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A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer.

Ferrand M, Galy A, Boisgerault F - Gene Ther. (2014)

Bottom Line: Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy.Following rAAV2/1 delivery of an immunogenic α-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates.Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.

View Article: PubMed Central - PubMed

Affiliation: 1] Inserm, U951, University of Evry, UMR_S951, Genethon, Evry, France [2] Genethon, Molecular Immunology and Innovative Biotherapies Group, Evry, France.

ABSTRACT
Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy. Thus, we compared dystrophic mice devoid of α-sarcoglycan with healthy mice to characterize immune cell activation and cellular populations contributing to the loss of gene-modified myofibers. Following rAAV2/1 delivery of an immunogenic α-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates. Selective cell subset depletion models revealed that CD4+ T cells were essential for transgene rejection in both healthy and pathologic mice, but macrophages and CD8+ T cells additionally contributed as effector cells of transgene rejection only in dystrophic mice. Vectors restricting transgene expression in antigen-presenting cells showed that endogenous presentation of transgene products was the sole mechanism responsible for T-cell priming in normal mice, whereas additional and protracted antigenic presentation occurred in dystrophic animals, leading to secondary CD4+ T-cell activation and failure to maintain transgene expression. Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.

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Contribution of effector CD8+ T cells and macrophages to transgene rejection. C57BL/6, CD8-/-, Sgca-/- and CD8-/- Sgca-/- mice were injected i.m. with PBS or with 5 × 109 vg of rAAV1_CMV_SGCA-HY vector in the left TA and transgene expression, and the muscle integrity were analyzed at day 14. Levels of SGCA mRNA expression in the TA of normal (a) and dystrophic (b) mice were measured by quantitative RT-PCR (qRT-PCR) and normalized to PO levels. The reference of 1.0 corresponds to therapeutics levels of SGCA. Results show average values obtained from six mice per group. (c) HE stain and histologic analysis of C57BL/6- and CD8-/- injected TA muscles. (d) Immunostaining of the α-sarcoglycan protein on cross-sections of Sgca-/- and CD8-/- Sgca-/- injected TA muscles. Images are representative from one experiment out of three, with three mice per group (scale bar=150 μm). In (e and f), C57BL/6 or Sgca-/- mice received a single i.m. injection of PBS or of 5 × 109 vg of rAAV1_CMV_SGCA-HY vector in the left TA and four injections intravenously of Clodrosome (lipos. clodronate) or of Encapsome as control (lipos.ctl). Levels of SGCA mRNA expression in the TA of normal (e) and dystrophic (f) mice were measured at different time points by qRT-PCR and normalized to PO levels. The reference of 1.0 corresponds to levels of SGCA known to be therapeutic in Sgca-/- mice. Data are representative of the average value obtained in six mice per group. (g) HE histologic analysis of the TA of C57BL/6 mice injected 14 days earlier with the vector. (h) Immunohistochemical analysis of α-sarcoglycan expression in the injected TA of Sgca-/- mice. Images are representative from one experiment out of three, with three mice per group (scale bar=150 μm). ***P<0.001, **P<0.01, *P<0.05, NS, not statistically significant.
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fig3: Contribution of effector CD8+ T cells and macrophages to transgene rejection. C57BL/6, CD8-/-, Sgca-/- and CD8-/- Sgca-/- mice were injected i.m. with PBS or with 5 × 109 vg of rAAV1_CMV_SGCA-HY vector in the left TA and transgene expression, and the muscle integrity were analyzed at day 14. Levels of SGCA mRNA expression in the TA of normal (a) and dystrophic (b) mice were measured by quantitative RT-PCR (qRT-PCR) and normalized to PO levels. The reference of 1.0 corresponds to therapeutics levels of SGCA. Results show average values obtained from six mice per group. (c) HE stain and histologic analysis of C57BL/6- and CD8-/- injected TA muscles. (d) Immunostaining of the α-sarcoglycan protein on cross-sections of Sgca-/- and CD8-/- Sgca-/- injected TA muscles. Images are representative from one experiment out of three, with three mice per group (scale bar=150 μm). In (e and f), C57BL/6 or Sgca-/- mice received a single i.m. injection of PBS or of 5 × 109 vg of rAAV1_CMV_SGCA-HY vector in the left TA and four injections intravenously of Clodrosome (lipos. clodronate) or of Encapsome as control (lipos.ctl). Levels of SGCA mRNA expression in the TA of normal (e) and dystrophic (f) mice were measured at different time points by qRT-PCR and normalized to PO levels. The reference of 1.0 corresponds to levels of SGCA known to be therapeutic in Sgca-/- mice. Data are representative of the average value obtained in six mice per group. (g) HE histologic analysis of the TA of C57BL/6 mice injected 14 days earlier with the vector. (h) Immunohistochemical analysis of α-sarcoglycan expression in the injected TA of Sgca-/- mice. Images are representative from one experiment out of three, with three mice per group (scale bar=150 μm). ***P<0.001, **P<0.01, *P<0.05, NS, not statistically significant.

