Figure 3. Characterization of cDC, pDC, T-cell, and NK-cell lineage progenies generated from PB HPCs.

(A) Representative flow cytograms of BDCA1 (CD1c), BDCA2(CD303), BDCA3 (CD141), and BDCA4 (CD304) expressions in HLA-DR⁺Lin⁻CD11c⁺ cDC- and HLA-DR⁺Lin⁻CD123⁺ pDC-lineage progenies generated from four-week mass-cultures of HPCs with OP9-DL1 cells in the presence of KL, FL, and IL-7 (upper panel). Cytokine production of cDC- and pDC-lineage progenies generated from four-week mass-cultures from 3 healthy donors, as described above (lower left panels). Productions of IL-6 and IFNα by cDC and pDC progenies were measured after five-day culture with or without GM-CSF and IL-3 (10 ng/ml), respectively, in the absence of stroma cells. Poly I:C and R848 were added to the cultures of cDC and pDC, respectively, at day 3. Cytokine measurement for each donor was independently performed. May-Giemsa staining confirmed that GM-CSF-stimulated HLA-DR⁺Lin⁻CD11c⁺ and IL-3-stimulated HLA-DR⁺Lin⁻CD123⁺ were mature cDCs and pDCs, respectively (lower right panels). The scale bars represent 10μm. Images were acquired by BIOREVO model BZ-9000 (Keyence Co.) with Plan Apo 100×/1.40 numerical aperture objective lens (Nikon) using BZ-II Viewer and BZ-II Analyzer software (right panels).

(B) Representative flow analyses and morphology of CD7⁺CD5⁺ T- and CD7⁺CD56⁺ NK- lineage cells generated in OP9-DL1 co-cultures. Flow cytograms represent CD4 and CD8 expressions in CD7⁺CD5⁺CD3⁺ and CD7⁺CD5⁺CD3⁻ cells generated in ten-week co-culture (upper left panels). Expressions of T-cell receptor αβ and γδ in CD7⁺CD5⁺ CD3⁺ cells was also confirmed (lower panels). Expressions of CD16 and CD127 were analyzed for CD7⁺CD56⁺ cells generated in five-week co-culture (upper and lower middle panel, respectively). May-Giemsa staining of CD7⁺CD5⁺ T- (upper right panel) and CD7⁺CD56⁺ NK- (lower right panel) lineage cells represents cycling lymphoid cell features.