- Email: [email protected]
Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome
Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome Marita Bosticardo, PhD,a Elena Draghici, MS,a Francesca Schena, PhD,b Aisha Vanessa Sauer, PhD,a Elena Fontana, PhD,c Maria Carmina Castiello, MS,a Marco Catucci, PhD,a Michela Locci, PhD,a Luigi Naldini, MD, PhD,a,d Alessandro Aiuti, MD, PhD,a,e Maria Grazia Roncarolo, MD,a,d Pietro Luigi Poliani, MD, PhD,c Elisabetta Traggiai, PhD,b and Anna Villa, MDa,f Milan, Genoa, Brescia, and Rome, Italy Background: Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, infections, autoimmunity, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical donors is curative, but it is not available to all patients. We have developed a gene therapy (GT) approach for WAS by using a lentiviral vector encoding for human WAS promoter/cDNA (w1.6W) and demonstrated its preclinical efficacy and safety. Objective: To evaluate B-cell reconstitution and correction of B-cell phenotype in GT-treated mice. Methods: We transplanted Was2/2 mice sublethally irradiated (700 rads) with lineage marker-depleted bone marrow wild-type cells, Was2/2 cells untransduced or transduced with the w1.6W lentiviral vector and analyzed B-cell reconstitution in bone marrow, spleen, and peritoneum. Results: Here we show that WAS protein1 B cells were present in central and peripheral B-cell compartments from GT-treated mice and displayed the strongest selective advantage in the splenic marginal zone and peritoneal B1 cell subsets. After GT, splenic architecture was improved and B-cell functions were restored, as demonstrated by the improved antibody response to pneumococcal antigens and the reduction of serum IgG autoantibodies. Conclusion: WAS GT leads to improvement of B-cell functions, even in the presence of a mixed chimerism, further validating the clinical application of the w1.6W lentiviral vector. (J Allergy Clin Immunol 2011;127:1376-84.) Key words: Gene therapy, lentiviral vectors, primary immunodeficiency, Wiskott-Aldrich syndrome, B cells, autoimmunity
Wiskott-Aldrich syndrome (WAS; Online Mendelian Inheritance in Man 301000) is caused by mutations impairing the From athe San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan; bthe Laboratory of Immunology and Rheumatic Disease, IGG, Genoa; cthe Department of Pathology, University of Brescia; dUniversita Vita-Salute San Raffaele, Milan; ethe University of Rome ‘‘Tor Vergata’’; and fCNR-IRGB, Milan. Supported by grants from the Italian Telethon Foundation (M.G.R., A.V.), and Ministero della Salute RF 2007-RF 2008 Giovani Ricercatori Grants (M.B.). Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. Received for publication August 31, 2010; revised March 22, 2011; accepted for publication March 24, 2011. Available online April 29, 2011. Reprint requests: Anna Villa, MD, HSR-TIGET, via Olgettina 58, 20132 Milan, Italy. E-mail: [email protected], [email protected]. 0091-6749/$36.00 Ó 2011 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2011.03.030
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Abbreviations used BM: Bone marrow dsDNA: Double-strand DNA FACS: Fluorescence-activated cell sorting FITC: Fluorescein isothiocyanate GT: Gene therapy HRP: Horseradish peroxidase HSC: Hematopoietic stem cell Lin2: Lineage marker–depleted LV: Lentiviral vector MARCO: Macrophage receptor with a collagenous structure MFI: Mean fluorescence intensity MoMa: Metallophilic macrophage MZ: Marginal zone MZM: Marginal zone macrophage PC: Peritoneal cavity VCN: Vector copy number WAS: Wiskott-Aldrich syndrome Was2/2: C57BL/6 Was2/2 WASp: Wiskott-Aldrich syndrome protein wt: Wild-type
expression or function of the hematopoietic-specific WAS protein (WASp), a key regulator of actin cytoskeleton remodeling.1 Life expectancy of patients with WAS is severely reduced unless they undergo hematopoietic stem cell (HSC) transplantation, which has a very high survival rate, when an HLA-matched donor is available.2-5 However, many patients with WAS lack an HLA-matched bone marrow (BM) donor, and transplantation with HSC from mismatched donors is still associated with an unsatisfactory survival rate.6 Even after transplant, autoimmunity can occur, and in many cases, this correlates with the degree of chimerism.6 We propose, as an alternative therapeutic approach, the administration of genecorrected autologous HSC, which can be used in all patients with WAS and has been proven safe and efficacious in correcting T-cell defects both in patients’ cells and in the Was2/2 mouse model.7-9 Nonetheless, to achieve full immunologic restoration and prevent autoimmune disorders, WASp expression must be attained in all hematopoietic cell lineages. In particular, much evidence highlighted an intrinsic defect of WAS B cells,10-14 confirmed by a strong selective advantage for WASp1 mature B cells in competitive settings.15,16 Moreover, the potential role of WASp-deficient B cells in inducing autoimmunity has been shown.17,18 Thus, we thoroughly evaluated B-cell reconstitution in gene therapy (GT)–treated mice to further support the efficacy of our gene transfer approach. We analyzed restoration of WASp expression in B lymphocyte subsets present in BM, spleen, and
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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 was2/2 (gray filled histograms) and GT mice (black clear histograms). In each panel, the positioning of the markers used to calculate frequency and MFI of WASp1 cells is also shown. Max, Maximum.
peritoneal cavity (PC) by flow cytometry, and we also analyzed Bcell follicle and marginal zone (MZ) architecture in splenic sections by immunohistochemistry. B-cell functionality was evaluated by an in vivo challenge with a vaccine containing pneumococcal antigens. Finally, we tested serum from GT-treated mice for the presence of IgG autoantibodies.
METHODS Mice
C57BL/6 Was2/2 (Was2/2) mice were kindly provided by K. A. Siminovitch (Toronto, Ontario, Canada).19 Control mice were purchased from Charles River Laboratories Inc (Calco, Italy). All mice were housed in specific pathogen free conditions and treated according to protocols approved by the
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FIG 2. Evidence of WASp expression in B-cell subsets from tissues of GT-treated mice. Frequency of WASp1 cells in Was2/2 mice transplanted with Lin2 BM wt cells (BMT wt), Was2/2 cells untransduced (BMT was2/2) or LV-transduced (GT). A, BM. Percentage of WASp1 cells among pre-B, immature B, and mature/recirculating (recirc) B cells. B, Peritoneal cavity. Percentage of WASp1 cells among B1a cells, B1b cells, and mature follicular (B2) cells. C, Spleen. Percentage of WASp1 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.
Animal Care and Use Committee of the San Raffaele Scientific Institute (Institutional Animal Care and Use Committee protocol no. 318).
GT protocol
Lineage marker–depleted (Lin2) BM cells were purified from wild-type (wt) and Was2/2 mice 8 to 12 weeks old by using the hematopoietic progenitor
enrichment kit (Stem Cell Technologies Inc, Vancouver, British Columbia, Canada). Lin2 BM cells were cultured overnight and transduced as previously described.7 Transduction was performed by culturing 1 3 106 Lin2 BM cells in the presence of 1 to 2 3 108/mL infectious viral genomes of the w1.6W lentiviral vector (LV; multiplicity of infection 5 100-200) for 12 hours. The w1.6W is a self-inactivating LV encoding for the human WAS cDNA under the control of a 1.6-kb fragment of the autologous proximal WAS
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TABLE I. Absolute B-cell numbers in spleens from the different treatment groups Cell subset
Total B2201 Transitional MZ FO
BMT wt (n 5 24)*
25.6 4.1 2.6 17
6 6 6 6
17.2 3.5 1.3 13.6
BMT was2/2 (n 5 17)*
9.3 1 1 5.8
6 6 6 6
10.6 1.1 0.8 7.3
GT (n 5 25)*
13.1 1.7 1.9 7.5
6 6 6 6
10.8à 1.7 § 1.4§ 7.2
FO, Follicular. *Median (3106) 6 SD. P < .001 vs BMT wt group. àP < .005 vs BMT wt group. §P < .005 vs BMT was2/2 group.
promoter.7,8,20,21 After transduction, Lin2 BM cells (0.25 3 106 cells/mouse) were transferred intravenously into sublethally irradiated (700 rad) Was2/2 recipient mice 6 to 8 weeks old. The mice analyzed in this study belong to three different groups: Was2/2 mice transplanted with Lin2 BM wt cells (called hereafter ‘‘bone marrow transplant [BMT] wt’’); Was2/2 mice transplanted with Lin2 BM Was2/2 cells untransduced (BMT Was2/2) and Was2/2mice transplanted with Lin2 BM Was2/2 cells transduced with the w1.6W LV (GT).
diaminobenzidine. Nuclei were counterstained with methyl green. IgD was revealed before incubation with HRP-conjugated sheep anti-FITC (1:500; Southern Biotech, Birmingham, Ala). Double immunofluorescence staining for B220 and WASp was carried out by using an FITC-conjugated mouse preabsorbed secondary antibody (1:200; Vector Laboratories) and the appropriate biotinylated secondary antibody followed by Streptavidin–Texas Red (1:100; Southern Biotech), respectively. Images were acquired with an Olympus DP70 digital camera mounted on an Olympus Bx60 microscope (Olympus Europa GmBH, Hamburg, Germany) using CellF imaging software (Soft Imaging System GmbH, M€unster, Germany). The data presented are representative of 5 BMT wt, 7 BMT was2/2, and 10 GT mice analyzed.
