Sylvie Da Rocha

Jérémy Bigot

Fanny Onodi

Jérémie Cosette

Guillaume Corre

Jérôme Poupiot

David Fenard

Bernard Gjata

Anne Galy
email: galy@genethon.fr

T M A Neildez-Nguyen

Thi My

Anh Neildez-Nguyen

Thi My Anh

Généthon

Temporary Reduction of Membrane CD4 with the Antioxidant MnTBAP Is Sufficient to Prevent Immune Responses Induced by Gene Transfer

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) is a cell-permeable synthetic antioxidant with superoxide dismutase-like activity. This molecule neutralizes superoxide [START_REF] Patel | Metalloporphyrin class of therapeutic catalytic antioxidants[END_REF] in both extracellular and intracellular compartments. [START_REF] Amarnath | Endogenous TGF-beta activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4+CD25-T cells[END_REF] Tested in vivo, MnTBAP is non immunogenic, non-toxic, and shows significant therapeutic benefits in diminishing inflammation in various model of injuries [START_REF] Liu | Mn (III) tetrakis (4-benzoic acid) porphyrin scavenges reactive species, reduces oxidative stress, and improves functional recovery after experimental spinal cord injury in rats: comparison with methylprednisolone[END_REF][START_REF] Suresh | The protective role of MnTBAP in oxidant-mediated injury and inflammation in a rat model of lung contusion[END_REF] or of organ transplantation, [START_REF] Cui | SOD mimetic improves the function, growth, and survival of small-size liver grafts after transplantation in rats[END_REF] or by reducing effector immune responses against a virus challenge. [START_REF] Crump | Antioxidant treatment regulates the humoral immune response during acute viral infection[END_REF] In such models, the effects of MnTBAP have been attributed to reactive oxygen species (ROS) inhibition, which is its main mechanism of action. However, previous results from our laboratory suggested that MnTBAP could also act on T cells directly. Indeed, we observed that the conversion of naïve CD4 + T lymphocytes into Foxp3 + regulatory T cells (Tregs) was hampered in the presence of 200 µM MnTBAP in vitro. [START_REF] Neildez-Nguyen | Hypoxic culture conditions enhance the generation of regulatory T cells[END_REF] This prompted us to examine in greater detail the effects of MnTBAP on CD4 + T cells. In the present study, we report for the first time that MnTBAP, can reversibly downregulate the CD4 molecules on T cells.

As a primary receptor for HIV-1, CD4 is one of the most studied molecules in the human body. The CD4 T cell surface glycoprotein mediates optimal TCR signaling during MHC class II (MHCII) antigenic activation by helping to recruit the Src-tyrosine kinase p56 Lck8 to facilitate activation of helper T cells.

Interactions between CD4 and p56 Lck stabilize CD4 at the plasma membrane by anchoring CD4 into distinct microvilli microdomains which constitute the primary contacts between T cells and antigenpresenting cells (APCs). [START_REF] Glatzova | Dual Role of CD4 in Peripheral T Lymphocytes[END_REF] A tight regulation of CD4 cell surface expression is essential for adaptive immunity as it ensures prospective scanning of MHCII by CD4 on the surface of APCs which is pivotal for T cell priming, [START_REF] Glatzova | Dual Role of CD4 in Peripheral T Lymphocytes[END_REF] and enables a productive signal transduction during T cell activation. It is well established that CD4 + T cells also regulate the pathogenesis of autoimmune diseases, allergies, graftversus host disease and rejection processes in organ transplant. [START_REF] Blazar | Advances in graft-versus-host disease biology and therapy[END_REF][START_REF] Islam | T cell homing to epithelial barriers in allergic disease[END_REF][START_REF] Palmer | Autoimmunity: increasing suspects in the CD4+ T cell lineup[END_REF][START_REF] Wood | Mechanisms of rejection: current perspectives[END_REF] Furthermore, CD4 + T cells which are essential for protective and memory immunity against viruses initiated by infection or by vaccination, [START_REF] Sant | Revealing the role of CD4(+) T cells in viral immunity[END_REF] are also key players in the induction of adverse immune responses following viral gene transfer therapies. [START_REF] Ferrand | A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer[END_REF][START_REF] Mays | Adeno-associated virus capsid structure drives CD4-dependent CD8+ T cell response to vector encoded proteins[END_REF] Thus, to gain insights in the effects of MnTBAP, we explored the mechanism of 4 CD4 deprivation at the cell surface of T cells upon MnTBAP treatment and examined the functional effects on CD4-dependent T cell functions. Indeed, we demonstrate here that MnTBAP rapidly and reversibly down-regulates CD4 on T cells, disrupting the CD4/p56 Lck complex. MnTBAP-treated cells cannot be transduced with gp120-mediated lentiviral vectors. A short MnTBAP treatment at the time of antigen-priming, provokes immune unresponsiveness to OVA peptide immunization, and reduces specific immunological responses to Adeno Associated Viral vector (AAV) gene transfer. This new immunomodulatory feature of MnTBAP could be exploited to provide short-term treatment for long-term benefit in a large set of therapeutic or immunopathological conditions.

5

RESULTS

CD4 at the cell surface of T cells is reversibly internalized upon MnTBAP treatment

