2), indicating that cell identity was altered due to the action of GM-CSF. In other words, GM-CSF changed the DC progeny of Flt3L cultured

BM progenitors. It is well accepted that GM-CSF and Flt3L mobilize different ACP-196 precursor cells for DC differentiation. Ly6Chigh monocytes are the final precursor stage en route to the generation of GM-DCs from total BM [24]; but not FL-DCs, whose immediate precursors are Ly6C− pro-DCs [22]. Therefore, the dominant nature of GM-CSF over Flt3L in BM culture raises further issues. What is the developmental fate of precursor cells for FL-DCs in the dual cytokine culture? Do FL-DCs die of neglect or is their differentiation hijacked by GM-CSF to divert fate to GM-DCs? Data both already published and uncovered in the current study support the latter scenario. First, as the Flt3L level present in the dual cytokine cultures was the same as it was in the Flt3L single cultures, there should not have been a lack of Flt3L stimulation. Second, DC progenitors, including pro-DCs, express GM-CSF receptor [16], which makes it physically possible for these cells to receive GM-CSF signaling during maturation. Third, Flt3L signaling in BM

progenitor cells activates the transcription factor STAT3, whereas GM-CSF signaling activates STAT1, STAT3, and STAT5 [25]. Therefore, when DC precursors meet both cytokines, the signaling pathway of GM-CSF can subsume that of Flt3L. Fourth, some hematopoietic cytokines, such as M-CSF and G-CSF have already been reported to govern lineage choice [26, 27]. Finally, when purified and cultured in the presence of both Flt3L and see more GM-CSF, FL-DC-committed precursor cells were diverted toward GM-DCs in spite of the presence of Flt3L. This is direct in vitro evidence for a lineage diversion role of GM-CSF. Although the incidence of macrophage colony-forming CHIR-99021 manufacturer cells remained around 5% in these pro-DCs [22], this may have

been skewed by the collection of only cells loosely attached to the substrate at the end of the culture, thus omitting most of the strongly adherent macrophage population. However, phenotypic analysis consistently indicated that the harvested cells were predominantly CD11chi and MHCII+ DCs (Fig. 5) and thus unlikely to be macrophages. Furthermore, it would be difficult to explain the more than twofold expansion in cell numbers in the cultures with dual cytokines compared with that of Flt3L alone to be due to a minor macrophage population (Fig. 5). For the above reasons, we do not think that the altered outcomes from the combined GM-CSF and Flt3L additions were due to outgrowth from distinct precursors within the enriched pro-DC population. Nevertheless, we tried to perform limiting analysis studies using GFP+ pro-DCs from mice transgenic for GFP under the promiscuous UBC promoter [15]. We seeded pro-DCs at either one or ten cells per well with 200,000 BM feeder cells in 96-well microtitre trays.