In vivo challenge with pneumococcal antigens
Treated mice were challenged intraperitoneally with 0.5 mg/mouse of a vaccine containing a mixture of highly purified capsular polysaccharides from the 23 most prevalent or invasive pneumococcal types of Streptococcus pneumoniae (Pneumovax23; Sanofi Pasteur MSD, Lyon, France) and then bled after 7 days. IgM antibodies specific for Pneumovax23 polysaccharidic components were evaluated by ELISA assay on serum after coating polystyrene plates with 5 mg/mL Pneumowax23. Serial dilution of serum samples (from 1:50 to 1:3200) were incubated on the plates for 2 hours, and antibodies were detected with alkaline phosphatase–conjugated goat antimouse IgM (Southern Biotech). OD values were evaluated at 405 nm.
Analysis of treated mice Between 4 and 5 months after transplantation, mononuclear cells were isolated from tissues of treated mice. mAbs used for immunophenotyping were as follows: anti-CD45R/B220 (clone RA3-6B2), anti-IgM (II/41), antiIgD (11-26), anti-CD21/35 (7G6), anti-CD23 (B3B4), anti-CD5 (53-7.3), anti-CD43 (S7), anti-CD11b/Mac1 (M1/70), and anti-CD3 (17A2; all from BD Pharmingen, San Diego, Calif). The anti-WASp antibody 503 (a kind gift from Profs H. D. Ochs, Seattle, Wash, and L. D. Notarangelo, Boston, Mass) was used for intracytoplasmic detection of WASp on cell fixation and permeabilization (Cytofix/Cytoperm kit; BD Pharmingen). Analysis of lentiviral vector copy number (VCN) per diploid genome was performed on genomic DNA extracted from total BM and spleen cells by using the QIAmp DNA Blood mini-kit (Qiagen, Hilden, Germany) as previously described.9
Immunohistochemistry of spleen sections Following sacrifice, spleens were either snap-frozen in isopentane precooled in liquid nitrogen or formalin-fixed and paraffin-embedded. Cryostat sections 5 mm thick were air-dried overnight at room temperature and fixed in acetone for 10 minutes before immunostaining. Paraffin sections (2 mm thick) underwent heat-based antigen retrieval treatment by using a thermostatic bath in 1.0 mmol/L EDTA buffer (pH 8.0). Hematoxylin and eosin stainings were used to assay basic histopathological changes. The following primary antibodies were used: rat anti–macrophage receptor with a collagenous structure (MARCO; clone ED31, IgG1, 1:50; AbD Serotec, Dusseldorf, Germany), rat anti-mouse metallophilic macrophages (clone MoMa–1, IgG2a, 1:100; Cederlane Laboratories, Burlington, Ontario, Canada), rat anti-B220 (clone RA3-6B2, IgG2a, 1:100; Caltag Laboratories, Burlingame, Calif), rabbit anti-WASp 503 (1:2500), rat-antimurine IgD fluorescein isothiocyanate (FITC)–conjugated rat-antimurine IgD (1:200; BD Pharmingen), and biotinylated goat-antimurine IgM (1:400; Vector Laboratories, Burlingame, Calif). Briefly, tissue sections were washed in blocking buffer (TRIS/1% BSA) before incubation with primary antibody for 2 hours at room temperature or overnight at 48C, washed again, and incubated for 30 minutes with the appropriate biotinylated secondary antibody (antirat 1:200, Vector Laboratories; antirabbit 1:150, DAKO, Glostrup, Denmark). The signal was revealed by ChemMATE Envision Rabbit/Mouse horseradish peroxidase (HRP)/ streptavidin-conjugated HRP (DAKO) followed by diaminobenzidine as chromogen and hematoxylin as counterstain. Double immunostains were performed by using the appropriate biotinylated secondary antibodies (DAKO) followed by either streptavidin-conjugated HRP or alkaline phosphatase (DAKO); chromogen reaction was developed by Ferangi Blue Chromogen Kits (Biocare Medical, Concord, Calif) and
Autoantibody evaluation Anti–double-strand DNA (dsDNA) antibodies were also evaluated by ELISA assay. Briefly, polystyrene plates were coated with poly-L-lysine (Sigma, St Louis, Mo) and DNA from calf thymus (Sigma); after coating with 50 mg/mL polyglutamic acid for 45 minutes and blocking with PBS 3% BSA, serial dilutions of serum from 1:20 to 1:1280 were incubated overnight. Bound antibodies were detected with alkaline phosphatase–conjugated goat antimouse IgG (Southern Biotech). OD values were calculated as follows: OD values of sera incubated on dsDNA-coated plates minus OD values of sera incubated on poly-L-lysine-coated plates to subtract the background. OD values higher than 2-fold the background were considered positive. The score of positivity was assigned to sera that were positive for dilution of 1:60 or higher. Indirect immunohistochemistry was performed on sections from recombination activating gene 2 (Rag2)2/2gc2/2 mice incubated with serum from treated mice. Autoantibodies were revealed by using a goat antimouse IgG-HRP (Molecular Probes; Invitrogen, Carlsbad, Calif) and diaminobenzidine1 (DAKO) followed by hematoxylin counterstaining (Sigma). Slides were examined on a Zeiss Axioplan2 microscope (Carl Zeiss Microimaging, Thornwood, NY). For each serum, we analyzed the reactivity to 3 tissues (salivary gland, thyroid, gastrointestinal tract). For statistical analysis, we fitted a logistic regression model within the generalized linear model framework, considering treatment groups as covariates.
RESULTS Evidence of WASp expression in all B-cell subsets in GT-treated Was2/2 mice We transplanted Was2/2 mice, sublethally irradiated, with Lin2 BM wt cells (BMT wt), Was2/2 cells untransduced (BMT was2/2) or transduced with the w1.6W LV (GT). Transduced cells were present in BM and spleen of all GT-treated mice (see this article’s Fig E1 in the Online Repository at www. jacionline.org), with values ranging from 0.2 to 5.3 VCN/cell in BM (median 6 SD, 1.3 6 1.2) and from 0.5 to 6.2 in spleen (1.6 6 1.6). Of note, in each mouse analyzed, VCN found in spleen was significantly higher than that found in BM, indicating an overall enrichment for transduced cells in peripheral tissues. We analyzed WASp expression in B-cell subsets located in different tissues (BM, spleen, and PC). Fig 1 shows representative
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FIG 3. MFI of WASp expression in B-cell subsets from tissues of GT-treated mice. MFI of WASp was evaluated in Was2/2 mice transplanted with Lin2 BM wt cells (BMT wt), Was2/2 cells untransduced (BMT was2/2) or LV-transduced (GT). In the graphs, MFI is expressed as fold increase in respect to values found in the BMT was2/2 group. In the GT group, MFI was calculated only on WASp1 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.
fluorescence-activated cell sorting (FACS) histograms for WASp staining in each B-cell subset analyzed in the 3 groups of mice (see this article’s Table E1 in the Online Repository at www. jacionline.org for the list of markers used in the FACS stainings). Frequencies of WASp1 cells were determined according to the markers displayed in each panel and are shown in Fig 2. We first evaluated B-cell subsets in BM of transplanted mice. As shown in Fig 2, A, WASp1 B lymphocytes were found in GT mice at all BM differentiation steps: pre (median frequency of WASp1 cells 6 SD, 19% 6 7.8%), immature (24.1% 6 11.4%), and mature/ recirculating B cells (20.3% 6 12.5%). We also confirmed WASp expression in B-cell subsets from peripheral tissues. We evaluated another subset of B lymphocytes, B1 cells, that are
enriched in the peritoneal cavity and can be classified as B1a or B1b cells on the basis of CD5 expression.22 Fig 2, B, shows high frequencies of WASp1 B1a and B1b cells in GT mice, with a median of 34.1% 6 20% B1a and 26.6% 6 16% B1b WASp1 cells, respectively. We observed a significantly higher frequency of WASp1 cells among B1a cells compared with conventional mature B cells (B2). We also evaluated the percentage of splenic WASp1 B cells in transitional and mature subpopulations (Fig 2, C), retrieving similar values (27.7% 6 17% vs 23.2% 6 10%, respectively). Importantly, significant enrichment in WASp1 B cells was found among splenic mature B cells in MZ cells compared with follicular cells (43.7% 6 21% vs 25.1% 6 12%, respectively; Fig 2, C). In addition, analysis of absolute
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FIG 4. Presence of WASp-expressing cells in splenic tissue sections from GT-treated Was2/2 mice. Spleen sections from BMT wt (A), BMT was2/2 (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 310 original magnification.