To study the effects of MnTBAP on CD4 + T cells, purified splenic CD4 + T cells from C57BL/6 mice were treated in vitro for 2 h with increasing doses of MnTBAP (100-400 µM). A gradual and dose-dependent decrease of the CD4 cell surface co-receptor was observed with almost complete disappearance of the CD4 + T cell population at the highest dose of MnTBAP tested (Figure 1A). At 400 µM, MnTBAP reduced by 8 fold the expression of CD4 at the surface of T lymphocytes (Figure 1B). This effect was not due to the induction of cell death as measured by eFluor 780 positivity (Figure S1A). Subsequent experiments were conducted with 400 µM of MnTBAP, which triggered consistently high and non-toxic loss of CD4 T cell surface molecules. The kinetics of CD4 down-modulation by MnTBAP, investigated by time-lapse microscopy recordings over a period of 4 h of treatment, showed a rapid drop of fluorescence starting sooner than 2 min with sustained decay (gray line) compared to control (black line) (Figure 1C). Downregulation was maximal by two hours of MnTBAP treatment, reaching a half-time (50% decrease of cell surface CD4) of approximately 30 min. The CD4 down-regulation was accompanied by the dissociation of the CD4/p56 Lck complex in the cells. Less p56 Lck was coprecipitated with CD4 in splenic T lymphocytes after 2 hours treatment with MnTBAP compared to controls (Figure 1D), whereas similar amounts of p56 Lck and CD4 were detected on immunoblots of whole-cell lysates ("Lysates") from cells treated with or without MnTBAP (Figure 1D). Indeed, more than 3 times less p56 Lck molecules were CD4-associated after MnTBAP treatment (Figure 1E). In naïve murine CD4 + T cells, p56 Lck was localized to the cytosol and at the plasma membrane and p56 Lck localization did not change with MnTBAP treatment (Figure 1F). However, the distribution of CD4 molecules on CD4 + T cells was dramatically affected by MnTBAP treatment. In sharp contrast with the control condition in which the CD4 molecules were globally distributed throughout the cell surface membrane (Figure S1B, None), MnTBAP induced the disappeareance of CD4 from the cell surface and caused its redistribution in vesicles near the cell membrane (white arrows) and in the cell center (red arrow) (Figure S1B, MnTBAP). The internalization of CD4 was accompanied by its incorporation into clathrin-coated pits, [START_REF] Foti | p56Lck anchors CD4 to distinct microdomains on microvilli[END_REF] as evidenced by CD4 and clathrin immunostaining and confocal microscopy analysis (Figure 1G). Untreated T lymphocytes displayed CD4 at the cell surface defining their shape (Figure 1G, left, red signal) whereas clathrin was on the inside face of the membrane (Figure 1G, left, green signal). In MnTBAP-treated cells, most cells had lost CD4 cell surface expression in favor of a colocalization with clathrin molecules (Figure 1G, right, yellow spots and Figure S1C, merge). Further evidence that the MnTBAP-induced CD4 internalization is dependent on the formation of clathrin-coated pits was provided by experiments in hypertonic conditions. Hypertonic cell culture medium containing 0.45 M sucrose blocks clathrin dependent endocytosis [START_REF] Heuser | Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation[END_REF] and prevented the CD4 down regulation induced by MnTBAP (Hypertonic, Figure 1H), whereas CD4 could be internalized in iso-osmotic medium (Normal, Figure 1H). Many of the receptors internalized by clathrin-coated pits are recycled to the cell surface and re-used up to several hundred times by the cells. [START_REF] Maxfield | Endocytic recycling[END_REF] Reportedly, in transfected 293T cells about 45% of internalized CD4 recycled back to the cell surface within 10 min. [START_REF] Piguet | Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes[END_REF] As expected from recycling biology, CD4 internalization into clathrin coated pits induced by MnTBAP was reversible (Figure 1I): while 2 h-treatment ("Treated") reduced almost 80% of CD4 cell surface expression, washing to remove MnTBAP and culture of T lymphocytes for additional 2 h ("2 h removed") or overnight ("O/N removed") permitted to recover gradually 53 and 83% of the initial CD4 expression, respectively.

MnTBAP dramatically reduces the cell surface CD4 on murine and human T lymphocytes and prevents gp120-mediated entry into human T cells

The levels of CD4 on murine and human T lymphocytes were similarly affected by two hours treatment with 400 µM MnTBAP with 92% and 85% decrease compared to control non-treated cells for murine and human T cells, respectively (Figures 2, A andB). MnTBAP also reduced the cell surface CD8 by 30% and 40% decrease compared to control non-treated cells for murine and human T cells, respectively.

No alteration of CD3 expressions was observed with MnTBAP treatment (Figures 2, A andB). Since CD4 is the primary entry receptor for HIV and mediated by gp120:CD4 interactions, we evaluated the functional consequences of MnTBAP induced-CD4 cell surface decline in a model of HIV infection. We tested the transduction of primary human CD3 + T lymphocytes by gp120HXB2-enveloped (gp120HXB2- env) lentiviral vectors encoding GFP which transduce specifically CD4 + T cells (None, Figure 2C, left panel). MnTBAP treatment strongly prevented virus transduction in all cells (MnTBAP, Figure 2C, right panel and Figures 2D) in coherence with the reduced membrane CD4 described above (Figure 2B).

Moreover, once again we emphasize the reversibility effect of MnTBAP, as shown above in Figure 1I, since 72 h after the MnTBAP removal, treated human T lymphocytes (MnTBAP, Figure 2C, right panel) expressed the cell surface CD4 to levels similar to the control non-treated cells (None, Figure 2C, left panel).

A Short MnTBAP treatment renders the immune system temporarily inoperative in vivo

To examine if MnTBAP could have a functional effect via CD4 in vivo, we used a well-characterized CD4-dependent murine immunization model with MHC class II-restricted OVA peptides. In addition, CD8 + T cell responses were also determined in this model by the use of MHC class I-specific peptides.

MnTBAP is injectable and has already been administered to mice for its anti-oxidant effects. [START_REF] Liu | Mn (III) tetrakis (4-benzoic acid) porphyrin scavenges reactive species, reduces oxidative stress, and improves functional recovery after experimental spinal cord injury in rats: comparison with methylprednisolone[END_REF][START_REF] Suresh | The protective role of MnTBAP in oxidant-mediated injury and inflammation in a rat model of lung contusion[END_REF][START_REF] Cui | SOD mimetic improves the function, growth, and survival of small-size liver grafts after transplantation in rats[END_REF] Prior published data from a patent (U.S. patent No. 8598150 B1, 2013) provided indication on the effective and toxic range of MnTBAP in mice. However, a protocol for the use of MnTBAP in vivo to downregulate CD4 was not known, and had to be established. Using an HPLC technique, [START_REF] Wu | Stability, disposition, and penetration of catalytic antioxidants Mn-porphyrin and Mn-salen and of methylprednisolone in spinal cord injury[END_REF] we determined that the plasma concentration of MnTBAP in C57BL/6 mice rapidly declined over time after intraperitoneal (i.p.) injection of 80 mg/kg (data not shown), prompting a multi-administration schedule. The first protocol tested aimed to inhibit CD4 + T cells prior to, and after exposure to the antigen, in order to demonstrate the possibility of interfering with CD4 + T cell-mediated responses after immunization. This protocol included one MnTBAP injection 2 h prior to immunization and four daily injections post-immunization (Figure 3A). Immunization was conducted through subcutaneous (s.c.) OVA peptides or PBS injections with an adjuvant (IFA) (Figure 3A). IFN-γ ELISPOT measurements of T cell activation were performed 8 days after OVA immunization, by in vitro stimulation of splenic cells with OVA 257-264 or OVA 323-339 , used to detect specifically cytolytic CD8 + and CD4 + T cell responses, respectively. In control mice not treated with MnTBAP (PBS + OVA / IFA), potent OVA-specific CD4 + and CD8 + IFN-γ-producing T cell responses were measured in the spleen of mice immunized with OVA as expected (Figure 3B). In contrast, no OVA-specific T cell immune responses were mounted in OVAimmunized mice treated with MnTBAP (MnTBAP + OVA / IFA), similar to control mice which were not immunized (PBS + PBS / IFA and MnTBAP + PBS / IFA). Thus, MnTBAP reduced both CD4 + and CD8 + T cell-mediated immunity following immunization. As these effects could be potentially immunosuppressive, a second protocol was designed to confirm the presumed reversible effect of MnTBAP by immunization with OVA outside the MnTBAP treatment window (Figure 3C). Mice injected with peptides 21 days post MnTBAP administration were capable of mounting OVA-specific CD4 + and CD8 + IFN-γ T cell responses as high as in the PBS group (Figure 3C and3D). Splenic cells from PBS-or MnTBAP-treated mice were assayed by flow cytometry for cell surface expression of CD3, CD4 and CD8 (Figures 3, E, F andD). Significant downregulation of CD4 and CD8 by MnTBAP were shown to occur in vivo at d4, with cell surface levels being ≈ 80% and 90% of those seen in PBS-treated mice, respectively (Figures 3, F andG). The CD4 and CD8 cell surface recovered at d8 and d16 to levels similar to those of control mice. Whereas the CD3 level remained unchanged with MnTBAP treatment throughout the study (Figure 3E).

