Autologous accessory cells enhance retroviral-mediated gene transfer into naive UC T cells.

Mononuclear cells from UC blood and PB were obtained by Ficoll-Hypaque density centrifugation. To obtain a highly purified T-cell population devoid of accessory cells such as monocytes, T cells were positively selected using an anti-CD3 antibody conjugated to magnetic beads (Dynabeads). These purified T cells or nonpurified T-lymphocyte populations were then stimulated for 2 days with immobilized anti-CD3 and anti-CD28 antibodies and exposed twice to LZRS-EGFP retrovirus on fibronectin-coated plates. (A) Transduction efficiency of purified and nonpurified PB and UC T cells derived from individual donors. All transductions were performed in duplicate on fibronectin-coated plates and the mean percentage of EGFP-expressing cells was monitored 48 hours after transduction. (B) The presence of the LZRS-EGFP provirus in the EGFP⁻ and EGFP⁺ UC and PB CD3⁺ subsets was monitored by Southern blot. CD3⁺ cells were purified by Dynal selection and transduced as described above. EGFP⁻ and EGFP⁺populations were FACS sorted, genomic DNA was isolated, digested withXbaI, and hybridized with the 1.3-kb EGFP probe. The PG13/LZRS-EGFP producer clone was used as a positive control. The 1.7-kb fragment encompassing EGFP and the 3′LTR is indicated. (C) FACS profile of nontransduced and transduced UC T cells. The percentage of EGFP⁺ cells as well as the level of EGFP expression (MFI) is indicated. (D) Relative gene transfer in UC T cells following purification by either a positive (Dynal) or negative selection method (Stemsep) is compared to that obtained in a nonpurified T-cell population. The mean ± SD of EGFP⁺ cells in 2 to 5 independent experiments is shown.