CLPF clonally give rise to B and T cells. (A) B-cell (top panel) and T-cell (bottom panel) progenitor frequency of CLPF was assessed by culturing them on either S17 (for B cells) and delta-like 1 expressing OP9 (for T cells) stroma cells in the presence of IL-7, SCF, and Flt3L for 10 days in 96-well plates at densities between 0 and 8 cells per well. The left panel shows the percentage of failure of detection of either CD19+ or Thy1.1+ progeny by FACS. The middle panel shows the number of progeny cells obtained from a single CLPF in these assays with the black bar marking the average burst size. Right panel shows a representative CD19/Thy1.1 FACS plot of a B- and T-cell culture. Horizontal bars in the middle panels represent means. Numbers on right plots are percentages of gated cells. (B) Limiting dilution analysis of donor cells after transfer of CLPF into sublethally irradiated hosts. For B cells, CLPF were intravenously injected and spleens analyzed at 4 weeks. For T cells, CLPF were intrathymically injected and thymi analyzed at 4 days after injection. Three to 5 mice per cell dose were analyzed and numbers shown within the plots represent the calculated limiting number. (C) Results of a clonal assay to simultaneously detect all 4 lymphoid lineages from a single cell (for experimental design and technical details, see Figure S3). Positive clones are symbolized by black circles and a negative result by white circles. Of the 17 clones analyzed, each derived from a single CLPF cell, 8 of 17 gave rise to B cells and 12 of 17 to T cells when cultured under appropriate conditions. Of these positive clones a total of 35% showed simultaneous development of T and B cells, demonstrating the bipotential of CLPF. Furthermore, 7 clones of the 17 underwent 3 or more cell divisions and therefore could also be analyzed for NK, DC, and myeloid potential. Whereas 4 of 7 showed NK cell and 6 of 7 DC potential, none of the clones generated any myeloid cells (0/7). Of the 7 clones analyzed for all 4 lymphoid lineages 2 (28%) simultaneously readout all 4 lymphoid lineages, proving that a single CLPF can possess the full lymphoid potential.
