The developmental progression of conventional DC has been quite well defined, yet the developmental pathway of monocyte-derived, GM-CSF-driven DC is less well understood. We addressed this issue by establishing an isolation strategy that identifies five distinct GM-CSF derived cell types. Expression of Ly6C and CD115 (Csf-1R) was used to identify and isolate four populations. One of the populations could be further separated based on CD11c expression, distinguishing five populations. We further defined these cells based on expression of transcription factors and markers of early and later stages of myeloid development. These discreet developmental stages corresponded well with previously defined populations: Common Myeloid Progenitors (CMP), Granulocyte/Macrophage Progenitors (GMP), Monocytes, as well as Monocyte-derived macrophages (moMac) and Monocyte-derived DC (moDC). Finally, within the moMac population we also identified moDC precursor activity (moDP) that could be distinguished from moMac and moDC based on their level of MHC class II expression and developmental plasticity.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

DC differentiated through culture with GM-CSF are used in many clinically promising applications including vaccines. However, the developmental timeline during which specific stimulatory or immunosuppressive functions are acquired by these cells is poorly understood. Here, we developed a method of isolating progenitor populations at specific stages of development to monitor their phenotype and function. Bone marrow cells were cultured in GM-CSF for three days, then sorted based on Ly6c and CSF-1R (CD115) expression, two hallmarks of myeloid cell development. We then monitored the developmental progression and differentiation of each population based on the expression of these proteins as well as several other hallmarks of the dendritic cell phenotype. We measured their gene expression profiles by qRT-PCR and their response to inflammatory stimuli at each stage of development. Through this analysis, we have identified five distinct cell types representative of Common Myeloid Progenitors (CMP), Granulocyte Macrophage Progenitors (GMP), Monocytes, inflammatory DC (iDC), and a novel dendritic cell precursor which is phenotypically and functionally distinct from previously described myeloid cells. Our findings suggest this novel cell type, Inflammatory DC precursor (IDP), arises from monocytes and serves as an immediate precursor to immature inflammatory DCs. These studies will aid in the refinement of protocols to acquire cells for specific clinical applications.

Myeloid cell progenitors have the potential to give rise to dendritic cells which have a high capacity to stimulate adaptive immune responses as well as to cells with potent immunosuppressive activity. However, the stage in their development in which these functions are gained is poorly defined. Here, we developed a method for isolating progenitor populations at specific stages of development. Bone marrow cells were lineage depleted and cultured in GM-CSF to drive their differentiation toward a myeloid lineage for three days. Cells were then sorted based on Ly6C and CSF-1R (CD115) expression, two markers whose expression is known to change throughout myeloid ontogeny. We then monitored the developmental progression of each population based on the expression of these markers as well as other hallmarks of dendritic cell or myeloid suppressor cell phenotypes. We also measured the function of each population relative to antigen uptake and degradation as well as their response to stimulation by TLR agonists. Collectively, our results indicate that myeloid progenitors progress along this pathway: Ly6C-CD115- -> Ly6C+CD115- -> Ly6C+CD115+ -> Ly6C-CD115+ -> Ly6C-CD115+. Phenotypically, these populations represent CMP, MDSC, monocytes, immature DC, and more mature DC, respectively. These findings not only enhance our understanding of myeloid cell development and differentiation driven by GM-CSF, but also inform the design of GM-CSF-derived DC based vaccine strategies.

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