Identification of a DC precursor subset among neonatal spleen cells. (A) Eight-day-old spleen cells were sorted to give 2 populations of c-kit⁺CD11c⁻ and c-kit⁻CD11c⁻ cells. (B) Equal numbers of cells were cocultured over STX3 to assess DC development, along with whole red blood cell (RBC)–lysed bone marrow (BM) as positive control, and STX3 stromal cells as negative control. Cocultures were observed under phase microscopy after 14 days. Bar represents 50 μm. (C) Total nonadherent progeny cells were collected from cocultures at 14 days and analyzed flow cytometrically. FSC and SSC parameters were used to distinguish large cells for analysis of relative cell yield and marker analysis. Cells were simultaneously stained for CD11c, CD11b, CD8α, and MHC-II to delineate DC subsets. Positive staining cells (dot plot) were gated using relevant isotype controls (density plot). Percentage of positive cells relative to all cells collected from cocultures is indicated in quadrants. (D) L-DCs produced in cocultures were tested for the capacity to activate purified CD8⁺ OT-I TCR-tg T cells or CD4⁺ OT-II TCR-tg T cells. L-DCs were collected from cocultures of CD11c⁻c-kit⁺ spleen after 21 days. Control f-DCs were isolated from spleen using CD11c⁺ MACS microbeads. Antigen-presenting cell subsets were pulsed with antigen (10 μg/mL OVA for OT-I, or 100 μg/mL OVA for OT-II) in vitro for 12 hours before addition of CFSE-labeled T cells. Cocultures were established at a 1:5 DC:T-cell ratio, in the presence and absence of LPS (10 μg/mL), which was washed off after 12 hours. HEL was control antigen. T-cell activation was measured flow cytometrically after gating live (PI⁻)CD11c⁻CD8⁺ OT-I T cells, or PI⁻CD11c⁻CD4⁺ OT-II T cells). Data are presented as percentage of T cells proliferating on the basis of reduction in CFSE intensity. Data are representative of 2 similar experiments.