B-cell numbers (Table I) showed a significant increase in the MZ subset of GT mice compared with BMT was2/2 mice. This increase was not observed in the follicular B-cell subset. These data confirm the critical role of WASp in MZ B cells, in accordance with data previously shown in patients with WAS11 and the Was2/2 mouse model.13,15,16 In Fig 3, we show mean fluorescence intensity (MFI) of WASp expression in B-cell subsets from BM, PC, and spleen in terms of fold increase in respect to BMT was2/2 mice (median values of MFI fold increase for each subset 6 SD are listed in this article’s Table E2 in the Online Repository at www.jacionline.org). It must be noted that the MFI of WASp expression in GT mice, although significantly higher than that observed in BMT was2/2 mice, never reaches a level comparable to that found in BMT wt mice for all B-cell subsets analyzed, with the exception of splenic transitional B cells. In summary, our data demonstrate that in our GT mice, WASp1 cells were detected in all B-cell subsets but also that we did not achieve a complete reconstitution of the B-cell compartment with transduced cells. However, when we performed an analysis of the frequency of WASp-positive cells in peripheral blood over time in a set of mice 3 and 9 months after GT, we could demonstrate a significant increase in B2201 WASp-expressing cells (see this article’s Fig E2 in the Online Repository at www. jacionline.org), indicating that an improvement in reconstitution may occur over time but with slower kinetics than in the case of heterozygous mice or competitive settings.15,16
Improvement of splenic MZ architecture in Was2/2 mice upon GT To understand whether the presence of high frequencies of WASp-expressing cells in the MZ B-cell subset in GT-treated mice resulted in the restoration of B-cell follicle and MZ architecture, we performed immunohistochemistry of spleen sections (Figs 4 and 5). First, we could demonstrate the presence of WASp-expressing cells in B-cell follicles from splenic tissue sections obtained from GT-treated mice (Fig 4, C; see this article’s Fig E3 in the Online Repository at www.jacionline.org), in
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contrast with BMT was2/2 mice (Fig 4, B), in which no positive staining for WASp could be detected. Nevertheless, it has to be noted that the intensity of WASp staining in GT-treated mice was reduced compared with BMT wt mice (Fig 4, A); this could be the result of the fact that not all cells express WASp in GT-treated mice and that the level of the protein expression in the single cell is lower than in wt cells (see also Fig 3). It has been shown that Was2/2 mice present a marked reduction of MZ B-cell numbers in addition to a decreased number of MZ macrophages (MZMs; MARCO1 or MoMa1).15 Mice transplanted with Was2/2 untransduced cells confirmed these data by showing a reduced proportion of MZ B cells (B2201IgMhiIgDlow/-) and MZMs in the spleen (Fig 5, B) compared with mice transplanted with wt cells (Fig 5, A). Spleens from GT-treated mice showed a significant improvement in terms of the number of MZ B cells and MZMs, with the architecture of the B-cell follicle closely resembling that of mice transplanted with wt cells (Fig 5, C).
B-cell response to T-independent antigens is improved in GT-treated Was2/2 mice To assess restoration of B-cell function in GT-treated mice, we evaluated the response to a challenge with a vaccine containing T-independent polysaccharidic antigens, shown to be defective both in Was2/2 mice (M.B., unpublished data, August 2007) and in patients with WAS.1 We performed the antigen challenge 4 to 5 months after GT and evaluated vaccine-specific IgM serum antibodies 7 days after immunization (Fig 6). The specific response to vaccine was found significantly reduced in BMT was2/2 mice compared with BMT wt mice (P < .001), whereas the antibody response was significantly improved in GT-treated mice (P < .001 vs BMT was2/2 mice), although lower than that observed in BMT wt mice (P < .05).
Decreased autoantibody titer and positivity in Was2/2 mice after GT treatment Humblet-Baron et al17 have provided the first evidence of an alteration of B-cell tolerance in Was2/2 mice, showing the presence of circulating autoantibodies against single-strand DNA and dsDNA in Was2/2 mice. We evaluated the presence of IgG circulating autoantibodies, specific for dsDNA (Fig 7, A and B) and against tissue antigens (Fig 7, C and D), in the serum of transplanted mice. Fig 7, A, shows that only 1 of the BMT wt mice presented serum autoantibodies against dsDNA (1/32; 3.1%), whereas half of BMT was2/2 mice had IgG circulating autoantibodies (16/32; 50.0%). GT treatment ameliorated this phenotype, because about 28% of treated mice were positive for serum autoantibodies (12/43; 27.9%). Moreover, the reduction in positivity for anti-dsDNA autoantibodies in GT mice was also accompanied by a decreased titer (Fig 7, B). These results were further confirmed by indirect immunohistochemistry performed on tissue sections from Rag22/2gc2/2 mice (Fig 7, C and D). However, despite the presence of circulating autoantibodies, we did not observe tissue damage (in particular, proteinuria consequent to kidney injury; data not shown) caused by autoimmune reactions in any group of mice analyzed in this study. Nevertheless, these data are in line with our previous observations of absence of tissue damage in Was2/2 mice followed for 1 year after GT.9
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FIG 5. Improvement of splenic B-cell follicle and MZ architecture in GT-treated Was2/2 mice. Spleen sections from BMT wt (A), BMT was2/2 (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; 310 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; 320 original magnification).
FIG 6. GT increases response to polysaccharide antigens in Was2/2 mice. In vivo challenge of treated mice was performed by intraperitoneal injection of 0.5 mg/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 5 23; black circles), BMT was2/2 (n 5 14; white circles), and GT mice (n 5 20; gray squares). Statistical analysis was performed by using a 2-way ANOVA test. *P < .05; ***P < .001.
DISCUSSION Gene therapy is emerging as a possible treatment for patients with WAS lacking a suitable bone marrow donor.23,24 Over the past few years, we have developed a protocol of GT for WAS using an HIV-based LV encoding for WAS cDNA under the control of a 1.6-kb fragment of the autologous promoter (w1.6W) and based on transplantation of gene-corrected HSCs in sublethally conditioned hosts. We have demonstrated, in the Was2/2 mouse preclinical model for GT, long-term restoration of WASp expression and functional correction of T, B, and dendritic cells, with a
selective advantage for transduced lymphoid cells, in the absence of adverse events.7,9 Despite earlier reports on WAS B cells were not indicating a clear functional impairment,19,25 there is much recent evidence pointing to an intrinsic defect of WASp-deficient B cells,10-14 and to their potential role in inducing autoimmunity.17 In addition, a strong selective advantage for WASp1 mature B cells in competitive settings and heterozygous mice has been recently demonstrated.15,16 We set out to analyze in depth B-cell reconstitution in our mouse model of WAS GT. Flow cytometric stainings on cells isolated from GT-treated mice demonstrated the presence of WASp-expressing B cells in all tissues analyzed (BM, PC, and spleen) and at all developmental stages. These data demonstrate the ability of LV-transduced hematopoietic progenitor cells to give rise to all different subsets of B cells. In addition, our results show that WASp1 cells are particularly enriched among mature MZ B cells in the spleen and B1a cells in the peritoneal cavity. These are interesting results, because B1 cells are a complex subset of B cells, originating from distinct fetal liver precursors or from follicular (B2) cells in response to B-cell receptor and other as yet not fully defined signals,22 and share many phenotypic characteristics of MZ cells, such as production of ‘‘natural’’ IgM antibodies and response to T-independent antigens. In addition, because the repertoire of B1 cells seems to be selected by self-antigens, they could be involved in the pathogenesis of autoimmune manifestations.26 These data suggest a role for WASp in the development and function of B1 cells and confirm the importance of WASp in the MZ B-cell subset as previously demonstrated both in human beings and mice.11,13,15,16 The presence of WASp1 MZ B cells in GT-treated mice likely contributes to the improvement of
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splenic B-cell follicles and MZ architecture, characterized by an increased frequency of MZ B cells and enrichment in metallophilic macrophages (MoMa1) and MZMs (MARCO1), and closely resembling the structures found in mice reconstituted with wt cells. The preferential reconstitution of the MZ B-cell compartment with WASp1 cells, and an increase in their absolute number, could also be the reason for the improved response to Pneumovax 23 challenge observed in GT-treated mice, because MZ B cells have a crucial role in the response to T-independent antigens.27 However, different from what has been reported in the literature in heterozygous mice and competitive settings,15,16 we did not observe a strong selective advantage for mature/recirculating BM B cells and splenic follicular B cells. Such a discrepancy could possibly be a result of the lower intensity of WASp expression induced by the LV compared with the wt protein, which might not confer selective advantage to all subsets of mature B cells, or require more time to be established. However, other factors could also be responsible for this difference, including partial conditioning and transduction efficiency. Moreover, a role of B cells in the induction of autoimmune manifestations has been suggested by several studies in human beings and mice6,17,18 (M.B., unpublished data, December 2007). In particular, in the Was2/2 mouse model, the presence of circulating autoantibodies suggests a loss of B-cell tolerance in the absence of WASp17,18 (M.B., unpublished data, December 2007). The reduced frequency of mice positive for the presence of autoantibodies against dsDNA and tissue antigens in GTtreated mice and the reduced titer in those remaining positive are then an indication that our treatment leads to a limited development of autoreactive B cells. Whether this is the result of a direct effect on B-cell development or of an increased, natural T regulatory cell-mediated, peripheral tolerance is still under investigation. Our data showed the presence of WASp-expressing cells in all subsets of B cells analyzed in tissues isolated from GT-treated mice. However, a substantial fraction of B cells, especially in the early developmental stages, in a variable proportion among the different treated mice and correlating with the transduction efficiency (data not shown), did not express WASp by flow cytometry. This situation could result from the fact that recipient mice are sublethally irradiated and retain a certain proportion of host B cells, as shown in our previous studies (range, 0% to 30%),9 so that we cannot exclude a role for residual host cells in
FIG 7. GT results in decreased autoantibody titer in Was2/2 mice. A, The presence of anti-dsDNA autoantibodies was evaluated by ELISA assay. _ 1:60) for anti-dsDNA The graph shows the frequency of positivity (titer > autoantibodies in the 3 groups of treated mice (BMT wt, black bar; BMT
was2/2, 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 < .001. 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 was2/2, n 5 10, white circles; GT, n 5 7, gray circles). Statistical analysis was performed by using a 2-way ANOVA test. **P < .005. C, Tissue-specific autoantibodies were detected by indirect immunohistochemistry on cryostat sections (7 mm) from adult Rag22/2gc2/2 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 < .001. D, The figure shows autoantibodies against salivary gland in mice representative of each group of treatment (BMT wt, BMT was2/2, and GT). Magnification 340.
