Human cFCs are present in clinical stem-cell sources and are distinct from conventional T lymphocytes. (A) Representative FACS analysis of CD8⁺TCR⁻CD3⁺ cFCs in mPB, intracellular (i.c.) TCRβ chain expression by cFCs. Numbers are percentage of total MNC (left plot) or of the gated population, as indicated (middle and right panels). (B) The content of cFCs shown as % of CD8⁺TCR⁻ cells or per 10⁶ MNC (mean ± SEM) in mPB (n = 24), BM (n = 3), CB (n = 3), and normal PB (NPB, n = 8). (C) FACS analysis (using FACS Calibur and CellQuest software, Beckton Dickinson Biosciences, San Jose, CA) of cell-surface markers expressed by cFCs (solid line) compared with conventional CD8⁺ T cells (shaded area); broken line, staining with IgG1 isotype control mAbs. Further phenotypic analysis of cFCs showed absence of early hematopoietic and lineage markers CD34, CD56, CD10, and CD19 (data not shown). (D) FACS analysis of cFCs purified by FACS sorting and cultured for 14 days on anti-CD3 mAb-coated plates in the presence of IL-7. The CD8⁺TCR⁻CD3⁺ phenotype was maintained in 80% to 90% of cultured cells. Numbers on plots are percentages of total cells. (E) Quantitative real-time RT-PCR analysis of IFN-γ mRNA (mean ± SEM) in conventional T cells and cFCs, resting (−) and after 3 days culture on anti-CD3 mAb-coated plates in the presence of IL-7 (+). IFN-γ mRNA expression level in relation to resting T cells, defined as 1. IFN-γ primer sequences: forward 5′-TCAGCTCTGCATCGTTTTGG-3′, reverse 5′-GTTCCATTATCCGCTACATCTGAA-3′. Levels of mRNAs encoding Flt3L, SCF, TGF-β, SDF-1, and IFN-α in resting cFCs and conventional T cells were not significantly different (not shown). (F) FACS analysis of cell-surface Flt3L by purified cFCs cultured with IL-7 for 5 days.