Differential expression of CD8 and CD8β chains on distinct subsets of human pre-T cells: Intracytoplasmic TCRβ expression and cell-cycle progression are associated with CD8β chain expression. (A) Large CD3⁻, large CD3^lowpT⁺, and small CD3⁻ pre-T cells, isolated as described in Materials and Methods, were analyzed by two-color flow cytometry for CD8 versus CD8β expression. (B) Large CD3⁻ CD4⁺CD8⁺ (DP) pre-T cells were fractionated by cell sorting into CD8⁺β⁻(top panels) and CD8⁺β⁺ (bottom panels) cells after labeling with the 2ST8-5H7 anti-CD8β MoAb plus PE-coupled goat antimouse IgG_2a. Reanalysis of surface CD8β expression postsorting is shown (shaded histograms). Sorted cells were then analyzed by flow cytometry for intracytoplasmic TCRβ chain (TCRβic) expression (shaded histograms) and DNA content. Cytoplasmic background fluorescence was determined on sorted cells stained with a nonreactive mouse IgG₁ MoAb plus PE-Cy5-coupled goat antimouse IgG₁ (unshaded histograms). Percentages of cycling cells (in S and G₂/M phases) are indicated. Results are representative of four independent experiments.