Taken together, we show that in mice, adaptive T cell immune responses to highly immunogenic antigens can be quelled by MnTBAP, provided that the vaccine is administrated within the MnTBAP treatment window in which a major effect on CD4 + T cells and weaker effect on CD8 + T cells are observed. These results prompted us to investigate whether MnTBAP could be exploited in a challenging and therapeutic application involving strong CD4 + T cell-mediated immune responses.

MnTBAP treatment prevents anti-transgene immune responses following intramuscular rAAV gene transfer in mice, reducing CD8 + T cell infiltration and muscle damage.

In gene therapy, cellular immune responses can be readily induced against transgene and vector following rAAV gene transfer in mice. These immune responses lead to the loss of transgeneexpressing cells and to the production of neutralizing antibodies against the rAAV capsid that prevent redosing the mice with the same vector. We previously established an immunogenic gene transfer model to follow transgene-specific CD4 + and CD8 + T cell responses following intramuscular (i.m.) rAAV1-mediated gene delivery in mice. In this model, the transgene is expressed at the membrane of skeletal muscle fibers and consists of the human α-sarcoglycan (SGCA) gene fused to the male HY gene sequences (SGCA-HY). When expressed from a CMV promoter, this transgene is highly immunogenic in female mice that are not tolerant to the male HY antigen. The i.m. injection of low doses of rAAV1 (2-5 x 10 9 viral genome (vg)/injection i.e. about 10 11 vg/kg) was sufficient to induce effector antitransgene and anti-capsid T cell responses that are highly dependent on MHC class II-dependent antigenic presentation and activation of transgene-specific CD4+ T cells. [START_REF] Ferrand | A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer[END_REF][START_REF] Boisgerault | Prolonged gene expression in muscle is achieved without active immune tolerance using microrRNA 142.3p-regulated rAAV gene transfer[END_REF] To determine if MnTBAP could affect T and B cell immune responses induced by rAAV1 i.m. gene transfer, we implemented the protocol of MnTBAP administration used for OVA immunization (Figure 4A). The CMV-driven SGCA-HY transgene (rAAV1_CMV_SGCA_HY, 2.5 x 10 9 vg per mouse) was i.m. delivered in left tibialis anterior (TA) muscle, in the time window of MnTBAP administration, two hours post the first MnTBAP injection (Figure 4A). Since MnTBAP is primarily a well-known antioxidant molecule, its contribution to reduce inflammation by the innate immune system, at the site of vector delivery, should also decrease the establishment of subsequent adaptive immunity. Indeed, there was no significant inflammation detected by light emission in the left TA of animals injected with PBS or MnTBAP alone, as shown in Figure S2A, following a chemical reaction of ROS with the luminol-based substrate L-012 (Figure 4A). Moreover MnTBAP-treated mice presented clearly a very low total ROS level at the intraperitoneal site of multi-injection, compared to PBS-treated mice (Figure S2A). In a similar way, rAAV1-injected muscles of MnTBAP-treated mice presented four days post vector injection, a reduced inflammation compared to their control counterparts (Figures S2, B andC). At the histological level 15 days after gene transfer, a reduced muscular inflammation in MnTBAP-treated mice (MnTBAP/rAAV1) coincided with the presence of high number of preserved myofibers shown by hematoxylin-eosin staining, strongly contrasting with the tissue destruction and generalized centro-nucleation of myofibers reflecting extensive tissue regeneration in control mice (PBS/rAAV1) (Figures 4,B, left panels and C). At the same time point, the i.m. administration of rAAV1_CMV_SGCA_HY vector in MnTBAP-treated mice led also to lower level of CD8 + T cell infiltrate, but to robust and persistent SGCA transgene expression (Figures 4, D andE, respectively), as measured by RT-PCR in TA of MnTBAP-treated mice, contrary to controls.

Muscle immunohistology confirmed the presence, in control mice, of a strong CD8 + T cell infiltrate detected as disseminated brown spots throughout the muscle, while very few or no spots could be seen in MnTBAP-treated and naïve mice (PBS/PBS), respectively (Figure 4B, right panels).

Concomitantly, spleens of mice were harvested to measure transgene-specific CD4 + and CD8 + T cell responses, by the frequency of Dby-or Uty-activated IFN-γ-producing cells, respectively. Preservation of muscle structure along with long-term maintenance muscle transduction in MnTBAP-treated mice correlated with the absence of transgene-specific CD4 + cell response detected at day 8 post vector injection, and persistence of very low transgene-specific CD4 + and CD8 + T cell immune responses at day 15, compared to control mice (Figures 4, F andG). Hence, MnTBAP treatment appears to be effective not only at preventing inflammation due to its intrinsic antioxidant property but also at the same time, by impairing CD4 + T cell responses and decreasing CD8 + T cell infiltrate and cytotoxicity against transgene. In this manner MnTBAP allowed muscle structure preservation and sustained transgene expression in skeletal muscle.

MnTBAP attenuates humoral response against capsid allowing effective transduction upon readministration of vectors of the same serotype

