Introduction

Wiskott–Aldrich syndrome (WAS) is a rare X-linked recessive primary immunodeficiency characterized by immune dysregulation, microthrombocytopaenia, eczema, and lymphoid malignancies.^1,2 Mutations in the WAS gene usually lead to an absence of WAS protein (WASp) and severe disease, or a reduction in protein levels and the milder phenotype of X-linked thrombocytopaenia or attenuated WAS.^3,4 The gene product, WASp, is a 502-amino acid multidomain hematopoietic restricted member of a family of proteins, involved in dynamic reorganization of the actin cytoskeleton through interaction with the ARP2/3 complex.⁵ A lack of WASp results in cytoskeletal defects that compromise multiple aspects of normal cellular activity including proliferation, phagocytosis, immune synapse formation, adhesion, and directed migration.^{6,7,8,9,10,11,12,13,14}

Although allogeneic hematopoietic stem cell transplantation is successful in treating WAS, the high morbidity and mortality associated with mismatched transplantation and lack of suitable donors for many patients makes somatic gene therapy a promising alternative treatment.¹⁵ Previous studies in vitro have shown that introduction of WASp can restore actin cytoskeletal structures such as filopodia in B cells¹⁶ and podosomes in macrophages,¹⁷ dendritic cells (DCs),⁸ and osteoclasts.¹⁸ Other studies in murine models have shown that retroviral and lentiviral vectors containing heterologous or endogenous regulatory domains have the capability of restoring WASp expression to all hematopoietic lineages following transplantation of transduced bone marrow stem cells.^19,20,21,22 Furthermore, this resulted in partial correction of T cell receptor–mediated and viral mediated T-cell responses, recovery of normal T-cell immune synapses, restoration of podosomes in DCs, and amelioration of colitis. Similarly, human hematopoietic stem cells transduced with a gammaretroviral vector encoding WASp achieved normal protein levels and podosome reconstitution in differentiated macrophages.^23,24

In this study, we have analyzed mice reconstituted with lentiviral vectors encoding an eGFP–WASp fusion protein. This allowed reconstitution of activity but also facilitated visualization and collection of transduced cells for functional analysis. We demonstrate successful long-term restoration of migratory activity in both myeloid and lymphoid lineages, providing further evidence that gene therapy is an effective strategy for treatment of WAS.

Results

Transduction of murine bone marrow

A self-inactivating lentiviral vector was constructed to express either enhanced green fluorescent protein (eGFP) or an eGFP–WASp fusion protein under the control of the spleen focus forming virus long-terminal repeat (Figure 1a). This vector incorporates central polypurine tract (cPPT) and woodchuck hepatitis virus post-transcriptional regulatory elements for enhanced transgene expression.²⁵ The virus was pseudotyped with a vesicular stomatitis virus G envelope and concentrated by ultracentrifugation. Cells transduced with this vector expressed an eGFP–WASp fusion protein detectable by Western blot at 90 kd in nonhematopoietic and hematopoietic cells, and which exhibited a cytosolic eGFP staining pattern similar to that previously described for WASp (Supplementary Figure S1). The activity of the eGFP–WASp fusion protein in WASp-deficient cells has been confirmed previously^8,17 (and Supplementary Figure S1).

Figure 1.

Reconstitution of Wiskott–Aldrich syndrome (WAS) micewith eGFP–WASp. (a) Schematic of lentiviral constructs used in this study. (b) WAS knockout (KO) mice were lethally irradiated and reconstituted with 2 10⁵ lin– cells transduced with vectors encoding enhanced green fluorescent protein (eGFP) (SEW) or eGFP–WASp (SEWW) as shown. Mice were killed 5–10 months after cell transfer and splenocytes stained for B220, CD3, and CD11b and analyzed by flow cytometry. Representative plots show live gated cells. (c) Secondary transplants and (d) WAS KO mice transplanted with wild-type (WT) cells transduced with SEW were analyzed 9 months and 5 months after cell transfer and splenocytes stained for B220, CD11b, CD4, and CD8 and analyzed by flow cytometry. Plots shown are gated on the eGFP positive fraction. Representative plots show live gated cells. cPPT, central polypurine tract; LTR, long-terminal repeat; SFFV, spleen focus forming virus; WASp, WAS protein; WPRE, woodchuck hepatitis virus post-transcriptional regulatory.