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the chimerism WASp-positive and WASp-negative cells. In addition, donor WASp-negative cells are also expected on the basis of the variability of transduction efficiency of infused cells (range, 20% to 77%) and the mixed chimerism in engrafted transduced progenitor cells in the bone marrow (data not shown). These data are of particular relevance and could give rise to concern because it is expected that mixed chimerism will occur in GT clinical trials using reduced intensity conditioning23,24,28 and that a mix of transduced and untransduced cells will be transferred. In allogeneic transplant settings, successful reconstitution of the hematopoietic compartment with donor cells is necessary to achieve clinical benefit and decrease the risk of autoimmunity, which can occur after transplant, and in many cases correlates with the degree of chimerism.6 However, the autologous setting of a GT approach may prevent many of the complications related to allogeneic transplantation without the need to achieve a full chimerism of gene corrected cells. In fact, even in the presence of mixed chimerism of WASp1 and WASp2 cells in our GTtreated mice, we observed a reduction in serum autoantibodies and found a significant improvement in the response of B cells to pneumococcal antigen challenge. In conclusion, we provide here much evidence supporting the efficacy of a GT approach in yielding WASp expression and improving functionality of B cells from Was2/2 mice, ultimately contributing to the validation of our GT for the treatment of WAS. We thank Alessandro Nonis and Chiara Brombin (University Center for Statistics in the Biomedical Sciences, Universita Vita-Salute San Raffaele, Milan, Italy) for help in statistical analyses.
Clinical implications: We provide evidence supporting the efficacy of GT in improving functionality of the B-cell compartment in Was2/2 mice and ultimately in the treatment of patients with WAS.
REFERENCES 1. Ochs HD, Thrasher AJ. The Wiskott-Aldrich syndrome. J Allergy Clin Immunol 2006;117:725-38. 2. Filipovich A, Stone J, Tomany S, Ireland M, Kollman C, Pelz C, et al. Impact of donor type on outcome of bone marrow transplantation for Wiskott-Aldrich syndrome: collaborative study of the International Bone Marrow Transplant Registry and the National Marrow Donor Program. Blood 2001;97:1598-603. 3. Antoine C, Muller S, Cant A, Cavazzana-Calvo M, Veys P, Vossen J, et al. Longterm survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience 1968-99. Lancet 2003;361:553-60. 4. Kobayashi R, Ariga T, Nonoyama S, Kanegane H, Tsuchiya S, Morio T, et al. Outcome in patients with Wiskott-Aldrich syndrome following stem cell transplantation: an analysis of 57 patients in Japan. Br J Haematol 2006; 135:362-6. 5. Ozsahin H, Le Deist F, Benkerrou M, Cavazzana-Calvo M, Gomez L, Griscelli C, et al. Bone marrow transplantation in 26 patients with Wiskott-Aldrich syndrome from a single center. J Pediatr 1996;129:238-44. 6. Ozsahin H, Cavazzana-Calvo M, Notarangelo LD, Schulz A, Thrasher AJ, Mazzolari E, et al. Long-term outcome following hematopoietic stem-cell transplantation in Wiskott-Aldrich syndrome: collaborative study of the European Society for Immunodeficiencies and European Group for Blood and Marrow Transplantation. Blood 2008;111:439-45.
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7. Dupre L, Marangoni F, Scaramuzza S, Trifari S, Hernandez RJ, Aiuti A, et al. Efficacy of gene therapy for Wiskott-Aldrich syndrome using a WAS promoter/ cDNA-containing lentiviral vector and nonlethal irradiation. Hum Gene Ther 2006;17:303-13. 8. Dupre L, Trifari S, Follenzi A, Marangoni F, Lain de Lera T, Bernad A, et al. Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction. Mol Ther 2004;10:903-15. 9. Marangoni F, Bosticardo M, Charrier S, Draghici E, Locci M, Scaramuzza S, et al. Evidence for long term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models. Mol Ther 2009;17:1073-82. 10. Park JY, Shcherbina A, Rosen FS, Prodeus AP, Remold-O’Donnell E. Phenotypic perturbation of B cells in the Wiskott-Aldrich syndrome. Clin Exp Immunol 2005; 139:297-305. 11. Vermi W, Blanzuoli L, Kraus MD, Grigolato P, Donato F, Loffredo G, et al. The spleen in the Wiskott-Aldrich syndrome: histopathologic abnormalities of the white pulp correlate with the clinical phenotype of the disease. Am J Surg Pathol 1999; 23:182-91. 12. Westerberg L, Greicius G, Snapper SB, Aspenstrom P, Severinson E. Cdc42, Rac1, and the Wiskott-Aldrich syndrome protein are involved in the cytoskeletal regulation of B lymphocytes. Blood 2001;98:1086-94. 13. Westerberg L, Larsson M, Hardy SJ, Fernandez C, Thrasher AJ, Severinson E. Wiskott-Aldrich syndrome protein deficiency leads to reduced B-cell adhesion, migration, and homing, and a delayed humoral immune response. Blood 2005; 105:1144-52. 14. Westerberg L, Wallin RP, Greicius G, Ljunggren HG, Severinson E. Efficient antigen presentation of soluble, but not particulate, antigen in the absence of Wiskott-Aldrich syndrome protein. Immunology 2003;109:384-91. 15. Westerberg LS, de la Fuente MA, Wermeling F, Ochs HD, Karlsson MC, Snapper SB, et al. WASP confers selective advantage for specific hematopoietic cell populations and serves a unique role in marginal zone B-cell homeostasis and function. Blood 2008;112:4139-47. 16. Meyer-Bahlburg A, Becker-Herman S, Humblet-Baron S, Khim S, Weber M, Bouma G, et al. Wiskott-Aldrich syndrome protein deficiency in B cells results in impaired peripheral homeostasis. Blood 2008;112:4158-69. 17. Humblet-Baron S, Sather B, Anover S, Becker-Herman S, Kasprowicz DJ, Khim S, et al. Wiskott-Aldrich syndrome protein is required for regulatory T cell homeostasis. J Clin Invest 2007;117:407-18. 18. Nikolov NP, Shimizu M, Cleland S, Bailey D, Aoki J, Strom T, et al. Systemic autoimmunity and defective Fas ligand secretion in the absence of the WiskottAldrich syndrome protein. Blood 2010;116:740-7. 19. Zhang J, Shehabeldin A, da Cruz LAG, Butler J, Somani A-K, McGavin M, et al. Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes. J Exp Med 1999; 190:1329-41. 20. Charrier S, Dupre L, Scaramuzza S, Jeanson-Leh L, Blundell MP, Danos O, et al. Lentiviral vectors targeting WASp expression to hematopoietic cells, efficiently transduce and correct cells from WAS patients. Gene Ther 2007;14:415-28. 21. Petrella A, Doti I, Agosti V, Giarrusso P, Vitale D, Bond H, et al. A 59 regulatory sequence containing two Ets motifs controls the expression of the Wiskott-Aldrich syndrome protein (WASP) gene in human hematopoietic cells. Blood 1998;91: 4554-60. 22. Berland R, Wortis HH. Origins and functions of B-1 cells with notes on the role of CD5. Annu Rev Immunol 2002;20:253-300. 23. Galy A, Roncarolo MG, Thrasher AJ. Development of lentiviral gene therapy for Wiskott Aldrich syndrome. Expert Opin Biol Ther 2008;8:181-90. 24. Boztug K, Schmidt M, Schwarzer A, Banerjee PP, Diez IA, Dewey RA, et al. Stemcell gene therapy for the Wiskott-Aldrich syndrome. N Engl J Med 2010;363: 1918-27. 25. Snapper SB, Rosen FS, Mizoguchi E, Cohen P, Khan W, Liu CH, et al. WiskottAldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation. Immunity 1998;9:81-91. 26. Martin F, Kearney JF. B1 cells: similarities and differences with other B cell subsets. Curr Opin Immunol 2001;13:195-201. 27. Martin F, Kearney JF. Marginal-zone B cells. Nat Rev Immunol 2002;2:323-35. 28. Bosticardo M, Marangoni F, Aiuti A, Villa A, Roncarolo MG. Recent advances in understanding the pathophysiology of Wiskott-Aldrich syndrome. Blood 2009;113: 6288-95.
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FIG E1. Presence of gene corrected cells in BM and spleen (SP) in Was2/2 mice reconstituted with LV-transduced Lin2 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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FIG E2. Analysis of WASp-expressing cell frequency in peripheral blood B2201 cells from GT-treated mice 3 and 9 months after transplant. **P < .005 (paired t test).
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FIG E3. Presence of WASp-expressing B cells in splenic tissue sections from GT-treated Was2/2 mice. Spleen sections from BMT wt (A), BMT was2/2 (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 320 original magnification.
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TABLE E1. Surface markers used to define B-cell subsets B-cell subset
BM Pre Immature Mature/recirculating Peritoneal cavity B1a B1b B2 Spleen Transitional Mature MZ FO FO, Follicular.
Markers
CD432B220loIgM2 CD432B220loIgM1 CD432B220hiIgM2 CD191IgM1CD51B220lo CD191IgM1CD52B220lo CD191IgM1CD52B220hi B2201IgMhiIgDhi B2201IgMloIgDhi B2201CD21hiCD23lo B2201CD21intCD23hi
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TABLE E2. MFI of WASp expression indicated as fold increase in respect to values found in the BMT was2/2 group Cell subset
BM Pre-B Immature B Mature/recirc B Cell subset
Peritoneal cavity B1a B1b B2 Cell subset
Spleen Transitional FO MZ FO, Follicular; recirc, recirculating. *Median 6 SD. P < .001 vs BMT wt group. àP < .005 vs BMT wt group. §P < .001 vs BMT was2/2 group.
