Skip to main content
. Author manuscript; available in PMC: 2015 Jul 1.
Published in final edited form as: Mucosal Immunol. 2014 Jun 18;8(1):141–151. doi: 10.1038/mi.2014.51

Figure 4. GM-CSF production by stromal cells is enhanced by DCs and required for the imprinting of DCs with high RALDH activity.

Figure 4

a Aldefluor staining of splenic DCs after 24 h treatment with SI LP CD45−Epcam−Pdpn+CD31− stromal cells (SCs) or with media supplemented with increasing concentrations of RA. Data from one representative experiment of three experiments with similar results

b Left panel shows aldefluor staining of a cell line established from sorted primary CD45−Pdpnhi splenic cells (Sp SCs) versus SI LP CD45−Epcam−Pdpn+CD31− stromal cells (SCs). Middle panel shows aldefluor staining of splenic DCs (CD45+CD11c+MHCIIhi gate) cultured for 24h with the aforementioned cell line in the presence or not of RA (100 nM). Right panel shows aldefluor staining of splenic DCs treated with RA plus concentrated sups from the aforementioned cell line. Sups were digested with proteinase K when indicated. Shown is the data from one representative experiment of two.

c left panel shows aldefluor staining of splenic DCs after 24h treatment with RA plus sequential fractions of the sups from Sp SCs (fractionation details within the material and methods section). Right panel shows an estimate of the size of the proteins contained within the fraction that gave us the most activity.

d left panel shows aldefluor staining of splenic DCs cultured for 24 hours alone or together with SCs from WT or GM-CSF KO mice. Anti-GM-CSF blocking antibody was added where indicated. Right panel shows the fold increase on aldefluor staining (mean fluorescence intensity, MFI) on splenic DCs co-cultured with SI LP CD45−Epcam−Pdpn+CD31− stromal cells (SCs) from WT vs GM-CSF KO mice. Data shows the results from three independent experiments * P < 0.05 (unpaired t-test)

e Aldefluor staining of splenic DCs after 24h culture with SI LP CD45−Epcam−Pdpn+CD31− stromal cells (SCs) in the same well or separated by a permeable membrane (transwell) Bar graphs shows data from three replicate wells within one experiment. Data shows one representative experiment out of two.

f shows GM-CSF by intracellular staining (left panel) or ELISA (right) on SI LP CD45−Epcam−Pdpn+CD31− stromal cells (SCs) cultured for 24h alone or together with splenic DCs. Left panel shows one representative experiment out of three. Right panel shows data analyzed corresponding to five independent experiments ** P <0.01 (unpaired t-test)