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Lineage-negative cells from male WAS knockout (KO) bone marrow were transduced with SEW or SEWW and injected back into lethally irradiated female recipients. The level of transduction was measured by flow cytometry 72 hours after infection and was found to be 51–76% for SEW (n = 5) and 45–63% for SEWW (n = 13). Single cell suspensions from thymus (ranges for gfp- positivity between different mice:18–35% and 0–10% for SEW and SEWW, respectively), peripheral blood (ranges 42–67% and 8–18% for SEW and SEWW, respectively) and spleen (ranges 14–44% and 6–20% for SEW and SEWW, respectively) were positive for eGFP or eGFP–WASp up to 10 months after engraftment by flow cytometry (data not shown) and the spleen was positive for eGFP–WASp by Western blot (Supplementary Figure S1d). Long-term multilineage engraftment in the spleen of WAS KO recipients was confirmed by antibody staining against B220, CD11b, and CD3 and flow cytometric analysis (Figure 1b). The integrated viral copy number in the spleen determined by real-time quantitative polymerase chain reaction (qPCR) ranged between two and five confirming the presence of an integrated transgene. The percentage of eGFP positive cells in each lineage was comparable for both SEW and SEWW treated mice (data not shown). To confirm transduction of multilineage progenitors, secondary transplants (n = 4) were performed using bone marrow cells from SEWW reconstituted mice 10 months after engraftment which were sorted on eGFP expression up to 91% purity. After a further 9 months, recipient mice were analyzed for the presence of eGFP–WASp. eGFP was detectable in three of four mice (range 3–12% ) and the eGFP positive fraction contained B cells, CD4 and CD8⁺ T cells, and myeloid cells showing that long-term stable expression is achievable (Figure 1c). As a positive control, wild-type (WT) lineage negative cells were transduced with SEW and injected into lethally irradiated recipients. eGFP was again detectable (range 6–80% ) and the eGFP positive fraction contained B cells, CD4 and CD8⁺ T cells, and myeloid cells (Figure 1d).

Improvement of BMDC podosome numbers and dynamics

Podosomes are specialized adhesion structures found at sites of close contact with the substratum in differentiated myeloid lineage cells in particular, and are thought to be important for effective localization and migration. Absence of podosomes is characteristic of WASp-deficiency.⁹ When DCs were differentiated from the bone marrow (BMDCs) of normal, WAS KO and SEW or SEWW reconstituted WAS KO mice the cells were scored for the presence or absence of these specialized actin structures. Normal mice had podosomes in 64 6.8% of the cells compared to 5.3 3% in WAS KO cells. SEW transduced DCs had similar levels of podosomes to WAS KO DC; however, SEWW treated mice had increased numbers (range 12.4–22.4, average 18.1 4.6% ) of cells with podosomes. In severe WAS complete absence of protein leads to very few, if any, podosomes. However, X-linked thrombocytopaenia patients have an attenuated phenotype and their macrophages have a reduced number of podosomes per cell.²⁶ Similarly, RNA interference to inhibit WASp expression has also shown that low levels of WASp lead to a reduction rather than absence of podosomes.²⁷ We therefore counted the number of podosomes per cell in BMDCs from treated mice. As expected, the WAS KO DCs had fewer podosomes per cell (in the few that had visible podosomes), compared to normal controls, being 14.9 and 31.4, respectively. BMDCs from SEWW reconstituted mice had an average of 23.3 podosomes per cell (Figure 2a), which was significantly higher than for WAS KO. Confocal microscopy of BMDCs from reconstituted mice revealed that the podosomes had a normal structure with an actin core surrounded by vinculin. The core also co localized with eGFP—WASp, suggesting normal podosome assembly (Figure 2b).

Figure 2.

Podosome recovery and interference reflection microscopy (IRM) on bone marrow dendritic cells (DCs). (a) Bone marrow–derived dendritic cells (BMDCs) containing podosomes had the number of podosomes per cell counted. Each point represents an individual cell. Fifty to two hundred cells were counted. (b) BMDCs were plated onto glass coverslips and confocal microscopy images captured to reveal the actin cytoskeleton. Arrows point to podosomes. (c) SEWW, (d) SEW, and (e) SEW WT BMDCs were imaged by time-lapse microscopy capturing both the enhanced green fluorescent protein (eGFP) fluorescence and IRM images. Panels show the movement over a time frame of 6.5 minutes, with the final cell position outlined in red over the starting image. (f) Turnover index of adhesion contacts in transduced and control DCs. ^*P < 0.05 (Students t-test) SEWW 1°SEWW 2° and SEW WT compared to WAS KO and SEW DCs and error shown is SEM. n = 5–18. KO, knockout; WAS, Wiskott–Aldrich syndrome; WT, wild-type.

