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

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

<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

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

Frozen Cord Blood Hematopoietic Stem Cells Differentiate into Higher Numbers of Functional Natural Killer Cells <i>In Vitro</i> than Mobilized Hematopoietic Stem Cells or Freshly Isolated Cord Blood Hematopoietic Stem Cells

<div><p>Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34⁺) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34⁺) and frozen PBCD34⁺ to generate NK cells <i>in vitro</i>. By modifying a previously published protocol, we showed that frozen CBCD34⁺ cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34⁺ cultures. NK cells generated from CBCD34⁺ and PBCD34⁺ expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34⁺-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 <i>in vitro</i> and <i>in vivo</i> as compared to PBCD34⁺-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34⁺ for the production of NK cells <i>in vitro</i> results in higher cell numbers than PBCD34⁺, without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells <i>in vitro</i> for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.</p></div

NK cell development in CBCD34⁺ and PBCD34⁺ cultures.

<p>NK cell stages 1–4 for one representative sample from CBCD34⁺ (n = 8) (<b>A</b>) and PBCD34⁺ (n = 6) (<b>B</b>) cultures. Percentages are gated from CD3⁻ cells according to the following NK cell stages: stage 1: CD34⁺CD117⁻CD94⁻, stage 2: CD34⁺CD117⁺CD94⁻, stage 3: CD34⁻CD117⁺CD94⁻ and stage 4: CD34⁻CD117^+/−CD94⁺. (<b>C</b>) NK cell stages are shown for one representative sample for CBCD34⁺ (n = 8, upper panel) and PBCD34⁺ (n = 6, bottom panel) cultures at different time points. Stage 1 and 2 are from the CD3⁻CD94⁻ gate and stages 3 and 4 from the CD3⁻CD34⁻ gate. Transcriptional analysis for each time point is shown for transcription factors involved in NK cell differentiation (left panel) and maturation (right panel) for CBCD34⁺ (n = 4) (<b>D</b>) and PBCD34⁺ (n = 3) (<b>E</b>) cultures. Values are normalized using three reference genes. Higher ratio values correspond to less mRNA expression.</p

Killing of K562 <i>in vivo</i> by NK cells from CBCD34⁺ and PBCD34⁺ cultures.

<p>NSG mice were injected with GFP-K562 cells followed by CBCD34⁺-NK cells or PBCD34⁺-NK cells 24 h later. (<b>A</b>) Percentage of GFP-K562 cells detected in the BM, liver, lungs and spleen of NSG mice. (<b>B</b>) Percentage of NK cells detected in the BM, liver, lungs and spleen of NSG mice. The statistical analysis was performed using Mann-Whitney test. * <i>P</i><0.05.</p

Effects of IL-12 stimulation on the function of the differentiated NK cells.

<p>NK cells were incubated with IL-12 for 4 h, 24 h or 40 h. (<b>A</b>) The figure illustrates the effect of IL-12 on the secretion of IFN-γ and (<b>B</b>) TNF-α measured by ELISA after incubation with PMA&Iono. (<b>C</b>) The graph depicts the intracellular expression of IFN-γ after incubation with PMA&Iono. (<b>D</b>) The graph shows CD107a degranulation after incubation with PMA&Iono. (<b>E</b>) NK cell killing capacity against ⁵¹Cr labeled K562 cells or (<b>F</b>) P815 cells coated with anti-CD16. The effector-target ratio used was 10∶1. Statistical analysis was performed using Mann-Whitney test. * <i>P</i><0.05, ** p<0.005.</p

Phenotype of NK cells differentiated from CBCD34⁺ and PBCD34⁺ cultures.

<p>(<b>A</b>) Surface antigens were detected by flow cytometry. A representative sample for the expression of each antigen in the CD56^bright and CD56^dim subsets for CBCD34⁺ and PBCD34⁺ cultures is shown. (<b>B</b>) Transcriptional analysis for KIR2DL1 and KIR2DL2 at days 21, 28 and 35 is shown for NK cells from CBCD34⁺ (n = 4) and PBCD34⁺ (n = 3) cultures. Values are normalized using three reference genes. (<b>C</b>) Graphs depict the surface expression at day 35 of IL-12β1 and CXCR4 on NK cell subsets from frozen CBCD34⁺ (n = 4–5) and PBCD34⁺ (n = 3) cultures. Mann-Whitney test was performed, * <i>P</i><0.05, ** <i>P</i><0.005.</p

NK cell production from CBCD34⁺ and PBCD34⁺.

<p>(<b>A</b>) Total fold expansion and (<b>B</b>) CD3⁻CD56⁺ NK cell number of CBCD34⁺ (n = 8) and PBCD34⁺ (n = 6) cultures at different time points. (<b>C</b>) Representative plot of CD56 <i>vs</i> CD16 from the lymphocyte gate for CBCD34⁺ and PBCD34⁺ cultures. (<b>D</b>) Representative side scatter <i>vs</i> CD56⁺ plots for CBCD34⁺ (upper row) and PBCD34⁺ (bottom row) cultures at days 7, 14, 21, 28, 35 and 42. Mann-Whitney test was performed, * <i>P</i><0.05, ** <i>P<</i>0.005.</p