Fig 1.
Expression of myeloid and dendritic cell markers by GM-CSF driven bone marrow cells over seven days in culture.
Bone marrow cells were cultured in GM-CSF for seven days. A) Expression of CD11b, CD115, Ly6C, and CD11c were monitored by flow cytometry each day. The percent of cells expressing each marker is depicted vs. day of culture. The mean and standard deviations of three independent experiments are shown. B) Flow cytometric plots of co-expression of Ly6C vs. CD115 by bone marrow cells cultured in GM-CSF over seven days. C) Compiled data from three independent experiments illustrating the relative percentages of each of the four populations over 7 days of culture.
Fig 2.
GM-CSF differentiated cells isolated based on expression of Ly6C and CD115 transition through a series of successive stages of development.
After 3 days of culture in GM-CSF, cells were sorted into four populations based on expression of Ly6C and CD115. Isolated populations were then re-cultured in GM-CSF and re-examined for expression of Ly6C and CD115 on the indicated days. A) Ly6C+CD115- B) Ly6C+CD115+ cells C) Ly6C-CD115- cells were sorted and monitored over three days post sorting. D) Ly6C-CD115- cells were first depleted of CD11c+ cells to remove contaminating DC. Then they were monitored over 16 days because no progression was evident within the three day time period of the other populations. Changes in the population became evident at day 7 of culture after sorting and continued through day 16.
Fig 3.
Developmental progression of GM-CSF driven differentiation in the presence of feeder cells in vitro or in vivo.
Bone marrow was harvested from Ptprcb (CD45.1) mice, cultured in GM-CSF supplemented media for 2 or 5 days, and sorted as previously described. A) 104 CD45.1+ sorted cells were co-cultured with 106 CD45.2+ fresh bone cells, and B-D) Ly6C/CD115 expression was analyzed for six days by flow cytometry. Adoptive transfers were performed by intraperitoneal injection of 106 (CD45.1+) E) Ly6C-CD115- or G) Ly6C+CD115+cells into CD45.2 mice, suspended in PBS with 200ng of GM-CSF. F) Composition of recovered CD45.1+ cells following CMP adoptive transfer 2, 4, 6, and 10 days post injection compiled from 3 independent experiments. Mice received daily injections of 200ng of GM-CSF. Peritoneal lavage was collected every 48 hours, and donor (CD45.1+) cells Ly6C/CD115 levels were evaluated by flow cytometry.
Fig 4.
Distinct cell surface marker and gene expression profiles in the five stages of inflammatory DC development.
A) 16 cell surface markers were measured by flow cytometry at each of the five stages of development. Empty histograms represent fluorescence minus-one controls. B) After three days of culture in GM-CSF, bone marrow cells were sorted into five populations based on expression of Ly6C, CD115 and CD11c. RNA was purified from each population and analyzed for expression of 25 genes plus controls using a custom qRT-PCR array. Relative levels of expression are depicted by intensity of color on the heat map with red being highest expression and green lowest. Results represent averages from three independent experiments.
Fig 5.
Early moMacs give rise to two cell types.
A) After five days of culture in GM-CSF, moMacs (Ly6C-CD115+) were purified and recultured with GM-CSF. CD115 expression was monitored by flow cytometry for 6 days. Six days post-initial sort, B) moDC (Ly6C-CD115-) and C) moMacs (Ly6C-CD115+) were purified and recultured with GM-CSF. CD115 expression was monitored by flow cytometry for the next 48 hours.
Fig 6.
MHC class II level distinguishes developmental stages within moMac phenotype.
Bone marrow cells were cultured in GM-CSF for 5 days. A) CD11c+ cells were sorted based on expression of CD11b and MHCII into three populations: MHCIILow, MHCIIInt, and MHCIIHigh. B) Expression of Ly6C and CD115 were analyzed post sort, and the isolated populations were re-cultured in GM-CSF. C) Changes in CD11b and MCHII expression were analyzed on day 2 and 4 post sort by flow cytometry. D) Resulting MHCIIInt and MHCIIHigh cells on Day 4 were further analyzed by Ly6C and CD115 expression by flow cytometry. E) Percent of cells exhibiting MHCIILow, MHCIIInt, or MHCIIHigh phenotypes post-sort (PS) and after re-culture for 2 or 4 days.
Fig 7.
Comprehensive model of GM-CSF driven DC development.
Transcriptional and phenotypic changes as cells progress through GM-CSF driven development. Common myeloid progenitor gives rise to granulocyte/monocyte progenitor (GMP), followed by monocytes, and monocyte-derived macrophages (moMac). moMacs are maintained long term and share a phenotype with a precursor of monocyte-derived DC (moDC). This precursor has been termed monocyte-derived DC progenitor (moDP).