High levels of Notch signaling reduces the frequency of TCR-αβ T-cell precursors. (A) Relative frequency of αβ or γδ lineage containing wells (left graph) or frequency of αβ-only, γδ-only, or bipotent αβ and γδ containing wells (right graph) within single-plated CD34⁺CD1⁻ (left) and CD34⁺CD1⁺ (right) thymocytes on OP-DL1 with 0 or 1 μM GSI that showed T-cell reconstitution. Data represent mean plus or minus SEM (n = 3), encompassing 101 and 45 positive wells, of 216 and 72 wells plated for 0 and 1 μM GSI cultures, respectively, for CD34⁺CD1⁻ thymocytes and 46 and 54 positive wells, of 168 and 72 wells plated for 0 and 1 μM GSI cultures, respectively, for CD34⁺CD1⁺ thymocytes. (B) Absolute frequency of αβ or γδ lineage containing wells (left graph) or frequency of αβ-only, γδ-only, or bipotent αβ and γδ containing wells (right graph) within all wells plated, derived from data as described in panel A. *Significant difference (P < .05). (C) Limiting dilution analysis to determine the TCR-αβ and TCR-γδ precursor frequency in CD34⁺CD1⁺ thymocytes after OP9-DL1 coculture in the absence or presence of 1 μM GSI. Data are presented as precursor frequency⁻¹ (95% confidence limits) and is the result of 3 independent experiments with a total of 60, 60, 60, and 96 replicate wells for 30, 10, 3, and 1 CD34⁺CD1⁺ thymocytes plated.