Exogenously added IFN-α reversibly interferes with the development of thymic CD34⁺CD1a^–precursors into T cells. Sorted CD34⁺CD1a^– thymic precursors were cultured on OP9-DL1 cells in the presence of IL-7 (5 ng/mL) and increasing concentrations of exogenously added IFN-α. At the time points indicated, percentages of cells expressing (A) CD1a, (B) CD4 and CD8 (ie, double-positive cells), (C) TCRαβ, (D) TCRγδ, and (E) CD3 (numbers indicate the percentages of cells in high and intermediate CD3 expression gates, respectively, on day 18 were determined by flow cytometry. (F) Inhibitory effects of IFN-α on T-cell development are reversible. Seven days after coculture in the presence of IFN-α (1000 U/mL), cells were washed, split, and cultured in the presence or absence of IFN-α. No IFN treatment was used as control. (G) V-DJ rearrangement of the TCRβ chain. Genomic DNA was isolated from CD34⁺CD1a^– thymocytes cultured for the indicated time periods in the presence or absence of IFN-α and analyzed for V to DJ rearrangement by multiplex PCR and GeneScan analysis. Results of “tube A” containing 23 different Vβ primers and 9 different Jβ primers are shown. Black lines represent Vβ-Jβ1 rearrangements; gray lines, Vβ-Jβ2 rearrangements. Genomic DNA of peripheral blood mononucleated cells (PBMCs) served as control for polyclonal V-DJ rearrangement. Results are representative of at least 4 independent experiments.