A notable obstacle for gene therapy using rAAV vectors is the induction in vivo of capsid-specific CD4 + and CD8 + T cell responses and anti-AAV neutralizing antibodies (NAbs), which may not only limit the duration of transgene expression following AAV gene transfer, but can completely prevent transduction of a target tissue when administering the same serotype. [START_REF] Ertl | Impact of AAV Capsid-Specific T-Cell Responses on Design and Outcome of Clinical Gene Transfer Trials with Recombinant Adeno-Associated Viral Vectors: An Evolving Controversy[END_REF] The induction of antibodies to the AAV caspid is dependent upon innate immune activation and involves TLR9-mediated CD4 + T cell activation into TH1 effector cells. [START_REF] Rogers | Unique Roles of TLR9-and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer[END_REF][START_REF] Sudres | MyD88 signaling in B cells regulates the production of Th1-dependent antibodies to AAV[END_REF] To assess the effects of MnTBAP on the induction of anticapsid antibodies following rAAV gene transfer, we designed a protocol in which the rAAV vector was this time intravenously (i.v.) injected, to favor a higher exposure to the vector material of lymphoid tissues, which are critically involved in antigen presentation and in initiation of the immune response to capsids, and rechallenged the mice with local i.m.injections of a rAAV bearing the same capsid serotype but a different transgene (Figure 5A). MnTBAP treatment for 5 consecutive days (d0 to d4) at the same time as vector administration (d0) successfully attenuated humoral immunity to viral capsid since MnTBAP-treated mice produced no or significantly less specific anti-rAAV IgG compared to the control group, respectively at day 21 and day 28 post vector injections (Figures 5, B andC). A second rAAV was injected 28 days after primary vector exposure (Figure 5A). The second vector bears the same serotype but encodes a secreted form of murine alkaline phosphatase (mSEAP) which can be quantified in blood samples as well as on tissue sections. The level of secondary-type anti-rAAV1 antibody titers, measured 10 days following vector reexposure (d38), in the MnTBAP cohort was significantly lower to that in the control PBS group (Figure 5D). In addition, a higher transduction efficiency of injected muscle was observed, since a significant amount of mSEAP positive fibers was detected, around 3 months after vector reexposure in the injected-TA muscle sections of MnTBAPtreated primed mice (TA MnTBAP/rAAV1) (Figure 5E). The amount of mSEAP positive fibers observed was comparable to that in the positive control group of PBS-treated mice that had not been exposed to rAAV1 vector before mSEAP vector administration (TA PBS/PBS) (Figure 5E). In contrast, similarly to contralateral non-injected muscles which did not stain at all (contralateral TA), only very few mSEAP positive fibers could be seen in muscle sections of PBS-treated mice that had been primed with rAAV1 vector (TA PBS/rAAV1) and reinjected with mSEAP vector (Figure 5E). As expected, the levels of circulating mSEAP correlated with the number of muscle fibers positive to mSEAP (Figure 5F). Likewise, significantly higher copy number of the readministrated viral genome was detected, 3 months after vector reexposure, in the injected-TA muscles of MnTBAP-treated mice compared to control mice (Figure 5G). Altogether, these results indicate that one short period of treatment with MnTBAP at the time of primary vector exposure appears to be effective at attenuating memory humoral immunity to rAA1 capsid proteins to levels that allow efficient preservation of second vector of the same serotype from neutralization upon reexposure. That was not the case for PBS-treated primed mice, in which upon secondary response to the same antigen, memory cells might have been rapidly activated [START_REF] Devarajan | Autoimmune effector memory T cells: the bad and the good[END_REF] to produce NAbs to bind to the AAV capsid, resulting in the reduction of transduced target cells (Figures 5, E andF).

DISCUSSION

Our data show for the first time that the synthetic anti-oxidant MnTBAP effectively, rapidly and reversibly deprives T cells of extracellular membrane CD4, and to a lesser extent of CD8, and that this has functional antiviral or immunomodulatory impacts. Indeed, MnTBAP treatment of T cells prevents CD4-dependent gp120-mediated lentiviral transduction. In vivo, repeated administration of MnTBAP to mice prevents the induction of antigen-specific TH1 T cell responses and the production of neutralizing antibodies to rAAV.

The effects of MnTBAP on the downregulation of CD4 and CD8 are novel since MnTBAP is otherwise known as a superoxide dismutase mimetic that prevents the induction of ROS. The mechanism involved here is rapid (maximum 2 min) and leads to internalization of cell surface CD4 into clathrincoated pits with possible reexpression at the membrane within hours up to a full recovery in less than a day. The mechanism concerns CD4 and CD8 but membrane levels of CD3 were unchanged. Upon antigenic recognition, TCR signaling is initiated by phosphorylation of TCR tyrosine residues by a pool of pre-activated p56 Lck present in the cells, [START_REF] Nika | Constitutively active Lck kinase in T cells drives antigen receptor signal transduction[END_REF] and subsequent protein-protein interactions between p56 Lck and CD4 promote the distribution of p56 Lck to the TCR-peptide-MHC complex in the immunological synapse. [START_REF] Rossy | How does the kinase Lck phosphorylate the T cell receptor? Spatial organization as a regulatory mechanism[END_REF] p56 Lck as well as its localization to the plasma membrane of T cells were not altered by MnTBAP treatment. However, the CD4/p56 Lck complex was disrupted. It is known that naïve peripheral T cells from Lck -/mice exhibit normal phenotype, with the exception of reduced levels of CD4 on the cell surface [START_REF] Lovatt | Lck regulates the threshold of activation in primary T cells, while both Lck and Fyn contribute to the magnitude of the extracellular signal-related kinase response[END_REF] and that myeloid cells lacking p56 Lck expression exhibit significant constitutive CD4 endocytosis. [START_REF] Raposo | Protein kinase C and NF-kappaB-dependent CD4 downregulation in macrophages induced by T cell-derived soluble factors: consequences for HIV-1 infection[END_REF] Therefore, it is not clear at this point if MnTBAP disrupts the CD4/p56 Lck which causes internalization of CD4, or if MnTBAP causes CD4 internalization which dissociates the complex.

MnTBAP was effective not only on murine cells but also on human cells, in reducing drastically cell surface CD4, and to a much lesser degree cell surface CD8. This differential effects of MnTBAP on CD4 and CD8 might be explained by a less extensive association of CD8 coreceptor to p56 Lck compared to CD4. [START_REF] Bachmann | Developmental regulation of Lck targeting to the CD8 coreceptor controls signaling in naive and memory T cells[END_REF] In mice treated with MnTBAP, the disappearance of CD4 and CD8 was observed at early time points (day 4 but not day 8 or later). Presumably the reduced CD4 and CD8 levels in T cells were also associated with disrupted interactions between CD4 or CD8 and p56 Lck which could have perturbed antigen-induced TCR-mediated signaling and immunological synpase formation. Furthermore, the loss of CD4 localization to microvilli may disturb the initial phase of T cell activation ensured by CD4 as a scanning machinery to select for cells that have MHCII on their surface. [START_REF] Glatzova | Dual Role of CD4 in Peripheral T Lymphocytes[END_REF] Therefore, MnTBAP is expected to inhibit the priming of T cells and the development of B cell responses with reduced effector T cells and antibody-secreting cells. The effects of MnTBAP on CD4 or CD8 downregulation have not yet been described elsewhere to our knowledge. However, MnTBAP is known to reduce acute viral infection when administered daily at a low dose of 5 mg/kg in a murine model of lymphocytic choriomeningitis virus (LCMV) infection. [START_REF] Crump | Antioxidant treatment regulates the humoral immune response during acute viral infection[END_REF] These effects were ascribed to CD4 + T cell death mediated by an altered redox regulation of MAP kinases. Our results suggest that LCMV priming defects could also have been induced by MnTBAP in this model.

