Characterization of expression and function of full-length Helios. (A) Increased frequencies of DN thymocytes among cells that express full-length Helios. Data represent mice at 10 to 16 weeks after transplantation from 6 separate experiments. Asterisks indicate statistically significant differences between GFP control (n = 17) and Helios mice (n = 16) (DN1 and DN2, P < .001; DN3 and DN4, P < .05). DN1 thymocytes were analyzed as CD4⁻CD8⁻CD25⁻B220⁻Mac-1⁻CD44⁺ (B) Real-time reverse transcription-PCR analysis of endogenous Helios expression in thymocyte subsets sorted by FACS. Data shown are representative of 3 independent cDNA preparations (2 independent preparations for DN1) using mRNA from double-sorted cells. DN1 thymocytes were gated as CD4⁻CD8⁻CD25⁻B220⁻Mac-1⁻IL-7Rα ⁻CD44⁺c-Kit⁺. cDNA from each subset was assayed in quadruplicate in each experiment. Error bars represent 1 SD from the mean. (C) CD4⁺ splenic T cells expressing either GFP or full-length Helios were stimulated in vitro with varying concentrations of anti-CD3 in the presence of irradiated APCs for 48 hours. Incorporation of tritiated thymidine into stimulated cells is shown. Error bars indicate ± SEM from quadruplicate wells from a representative experiment performed in triplicate.