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Although we and other groups²⁰ have reported the recovery of podosomes after introduction of WASp, the ability to restore their functionality in terms of turnover has not been measured in an in vivo model. In vitro, splenic WAS KO DCs have been shown to have decreased turnover compared to controls, which can be restored upon introduction of eGFP–WASp.²⁸ Therefore BMDCs from WAS KO, SEW, and SEWW mice were observed by time-lapse interference reflection microscopy, to determine areas of adhesion with simultaneous confocal microscopy to locate eGFP–WASp. Untransduced WAS KO and SEW transduced BMDCs showed little movement or change in their adhesion activity (Figure 2d and Supplementary Video S1) whereas SEWW and SEW WT transduced BMDCs formed protrusions over time within which podosomes were assembled and disassembled (Figure 2c and e and Supplementary Videos S2 and S3). The adhesion turnover was quantified as a turnover index.²⁸ In both SEWW primary and secondary transplanted mice, the turnover index was found to be twice that observed in untransduced or SEW transduced BMDCs and comparable to that seen for SEW transduced WT cells transplanted into WAS KO animals (Figure 2f).

Restoration of migratory defects

Chemotaxis and chemokinesis have been shown to be deficient in WAS KO leukocytes;^{10,11,14,29,30,31} therefore we wanted to show whether introduction of eGFP–WASp could restore this response. Splenocytes from SEW and SEWW reconstituted mice were plated into transwells and their ability to migrate through a filter toward a chemotactic gradient was measured. Migrated cells were surface stained for lineage markers to determine lineage specific migration. eGFP-positive cell fractions were analyzed separately, with eGFP-negative fractions compared as internal controls. SEWW reconstituted mice showed significant B cell (Figure 3a) and DC (Figure 3b) chemotaxis toward CXCL12 and CCL21, which was not observed in the eGFP negative fraction. Looking at this data in a different way, by comparing eGFP positive cell migration to eGFP- cell migration from the same animal, there was a significant selective advantage within SEWW treated animals (Supplementary Figure S2). Unfortunately, the low cell numbers precluded potential significance from being determined in the T-cell fraction (Figure 3c and Supplementary Figure S2). Interestingly, migration in response to either medium or chemokine control (chemokine in both compartments) was greater in SEWW reconstituted cells compared to SEW reconstituted cells, suggesting a general increase in motility. To test this observation we assessed migration of BMDCs from WT, WAS KO, or SEW and SEWW treated mice in a Dunn chamber to measure the velocity of movement in the presence of a chemokine gradient. There was a pronounced reduction in speed in WAS KO cells compared to normal controls which was partially restored in SEWW (counting eGFP-positive cells only) but not in SEW transduced cells (Figure 4a).

Figure 3.

Enhancement of migration in eGFP–WASp transduced splenocytes. Splenocytes from reconstituted mice were incubated in transwells and allowed to migrate for 3 hours at 37 °C. After surface staining, (a) B cell (b) dendritic cell (DC), and (c) T-cell migration were measured by flow cytometry for both the enhanced green fluorescent protein (eGFP) positive (eGFP+) and negative (eGFP^- ) fractions. Plots shown are the total number of transduced cells migrated compared to the total number of transduced cells added, expressed as a percentage of the input cells. SEW n = 3, SEWW n = 4. Error shown is 1 SD. ^*P < 0.01, ^**P < 0.02 Students t-test. WASp, Wiskott–Aldrich syndrome protein.

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Figure 4.

Correction of antigen presenting cell motility in SEWW transduced mice. (a) Dendritic cells (DCs) from wild-type (WT), Wiskott–Aldrich syndrome (WAS) knockout (KO), SEW, SEWW, or SEW WT transduced mice were plated onto coverslips and filmed in a Dunn chamber in media containing CCL21. Cells were tracked by marking the leading edge of individual cells for each frame of film. In reconstituted mice, only enhanced green fluorescent protein (eGFP)+ transduced cells were tracked. The boxplot shown is the interquartile range with the median marked and error bars shown are 5th/95th percentile. ^*P < 0.001 (Students t-test) for WT, SEWW, and WT SEW compared to both WAS KO and SEW treated mice. (b) Ears from WT, WAS KO, SEW, SEWW, and SEW WT transduced mice were split into dorsal and ventral sides and cultured in the presence of tumor necrosis factor- for 24 hours. The surface area of the ear was measured and Langerhans cell (LC) migration into the surrounding tissue culture wells was assessed by flow cytometry to give the number of cells per millimeter square. Each point represents the average for both the ventral and dorsal sides per mouse. ^*P < 0.05 (Students t-test) for WT, SEWW, and SEW WT compared to WAS KO mice.