The induction of immune tolerance is a daunting challenge in many conditions including solid organ and hematopoietic stem cell transplantation, [START_REF] Mcdonald-Hyman | Advances and challenges in immunotherapy for solid organ and hematopoietic stem cell transplantation[END_REF] as well as in gene therapy. [START_REF] Mingozzi | Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape[END_REF] Our results provide a novel target for immunomodulatory strategies in gene therapy. In view of the crucial role of the CD4 coreceptor as a target for the onset and the maintenance (memory formation signal) of immune responses, [START_REF] Phares | CD4 T cells promote CD8 T cell immunity at the priming and effector site during viral encephalitis[END_REF][START_REF] Zhang | The role of CD4 T cell help for CD8 CTL activation[END_REF] the possibility to render this target surface molecule inaccessible during the priming of immune responses, as was shown here with MnTBAP, could be useful. As shown with OVA immunization, the MnTBAP treatment can effectively prevent T cell-mediated immunity but is transient and functionally reversible. Therefore, selective and not generalized immunosuppressive treatment could be devised, which is useful for gene therapy applications. MnTBAP could thus still allow the body to react to opportunistic infections contrary to common immunosuppressive drugs, [START_REF] Meliani | Antigenselective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration[END_REF] which penalize the entire immune system for unwanted activity of just a small fraction of its lymphocytes. Transient CD4 co-receptor modulation by MnTBAP was evaluated in the context of AAVmediated gene transfer. Recent studies documented rAAVs as a tool of choice for gene therapy to treat genetic disorders because of their efficiency in vivo, leading to the market approval in Europe and US of several gene therapy drugs including a rAAV1-based gene delivery in muscle. [START_REF] Colella | Emerging Issues in AAV-Mediated In Vivo Gene Therapy[END_REF] rAAVs are non-inflammatory vectors but trigger immune responses to the AAV capsid and/or transgene capable of compromising long-term therapeutic efficacy. Novel strategies are therefore needed to be able to readminister the vector after a period of time to maintain therapeutic levels of transgene product in patients. [START_REF] Tse | Strategies to circumvent humoral immunity to adeno-associated viral vectors[END_REF] However, the neutralizing anti-capsid antibodies generated by the first rAAV vector administration limit vector redosing. [START_REF] Tse | Strategies to circumvent humoral immunity to adeno-associated viral vectors[END_REF] Immunomodulatory or immunosuppressive treatments which enable rAAV redosing present a great interest for gene therapy. Novel immunosuppressive agents such as nanoparticles loaded with rapamycin have been successfully employed with rAAV gene delivery preclinical models to reduce rAAV capsid immunogenicity, and to enable effective vector readministration. [START_REF] Meliani | Antigenselective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration[END_REF] Our study provides another alternative to reduce immune responses induced by rAAV gene transfer using a non-toxic, non-immunosuppressive agent at the time of vector delivery.

MnTBAP not only reduced anti-transgene CD4 and CD8 T cell responses preventing muscle fiber destruction, but it also prevented the establishment of T cell and B cell immune responses against the rAAV1 capsid, enabling redosing with the same serotype. The control of anti-SGCA-HY immune responses obtained with MnTBAP was as efficient as previously obtained in this model by regulating direct antigenic presentation of the transgene using a microRNA 142.3p-regulated cassette. [START_REF] Boisgerault | Prolonged gene expression in muscle is achieved without active immune tolerance using microrRNA 142.3p-regulated rAAV gene transfer[END_REF] This further argues that MnTBAP acts by blocking the priming of T cell responses against the AAV1 capsid and against the SGCA-HY transgene peptides. Advantageously, a brief period of MnTBAP treatment was sufficient to prevent the establishment of T cell immune memory allowing for antigen reexposure.

MnTBAP is not an approved drug for human use. Thus, further work is warranted to explore the pharmacological and therapeutic properties of MnTBAP or similar compounds, as well as other strategies leading to temporary CD4 down-regulation. In addition, the appealing properties of MnTBAP are not only potentially useful for gene therapy but it may also be exploited for autoimmunity or in any form of active immunopathology associated with excessive or undesirable immune responses. [START_REF] Devarajan | Autoimmune effector memory T cells: the bad and the good[END_REF]

MATERIALS AND METHODS

Animals

Six-to eight-week-old female C57BL/6 mice were purchased from Charles River Laboratories (L'Arbresle, France). Mice were housed under specific pathogen-free conditions and handled in accordance with French and European directives. Protocols were conducted according to national and international ethical standards and were approved by the local ethics committee (C2EA-51).

In vitro and in vivo MnTBAP treatments

MnTBAP (Calbiochem, San Diego, USA or Enzo Life Sciences, Farmingdale, USA) was dissolved in 75 mM NaOH to constitute a 20 mM stock solution. MnTBAP was dissolved in the complete medium, used for in vitro treatment, containing RPMI-1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin/streptomycin, 2 mM glutamax and 50 µM 2-ME. Hypertonic medium, used to block clathrin dependent endocytosis, is complete medium supplemented with 0.45 M sucrose. For in vivo treatment, the stock solution was diluted with PBS and administered at 80 mg/kg intraperitoneally daily during five days. Control animals were injected with equivalent volume of PBS.

Splenic CD4 + T cells isolation and flow cytometry analysis

After mechanic disruption of the spleen, erythrocytes were eliminated by hypotonic shock with ammonium chloride potassium (ACK) lysing buffer. When indicated, splenic cell suspensions were directly submitted, without further purification, to staining with antibodies. CD4 + T cells were isolated from splenic cell suspensions by negative selection using a CD4 + T cell isolation kit (Miltenyi Biotec, Paris, France) followed by auto-MACS (Miltenyi Biotec) magnetic sorting, according to the manufacturer's instructions. Purity of isolated CD4 + T cells was usually > 95%.

For flow cytometry analysis, cell suspensions were first incubated, for murine cells with anti-FcγRIII/II (Fc Block 2.4G2, BD Biosciences, Le Pont de Claix, France) monoclonal antibodies (mAb), and for human cells with γ-globulins from human blood (Sigma-Aldrich, St-Quentin-Fallavier, France) for 10 minutes at 4°C and then stained for 30 minutes at 4°C in PBS, 0.1% bovine serum albumin (BSA) with Fc Block or γ-globulins present, using saturating amounts of the following mAbs: rat anti-mouse CD4-Pacific Blue (RM4-5, BD Biosciences), hamster anti-mouse CD3-APC (145-2C11, BD Biosciences), rat antimouse CD8-Alexa fluor 700 (53-6.7, eBioscience, Paris, France), mouse anti-human CD3-FITC (HIT3a, BD Biosciences), mouse anti-human CD4-APC (RPA-T4, BD Biosciences), mouse anti-human CD8-Pacific Blue (RPA-T8, BD Biosciences). Gates for the different cell populations were set according to their corresponding isotype controls. Cell viability was determined by staining with 7-aminoactinomycin D (7-AAD; Sigma-Aldrich) or Fixable Viability Dye eFluor 780 (eBioscience). When indicated, samples were fixed with 1% paraformaldehyde. Samples were acquired on an LSR II flow cytometer (BD Biosciences) and data were analyzed with DIVA software (BD Biosciences).

