Fig 1.
Two distinct populations developing in GM-CSF culture.
For in vitro generation of dendritic cells, bone marrow cells from gnotobiotic miniature pigs were cultured with GM-CSF for 10 days. (A) After GM-CSF culturing, MHCII expression was confirmed using flow cytometry, and two distinct populations were sorted based on differential expression of MHCII (MHCIIhigh and MHCIIlow). Allophycocyanin (APC)-conjugated goat anti-mouse IgG was used as secondary antibody. (B) The morphology of MHCIIhigh and MHCIIlow cells was observed using an AxioVert200 inverted microscope at 10×, 20×, and 40× magnification. The blue arrow heads denote cluster formation.
Fig 2.
The phenotype of MHCIIhigh and MHCIIlow cells in GM-CSF culture.
The phenotype of cells was analyzed for the expression of the markers of interest by flow cytometry. MHCIIhigh and MHCIIlow cells were sorted by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG as secondary antibodies. The blue-filled histogram shows the MHCIIhigh population and the green-filled histogram shows the MHCIIlow population. The control represents cells stained only with allophycocyanin (APC)-conjugated goat anti-mouse IgG as secondary antibodies.
Fig 3.
Transcriptional profiling of MHCIIhigh and MHCIIlow cells in GM-CSF culture.
(A) A heat map of hierarchical clustering shows significant transcripts in three independent samples of hematopoietic stem cells (HSCs), MHCIIhigh cells, and MHCIIlow cells, based on the Euclidian distance with the complete linkage method. (B) A multidimensional scaling plot shows the relationships of HSCs, MHCIIhigh, and MHCIIlow cells in the total gene expression map shown in (A). Each dot represents data from one animal. (C) Heat map of selected transcripts in three independent samples of MHCIIhigh and MHCIIlow cells. (D) The expression levels of the indicated DC signature genes were analyzed by qPCR. Data are expressed as the mean ± SEM, derived from multiple tests (n = 3). *P < 0.05; **P < 0.005; ***P < 0.0005.
Fig 4.
Mixed lymphocytes reaction of MHCIIhigh and MHCIIlow cells.
(A) Splenocytes stained with carboxyfluorescein succinimidyl ester (CFSE) cultured alone for 5 days and analyzed their proliferation. (B, C) CFSE-labelled allogeneic splenocytes were cultured with MHCIIhigh and MHCIIlow cells at the indicated ratios for 5 days. (B) The blue histogram denotes allogenic splenocytes cultured with MHCIIhigh cells, and the green histogram shows splenocytes cultured with MHCIIlow cells. The gate indicates the percentage of proliferated T cells.
Fig 5.
Phagocytosis by MHCIIhigh and MHCIIlow cells.
(A) For analysis of phagocytic bead uptake, MHCIIhigh and MHCIIlow cells were incubated with fluorescein isothiocyanate (FITC)-tagged latex beads for 3 h. Internalized beads were analyzed by flow cytometry. (B, C) FITC-tagged latex beads were incubated with phagocytes for the indicated times. (B) The blue histogram represents MHCIIhigh cells and the green histogram represents MHCIIlow cells.