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To further investigate the migratory potential of resident antigen presenting cells of reconstituted mice, ears were split into ventral and dorsal sides, cultured in vitro and the Langerhans cells (LCs), DCs, and dermal macrophages allowed to migrate out into the culture medium following tumor necrosis factor- stimulation. The cells that migrated out were surface stained for CD207, CD11c, and major histocompatibility complex II to distinguish different cell populations and an absolute count obtained. By measuring the surface area of the ear, the number of LCs able to migrate per millimeter square of ear was determined. There was a significantly reduced number of WAS KO LCs (Figure 4b), DCs and macrophages (data not shown) able to migrate compared to normal. This defect was restored to normal levels in the SEWW treated mice but not in the SEW mice.

Restoration of contact hypersensitivity response

We next evaluated the ability of reconstituted mice to mount a type IV inflammatory response to an antigen. To elicit such a response, mice were sensitized to dinitrofluorobenzene (DNFB) in a 4:1 acetone/olive oil vector mix ectopically applied. After 5 days the mice were rechallenged by application to the right ear and the inflammatory response measured by comparing the thickness of DNFB-treated right ears compared to vector (alone) treated left ears on the same animal. In 8 hours there was a detectable inflammatory response in the WT mice that was greater than that of WAS KO and SEWW treated mice. However, in 24 hours the SEWW treated mice had managed to mount a response that was almost as strong as that observed in normal mice whereas WAS KO mice still had not mounted a response. WAS KO mice were eventually able to mount an inflammatory response in 48 hours, by which time the response was resolving in normal mice (Figure 5a). Furthermore eGFP–WASp positive cells could be observed in cryosections of the dermal layers that were also positive for major histocompatibility complex II, F4/80, and CD11c (Figure 5b), confirming that transduced antigen presenting cells were present.

Figure 5.

Contact hypersensitivity response kinetics to dinitrofluorobenzene (DNFB) are restored in SEWW reconstituted mice. (a). Micewere sensitized to DNFB by topical application and rechallenged by application to the right ear 5 days after the initial challenge. Left ears were treated with a vector lacking DNFB as a control. The thickness of the ears was measured using engineer's micrometers. The graph represents the difference in thickness between DNFB treated and vector (alone) treated ears over time. Each point gives the average from eight mice. Error shown is SEM. ^*P < 0.02 (sv129 compared to both Wiskott–Aldrich syndrome (WAS) knockout (KO) and SEWW), ^**P < 0.05 (sv129 compared to WAS KO), ^***P < 0.05 (SEWW compared to WAS KO).(b) Ears from SEWW transduced mice were frozen in optimal cutting temperature and 7 m cryosections antibody stained for major histocompatibility complex class II, F4/80, or CD11c. Enhanced green fluorescent protein (eGFP) positive cells are visible within the dermal layer and co-stainwith all three markers (marked by arrows). Bars = 50 m.

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Partial restoration of marginal zone B cells

Splenic architecture in WAS KO mice has been shown to be abnormal compared to control mice. The marginal zone CD21^hi/CD23^int B (MZB) cell population, which is normally found surrounding a layer of MOMA-1⁺ metallophilic macrophages, in particular is reduced in size.^11,29 Spleen sections from WAS KO, WT and reconstituted mice were stained for MOMA-1(metallophillic macrophages) and B220. eGFP fluorescence co-localized in SEW, SEWW 1° and 2° and SEW transduced WT transplanted WAS KO mice to MOMA-1 positive cells and B220 positive cells (Figure 6). MZB cells were clearly visible in control sections but were largely absent from WAS KO sections, often in conjunction with a reduced thickness of the macrophage layer (Figure 7a). The thickness of the MZB cell layer was quantified using ImageJ software for WT, WAS KO and reconstituted mice as described in the Materials and Methods (MZB is shaded yellow in Figure 7b). The MZB area was then graphically represented as a percentage of the total follicle size to correct for the generally smaller follicles seen in reconstituted mice. In four of seven SEWW reconstituted mice that were able to be analyzed there was an increase in the thickness of the layer of B220 positive cells surrounding the macrophage layer compared to WAS KO animals (Figure 7c). This increase was similar to that observed when SEW transduced WT cells were transplanted into WAS KO recipients, but did not reach the levels observed in WT untreated mice for both groups (P < 0.05). Furthermore, flow cytometry of single cell suspensions surface stained for CD21^hi/CD23^int, although not as high as the levels in WT untreated animals, demonstrated enrichment for eGFP–WASp expressing cells in seven of nine treated animals. Although this enrichment did not reach statistical significance (P = 0.08), the trend is highly suggestive of the ability of successfully transduced SEWW positive cells to reconstitute the MZB cell layer (Figure 7d and e). Furthermore, there was no enrichment for eGFP positive cells in SEW WT transduced mice.

