Characterization of human β-selection. (A) Flow cytometric analysis of human postnatal thymocytes, showing intracellular TCR-β staining versus surface CD28 and CD71, gated on CD4⁺CD8α⁻CD8β⁻CD3⁻ (top) or CD4⁺CD8α^−/+CD8β⁻CD3⁻ (bottom) cells. Numbers in quadrants indicate the percentage of the corresponding population. Results shown are representative for 3 independent stainings. (B) Flow cytometric analysis of TCR-β⁺ gated CD4⁺CD8α⁻CD3⁻ thymocytes, showing coexpression of CD28 and CD71. Numbers in quadrants indicate the percentage of the corresponding population. (C) Relative cell size difference in CD4⁺CD3⁻CD8α⁻CD28⁻ (CD4⁺CD28⁻, black histogram) versus CD4⁺CD3⁻CD8α⁻CD28⁺ (CD4⁺CD28⁺, gray histogram) thymocytes as shown by FSC analysis. (D) Dot plot shows flow cytometric analysis of CD3⁻CD8α⁻ human postnatal thymocytes, showing CD4 versus CD28 expression. The histogram underneath highlights the CD4 expression level difference in CD4⁺CD3⁻CD8α⁻CD28⁻ (CD4⁺CD28⁻, black histogram) versus CD4⁺CD3⁻CD8α⁻CD28⁺ (CD4⁺CD28⁺, gray histogram) thymocytes. Data shown are representative of at least 3 independent analyses. (E) Quantitative real-time RT-PCR gene expression analysis of Notch and T cell–related genes in early human postnatal thymocyte subsets, ordered according to developmental progression along the T-cell pathway. Data shown are the average (± SEM) from 2 sets of independent samples, relative to β-actin levels.