Efficient retroviral-mediated gene transfer in naive CD45RA⁺ UC T cells upon IL-7 stimulation.

(A) CD45RA and CD45RO expression on naive UC T cells stimulated with IL-7 for 10 days. (B) Naive UC T cells were cultured for 4 days in the presence of IL-7 and then transduced for 2 consecutive days with LZRS-EGFP retroviral supernatants. EGFP expression was monitored by FACS analysis 2 days following transduction. (C) Cell cycle entry of the UC T cells used for transduction was determined by PI staining at day 4 of cytokine stimulation. (D) Comparison of T-cell receptor CDR3 size distribution (Immunoscope profiles) of nontransduced (EGFP⁻) and transduced (EGFP⁺) IL-7-stimulated UC T cells. Twenty-four PCR products were generated by reverse transcriptase-PCR with 24 different TCRVB subfamily-specific primers and 1 Cβ consensus primer, followed by a run-off reaction with a fluorescent Cβ primer. The graphs represent fluorescence intensity in arbitrary units (y-axis) plotted against CDR3 size (x-axis). Representative results for TCRVB 2, TCRVB 3, TCRVB 6b, TCRVB 7, and TCRVB 9 are shown. A gaussian-like profile is observed for the first 4 TCRVB families; there is a skewed profile for TCRBV 9 in the nontransduced population.