65]
Summary
Characterization of dendritic cell (DC) subsets in spleen has progressed rapidly in terms of phenotype and function; other splenic myeloid subsets are less clearly defined. The term “myeloid” has been used as an umbrella term describing DC, granulocytes, and macrophages/monocytes, which descend from a common myeloid progenitor (CMP) in bone marrow [1]. Granulocytes comprise neutrophils, eosinophils, basophils, and mast cells [2], while monocytes have been classified into distinct resident and inflammatory populations, in line with monocyte subsets described in peripheral blood [3]. Recent definition of a common dendritic progenitor (CDP) separates the development of conventional (c) and plasmacytoid (p) DC from other myeloid cell types [4, 5]. Studies focusing on specific subsets do not compare one subset against another to ensure pure populations and do not achieve a comprehensive picture
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