3), this redistribution of DC subsets indicates that DC differentiation in the spleen may be skewed away from pDC and CD8+ DC production in the GM-CSF transgenic in vivo model. Finally, to investigate the effect of GM-CSF on CD8+ DC development in an actual inflammation/infection setting, B6 mice were infected intravenously with 2 × 104 Listeria monocytogenes, click here a pathogen known to increase serum GM-CSF levels [17]. Spleens were harvested from mice after 1–3 days of infection and DCs were enriched by density centrifugation. Infection gradually reduced the percentage

of CD8+ DCs within the resident DC population. By day 2 and day 3 after infection, when the CD8+ DCs have turned over in the spleen, the reduction of CD8+ DCs was significant when compared to uninfected mice (Fig. 6). Despite an overall increase Selleckchem Navitoclax in spleen cellularity after infection (mainly monocytes, monocyte-derived DCs, and neutrophils) [9, 23], the numbers of CD8+ DCs in the spleen were significantly reduced by 3 days after infection while CD8−

DCs resident DCs (characterized by CD11chighGR1−) remained unchanged (Fig. 7B). In this study, we demonstrated that GM-CSF overcomes the effect of Flt3L in promoting DC differentiation. We revealed that the addition of GM-CSF to Flt3L supplemented culture drives the development of BM cells to a unique DC population that lacks pDCs and CD8eDCs. The diversion of DC differentiation by GM-CSF happens at the precursor stage, affecting already-committed precursors. This effect of GM-CSF seems to have correlates in vivo. Mice defective in GM-CSF or GM-CSFR have increased numbers FAD of CD8+ DCs in steady state, whereas reduction of this DC subset was evident in the mice overexpressing GM-CSF or during Listeria infection when serum GM-CSF levels were elevated. GM-CSF and Flt3L are two critical cytokines that drive DC differentiation. It has been reported that GM-CSF inhibits IRF8-dependent pDC development in Flt3L culture via Stat5 [20]. However, the characterization of the DC population after inhibition

was not performed in that study. Apart from the disappearance of pDCs in the BM culture supplemented with both Flt3L and GM-CSF, we also found impaired development of another IRF8-dependent subset, CD8eDCs. This impairment occurred at earlier time points (Fig. 1). Therefore, this result poses the question: does GM-CSF selectively suppress IRF8 transcription, critical for the development of pDCs and CD8eDCs, but still allow the development of Sirpα+ DCs, the in vitro counterpart of CD11b+CD8− resident DCs? Comparison of the remaining Sirpα+ subset in the Flt3L culture following GM-CSF inhibition with the original Sirpα+ subset in the Flt3L culture without addition of GM-CSF demonstrated a difference in cell size, granularity, and intracellular levels of ROS between the two populations.