BMT wt (n 5 15)*
BMT was2/2 (n 5 20)*
GT WASp1 (n 5 24)*
3.7 6 0.9 5 6 1.6 6.1 6 3.6
1 6 0.2 1 6 0.2 1 6 0.2
2.9 6 0.4 § 2.8 6 0.6 § 2.7 6 0.7 §
BMT wt (n 5 21)*
BMT was2/2 (n 5 15)*
GT WASp1 (n 5 22)*
3.9 6 1.2 3.8 6 0.9 4.4 6 1.8
1 6 0.1 1 6 0.2 1 6 0.1
3.4 6 0.6ৠ3.1 6 0.5ৠ3.5 6 0.8à§
BMT wt (n 5 30)*
BMT was2/2 (n 5 23)*
GT WASp1 (n 5 32)*
5 6 1.9 5.5 6 3.6 5.2 6 3.2
1 6 0.3 1 6 0.4 1 6 0.4
4.5 6 1.7§ 3.1 6 1.2 § 2.9 6 1.4 §
Wiskott-Aldrich syndrome (WAS; Online Mendelian Inheritance in Man 301000) is caused by mutations impairing the From athe San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan; bthe Laboratory of Immunology and Rheumatic Disease, IGG, Genoa; cthe Department of Pathology, University of Brescia; dUniversita Vita-Salute San Raffaele, Milan; ethe University of Rome ‘‘Tor Vergata’’; and fCNR-IRGB, Milan. Supported by grants from the Italian Telethon Foundation (M.G.R., A.V.), and Ministero della Salute RF 2007-RF 2008 Giovani Ricercatori Grants (M.B.). Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. Received for publication August 31, 2010; revised March 22, 2011; accepted for publication March 24, 2011. Available online April 29, 2011. Reprint requests: Anna Villa, MD, HSR-TIGET, via Olgettina 58, 20132 Milan, Italy. E-mail: [email protected], [email protected]. 0091-6749/$36.00 Ó 2011 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2011.03.030
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Abbreviations used BM: Bone marrow dsDNA: Double-strand DNA FACS: Fluorescence-activated cell sorting FITC: Fluorescein isothiocyanate GT: Gene therapy HRP: Horseradish peroxidase HSC: Hematopoietic stem cell Lin2: Lineage marker–depleted LV: Lentiviral vector MARCO: Macrophage receptor with a collagenous structure MFI: Mean fluorescence intensity MoMa: Metallophilic macrophage MZ: Marginal zone MZM: Marginal zone macrophage PC: Peritoneal cavity VCN: Vector copy number WAS: Wiskott-Aldrich syndrome Was2/2: C57BL/6 Was2/2 WASp: Wiskott-Aldrich syndrome protein wt: Wild-type
expression or function of the hematopoietic-specific WAS protein (WASp), a key regulator of actin cytoskeleton remodeling.1 Life expectancy of patients with WAS is severely reduced unless they undergo hematopoietic stem cell (HSC) transplantation, which has a very high survival rate, when an HLA-matched donor is available.2-5 However, many patients with WAS lack an HLA-matched bone marrow (BM) donor, and transplantation with HSC from mismatched donors is still associated with an unsatisfactory survival rate.6 Even after transplant, autoimmunity can occur, and in many cases, this correlates with the degree of chimerism.6 We propose, as an alternative therapeutic approach, the administration of genecorrected autologous HSC, which can be used in all patients with WAS and has been proven safe and efficacious in correcting T-cell defects both in patients’ cells and in the Was2/2 mouse model.7-9 Nonetheless, to achieve full immunologic restoration and prevent autoimmune disorders, WASp expression must be attained in all hematopoietic cell lineages. In particular, much evidence highlighted an intrinsic defect of WAS B cells,10-14 confirmed by a strong selective advantage for WASp1 mature B cells in competitive settings.15,16 Moreover, the potential role of WASp-deficient B cells in inducing autoimmunity has been shown.17,18 Thus, we thoroughly evaluated B-cell reconstitution in gene therapy (GT)–treated mice to further support the efficacy of our gene transfer approach. We analyzed restoration of WASp expression in B lymphocyte subsets present in BM, spleen, and
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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 was2/2 (gray filled histograms) and GT mice (black clear histograms). In each panel, the positioning of the markers used to calculate frequency and MFI of WASp1 cells is also shown. Max, Maximum.
peritoneal cavity (PC) by flow cytometry, and we also analyzed Bcell follicle and marginal zone (MZ) architecture in splenic sections by immunohistochemistry. B-cell functionality was evaluated by an in vivo challenge with a vaccine containing pneumococcal antigens. Finally, we tested serum from GT-treated mice for the presence of IgG autoantibodies.
METHODS Mice
C57BL/6 Was2/2 (Was2/2) mice were kindly provided by K. A. Siminovitch (Toronto, Ontario, Canada).19 Control mice were purchased from Charles River Laboratories Inc (Calco, Italy). All mice were housed in specific pathogen free conditions and treated according to protocols approved by the
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FIG 2. Evidence of WASp expression in B-cell subsets from tissues of GT-treated mice. Frequency of WASp1 cells in Was2/2 mice transplanted with Lin2 BM wt cells (BMT wt), Was2/2 cells untransduced (BMT was2/2) or LV-transduced (GT). A, BM. Percentage of WASp1 cells among pre-B, immature B, and mature/recirculating (recirc) B cells. B, Peritoneal cavity. Percentage of WASp1 cells among B1a cells, B1b cells, and mature follicular (B2) cells. C, Spleen. Percentage of WASp1 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.
Animal Care and Use Committee of the San Raffaele Scientific Institute (Institutional Animal Care and Use Committee protocol no. 318).
GT protocol
Lineage marker–depleted (Lin2) BM cells were purified from wild-type (wt) and Was2/2 mice 8 to 12 weeks old by using the hematopoietic progenitor
enrichment kit (Stem Cell Technologies Inc, Vancouver, British Columbia, Canada). Lin2 BM cells were cultured overnight and transduced as previously described.7 Transduction was performed by culturing 1 3 106 Lin2 BM cells in the presence of 1 to 2 3 108/mL infectious viral genomes of the w1.6W lentiviral vector (LV; multiplicity of infection 5 100-200) for 12 hours. The w1.6W is a self-inactivating LV encoding for the human WAS cDNA under the control of a 1.6-kb fragment of the autologous proximal WAS
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TABLE I. Absolute B-cell numbers in spleens from the different treatment groups Cell subset
Total B2201 Transitional MZ FO
BMT wt (n 5 24)*
25.6 4.1 2.6 17
6 6 6 6
17.2 3.5 1.3 13.6
BMT was2/2 (n 5 17)*
9.3 1 1 5.8
6 6 6 6
10.6 1.1 0.8 7.3
GT (n 5 25)*
13.1 1.7 1.9 7.5
6 6 6 6
10.8à 1.7 § 1.4§ 7.2
FO, Follicular. *Median (3106) 6 SD. P < .001 vs BMT wt group. àP < .005 vs BMT wt group. §P < .005 vs BMT was2/2 group.
promoter.7,8,20,21 After transduction, Lin2 BM cells (0.25 3 106 cells/mouse) were transferred intravenously into sublethally irradiated (700 rad) Was2/2 recipient mice 6 to 8 weeks old. The mice analyzed in this study belong to three different groups: Was2/2 mice transplanted with Lin2 BM wt cells (called hereafter ‘‘bone marrow transplant [BMT] wt’’); Was2/2 mice transplanted with Lin2 BM Was2/2 cells untransduced (BMT Was2/2) and Was2/2mice transplanted with Lin2 BM Was2/2 cells transduced with the w1.6W LV (GT).
diaminobenzidine. Nuclei were counterstained with methyl green. IgD was revealed before incubation with HRP-conjugated sheep anti-FITC (1:500; Southern Biotech, Birmingham, Ala). Double immunofluorescence staining for B220 and WASp was carried out by using an FITC-conjugated mouse preabsorbed secondary antibody (1:200; Vector Laboratories) and the appropriate biotinylated secondary antibody followed by Streptavidin–Texas Red (1:100; Southern Biotech), respectively. Images were acquired with an Olympus DP70 digital camera mounted on an Olympus Bx60 microscope (Olympus Europa GmBH, Hamburg, Germany) using CellF imaging software (Soft Imaging System GmbH, M€unster, Germany). The data presented are representative of 5 BMT wt, 7 BMT was2/2, and 10 GT mice analyzed.
In vivo challenge with pneumococcal antigens
Treated mice were challenged intraperitoneally with 0.5 mg/mouse of a vaccine containing a mixture of highly purified capsular polysaccharides from the 23 most prevalent or invasive pneumococcal types of Streptococcus pneumoniae (Pneumovax23; Sanofi Pasteur MSD, Lyon, France) and then bled after 7 days. IgM antibodies specific for Pneumovax23 polysaccharidic components were evaluated by ELISA assay on serum after coating polystyrene plates with 5 mg/mL Pneumowax23. Serial dilution of serum samples (from 1:50 to 1:3200) were incubated on the plates for 2 hours, and antibodies were detected with alkaline phosphatase–conjugated goat antimouse IgM (Southern Biotech). OD values were evaluated at 405 nm.