Figure 6.

Cryosections of spleen from reconstituted mice have enhanced green fluorescent protein (eGFP) present in all areas of the B-cell follicle. Spleens from (a) SEW, (b) SEWW 1° (c) SEWW 2°, and (d) SEW wild-type (WT) transduced mice were frozen in optimal cutting temperature and 7 m sections stained for B220 (blue) and metallophillic macrophage-1 (MOMA-1) (red). eGFP positive cells (green) are visible in B cells in the follicle (marked by asterisks) in the MOMA-1 layer (arrows) and in the marginal zone B (MZB) cells layer surrounding the MOMA-1 layer (arrowheads). Bars = 100 m.

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Figure 7.

Quantification of the marginal zone B (MZB) cell layer in spleens of reconstituted mice. (a) Spleen samples from wild-type (WT), Wiskott–Aldrich syndrome (WAS) knockout (KO), SEWW, and SEW WT reconstituted mice were frozen in optimal cutting temperature and 7 m sections stained for B220 (blue) and metallophillic macrophage-1 (MOMA-1) (red) to show follicles and MZB cells. (b) The area of MZB cells was marked (yellow shading) and (c) quantified using ImageJ software to give a percentage of MZB per follicle. Each point represents the average taken from 5–15 images per mouse with SEWW 1° (open triangles) and SEWW 2° (closed squares). (d) Single cell suspensions of spleen from WT, WAS KO, SEWW 1°, SEWW 2°, and SEW WT reconstituted mice were stained for CD21 and CD23. The total cell population and eGFP positive follicular B (FB) cells CD23^hi/CD21^int and CD21^hi/CD23^int MZB cells are marked on representative plots gated on the lymphocyte population based on forward and side scatter. (e) Table showing the percentage of cells within the MZB fraction for total cell and eGFP positive fraction determined from the flow cytometry, with the fold enrichment for each mouse is also shown.

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Discussion

In this study, we have demonstrated the ability of lentiviral vectors encoding an eGFP–WASp fusion protein to restore several aspects of the WAS cellular phenotype. In particular we have shown for the first time that hematopoietic stem cell gene therapy in vivo results in partial correction leading to an improvement in migratory deficiencies in multiple immune cell lineages.

Previous studies have demonstrated reconstitution of T-cell function for proliferative response and synapse formation, but functional recovery in other lineages following in vivo gene therapy has not been reported in detail.^19,20,21 Considerable evidence now exists about the significant contribution from trafficking defects arising from migratory and localizing abnormalities to the immunopathology of WAS. For example, deficient DC migration is likely to contribute to the inability of CD4 T cells to prime normally, while inefficient B-cell homing may contribute to delayed initiation of humoral immune responses.^11,14,29 The overall consequence of these trafficking defects which arise from intrinsic cellular disturbances of adhesion, chemotaxis, and general motility, are less easy to define due to the multilineage nature of the disease. However, it seems likely that successful correction of the phenotype will depend on partial or complete restoration of many of these diverse activities. Demonstration that this is possible in preclinical model systems is therefore of considerable importance.

In line with other previously reported studies we have achieved successful restoration of podosomes in BMDCs. The number of podosomes per cell has been closely linked to severity of disease, as patients with X-linked thrombocytopaenia or attenuated WAS have numbers that are intermediate between a classical null phenotype and normal.²⁶ We have shown that SEWW reconstituted DCs have significantly increased numbers of podosomes per cell, but more importantly that they have a dynamic turnover characteristic of normal cells. This is likely to be important for recovery of normal motility as formation and dissolution of podosomes allows the cell to anchor leading edge protrusions, yet detach quickly as the cell moves forward. We have also shown an increase in the migratory potential of splenic B cells and DCs from reconstituted mice both in a transwell and Dunn chamber, and enhanced migration of LCs out of epidermal sheets in response to tumor necrosis factor- stimulation.

