1. Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome 
Marita Bosticardo, PhD, Elena Draghici, MS, Francesca Schena, PhD, Aisha Vanessa Sauer, PhD, Elena Fontana, PhD, Maria Carmina Castiello, MS, Marco Catucci, PhD, Michela Locci, PhD, Luigi Naldini, MD, PhD, Alessandro Aiuti, MD, PhD, Maria Grazia Roncarolo, MD, Pietro Luigi Poliani, MD, PhD, Elisabetta Traggiai, PhD, Anna Villa, MD 
Journal of Allergy and Clinical Immunology 
Volume 127, Issue 6, Pages e5 (June 2011)
DOI: /j.jaci
Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Representative FACS histograms showing WASp staining in each B-cell subset analyzed in BM (A, from left to right: pre–B-cell, immature B-cell, and mature/recirculating B-cell subsets), peritoneal cavity (B, from left to right: B1a-cell, B1b-cell, and B2-cell subsets), and spleen (C, from left to right: transitional B-cell, follicular B-cell, and MZ B-cell subsets) of BMT wt (gray clear histograms), BMT was−/− (gray filled histograms) and GT mice (black clear histograms). In each panel, the positioning of the markers used to calculate frequency and MFI of WASp+ cells is also shown. Max, Maximum.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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3. Fig 2
Evidence of WASp expression in B-cell subsets from tissues of GT-treated mice. Frequency of WASp+ cells in Was−/− mice transplanted with Lin− BM wt cells (BMT wt), Was−/− cells untransduced (BMT was−/−) or LV-transduced (GT). A, BM. Percentage of WASp+ cells among pre-B, immature B, and mature/recirculating (recirc) B cells. B, Peritoneal cavity. Percentage of WASp+ cells among B1a cells, B1b cells, and mature follicular (B2) cells. C, Spleen. Percentage of WASp+ cells among transitional and mature B cells (MZ and follicular). Results shown were obtained by analyzing 15 to 34 mice per group. Median values are shown per each group. The Mann-Whitney test was used to perform statistical analysis of data. ∗P < .05; ∗∗∗P < .001.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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4. Fig 3
MFI of WASp expression in B-cell subsets from tissues of GT-treated mice. MFI of WASp was evaluated in Was−/− mice transplanted with Lin− BM wt cells (BMT wt), Was−/− cells untransduced (BMT was−/−) or LV-transduced (GT). In the graphs, MFI is expressed as fold increase in respect to values found in the BMT was−/− group. In the GT group, MFI was calculated only on WASp+ cells. A, BM (pre B, immature B, and mature/recirculating [recirc] B-cell subsets). B, Peritoneal cavity (B1a cells, B1b cells, and B2 cells). C, Spleen (transitional, follicular [FO], and MZ B cells). Results shown were obtained by analyzing 15 to 32 mice per group. Median values are shown for each group. The Mann-Whitney test was used to perform statistical analysis of data. ∗∗P < .005; ∗∗∗P < .001.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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5. Fig 4
Presence of WASp-expressing cells in splenic tissue sections from GT-treated Was−/− mice. Spleen sections from BMT wt (A), BMT was−/− (B), and GT mice (C) were stained with hematoxylin and eosin (H&E; left panels), anti-WASp (middle panels), or anti-B220 antibody (right panels). All images are ×10 original magnification.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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6. Fig 5
Improvement of splenic B-cell follicle and MZ architecture in GT-treated Was−/− mice. Spleen sections from BMT wt (A), BMT was−/− (B), and GT mice (C) were stained for antimacrophage scavenger receptor MARCO to identify MZMs surrounding spleen follicles, highlighted by anti-B220 immunostaining (left panels; ×10 original magnification). MZ B cells (IgMhiIgDlow/-, indicated by arrows) have been highlighted by double immunostainings for anti-MoMa (blue signal) combined with either IgD or IgM (brown signals; right panels; ×20 original magnification).
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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7. Fig 6
GT increases response to polysaccharide antigens in Was−/− mice. In vivo challenge of treated mice was performed by intraperitoneal injection of 0.5 μg/mouse Pneumovax23. IgM Pneumovax23-specific antibodies were evaluated by ELISA assay on serum collected 7 days after immunization. The results shown in the graph were obtained from BMT wt (n = 23; black circles), BMT was−/− (n = 14; white circles), and GT mice (n = 20; gray squares). Statistical analysis was performed by using a 2-way ANOVA test. ∗P < .05; ∗∗∗P < .001.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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8. Fig 7
GT results in decreased autoantibody titer in Was−/− mice. A, The presence of anti-dsDNA autoantibodies was evaluated by ELISA assay. The graph shows the frequency of positivity (titer ≥ 1:60) for anti-dsDNA autoantibodies in the 3 groups of treated mice (BMT wt, black bar; BMT was−/−, white bar; GT, gray bar). The number of mice analyzed per group is shown in brackets below each bar. Statistical analysis was performed using a Fisher exact t test. ∗P < .05; ∗∗∗P < B, The graph shows the OD values at serial dilutions (from 1:20 to 1:640) restricted to serum samples that were positive for the presence of anti-dsDNA autoantibodies (BMT was−/−, n = 10, white circles; GT, n = 7, gray circles). Statistical analysis was performed by using a 2-way ANOVA test. ∗∗P < C, Tissue-specific autoantibodies were detected by indirect immunohistochemistry on cryostat sections (7 μm) from adult Rag2−/−γc−/− mice. Each serum was analyzed for the reactivity to 3 tissues (salivary gland, thyroid, gastrointestinal tract). The graph shows the frequency of serum samples reactive to 0, 1, 2, or 3 tissues for the 3 groups of mice. The number of samples analyzed is indicated in brackets below each bar. ∗∗∗P < D, The figure shows autoantibodies against salivary gland in mice representative of each group of treatment (BMT wt, BMT was−/−, and GT). Magnification ×40.
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9. Fig E1
Presence of gene corrected cells in BM and spleen (SP) in Was−/− mice reconstituted with LV-transduced Lin− BM cells. Lentiviral VCN/cell was evaluated on total BM and SP cells. Results shown were obtained analyzing 38 GT mice. Median value is shown per each group. Wilcoxon signed-rank test was used to perform statistical analysis of data. ∗∗∗P < .001.
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10. Fig E2
Analysis of WASp-expressing cell frequency in peripheral blood B220+ cells from GT-treated mice 3 and 9 months after transplant. ∗∗P < .005 (paired t test).
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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11. Fig E3
Presence of WASp-expressing B cells in splenic tissue sections from GT-treated Was−/− mice. Spleen sections from BMT wt (A), BMT was−/− (B), and GT mice (C) have been stained with anti-B220 antibody (left panels; green) and anti-WASp (middle panels; red). Right panels show merging of B220 and WASp stainings (yellow). All images are from ×20 original magnification.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions