Abstract
Dendritic cells (DCs) regulate innate and acquired immunity through their roles as antigen-presenting cells. Specific subsets of mature DCs, including monocyte-derived and lymphoid-derived DCs, can be distinguished based on distinct immunophenotypes and functional properties. The leukocyte integrin, CD11c, is considered a specific marker for DCs and it is expressed by all DC subsets. We created a strain of mice in which DCs and their progenitors could be lineage traced based on activity of the CD11c proximal promoter. Surprisingly, we observed levels of CD11c promoter activity that were similar in DCs and in other mature leukocytes, including monocytes, granulocytes, and lymphocytes. We sought to identify DNA elements and transcription factors that regulate DC-associated expression of CD11c. The ets transcription factor, PU.1, is a key regulator of DC development, and expression of PU.1 varies in different DC subsets. GM-CSF increased monocyte-derived DCs in mice and from mouse bone marrow cultured in vitro, but it did not increase CD8+ lymphoid-derived DCs or B220+ plasmacytoid DCs. FLT3L increased both monocyte-derived DCs and lymphoid-derived DCs from mouse bone marrow cultured in vitro. GM-CSF increased the 5.3 Kb CD11c proximal promoter activity in monocyte-derived DCs and CD8+ lymphoid-derived DCs, but not in B220+ plasmacytoid DCs. In contrast, FLT3L increased the CD11c proximal promoter activity in both monocyte-derived DCs and B220+ plasmacytoid DCs. We used shRNA gene knockdown and chromatin immunoprecipitation to demonstrate that PU.1 is required for the effects of GM-CSF or FLT3L on monocyte-derived DCs. We conclude that both GM-CSF and FLT3L act through PU.1 to activate the 5.3 Kb CD11c proximal promoter in DCs and to induce differentiation of monocyte-derived DCs. We also confirm that the CD11c proximal promoter is not sufficient to direct lineage specificity of CD11c expression, and that additional DNA elements are required for lineage-specific CD11c expression.
Highlights
Dendritic cells (DCs) are bone marrow-derived cells that play crucial roles in regulating and integrating innate and adaptive immune responses
Reflects CD11c promoter activity, one would expect the percentage of green fluorescent protein (GFP)+ cells to be higher in CD11c+ DC subsets than in the total cell population or in other cell types
In this study we demonstrated that expression of GFP driven by the 5.3 kb proximal promoter of mouse CD11c did not accurately reflect expression of native CD11c
Summary
Dendritic cells (DCs) are bone marrow-derived cells that play crucial roles in regulating and integrating innate and adaptive immune responses. DCs sample the environment, recognize invading pathogens through pattern recognition Toll-like receptors (TLRs), and initiate protective T cell responses by presenting antigens to lymphocytes. Because of their highly developed antigen presenting capacity, DCs have drawn attention for use in cell therapy [1,2]. DCs are a heterogeneous group of cells that have been classified into distinct subsets, primarily based on patterns of cell surface antigen expression. The integrin aL chain, CD11c, is considered relatively specific for DCs. CD11c is expressed on all mature DC subsets in mouse, and its expression increases as DCs mature from DC progenitors. Subsets of mature DCs in blood and the lymphatic system include conventional DCs
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