Analysis of treated mice Between 4 and 5 months after transplantation, mononuclear cells were isolated from tissues of treated mice. mAbs used for immunophenotyping were as follows: anti-CD45R/B220 (clone RA3-6B2), anti-IgM (II/41), antiIgD (11-26), anti-CD21/35 (7G6), anti-CD23 (B3B4), anti-CD5 (53-7.3), anti-CD43 (S7), anti-CD11b/Mac1 (M1/70), and anti-CD3 (17A2; all from BD Pharmingen, San Diego, Calif). The anti-WASp antibody 503 (a kind gift from Profs H. D. Ochs, Seattle, Wash, and L. D. Notarangelo, Boston, Mass) was used for intracytoplasmic detection of WASp on cell fixation and permeabilization (Cytofix/Cytoperm kit; BD Pharmingen). Analysis of lentiviral vector copy number (VCN) per diploid genome was performed on genomic DNA extracted from total BM and spleen cells by using the QIAmp DNA Blood mini-kit (Qiagen, Hilden, Germany) as previously described.9
Immunohistochemistry of spleen sections Following sacrifice, spleens were either snap-frozen in isopentane precooled in liquid nitrogen or formalin-fixed and paraffin-embedded. Cryostat sections 5 mm thick were air-dried overnight at room temperature and fixed in acetone for 10 minutes before immunostaining. Paraffin sections (2 mm thick) underwent heat-based antigen retrieval treatment by using a thermostatic bath in 1.0 mmol/L EDTA buffer (pH 8.0). Hematoxylin and eosin stainings were used to assay basic histopathological changes. The following primary antibodies were used: rat anti–macrophage receptor with a collagenous structure (MARCO; clone ED31, IgG1, 1:50; AbD Serotec, Dusseldorf, Germany), rat anti-mouse metallophilic macrophages (clone MoMa–1, IgG2a, 1:100; Cederlane Laboratories, Burlington, Ontario, Canada), rat anti-B220 (clone RA3-6B2, IgG2a, 1:100; Caltag Laboratories, Burlingame, Calif), rabbit anti-WASp 503 (1:2500), rat-antimurine IgD fluorescein isothiocyanate (FITC)–conjugated rat-antimurine IgD (1:200; BD Pharmingen), and biotinylated goat-antimurine IgM (1:400; Vector Laboratories, Burlingame, Calif). Briefly, tissue sections were washed in blocking buffer (TRIS/1% BSA) before incubation with primary antibody for 2 hours at room temperature or overnight at 48C, washed again, and incubated for 30 minutes with the appropriate biotinylated secondary antibody (antirat 1:200, Vector Laboratories; antirabbit 1:150, DAKO, Glostrup, Denmark). The signal was revealed by ChemMATE Envision Rabbit/Mouse horseradish peroxidase (HRP)/ streptavidin-conjugated HRP (DAKO) followed by diaminobenzidine as chromogen and hematoxylin as counterstain. Double immunostains were performed by using the appropriate biotinylated secondary antibodies (DAKO) followed by either streptavidin-conjugated HRP or alkaline phosphatase (DAKO); chromogen reaction was developed by Ferangi Blue Chromogen Kits (Biocare Medical, Concord, Calif) and
Autoantibody evaluation Anti–double-strand DNA (dsDNA) antibodies were also evaluated by ELISA assay. Briefly, polystyrene plates were coated with poly-L-lysine (Sigma, St Louis, Mo) and DNA from calf thymus (Sigma); after coating with 50 mg/mL polyglutamic acid for 45 minutes and blocking with PBS 3% BSA, serial dilutions of serum from 1:20 to 1:1280 were incubated overnight. Bound antibodies were detected with alkaline phosphatase–conjugated goat antimouse IgG (Southern Biotech). OD values were calculated as follows: OD values of sera incubated on dsDNA-coated plates minus OD values of sera incubated on poly-L-lysine-coated plates to subtract the background. OD values higher than 2-fold the background were considered positive. The score of positivity was assigned to sera that were positive for dilution of 1:60 or higher. Indirect immunohistochemistry was performed on sections from recombination activating gene 2 (Rag2)2/2gc2/2 mice incubated with serum from treated mice. Autoantibodies were revealed by using a goat antimouse IgG-HRP (Molecular Probes; Invitrogen, Carlsbad, Calif) and diaminobenzidine1 (DAKO) followed by hematoxylin counterstaining (Sigma). Slides were examined on a Zeiss Axioplan2 microscope (Carl Zeiss Microimaging, Thornwood, NY). For each serum, we analyzed the reactivity to 3 tissues (salivary gland, thyroid, gastrointestinal tract). For statistical analysis, we fitted a logistic regression model within the generalized linear model framework, considering treatment groups as covariates.
RESULTS Evidence of WASp expression in all B-cell subsets in GT-treated Was2/2 mice We transplanted Was2/2 mice, sublethally irradiated, with Lin2 BM wt cells (BMT wt), Was2/2 cells untransduced (BMT was2/2) or transduced with the w1.6W LV (GT). Transduced cells were present in BM and spleen of all GT-treated mice (see this article’s Fig E1 in the Online Repository at www. jacionline.org), with values ranging from 0.2 to 5.3 VCN/cell in BM (median 6 SD, 1.3 6 1.2) and from 0.5 to 6.2 in spleen (1.6 6 1.6). Of note, in each mouse analyzed, VCN found in spleen was significantly higher than that found in BM, indicating an overall enrichment for transduced cells in peripheral tissues. We analyzed WASp expression in B-cell subsets located in different tissues (BM, spleen, and PC). Fig 1 shows representative
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FIG 3. MFI of WASp expression in B-cell subsets from tissues of GT-treated mice. MFI of WASp was evaluated in Was2/2 mice transplanted with Lin2 BM wt cells (BMT wt), Was2/2 cells untransduced (BMT was2/2) or LV-transduced (GT). In the graphs, MFI is expressed as fold increase in respect to values found in the BMT was2/2 group. In the GT group, MFI was calculated only on WASp1 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.
fluorescence-activated cell sorting (FACS) histograms for WASp staining in each B-cell subset analyzed in the 3 groups of mice (see this article’s Table E1 in the Online Repository at www. jacionline.org for the list of markers used in the FACS stainings). Frequencies of WASp1 cells were determined according to the markers displayed in each panel and are shown in Fig 2. We first evaluated B-cell subsets in BM of transplanted mice. As shown in Fig 2, A, WASp1 B lymphocytes were found in GT mice at all BM differentiation steps: pre (median frequency of WASp1 cells 6 SD, 19% 6 7.8%), immature (24.1% 6 11.4%), and mature/ recirculating B cells (20.3% 6 12.5%). We also confirmed WASp expression in B-cell subsets from peripheral tissues. We evaluated another subset of B lymphocytes, B1 cells, that are
enriched in the peritoneal cavity and can be classified as B1a or B1b cells on the basis of CD5 expression.22 Fig 2, B, shows high frequencies of WASp1 B1a and B1b cells in GT mice, with a median of 34.1% 6 20% B1a and 26.6% 6 16% B1b WASp1 cells, respectively. We observed a significantly higher frequency of WASp1 cells among B1a cells compared with conventional mature B cells (B2). We also evaluated the percentage of splenic WASp1 B cells in transitional and mature subpopulations (Fig 2, C), retrieving similar values (27.7% 6 17% vs 23.2% 6 10%, respectively). Importantly, significant enrichment in WASp1 B cells was found among splenic mature B cells in MZ cells compared with follicular cells (43.7% 6 21% vs 25.1% 6 12%, respectively; Fig 2, C). In addition, analysis of absolute
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FIG 4. Presence of WASp-expressing cells in splenic tissue sections from GT-treated Was2/2 mice. Spleen sections from BMT wt (A), BMT was2/2 (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 310 original magnification.
B-cell numbers (Table I) showed a significant increase in the MZ subset of GT mice compared with BMT was2/2 mice. This increase was not observed in the follicular B-cell subset. These data confirm the critical role of WASp in MZ B cells, in accordance with data previously shown in patients with WAS11 and the Was2/2 mouse model.13,15,16 In Fig 3, we show mean fluorescence intensity (MFI) of WASp expression in B-cell subsets from BM, PC, and spleen in terms of fold increase in respect to BMT was2/2 mice (median values of MFI fold increase for each subset 6 SD are listed in this article’s Table E2 in the Online Repository at www.jacionline.org). It must be noted that the MFI of WASp expression in GT mice, although significantly higher than that observed in BMT was2/2 mice, never reaches a level comparable to that found in BMT wt mice for all B-cell subsets analyzed, with the exception of splenic transitional B cells. In summary, our data demonstrate that in our GT mice, WASp1 cells were detected in all B-cell subsets but also that we did not achieve a complete reconstitution of the B-cell compartment with transduced cells. However, when we performed an analysis of the frequency of WASp-positive cells in peripheral blood over time in a set of mice 3 and 9 months after GT, we could demonstrate a significant increase in B2201 WASp-expressing cells (see this article’s Fig E2 in the Online Repository at www. jacionline.org), indicating that an improvement in reconstitution may occur over time but with slower kinetics than in the case of heterozygous mice or competitive settings.15,16
Improvement of splenic MZ architecture in Was2/2 mice upon GT To understand whether the presence of high frequencies of WASp-expressing cells in the MZ B-cell subset in GT-treated mice resulted in the restoration of B-cell follicle and MZ architecture, we performed immunohistochemistry of spleen sections (Figs 4 and 5). First, we could demonstrate the presence of WASp-expressing cells in B-cell follicles from splenic tissue sections obtained from GT-treated mice (Fig 4, C; see this article’s Fig E3 in the Online Repository at www.jacionline.org), in
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contrast with BMT was2/2 mice (Fig 4, B), in which no positive staining for WASp could be detected. Nevertheless, it has to be noted that the intensity of WASp staining in GT-treated mice was reduced compared with BMT wt mice (Fig 4, A); this could be the result of the fact that not all cells express WASp in GT-treated mice and that the level of the protein expression in the single cell is lower than in wt cells (see also Fig 3). It has been shown that Was2/2 mice present a marked reduction of MZ B-cell numbers in addition to a decreased number of MZ macrophages (MZMs; MARCO1 or MoMa1).15 Mice transplanted with Was2/2 untransduced cells confirmed these data by showing a reduced proportion of MZ B cells (B2201IgMhiIgDlow/-) and MZMs in the spleen (Fig 5, B) compared with mice transplanted with wt cells (Fig 5, A). Spleens from GT-treated mice showed a significant improvement in terms of the number of MZ B cells and MZMs, with the architecture of the B-cell follicle closely resembling that of mice transplanted with wt cells (Fig 5, C).