As a measure of functionality we have shown that a DNFB-mediated contact hypersensitivity (CHS) response can be restored in vivo. This multifactorial immune response requires efficient take up and processing of the hapten by skin antigen presenting cells followed by migration and presentation to T cells in the draining lymph node. Both CXCL12 and CCL21 are known to be important for antigen presenting cell migration with neutralization of CCL21 resulting in impaired CHS.³² Furthermore natural killer cells,³³ B cells,³⁴ CD8⁺ and CD4⁺ T reg cells³⁵ have all been implicated in elicitation and resolution of this response, suggesting that multiple arms of the immune response can be restored by gene transfer.

WAS KO mice and humans have been shown to be lacking in MZB cells, possibly due to migratory defects and/or localization responses.²⁹ This is likely to explain the particular deficiency in humoral response to polysaccharide T-independent antigens. The ability of gene transfer or even of hematopoietic stem cell transplantation to restore the MZB compartment has not been explored, but in humans is likely to have significant implications for persisting susceptibility to infection from encapsulated bacteria. Encouragingly therefore, we were able to demonstrate that MZB cells are partially restored following hematopoietic stem cell gene therapy. The apparent variability of correction may reflect difficulty in accurately quantifying the cells, but also may reflect confounding factors including low-level chimerism, the choice of promoter used, prior irradiation of the mice, and potentially a time-dependent window outside of which reconstitution may not be possible, as SEW transduced WT cells were not able to restore MZB levels to that seen in unmanipulated WT mice.

In summary, we have demonstrated long-term multilineage correction of cytoskeletal and migratory defects in a murine model of hematopoietic stem cell gene therapy for WAS. These data support the ongoing and future development of clinical trials in the WAS.

Materials and Methods

Lentivirus production. A 2.3-kilobase complementary DNA sequence for N-terminal fusion protein of eGFP and WASp (described ref. 36) was subcloned from a pBluescript (Invitrogen) intermediate vector replacing the eGFP in the pHR'sin-based lentiviral vector pHR'SIN-cPPT-SE.²⁵ using BamHI/KpnI restriction sites. Lentiviral particles expressing eGFP (SEW), and eGFP fused to human WASp (SEWW), (Figure 1a) were prepared as previously described.²⁵ Briefly the transfer, packaging, and vesicular stomatitis virus G pseudotyped envelope plasmids were transfected into 293T cells and viral supernatant concentrated by ultracentrifugation before being stored at - 80 °C until used. Virus was titered by transducing HeLa cells with serial dilutions of viral supernatant and measuring transduction efficiency by the percentage of cells expressing eGFP by flow cytometry.

Animal engraftment. WAS KO mice (original stocks kindly supplied by S. Snapper, Boston, MA) were housed in specified pathogen-free conditions in individually ventilated cages (Tecniplast, Italy) and supplied with sterile food, water, and bedding. Recipient female mice were lethally irradiated (10 Gy, Cs 137) in a split dose 24 and 2 hours prior to infusion with 2 10⁵ to 3 10⁵ transduced lineage negative cells from male recipients.

Preparation and transduction of lineage negative cells. Hematopoietic progenitors were purified from WAS KO or sv129 WT bone marrow by magnetic bead lineage depletion using Stemsep Mouse Hematopoietic Progenitor Enrichment cocktail (StemCell Technologies). Lineage negative cells were placed into culture at 1 10⁶/ml in Stem span (StemCell Technologies), supplemented with stem cell factor (100 ng/ml), Flt-3 ligand (20 ng/ml), and IL-6 (20 ng/ml) (all from Peprotech, UK) at 37 °C, 5% CO₂ for 2 hours. WAS KO or WT lineage negative cells were transduced with 1 10⁸ virus particles/ml (equivalent to a multiplicity of infection of 100) of SEW or SEWW for 16–18 hours in the presence of cytokines. The cells were washed prior to infusion of 2–3 10⁵ cells per recipient. A sample was retained and further cultured for 72 hours to determine transduction efficiency based upon eGFP expression and qPCR. Animals were killed by using CO₂ for inhalation 5–10 months after engraftment. Thymus, bone marrow, spleen, peripheral blood, and ears were removed and single cell suspensions made where required.

