WT1-TCR vector constructs.

<p>A. Schematic representation of the pSIN second-generation lentiviral (Lv) vector encoding the codon-optimized, murinized hybrid HLA-A0201–restricted pWT126-specific additional cysteines modified TCR α- and β-chain genes separated by a self-cleaving porcine teschovirus 2A sequence (P2A). LTR indicates long terminal repeat; m, murine SFFV, spleen-forming focus virus; and WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. B. Schematic representation of the plasmid Sleeping Beauty (SB) vector consisting of a transgene expression cassette, encoding the codon-optimized, murinized hybrid HLA-A0201–restricted pWT126-specific additional cysteines modified TCR α- and β-chain genes separated by a self-cleaving porcine teschovirus 2A sequence (P2A), flanked by inverted repeats; and the hyperactive SB transposase SB100X. CMV indicates cytomegalovirus promoter; IL, left inverted repeat; IR, right inverted repeat; m, murine; SFFV, spleen-forming focus virus.</p

<i>In vivo</i> cytotoxicity of murine primary T cells.

<p><i>In vivo</i> cytotoxicity of modified mouse splenocytes against CFSE-labelled peptide-loaded mouse target B cells. A2K^b Tg mice, 2 days after intravenous injection of syngeneic effector cells modified with Sleeping Beauty (SB) WT1-TCR vector system, were again intravenously injected with a 1∶1 mix of relevant: irrelevant peptide-loaded A2K^b Tg target B cells, differentially labelled with CFSE (specific WT126 peptide, 1.5 µM CFSE, and irrelevant WT235 peptide for WT1-TCR, 0.15 µM CFSE). Eighteen hours later, splenocytes of injected animals were harvested and analysed by FACS to identify CFSE-labelled cells. Control A2K^b Tg mice were injected with only CFSE-labelled peptide-loaded target B cells or with unmodified splenocytes as effectors. Antigen-specific cytotoxicity was calculated as [1- (number of relevant peptide-loaded targets in experimental mice/number of irrelevant peptide-loaded targets in experimental mice)/(number of relevant peptide-loaded targets in control mice/number of irrelevant peptide-loaded targets in control mice )] ×100.</p

Integration profiles in human primary T cells by LM-PCR.

<p>CD3/CD28 microbeads-activated PBMCs were transduced at a multiplicity of infection of 20 (Lv) or nucleofected with both 5 µgs transposon and transposase plasmids (SB). Both vectors encoded the identical WT1-TCR transgene. Genomic DNA was extracted and lentivirus-chromosome or transposon-chromosome junctions were recovered by ligation-mediated PCR and sequenced. A. Genome-wide mapping of vector integrations at the chromosome level. Sequences were mapped to the University of California at Santa Cruz (UCSC) human genome by BLAT search and integration sites were depicted relative to chromosomes using the UCSC Genome Graphs tool. Lv in Blue, SB in Red. B. Frequency of integration sites of vectors within RefSeq genes. 1,000 random integration sites were generated by bioinformatics, as already described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068201#pone.0068201-Vink1" target="_blank">[21]</a>. C. Proximity of integration sites to transcription start sites (TSS). RefSeq genes containing integration sites were divided by length into 4 equally sized regions and 1 upstream region (0–5 kb), and the proportion of integration sites within each region was counted. To allow statistical comparison of integration preferences with average genomic content, 1,000 random chromosomal sites were generated by multiplying the total length of the genome by a random number between 0 and 1 and converting this value to a chromosomal coordinate. Vector integration frequencies are expressed relative to the proportion of random sites within each region.</p

WT1-TCR expression and function in human primary T cells.

<p>A. WT1-TCR cell surface expression. Percentages of Vβ2.1 positive cells in CD8+ T cells for each HLA-A2+ donor are shown after WT1-TCR lentiviral (Lv) or Sleeping Beauty (SB) vector gene transfers. CD3/CD28 microbeads-activated PBMCs were transduced at a multiplicity of infection of 20 or nucleofected with 5 µgs transposon and transposase plasmids. *p<0.05. B. Antigen-specific effector function. Mean percentages of IFNγ positive cells in CD8+ T cells from HLA-A2+ donors assayed by MACS Cytokine Secretion Assay of human primary CD8+ T cells, after WT1-TCR lentiviral (Lv) or Sleeping Beauty (SB) vector gene transfer, restimulated with either the specific WT1 peptide, anti-CD3 Ab (positive control) or irrelevant peptide (negative control), and autologous PBMCs (top). Representative dot plots of IFNγ secretion are also shown after WT1-TCR Lv (bottom left) or SB (bottom right) gene transfer.</p