B-cell response to T-independent antigens is improved in GT-treated Was2/2 mice To assess restoration of B-cell function in GT-treated mice, we evaluated the response to a challenge with a vaccine containing T-independent polysaccharidic antigens, shown to be defective both in Was2/2 mice (M.B., unpublished data, August 2007) and in patients with WAS.1 We performed the antigen challenge 4 to 5 months after GT and evaluated vaccine-specific IgM serum antibodies 7 days after immunization (Fig 6). The specific response to vaccine was found significantly reduced in BMT was2/2 mice compared with BMT wt mice (P < .001), whereas the antibody response was significantly improved in GT-treated mice (P < .001 vs BMT was2/2 mice), although lower than that observed in BMT wt mice (P < .05).
Decreased autoantibody titer and positivity in Was2/2 mice after GT treatment Humblet-Baron et al17 have provided the first evidence of an alteration of B-cell tolerance in Was2/2 mice, showing the presence of circulating autoantibodies against single-strand DNA and dsDNA in Was2/2 mice. We evaluated the presence of IgG circulating autoantibodies, specific for dsDNA (Fig 7, A and B) and against tissue antigens (Fig 7, C and D), in the serum of transplanted mice. Fig 7, A, shows that only 1 of the BMT wt mice presented serum autoantibodies against dsDNA (1/32; 3.1%), whereas half of BMT was2/2 mice had IgG circulating autoantibodies (16/32; 50.0%). GT treatment ameliorated this phenotype, because about 28% of treated mice were positive for serum autoantibodies (12/43; 27.9%). Moreover, the reduction in positivity for anti-dsDNA autoantibodies in GT mice was also accompanied by a decreased titer (Fig 7, B). These results were further confirmed by indirect immunohistochemistry performed on tissue sections from Rag22/2gc2/2 mice (Fig 7, C and D). However, despite the presence of circulating autoantibodies, we did not observe tissue damage (in particular, proteinuria consequent to kidney injury; data not shown) caused by autoimmune reactions in any group of mice analyzed in this study. Nevertheless, these data are in line with our previous observations of absence of tissue damage in Was2/2 mice followed for 1 year after GT.9
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FIG 5. Improvement of splenic B-cell follicle and MZ architecture in GT-treated Was2/2 mice. Spleen sections from BMT wt (A), BMT was2/2 (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; 310 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; 320 original magnification).
FIG 6. GT increases response to polysaccharide antigens in Was2/2 mice. In vivo challenge of treated mice was performed by intraperitoneal injection of 0.5 mg/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 5 23; black circles), BMT was2/2 (n 5 14; white circles), and GT mice (n 5 20; gray squares). Statistical analysis was performed by using a 2-way ANOVA test. *P < .05; ***P < .001.
DISCUSSION Gene therapy is emerging as a possible treatment for patients with WAS lacking a suitable bone marrow donor.23,24 Over the past few years, we have developed a protocol of GT for WAS using an HIV-based LV encoding for WAS cDNA under the control of a 1.6-kb fragment of the autologous promoter (w1.6W) and based on transplantation of gene-corrected HSCs in sublethally conditioned hosts. We have demonstrated, in the Was2/2 mouse preclinical model for GT, long-term restoration of WASp expression and functional correction of T, B, and dendritic cells, with a
selective advantage for transduced lymphoid cells, in the absence of adverse events.7,9 Despite earlier reports on WAS B cells were not indicating a clear functional impairment,19,25 there is much recent evidence pointing to an intrinsic defect of WASp-deficient B cells,10-14 and to their potential role in inducing autoimmunity.17 In addition, a strong selective advantage for WASp1 mature B cells in competitive settings and heterozygous mice has been recently demonstrated.15,16 We set out to analyze in depth B-cell reconstitution in our mouse model of WAS GT. Flow cytometric stainings on cells isolated from GT-treated mice demonstrated the presence of WASp-expressing B cells in all tissues analyzed (BM, PC, and spleen) and at all developmental stages. These data demonstrate the ability of LV-transduced hematopoietic progenitor cells to give rise to all different subsets of B cells. In addition, our results show that WASp1 cells are particularly enriched among mature MZ B cells in the spleen and B1a cells in the peritoneal cavity. These are interesting results, because B1 cells are a complex subset of B cells, originating from distinct fetal liver precursors or from follicular (B2) cells in response to B-cell receptor and other as yet not fully defined signals,22 and share many phenotypic characteristics of MZ cells, such as production of ‘‘natural’’ IgM antibodies and response to T-independent antigens. In addition, because the repertoire of B1 cells seems to be selected by self-antigens, they could be involved in the pathogenesis of autoimmune manifestations.26 These data suggest a role for WASp in the development and function of B1 cells and confirm the importance of WASp in the MZ B-cell subset as previously demonstrated both in human beings and mice.11,13,15,16 The presence of WASp1 MZ B cells in GT-treated mice likely contributes to the improvement of
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splenic B-cell follicles and MZ architecture, characterized by an increased frequency of MZ B cells and enrichment in metallophilic macrophages (MoMa1) and MZMs (MARCO1), and closely resembling the structures found in mice reconstituted with wt cells. The preferential reconstitution of the MZ B-cell compartment with WASp1 cells, and an increase in their absolute number, could also be the reason for the improved response to Pneumovax 23 challenge observed in GT-treated mice, because MZ B cells have a crucial role in the response to T-independent antigens.27 However, different from what has been reported in the literature in heterozygous mice and competitive settings,15,16 we did not observe a strong selective advantage for mature/recirculating BM B cells and splenic follicular B cells. Such a discrepancy could possibly be a result of the lower intensity of WASp expression induced by the LV compared with the wt protein, which might not confer selective advantage to all subsets of mature B cells, or require more time to be established. However, other factors could also be responsible for this difference, including partial conditioning and transduction efficiency. Moreover, a role of B cells in the induction of autoimmune manifestations has been suggested by several studies in human beings and mice6,17,18 (M.B., unpublished data, December 2007). In particular, in the Was2/2 mouse model, the presence of circulating autoantibodies suggests a loss of B-cell tolerance in the absence of WASp17,18 (M.B., unpublished data, December 2007). The reduced frequency of mice positive for the presence of autoantibodies against dsDNA and tissue antigens in GTtreated mice and the reduced titer in those remaining positive are then an indication that our treatment leads to a limited development of autoreactive B cells. Whether this is the result of a direct effect on B-cell development or of an increased, natural T regulatory cell-mediated, peripheral tolerance is still under investigation. Our data showed the presence of WASp-expressing cells in all subsets of B cells analyzed in tissues isolated from GT-treated mice. However, a substantial fraction of B cells, especially in the early developmental stages, in a variable proportion among the different treated mice and correlating with the transduction efficiency (data not shown), did not express WASp by flow cytometry. This situation could result from the fact that recipient mice are sublethally irradiated and retain a certain proportion of host B cells, as shown in our previous studies (range, 0% to 30%),9 so that we cannot exclude a role for residual host cells in
FIG 7. GT results in decreased autoantibody titer in Was2/2 mice. A, The presence of anti-dsDNA autoantibodies was evaluated by ELISA assay. _ 1:60) for anti-dsDNA The graph shows the frequency of positivity (titer > autoantibodies in the 3 groups of treated mice (BMT wt, black bar; BMT
was2/2, 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 < .001. 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 was2/2, n 5 10, white circles; GT, n 5 7, gray circles). Statistical analysis was performed by using a 2-way ANOVA test. **P < .005. C, Tissue-specific autoantibodies were detected by indirect immunohistochemistry on cryostat sections (7 mm) from adult Rag22/2gc2/2 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 < .001. D, The figure shows autoantibodies against salivary gland in mice representative of each group of treatment (BMT wt, BMT was2/2, and GT). Magnification 340.
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the chimerism WASp-positive and WASp-negative cells. In addition, donor WASp-negative cells are also expected on the basis of the variability of transduction efficiency of infused cells (range, 20% to 77%) and the mixed chimerism in engrafted transduced progenitor cells in the bone marrow (data not shown). These data are of particular relevance and could give rise to concern because it is expected that mixed chimerism will occur in GT clinical trials using reduced intensity conditioning23,24,28 and that a mix of transduced and untransduced cells will be transferred. In allogeneic transplant settings, successful reconstitution of the hematopoietic compartment with donor cells is necessary to achieve clinical benefit and decrease the risk of autoimmunity, which can occur after transplant, and in many cases correlates with the degree of chimerism.6 However, the autologous setting of a GT approach may prevent many of the complications related to allogeneic transplantation without the need to achieve a full chimerism of gene corrected cells. In fact, even in the presence of mixed chimerism of WASp1 and WASp2 cells in our GTtreated mice, we observed a reduction in serum autoantibodies and found a significant improvement in the response of B cells to pneumococcal antigen challenge. In conclusion, we provide here much evidence supporting the efficacy of a GT approach in yielding WASp expression and improving functionality of B cells from Was2/2 mice, ultimately contributing to the validation of our GT for the treatment of WAS. We thank Alessandro Nonis and Chiara Brombin (University Center for Statistics in the Biomedical Sciences, Universita Vita-Salute San Raffaele, Milan, Italy) for help in statistical analyses.