For secondary transplants, bone marrow from primary recipients was sorted on the basis of eGFP expression and 3 10⁵ cells injected back into lethally irradiated WAS KO recipients.

Purification of genomic DNA and qPCR. Genomic DNA was purified from 1 10⁶ cells by NP40 lysis followed by incubation with proteinase K for 2 hours at 56 °C. The proteinase K was inactivated at 95 °C for 15 minutes and the supernatant containing the genomic DNA frozen at - 20 °C until used.

qPCR has been previously described²⁰ and was performed using oligonucleotide primers and Taqman probes designed and used previously²⁰ using modified methods on an ABI 7000 sequence detector (ABI). Briefly, 5 l of genomic DNA (equivalent to 2.5 10⁴ cells) was amplified with 20 l of qPCR supermix-UDG with ROX (Invitrogen) containing 200 mol/l forward and reverse primers (Sigma) and 100 mol/l probe (MWG) for 40 cycles at 95 °C (15 seconds) then 60 °C (1 minute). Serially diluted plasmid DNA containing the relevant sequences was used as a standard curve, with all measurements performed in triplicate.

Immunofluorescence staining and imaging. For flow cytometry 1 10⁶ splenocytes were placed in phosphate-buffered saline containing 1% bovine serum albumin, washed twice and incubated with 1:100 dilutions of anti-CD3-PE, B220 PE, B220 PE-Cy5 (eBioscience, San Diego, CA) CD11b PE, CD4 PE-Cy5 (eBioscience), CD8 PE, CD21PE, CD23-biotin, and streptavidin PE-Cy5 for 30 minutes at 4 °C. Antibodies were from Pharmingen (Oxford, UK), unless stated otherwise. Stained cells were analyzed using an EPICS XL (Coulter) and Summit (Dako) software. BMDCs were prepared as previously described,¹¹ sorted on the basis of eGFP positivity, plated onto glass coverslips and fixed in 4% paraformaldehyde for 10 minutes followed by permeabilization with 0.1% Triton X-100 in phosphate-buffered saline for 3 minutes. The coverslips were washed and blocked in phosphate-buffered saline 1% bovine serum albumin for 30 minutes prior to incubating with rhodamine phalloidin (1:250; Invitrogen) to detect F-actin and mouse immunoglobulin G1 anti-vinculin (hVIN-1, ascetic fluid used at 1:100 dilution; Sigma) followed by goat anti-mouse immunoglobulin G conjugated to Cy-5 (Jackson Immunoresearch, West Grove, PA) to detect vinculin. Images were captured on a confocal Leica (TCS-SP2) microscope. Images shown were processed in Adobe Photoshop and are projections of 6–12 z sections. Five to ten random fields of view were captured to obtain images of 100–200 cells and scored for the presence or absence of podosomes followed by enumeration in the cells deemed to have podosomes. Samples of spleen or ears were immersed in optimal cutting temperature and frozen in liquid nitrogen. Seven micrometer cryosections were adhered to slides and fixed in 4% paraformaldehyde prior to staining for eGFP and cell surface antigens. Briefly, the sections were blocked with 2% mouse serum, avidin, and biotin prior to incubation with 1:1,000 dilution of rabbit anti-eGFP antibody (Invitrogen) in combination with 1:100 dilutions of CD11c-biotin (N418; BMA), B220 (eBioscience), MOMA-1-biotin (BACHEM), major histocompatibility complex class II-biotin (I-Ab; Pharmingen) or F 4/80 (Serotec) for 1 hour at room temperature. After washing, sections were incubated with goat anti-rabbit Alexa 488, streptavidin Alexa Fluor 555 (Invitrogen), or mouse anti-rat Cy5 (Jackson Immunoresearch). When B220 was detected in combination with other antigens, an additional block with 2% rat serum was performed in-between incubations.

Analysis of adhesion turnover. The podosome turnover in DCs expressing eGFP constructs was performed by simultaneously visualizing GFP signal and adhesion-substratum interface using a Zeiss LSM 510 Meta confocal scanning head as previously described,²⁸ using the 488-nm line of an Argon laser and a 470–500 nm band pass filter to detect the eGFP signal and a 505-nm long pass filter to detect the interference reflection signal. To analyze the persistence of adhesion sites, 10 images taken 30 seconds apart were overlapped using the difference function in Adobe Photoshop. We thus obtained a composite image with 10 relevant gray levels. The areas of light gray color pixels represent dynamic adhesions whereas areas of dark gray and black color pixels represent increasingly stable adhesions during the selected time course of measurement. Using the histogram function of Adobe Photoshop, we could quantify the percentage of pixels per image corresponding to each gray level, which allowed us to calculate a turnover index by dividing the percentage of pixels present in frames 1–5 by the percentage of pixels present in frames 6–10 (M.E. Holt, Y. Calle, D.M. Sutton, D.R. Critchly, G.E. Jones and G.A. Dunn, manuscript submitted). The Students t-test was used to assess the statistical significance of experimental results (^*P < 0.05).

