HTLV T-lymphocyte immortalization assays. Human PBMCs were isolated by Ficoll/Paque and cocultivated with irradiated (100 Gy [10 000 rad]) 729 stable cell lines. PBMCs (2 × 10⁶) were cultured with irradiated donor cells (1 × 10⁶) in 24-well plates as indicated. Representative growth curves for HTLV-1 (A) and HTLV-2 (B) infected cells are shown. Cell viability was determined weekly by trypan blue exclusion (0-11 weeks after cocultivation). The mean and SD of each time point were determined from 3 independent samples. (C) Quantification of viral protein expression from several immortalized T-lymphocyte lines established from panels A and B (20 weeks in culture). HTLV-immortalized T cells were plated in 24-well plates at 10⁶ cells/mL/well. Culture supernatants were collected at 24 hours and tested for p19 Gag output by ELISA. The values represent p19 production per cell per day. The mean and SD were determined from 4 replicates of 2 independent samples. (D-E) Detection of HTLV sequences in immortalized T lymphocytes by DNA PCR. Although fragment size is consistent with the expected deletion or insertion, diagnostic restriction enzyme digestion and DNA sequencing also were performed to confirm the presence of the mutations (data not shown).