Mentions: Unexpectedly, the lack of CD8+ T cells did not prevent the loss of transgene (Figure 3a and Supplementary Figure S2a). Injection of the vector in muscles of CD8-/- mice caused destruction and regeneration with a marked cellular infiltration only marginally reduced compared with C57/BL6 controls (Figure 3c). Similar results were obtained in β2M-/- mice (data not shown). As expected, anti-transgene CD8 T-cell responses were completely abolished in β2M-/- mice or in CD8-/- mice while anti-transgene CD4+ T-cell responses were maintained (Supplementary Figures S2b and S3a). These data suggest that CD8+ T cells are not the central effector cells associated with transgene rejection in our model. To evaluate the impact of a dystrophic context on CD8+ T-cell responses, we crossed CD8-/- mice with Sgca-/- mice to obtain CD8-/- Sgca-/- mice. Those double-knockout mice also displayed early signs of dystrophy characterized by a regenerating phenotype (Supplementary Figure S3b). Such mice failed to develop transgene-specific CD8+ T-cell response but retained the same level of CD4+ T-cell response as Sgca-/- mice (Supplementary Figure S3a). In the dystrophic background, the lack of CD8+ T cells did not prevent transgene rejection but mitigated this loss. The disappearance of transgene was documented at the mRNA level, but yet significantly higher levels of transgene mRNA remained after gene transfer in CD8-/- Sgca-/- mice compared with Sgca-/- mice (Figure 3b). In agreement with this finding, about 15% of SGCA+ myofibers were detected in muscles of CD8-/- Sgca-/- mice, while transgene marking was completely lost in Sgca-/- mice (Figure 3d). Thus, CD8+ T cells contribute to muscle destruction without being major effector cells.


A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer.

Ferrand M, Galy A, Boisgerault F - Gene Ther. (2014)