In vitro migration. For transwell migration, transwells of 6.5 mm diameter, with 5 m pore filters (24 wells; Costar, Corning, NY) were coated with fibronectin (10 g/ml; Sigma) for at least 2 hours at 37 °C. Splenocytes (5 10⁶ cells) were added to the upper compartment of the transwell and CXCL12 or CCL21 (both 100 ng/ml; Peprotech, London, UK) added to the lower compartment. After incubation for 90 minutes at 37 °C, the cells from the lower compartment were collected, stained with PE-CD3, PE-Cy5-B220, and APC-CD11c (Pharmingen). Flowcount beads (Beckman Coulter, High Wycombe, UK) were added for quantification and the cells counted on a Cyan cytometer (DakoCytomation, Glostrup, Denmark). The assay was performed in duplicate and negative (no chemokine in lower compartment) and chemokinesis (chemokine in both compartments) controls included. For migration with Dunn chambers (Hawksley, Lancing, UK), DCs (25 10³) were allowed to adhere for at least 1 hour to fibronectin-coated coverslips at 37 °C. Inner wells of the Dunn chambers were filled with culture medium without chemokine and outer wells with chemokine (CCL3). Dunn chambers were maintained at 37 °C on a microscope stage of an inverted microscope (Zeiss Axiovert 135; Zeiss, Hertfordshire, UK) and cell motility recorded using a 10 phase contrast lens (numerical aperture 0.25; Zeiss). Images were acquired every 10 minutes over a 5-hour period using a Hamamatsu digital camera (C4742-95) and Openlab 4.04 software (Improvision, Coventry, UK). Images were analyzed using Volocity 4.0 software (Improvision). Migration was determined by tracing the leading edge of individual cells for each frame. LC migration was assessed by quantifying the number of cells emigrating from skin sections over 48 hours in response to tumor necrosis factor-. Briefly, ears were removed from transduced mice and split into ventral and dorsal halves. Both halves were cultured for 24 hours in 25 ng/ml tumor necrosis factor- (R&D Systems) and supernatant collected. Cells in supernatant were stained with Alexa Fluor 647-CD207 (eBioscience), APC-CD11c (Pharmingen) and PE-MHC class II (IA/IE; Pharmingen), flowcount beads added for quantification and the cells counted on a Cyan cytometer. The surface area of each ear half was measured and data expressed as CD207+ cells per millimeter square of ear.

CHS. Sv129 WT, WAS KO and reconstituted mice were sensitized to DNFB by ectopic application of 50 l of 0.5% DNFB in 4:1 acetone:olive oil (vol/vol) onto the abdomen and 5 l on each footpad. After 5 days, mice were challenged by the application of 10 l of DNFB (0.2% ) on each side of the right ear with the left ear treated with vehicle alone. The type IV T-cell dependent response was measured by determining the thickness of the right and left ears measured using engineer's micrometers (Sealey, Bury St Edmunds, Suffolk) at 1, 4, 8, 24, and 48 hours after sensitization.

Determination of marginal zone thickness. Spleen sections were stained for eGFP, B220, and MOMA-1 as described earlier. They were then imaged by confocal microscopy and analyzed in ImageJ software. The total pixelated area arising from B220 staining was determined, and a measurement of the MZB derived by subtraction of the signal from follicular cells inside the macrophage cell layer. The area was then expressed as a percentage of the total follicle size.

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Acknowledgments

We thank the plasmid factory (Bielefeld, Germany) for production of plasmids pCMVR8.91 and pMD.G2, Anne Galy (Genethon, Paris, France) for donation of plasmid for generation of a standard curve and Joanne Sinclair for assistance with flow cytometry. These studies were supported by European Union Grant: EURO-POLICY-PID SP23-CT-2005-006411 (M.P.B.) the Wellcome Trust (A.J.T., G.B., and G.E.J.) and the MRC (G.E.J.).