CD4 Immunoprecipitation

Cells, harvested after 2 h culture with or without MnTBAP, were resuspended in solubilization buffer containing 1% NP-40 (Sigma-Aldrich), 0.1 M (NH4)2SO4, 20 mM Tris (pH 7.5), 10% glycerol, 25 mM NaF (Sigma-Aldrich), 1 mM Na3VO4 (Thermo Fisher Scientific, Villebon-sur-Yvette, France) and 1x protease inhibitor mixture (Roche, Basel, Switzerland). After 30 min of incubation under gentle agitation, cell lysates were centrifuged at 14,000 x g for 30 min. Lysates were frozen in aliquots at -20°C or used immediately. Immunoprecipitation was performed by incubation overnight of lysates with rat antimouse CD4 (GK 1.5, Biolegend, San Diego, CA, USA) under gentle agitation at 4°C. The antigen-antibody complex was then added to Protein A/G resin slurry (Pierce, Thermo Fisher scientific), and incubated with gentle mixing for 2 hours at room temperature, and finally microcentrifuged for 30 seconds. The pellet was washed five times with solubilization buffer, and the immunoprecipitated material was eluted by incubating the complex for for 2-5 minutes at 95-100°C in Laemmli sample buffer prepared without 2-ME. The immunoprecipitates and whole-cell lysates ("lysates") were electrophoresed in precast SDS-PAGE (10% polyacrylamide) gels (Bio-Rad Laboratories, Hercules, CA, USA), transferred to PVDF membrane (Merck, Darmstadt, Germany), and probed with anti-p56 Lck rabbit antibody (Santa Cruz Biotechnology, Nanterre, France) followed by IRDye conjugated secondary antibody according to the manufacturer's instructions (LI-COR Biosciences, Lincoln, NE). Blots of whole-cell lysates ("lysates") were stripped and reprobed with anti-CD4 (GK 1.5, Biolegend) antibody, and detected by the ECL method with secondary antibodies coupled to horseradish peroxidase (Pierce). Chemiluminescent signals were digitally acquired with a Chemidoc Imaging System (Biorad, Marnes-la-Coquette, France).

Infrared fluorescence of the secondary antibodies was read on an Odyssey Imaging System, and quantification of bands was performed using the ImageStudio Lite software (LI-COR Biosciences).

Immunofluorescence microscopy

After culture in complete medium with or without MnTBAP at 400 µM, murine splenic CD4 + T cells were collected and subjected to immunostaining as previously described. [START_REF] Neildez-Nguyen | Hypoxic culture conditions enhance the generation of regulatory T cells[END_REF] Briefly, after adhesion on poly-L-lysine-coated slides (Thermo Fisher Scientific), cells were fixed in 4% paraformaldehyde for 5 min at 4°C and for 10 min at room temperature, and permeabilized with methanol for 6 min at -20°C.

Cells were stained overnight with rat anti-mouse CD4-APC (BD Biosciences), or with unlabeled primary rabbit anti-mouse clathrin (Abcam, Paris, France) or p56 Lck (Santa Cruz) antibodies. These primary antibodies were then detected by staining with the goat anti-rabbit Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (Invitrogen, Carlsbad, USA) for 30 min at room temperature. Negative controls were performed with the corresponding isotype controls of primary antibodies used. The slides were mounted with Fluoromount/Dapi (SouthernBiotech, Montrouge, France). Images were acquired with a Leica TCS SP2 DMRE confocal-scanning microscope (Leica Microsystems, Nanterre, France) equipped with 40 X and 63 X immersion objectives. CD3 + T cells, from peripheral blood of healthy individual donors, obtained from the French blood establishment (EFS, Evry, France) following institutional agreements, were purified by positive selection via MACS beads using a magnetic cell sorting system (Miltenyi Biotec). CD3 + T cells were activated for 3 days at 37°C, 5% CO2 in plates coated with anti-CD3 (10 µg/ml) and anti-CD28 (10 µg/ml) -antibodies (both from Miltenyi Biotec) and filled with RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin/streptomycin, 2 mM glutamax, 100 IU/ml IL-2 (Miltenyi Biotec). Cells were then transferred on retronectin (20 μg/cm 2 ) coated-plates, in the absence or presence of 400 µM MnTBAP, at a density of 2.5 x 10 5 cells/ml for transduction during 6 h with 6 x 10 7 physical particles (pp)/ml of gp120HXB2-env pseudotyped lentiviral vectors (generous gift of Dr. D. Fenard) expressing GFP reporter gene under the control of hPGK (human phosphoglycerate kinase 1).

After transduction, cells were washed and further incubated for 72 h in fresh culture medium (RPMI 1640 medium supplemented as described above). Cells were then harvested for CD4 (mouse antihuman CD4-PE, M-T466, Miltenyi Biotec) and GFP expression analysis by FACS. Transduction efficiency is determined as the percentage of viable (7-AAD -) lymphoid cells expressing GFP (7-AAD -GFP + ).The data were normalized to be a percentage of the control value (None) from each experiment.

rAAV1 vector production

Recombinant non-replicative, adenovirus-free rAAV1_CMV_SGCA_HY and rAAV1_CMV_mSEAP vector preparations were generated by transient transfection of HEK293 cells with three endotoxin-free plasmids (Macherey-Nagel, Hoerdt, France): (1) the plasmid packaging (pLTR CO2) suppling the AAV2 rep and cap genes of AAV1 encoding the serotype 1 capsid, (2) the plasmid helper (pXX6) providing the adenovirus helper functions and (3) the plasmid encoding the transgene. For rAAV1_CMV_SGCA_HY vector, a chimeric transgene (pGG1 SGCA HY) was used, consisting of the human alpha-sarcoglycan (SGCA) gene fused to sequences encoding T cell epitopes of the murine HY gene (Dby peptide (NAGFNSNRANSSRSS) presented by I-A b and the Uty peptide (WMHHNMDLI) presented by H2-D b ) and expressed from the cytomegalovirus (CMV) promoter. [START_REF] Boisgerault | Prolonged gene expression in muscle is achieved without active immune tolerance using microrRNA 142.3p-regulated rAAV gene transfer[END_REF] For rAAV1_CMV_mSEAP vector, pGG2-CMV-muSeAP plasmid encoding the secreted form of the murine alkaline phosphatase under the control of the CMV-IE promoter was used. 39 rAAV1 vectors were purified by centrifugation over a CsCl gradient, and tittered in vg by real-time PCR as previously described. 39 Vector final suspension was tested for endotoxin level and found to be < 10 EU/ml.

OVA peptides subcutaneous immunization

Histologic analysis

Mice were killed at indicated time points. Injected muscles were harvested, snap frozen in isopentane previously chilled in liquid nitrogen and kept at -80°C until processing. Transverse cryosections (8 µm thick) of the muscles were either stained with H&E to evaluate inflammation and muscle integrity or used for colorimetric immunostaining or for mSEAP detection.

For colorimetric immunodetection of CD8a, cryosections were rehydrated with PBS for 5 min and then incubated 20 min with H 2 O 2 to inhibit endogenous peroxidases at room temperature (RT). After washing with PBS, sections were blocked with PBS, 10% goat serum for 30 min and then incubated with 1/40 dilution of a rat monoclonal primary antibody directed against CD8a (Thermo Fisher scientific), 1-2 h at RT. After washing with PBS, sections were incubated with secondary antibody goat anti-rat conjugated with horseradish peroxidase (HRP) (Invitrogen) diluted 1/600 for 1 h at RT. Sections were washed three times with PBS and then incubated with diluted diaminobenzidine (DAKO, Trappes, France) for 2-5 min. Then, sections were successively treated with ethanol (5 min), twice in xylene (5 min) and mounted with Eukkit (Labonord, VWR Internatinal, Fontenay-sous-BoisFrance). The slides were examined with a Nikon microscope (Champigny sur Marne, France). Digital images were captured using CCD camera (Sony) and processed using Cartograph 5.5 (Microvision Software; Microvision, Evry, France).

