Journal: Gene therapy

Article Title: Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

doi: 10.1038/gt.2010.4

Figure Lengend Snippet: Figure 4 Cytokine-stimulated, TCR-transduced human T-cells maintain CD28 and CD62L expression. CD8+ sorted T-cells were transduced with the lentiviral WT1-TCR after activation with OKT3+IL2, IL2 alone, IL7 alone, IL15 alone, IL15+IL7, IL15+IL21, IL7+IL21 and IL2+IL21 (a). The freshly transduced T-cell populations were then re-stimulated with T2 cells loaded with the specific (pWT126) peptide and irradiated feeder cells in the presence of the same cytokine/s used for transduction. After two rounds of peptide/cytokine stimulation the transduced T- cells were sorted into a Vb2.1-positive population using phycoerythrin-labelled anti-Vb2.1 antibodies and anti-phycoerythrin beads (Miltenyi Biotech, Germany). The purified CD8+ Vb2.1+ T-cells were stimulated for an additional 9 days with pWT126 following which expression of CD28 and CD62L was analysed by FACS. These experiments were representative of three independent experiments. (b) The mean fold increase in the mean fluorescence intensity of CD28 expression following transduction in the presence of IL15 alone or IL15+IL21 as compared with OKT3+IL2 is shown. Statistical significance was determined as Po0.05 using a two-tailed t-test. FACS, fluorescence-activated cell sorting; IL, interleukin; TCR, T-cell receptor.

Article Snippet: The concentration of common g-chain cytokines used either alone or in combination as stated was as follows: 20 U ml 1 of human recombinant IL2 (Roche, Basel, Switzerland), 5 ng ml 1 human recombinant IL7 (R&D Systems, Abingdon, Oxfordshire, UK), 10 ng ml 1 human recombinant IL15 (R&D Systems) and 30 ng ml 1 mouse recombinant IL21 (R&D Systems).

Techniques: Expressing, Transduction, Activation Assay, Irradiation, Two Tailed Test, FACS