Contribution of effector CD8+ T cells and macrophages to transgene rejection. C57BL/6, CD8-/-, Sgca-/- and CD8-/- Sgca-/- mice were injected i.m. with PBS or with 5 × 109 vg of rAAV1_CMV_SGCA-HY vector in the left TA and transgene expression, and the muscle integrity were analyzed at day 14. Levels of SGCA mRNA expression in the TA of normal (a) and dystrophic (b) mice were measured by quantitative RT-PCR (qRT-PCR) and normalized to PO levels. The reference of 1.0 corresponds to therapeutics levels of SGCA. Results show average values obtained from six mice per group. (c) HE stain and histologic analysis of C57BL/6- and CD8-/- injected TA muscles. (d) Immunostaining of the α-sarcoglycan protein on cross-sections of Sgca-/- and CD8-/- Sgca-/- injected TA muscles. Images are representative from one experiment out of three, with three mice per group (scale bar=150 μm). In (e and f), C57BL/6 or Sgca-/- mice received a single i.m. injection of PBS or of 5 × 109 vg of rAAV1_CMV_SGCA-HY vector in the left TA and four injections intravenously of Clodrosome (lipos. clodronate) or of Encapsome as control (lipos.ctl). Levels of SGCA mRNA expression in the TA of normal (e) and dystrophic (f) mice were measured at different time points by qRT-PCR and normalized to PO levels. The reference of 1.0 corresponds to levels of SGCA known to be therapeutic in Sgca-/- mice. Data are representative of the average value obtained in six mice per group. (g) HE histologic analysis of the TA of C57BL/6 mice injected 14 days earlier with the vector. (h) Immunohistochemical analysis of α-sarcoglycan expression in the injected TA of Sgca-/- mice. Images are representative from one experiment out of three, with three mice per group (scale bar=150 μm). ***P<0.001, **P<0.01, *P<0.05, NS, not statistically significant.
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fig3: Contribution of effector CD8+ T cells and macrophages to transgene rejection. C57BL/6, CD8-/-, Sgca-/- and CD8-/- Sgca-/- mice were injected i.m. with PBS or with 5 × 109 vg of rAAV1_CMV_SGCA-HY vector in the left TA and transgene expression, and the muscle integrity were analyzed at day 14. Levels of SGCA mRNA expression in the TA of normal (a) and dystrophic (b) mice were measured by quantitative RT-PCR (qRT-PCR) and normalized to PO levels. The reference of 1.0 corresponds to therapeutics levels of SGCA. Results show average values obtained from six mice per group. (c) HE stain and histologic analysis of C57BL/6- and CD8-/- injected TA muscles. (d) Immunostaining of the α-sarcoglycan protein on cross-sections of Sgca-/- and CD8-/- Sgca-/- injected TA muscles. Images are representative from one experiment out of three, with three mice per group (scale bar=150 μm). In (e and f), C57BL/6 or Sgca-/- mice received a single i.m. injection of PBS or of 5 × 109 vg of rAAV1_CMV_SGCA-HY vector in the left TA and four injections intravenously of Clodrosome (lipos. clodronate) or of Encapsome as control (lipos.ctl). Levels of SGCA mRNA expression in the TA of normal (e) and dystrophic (f) mice were measured at different time points by qRT-PCR and normalized to PO levels. The reference of 1.0 corresponds to levels of SGCA known to be therapeutic in Sgca-/- mice. Data are representative of the average value obtained in six mice per group. (g) HE histologic analysis of the TA of C57BL/6 mice injected 14 days earlier with the vector. (h) Immunohistochemical analysis of α-sarcoglycan expression in the injected TA of Sgca-/- mice. Images are representative from one experiment out of three, with three mice per group (scale bar=150 μm). ***P<0.001, **P<0.01, *P<0.05, NS, not statistically significant.
Mentions: Unexpectedly, the lack of CD8+ T cells did not prevent the loss of transgene (Figure 3a and Supplementary Figure S2a). Injection of the vector in muscles of CD8-/- mice caused destruction and regeneration with a marked cellular infiltration only marginally reduced compared with C57/BL6 controls (Figure 3c). Similar results were obtained in β2M-/- mice (data not shown). As expected, anti-transgene CD8 T-cell responses were completely abolished in β2M-/- mice or in CD8-/- mice while anti-transgene CD4+ T-cell responses were maintained (Supplementary Figures S2b and S3a). These data suggest that CD8+ T cells are not the central effector cells associated with transgene rejection in our model. To evaluate the impact of a dystrophic context on CD8+ T-cell responses, we crossed CD8-/- mice with Sgca-/- mice to obtain CD8-/- Sgca-/- mice. Those double-knockout mice also displayed early signs of dystrophy characterized by a regenerating phenotype (Supplementary Figure S3b). Such mice failed to develop transgene-specific CD8+ T-cell response but retained the same level of CD4+ T-cell response as Sgca-/- mice (Supplementary Figure S3a). In the dystrophic background, the lack of CD8+ T cells did not prevent transgene rejection but mitigated this loss. The disappearance of transgene was documented at the mRNA level, but yet significantly higher levels of transgene mRNA remained after gene transfer in CD8-/- Sgca-/- mice compared with Sgca-/- mice (Figure 3b). In agreement with this finding, about 15% of SGCA+ myofibers were detected in muscles of CD8-/- Sgca-/- mice, while transgene marking was completely lost in Sgca-/- mice (Figure 3d). Thus, CD8+ T cells contribute to muscle destruction without being major effector cells.

Bottom Line: Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy.Following rAAV2/1 delivery of an immunogenic α-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates.Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.

View Article: PubMed Central - PubMed

Affiliation: 1] Inserm, U951, University of Evry, UMR_S951, Genethon, Evry, France [2] Genethon, Molecular Immunology and Innovative Biotherapies Group, Evry, France.

ABSTRACT
Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy. Thus, we compared dystrophic mice devoid of α-sarcoglycan with healthy mice to characterize immune cell activation and cellular populations contributing to the loss of gene-modified myofibers. Following rAAV2/1 delivery of an immunogenic α-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates. Selective cell subset depletion models revealed that CD4+ T cells were essential for transgene rejection in both healthy and pathologic mice, but macrophages and CD8+ T cells additionally contributed as effector cells of transgene rejection only in dystrophic mice. Vectors restricting transgene expression in antigen-presenting cells showed that endogenous presentation of transgene products was the sole mechanism responsible for T-cell priming in normal mice, whereas additional and protracted antigenic presentation occurred in dystrophic animals, leading to secondary CD4+ T-cell activation and failure to maintain transgene expression. Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.

Show MeSH
Related in: MedlinePlus