Clinical implications: We provide evidence supporting the efficacy of GT in improving functionality of the B-cell compartment in Was2/2 mice and ultimately in the treatment of patients with WAS.
REFERENCES 1. Ochs HD, Thrasher AJ. The Wiskott-Aldrich syndrome. J Allergy Clin Immunol 2006;117:725-38. 2. Filipovich A, Stone J, Tomany S, Ireland M, Kollman C, Pelz C, et al. Impact of donor type on outcome of bone marrow transplantation for Wiskott-Aldrich syndrome: collaborative study of the International Bone Marrow Transplant Registry and the National Marrow Donor Program. Blood 2001;97:1598-603. 3. Antoine C, Muller S, Cant A, Cavazzana-Calvo M, Veys P, Vossen J, et al. Longterm survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience 1968-99. Lancet 2003;361:553-60. 4. Kobayashi R, Ariga T, Nonoyama S, Kanegane H, Tsuchiya S, Morio T, et al. Outcome in patients with Wiskott-Aldrich syndrome following stem cell transplantation: an analysis of 57 patients in Japan. Br J Haematol 2006; 135:362-6. 5. Ozsahin H, Le Deist F, Benkerrou M, Cavazzana-Calvo M, Gomez L, Griscelli C, et al. Bone marrow transplantation in 26 patients with Wiskott-Aldrich syndrome from a single center. J Pediatr 1996;129:238-44. 6. Ozsahin H, Cavazzana-Calvo M, Notarangelo LD, Schulz A, Thrasher AJ, Mazzolari E, et al. Long-term outcome following hematopoietic stem-cell transplantation in Wiskott-Aldrich syndrome: collaborative study of the European Society for Immunodeficiencies and European Group for Blood and Marrow Transplantation. Blood 2008;111:439-45.
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7. Dupre L, Marangoni F, Scaramuzza S, Trifari S, Hernandez RJ, Aiuti A, et al. Efficacy of gene therapy for Wiskott-Aldrich syndrome using a WAS promoter/ cDNA-containing lentiviral vector and nonlethal irradiation. Hum Gene Ther 2006;17:303-13. 8. Dupre L, Trifari S, Follenzi A, Marangoni F, Lain de Lera T, Bernad A, et al. Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction. Mol Ther 2004;10:903-15. 9. Marangoni F, Bosticardo M, Charrier S, Draghici E, Locci M, Scaramuzza S, et al. Evidence for long term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models. Mol Ther 2009;17:1073-82. 10. Park JY, Shcherbina A, Rosen FS, Prodeus AP, Remold-O’Donnell E. Phenotypic perturbation of B cells in the Wiskott-Aldrich syndrome. Clin Exp Immunol 2005; 139:297-305. 11. Vermi W, Blanzuoli L, Kraus MD, Grigolato P, Donato F, Loffredo G, et al. The spleen in the Wiskott-Aldrich syndrome: histopathologic abnormalities of the white pulp correlate with the clinical phenotype of the disease. Am J Surg Pathol 1999; 23:182-91. 12. Westerberg L, Greicius G, Snapper SB, Aspenstrom P, Severinson E. Cdc42, Rac1, and the Wiskott-Aldrich syndrome protein are involved in the cytoskeletal regulation of B lymphocytes. Blood 2001;98:1086-94. 13. Westerberg L, Larsson M, Hardy SJ, Fernandez C, Thrasher AJ, Severinson E. Wiskott-Aldrich syndrome protein deficiency leads to reduced B-cell adhesion, migration, and homing, and a delayed humoral immune response. Blood 2005; 105:1144-52. 14. Westerberg L, Wallin RP, Greicius G, Ljunggren HG, Severinson E. Efficient antigen presentation of soluble, but not particulate, antigen in the absence of Wiskott-Aldrich syndrome protein. Immunology 2003;109:384-91. 15. Westerberg LS, de la Fuente MA, Wermeling F, Ochs HD, Karlsson MC, Snapper SB, et al. WASP confers selective advantage for specific hematopoietic cell populations and serves a unique role in marginal zone B-cell homeostasis and function. Blood 2008;112:4139-47. 16. Meyer-Bahlburg A, Becker-Herman S, Humblet-Baron S, Khim S, Weber M, Bouma G, et al. Wiskott-Aldrich syndrome protein deficiency in B cells results in impaired peripheral homeostasis. Blood 2008;112:4158-69. 17. Humblet-Baron S, Sather B, Anover S, Becker-Herman S, Kasprowicz DJ, Khim S, et al. Wiskott-Aldrich syndrome protein is required for regulatory T cell homeostasis. J Clin Invest 2007;117:407-18. 18. Nikolov NP, Shimizu M, Cleland S, Bailey D, Aoki J, Strom T, et al. Systemic autoimmunity and defective Fas ligand secretion in the absence of the WiskottAldrich syndrome protein. Blood 2010;116:740-7. 19. Zhang J, Shehabeldin A, da Cruz LAG, Butler J, Somani A-K, McGavin M, et al. Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes. J Exp Med 1999; 190:1329-41. 20. Charrier S, Dupre L, Scaramuzza S, Jeanson-Leh L, Blundell MP, Danos O, et al. Lentiviral vectors targeting WASp expression to hematopoietic cells, efficiently transduce and correct cells from WAS patients. Gene Ther 2007;14:415-28. 21. Petrella A, Doti I, Agosti V, Giarrusso P, Vitale D, Bond H, et al. A 59 regulatory sequence containing two Ets motifs controls the expression of the Wiskott-Aldrich syndrome protein (WASP) gene in human hematopoietic cells. Blood 1998;91: 4554-60. 22. Berland R, Wortis HH. Origins and functions of B-1 cells with notes on the role of CD5. Annu Rev Immunol 2002;20:253-300. 23. Galy A, Roncarolo MG, Thrasher AJ. Development of lentiviral gene therapy for Wiskott Aldrich syndrome. Expert Opin Biol Ther 2008;8:181-90. 24. Boztug K, Schmidt M, Schwarzer A, Banerjee PP, Diez IA, Dewey RA, et al. Stemcell gene therapy for the Wiskott-Aldrich syndrome. N Engl J Med 2010;363: 1918-27. 25. Snapper SB, Rosen FS, Mizoguchi E, Cohen P, Khan W, Liu CH, et al. WiskottAldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation. Immunity 1998;9:81-91. 26. Martin F, Kearney JF. B1 cells: similarities and differences with other B cell subsets. Curr Opin Immunol 2001;13:195-201. 27. Martin F, Kearney JF. Marginal-zone B cells. Nat Rev Immunol 2002;2:323-35. 28. Bosticardo M, Marangoni F, Aiuti A, Villa A, Roncarolo MG. Recent advances in understanding the pathophysiology of Wiskott-Aldrich syndrome. Blood 2009;113: 6288-95.
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FIG E1. Presence of gene corrected cells in BM and spleen (SP) in Was2/2 mice reconstituted with LV-transduced Lin2 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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FIG E2. Analysis of WASp-expressing cell frequency in peripheral blood B2201 cells from GT-treated mice 3 and 9 months after transplant. **P < .005 (paired t test).
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FIG E3. Presence of WASp-expressing B cells in splenic tissue sections from GT-treated Was2/2 mice. Spleen sections from BMT wt (A), BMT was2/2 (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 320 original magnification.
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TABLE E1. Surface markers used to define B-cell subsets B-cell subset
BM Pre Immature Mature/recirculating Peritoneal cavity B1a B1b B2 Spleen Transitional Mature MZ FO FO, Follicular.
Markers
CD432B220loIgM2 CD432B220loIgM1 CD432B220hiIgM2 CD191IgM1CD51B220lo CD191IgM1CD52B220lo CD191IgM1CD52B220hi B2201IgMhiIgDhi B2201IgMloIgDhi B2201CD21hiCD23lo B2201CD21intCD23hi
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TABLE E2. MFI of WASp expression indicated as fold increase in respect to values found in the BMT was2/2 group Cell subset
BM Pre-B Immature B Mature/recirc B Cell subset
Peritoneal cavity B1a B1b B2 Cell subset
Spleen Transitional FO MZ FO, Follicular; recirc, recirculating. *Median 6 SD. P < .001 vs BMT wt group. àP < .005 vs BMT wt group. §P < .001 vs BMT was2/2 group.
BMT wt (n 5 15)*
BMT was2/2 (n 5 20)*
GT WASp1 (n 5 24)*
3.7 6 0.9 5 6 1.6 6.1 6 3.6
1 6 0.2 1 6 0.2 1 6 0.2
2.9 6 0.4 § 2.8 6 0.6 § 2.7 6 0.7 §
BMT wt (n 5 21)*
BMT was2/2 (n 5 15)*
GT WASp1 (n 5 22)*
3.9 6 1.2 3.8 6 0.9 4.4 6 1.8
1 6 0.1 1 6 0.2 1 6 0.1
3.4 6 0.6ৠ3.1 6 0.5ৠ3.5 6 0.8à§
BMT wt (n 5 30)*
BMT was2/2 (n 5 23)*
GT WASp1 (n 5 32)*
5 6 1.9 5.5 6 3.6 5.2 6 3.2
1 6 0.3 1 6 0.4 1 6 0.4
4.5 6 1.7§ 3.1 6 1.2 § 2.9 6 1.4 §