For mSEAP histochemical detection, transverse cryosections of TA muscles were fixed in 0.5% glutaraldehyde and washed twice with PBS. Endogenous alkaline phosphatase was heat-inactivated for 30 min at 65°C. Sections were then incubated for 5 h at 37°C in 0.165 mg/ml 5-bromo-4-chloro-3indolyl-phosphate and 0.33 mg/ml of nitroblue tetrazolium in 100 mM Tris-HCl, 100 mM NaCl and 50 mM MgCl 2 . Sections were counterstained with nuclear fast red.

Measure of transgene and CD8 expression by real-time reverse transcriptase-PCR (RT-PCR)

Total RNA was extracted from muscles using Trizol (Invitrogen). Residual DNA was removed from the samples using the Free DNA removal kit (Ambion, Courtaboeuf, France). cDNA was synthesized from 1 µg of RNA using a mix of random hexamers and anchored oligo dT 3:1 according to the protocol Thermo Scientific Verso cDNA Synthesis kit (Thermo Fisher Scientific). Real-time PCR was performed using ABI PRISM 7900 system (Life Technology, Saint Aubin, France) with 0.2 µM of each primer and 0.1 µM of the probe according to the protocol Thermo Scientific Absolute qPCR ROX Mix (Thermo Fisher Scientific). The primer pairs and Taqman probes used for ha-sarcoglycan amplification were: 920hasarco.F:5'-TGCTGGCCTATGTCATGTGC-3', 991hasarco.R:5'TCTGGATGTCGGAGGTAGCC-3', and 946hasarco.P:5'-CGGGAGGGAAGGCTGAAGAGAGACC-3'.

The ubiquitous acidic ribosomal phosphoprotein gene (PO) was used to normalize the data across samples. The primer pairs and Taqman probe used for PO mRNA amplification were: m181PO.F:5'-CTCCAAGCAGATGCAGCAGA-3', m267PO.R:5'-ACCATGATGCgCAAGGCCAT-3', and m225PO.P:5'-CCGTGGTGCTGATGGGCAAGAA-3'. The primer pairs and Taqman probes for the mouse CD8a were Mm01182107_g1 (Thermo Fisher Scientific).

Genomic DNA extraction and real-time quantitative-PCR

Genomic DNA was extracted from frozen muscles using MagNA Pure 96 Instrument (Roche). Real-time PCR to amplify CMV in relation to the titin gene was performed using ABI PRISM 7900 system (Life Technology, Saint Aubin, France) with 0.1 µM of each primer and 0.1 mM of the probe according to the protocol Absolute QPCR Rox Mix (ABgene, Cambridge, UK). The primer pairs and Taqman probes used for CMV and titin gene amplification were: mTitin-F: 5'-AAAACGAGCAGTGACGTGAGC-3', mTitin-R: 5'-TTCAGTCATGCTGCTAGCGC-3', mTitin-P: 5'-TGCACGGAAGCGTCTCGTCTCAGTC-3', mCMV-F: 5'-CATCAATGGGCGTGGATAGC-3', mCMV-R: 5'-GGAGTTGTTACGACATTTTGGAAA-3', mCMV-P: France) at 2µM or in presence of OVA peptides at 10 µg/ml. As a positive control, cells were stimulated with Concanavalin A (Sigma-Aldrich) at 1µg/ml in each test. After 24h, plates were washed three times with PBS, 0.05% Tween 20 and three times with PBS alone before 2 hours incubation at RT with 1 µg/ml biotinylated anti-mouse IFN-γ mAb in PBS/0.1%BSA. Plates were washed three times with PBS/0.05% Tween 20 and three times with PBS alone before 1 hour 30 incubation with 0.66U/ml streptavidinalkaline phosphatase (AP) conjugate (Roche Diagnostics GmbH, Mannheim, Germany) at room temperature. After wash step with PBS/0.05% Tween 20, and with PBS alone, spot were developed using BCIP/NBT-plus (Mabtech AB, Nacka Strand, Sweden). Spots were counted using an AID reader (Cepheid Benelux, Louven, Belgium). Spot forming units (SFU) per million cells for each mouse were calculated as the average value of duplicate measures after substraction of background values obtained with in vitro unstimulated splenocytes.

Measurement of anti-AAV1 antibody titers

rAAV1-specific serum antibody titers were evaluated by ELISA. F96 Maxisorp Nunc Immuno plates (Thermo Fisher Scientific) were coated overnight at 4°C with 0.2 x 10 9 vg of AAV1 vector diluted in 0.1M carbonate coating buffer (pH 9.6). Plates were washed three times and blocked with a solution of 6% milk in PBS for 2 hours at room temperature. Serial dilutions of samples in this blocking buffer (10-fold dilutions starting 10 -2 to 10 -5 were tested) were incubated on the plates for 1h at 37°C. Plates were washed three times, incubated with a horseradish peroxidase-conjugated goat antibody directed against mouse IgG (Southern Biotech, Birmingham, AL) and revealed by TMB Substrate reagent Set (BD Biosciences). Reaction was stopped with an H2SO4 solution and the optical density at 450 nm was determined using a luminometer (Discovery HT-R, BioServ, Thiais, France) with KC4 software. The area under the curve (AUC) values obtained from the optical densities at four dilution points were calculated for statistical analysis using GraphPad Prism software (Logi Labo, Paris, France).

Quantification of circulating mSEAP

Determination of alkaline phosphatase concentration in immunized mice sera was performed by chemiluminescence analysis. 39 Blood samples were centrifuged for 10 min at 8000 rpm to collect sera, endogenous alkaline phosphatase was heat inactivated for 5 min at 65°C and the heat-resistant mSEAP was measured by addition of the reaction buffer and CSPD® chemiluminescent substrate, according to the manufacturer's instructions (Tropix, Applied Biosystems, Forster City, USA). Chemiluminescence was quantified using a luminometer, Enspire (Perkin Elmer, Massachusetts, USA). Expression levels are expressed as ng of mSEAP per ml of serum using a standard curve of purified human placental alkaline phosphatase.

Statistics

Statistical analyses were performed using Graphpad Prism 5 software. For antibody production, differences were analyzed by comparing the average values of the area-under-the curve for the titration of each mouse serum.

