Phenotypic analysis of the cellular progeny generated after differentiation of CD4⁺ CD8⁺human pre-T cells in FTOC. (A) CD4⁺CD8⁺ pre-T cells, isolated by cell sorting as described in Materials and Methods, were cultured in a hybrid hu/mo FTOC and analyzed after 5 and 14 days for the expression of CD8, CD8β, TCRβ, and CD3. Analysis of TCRβ versus CD3 was performed by three-color flow cytometry after electronic gating on the CD8β⁺ progeny. (B) Intracytoplasmic TCRβ (TCRβic) and surface pT expression (shaded areas) was analyzed after gating on the CD3^low progeny recovered by day 5. Background fluorescence was determined with isotype-matched irrelevant MoAb and with a rabbit preimmune serum. A representative experiment out of three is shown.