A

  mixture OVA peptides was s.c. administrated in mice anesthetized by i.p. injection of xylazine (10 mg/kg) and ketamine (100 mg/kg). OVA 257-264 (SIINFEKL) (InvivoGen, San Diego, USA) is a class I (Kb)restricted peptide epitope of OVA presented by the class I MHC molecule (CMH) H-2Kb. OVA 323-339 (ISQAVHAAHAEINEAGR) (AnaSpec, San Jose, USA) is an H-2b-restricted OVA class II epitope. 100 µg of each peptide emulsified with the same volume of IFA (Sigma-Aldrich) were administrated. Controls were injected with PBS-IFA complexes. OVA peptides-and PBS-IFA complexes were injected two hours or 21 days post first i.p. injection of MnTBAP (or PBS). Eight days post OVA peptides injection, mice were sacrificed and spleens harvested to analyze OVA-specific T cell response. rAAV1 vector delivery Intramuscular injection: Two hours post first i.p. injection of MnTBAP, mice were anesthetized by i.p. injection of xylazine (10 mg/kg) and ketamine (100 mg/kg) and 25 µl of a rAAV1_CMV_SGCA_HY viral 9 preparation containing 2.5.10 vg was injected in the left TA muscle. Controls were injected with PBS. Mice were sacrificed 15 days after vector injection and the injected muscles were harvested to perform histologic analysis. Spleens were harvested to analyze transgene-specific T cell response. Intravenous injection: Two hours post first i.p. injection of MnTBAP, mice were anesthetized by i.p. injection of xylazine (10 mg/kg) and ketamine (100 mg/kg), and 200 µl of a rAAV1_CMV_SGCA_HY viral 9 preparation containing 2.5.10 vg was injected into retro orbital sinus or into tail vein. Controls were injected with PBS. Serum samples were harvested at D21 and D28 post vector injection to analyze capsid-specific humoral response. Readministration protocol: After the initial i.v. injection of rAAV1_CMV_SGCA_HY vector at D0 (two 9 hours post first i.p. injection of MnTBAP) the second rAAV1 injection (2.10 vg of rAAV1_CMV_mSEAP) was performed 28 days later in the left TA muscle. Mice were followed up to 93 days after readministration to analyze capsid-specific humoral response and to measure circulating mSEAP from serum samples. Mice were killed 93 days after second injection of vector. Left and right TA muscles were harvested for histological and enzymatic assay.

  5'-ATTTCCAAGTCTCCACCC-3'. Results were calculated from a standard curve based on dilutions of 2 plasmids containing either the Titin or CMV sequences. Background levels ranged between 36-40 CT values. IFNγ enzyme-linked immunospot assay (IFNγ ELISPOT) Spleens were harvested to analyze the cellular immune anti-transgene response by T cell cytokine response assay. Multiscreen-HA Assay System 96-well Filtration plates (Merck) were coated overnight with 2.5 µg/ml of anti-mouse IFN-γ purified mAb (eBioscience) at 4°C, washed, and then blocked with RPMI 1640 medium (Life technologies, Saint Aubain, France) supplemented with 10% heat inactivated fetal bovine serum (FCS) (Life technologies,), 100U/ml penicillin/streptomycin (Thermo Fisher Scientific) and 2mM GlutaMAX TM -I (Life technologies,) for 2 hours at 37°C. Plates were washed three times with RPMI 1640 medium without FCS. Splenocytes were added in duplicate to the IFN-γ-coated well (1 or 0.5 x 10 6 cells/well) and cultured in presence of Dby and Uty peptide (Genepep, Montpellier,

Figure 1 .

 1 Figure 1. MnTBAP-induced CD4 reversible internalization mechanism. (A) CD4 + T cells isolated by

Figure 2 .

 2 Figure 2. MnTBAP induces dramatic loss of CD4 on the surface of murine and human T lymphocytes

Figure 3 .

 3 Figure 3. Temporary in vivo inhibition of the immune system by MnTBAP. (A) Schema of OVA peptide

Figure 4 .

 4 Figure 4. MnTBAP counteracts the adverse effects of rAAV-mediated i.m. gene delivery. (A) Protocol

Figure 5 .

 5 Figure 5. MnTBAP inhibits primary and memory humoral response against capsid allowing efficient

FFig. 5

 5 Fig.2

  Danos, O, and Douar, AM (2006). Long-term expression and repeated administration of AAV type 1, 2 and 5 vectors in skeletal muscle of immunocompetent adult mice. Gene therapy 13, 1300-1308.

	39. Riviere, C, ABBREVIATIONS
	AUC, area under the curve; rAAV, recombinant adeno-associated viral vector; i.m., intramuscular;
	i.p., intraperitoneal; i.v., intravenous; MHCII, MHC class II; mSEAP, murine alkaline phosphatase;
	NAbs, neutralizing antibodies; ROS, reactive oxygen species; SGCA-HY, human α-sarcoglycan gene
	fused to HY gene sequences encoding Uty and Dby male epitopes; s.c., subcutaneous; TA, tibialis
	anterior; Tregs, Foxp3 + regulatory T cells; vg, viral genome.
	Significant differences were defined with probability values inferior to
	0.05 (*P < 0.05, ** P < 0.005, *** P < 0.001).

ACKNOWLEDGMENTS

We are grateful to Genethon support groups for help with animal care, animal experiments, histology, and rAAV vector production. We would like to thank Armelle Viornery, Maxime Ferrand, Julie Vendomele, Christophe Georger and Ababacar Khalil Seye for technical assistance, and Andràs Paldi for helpful discussions.

The authors declare no competing financial interests.

Time-lapse microscopy

Murine splenic CD4 + T cells were first stained with CFSE, according to the manufacturer's recommendations (Invitrogen), and then with rat anti-mouse CD4-APC (RM4-5, BD Biosciences) mAb.

After staining, cells were incubated in Ibidi's µ-chambers culture dish (Ibidi, Nanterre, France) in the thermo-regulated chamber of the Zeiss Axiovert 100M confocal microscope. A time lapse acquisition was performed with a delay of 120 sec between frames. Each frame was illuminated using a 488nm and 633 nm lasers, and fluorescence collected using 505-580nm and 644-719nm filters. The magnification was 40x/1.3 oil and images were saved as 8bits. The image stack was splitted using ImageJ-1.43, and images analyzed using CellProfiler 2.0.10415. The cell segmentation was performed on the CFSE fluorescence, and the fluorescence of the CD4 staining analyzed using the R software.

Intensity was normalized to the maximum fluorescence measured at T0 for each condition.

In vivo chemiluminescence imaging of localized inflammation

After mice isofluorane anaesthesia, 100 µl of L-012 solution (15 mg/ml) (Wako, Osaka, Japan) were administered intravenously in retro-orbital sinus 4 days after first MnTBAP injection. 2 minutes after L-012 administration, mice were placed in an in vivo imaging system (IVIS Lumina, Perkin Elmer, USA), and chemiluminescence images were acquired for each mouse with a typical exposure time of 5 minutes. Images were quantified using LivingImage (Perkin Elmer, USA) software, by drawing a Region of Interest (ROI) around the left inferior limb of the mouse. Radiance (ph/s/cm²/sr) was measured for each ROI.

Culture and Transduction of Human Primary Cells

SUPPLEMENTAL INFORMATION

Supplemental Information includes two figures.