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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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Transgene-specific CD4+ T-cell responses using Mir142T-regulated rAAV2/1 vectors. (a) C57BL/6 and (b) Sgca-/- mice were injected i.m. into the TA with PBS, 5 × 109 vg of rAAV1_CMV_SGCA-HY or rAAV1_CMV_SGCA-HY-Mir142T vectors. After 14, 21 and 30 days, spleen cells were harvested to measure Dby-specific CD4+ T-cell response by interferon-γ enzyme-linked immunospot assays following Dby peptide in vitro stimulation. Data represent four independent experiments in C57BL/6 mice and three independent experiments in Sgca-/- mice, each including three mice per group. Dots represent individual mouse data and horizontal bars indicate the average value. ***P<0.001, **P<0.01, *P<0.05, NS, not statistically significant.
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fig5: Transgene-specific CD4+ T-cell responses using Mir142T-regulated rAAV2/1 vectors. (a) C57BL/6 and (b) Sgca-/- mice were injected i.m. into the TA with PBS, 5 × 109 vg of rAAV1_CMV_SGCA-HY or rAAV1_CMV_SGCA-HY-Mir142T vectors. After 14, 21 and 30 days, spleen cells were harvested to measure Dby-specific CD4+ T-cell response by interferon-γ enzyme-linked immunospot assays following Dby peptide in vitro stimulation. Data represent four independent experiments in C57BL/6 mice and three independent experiments in Sgca-/- mice, each including three mice per group. Dots represent individual mouse data and horizontal bars indicate the average value. ***P<0.001, **P<0.01, *P<0.05, NS, not statistically significant.

Mentions: Administration of the Mir142T-regulated vector to C57BL/6 mice failed to induce CD4-mediated immune responses to the transgene in a durable manner compared with the non-regulated vector (Figure 5a). In contrast, in Sgca-/- mice, the Mir142T-regulated vector induced a potent CD4+ T-cell-mediated immune response reaching after 14 days the same levels of response as the non-regulated vector (Figure 5b). The levels of transgene-specific T cells in the animal can be tracked over time following the injection of congenic (CD45.1+) transgene-specific CD4+ T cells before gene transfer vector administration. With the non-regulated vector, the kinetics of transgene-specific blood CD4+ T-cell levels were similar in C57BL/6 (Figure 6a) and Sgca-/- mice (Figure 6b), starting day 6, peaking around 8 days even though the retraction phase was slightly different. Administration of the Mir142T-regulated vector in C57BL/6 mice did not result in a significant amplification of T cells as compared with PBS. On the contrary, in Sgca-/- mice T-cell responses were not controlled by the Mir142.3p-regulated vector, which induced a striking expansion of CD4+ T cells that peaked at day 11, beyond the peak time of the non-regulated immune response.


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)

Transgene-specific CD4+ T-cell responses using Mir142T-regulated rAAV2/1 vectors. (a) C57BL/6 and (b) Sgca-/- mice were injected i.m. into the TA with PBS, 5 × 109 vg of rAAV1_CMV_SGCA-HY or rAAV1_CMV_SGCA-HY-Mir142T vectors. After 14, 21 and 30 days, spleen cells were harvested to measure Dby-specific CD4+ T-cell response by interferon-γ enzyme-linked immunospot assays following Dby peptide in vitro stimulation. Data represent four independent experiments in C57BL/6 mice and three independent experiments in Sgca-/- mice, each including three mice per group. Dots represent individual mouse data and horizontal bars indicate the average value. ***P<0.001, **P<0.01, *P<0.05, NS, not statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4283385&req=5

fig5: Transgene-specific CD4+ T-cell responses using Mir142T-regulated rAAV2/1 vectors. (a) C57BL/6 and (b) Sgca-/- mice were injected i.m. into the TA with PBS, 5 × 109 vg of rAAV1_CMV_SGCA-HY or rAAV1_CMV_SGCA-HY-Mir142T vectors. After 14, 21 and 30 days, spleen cells were harvested to measure Dby-specific CD4+ T-cell response by interferon-γ enzyme-linked immunospot assays following Dby peptide in vitro stimulation. Data represent four independent experiments in C57BL/6 mice and three independent experiments in Sgca-/- mice, each including three mice per group. Dots represent individual mouse data and horizontal bars indicate the average value. ***P<0.001, **P<0.01, *P<0.05, NS, not statistically significant.
Mentions: Administration of the Mir142T-regulated vector to C57BL/6 mice failed to induce CD4-mediated immune responses to the transgene in a durable manner compared with the non-regulated vector (Figure 5a). In contrast, in Sgca-/- mice, the Mir142T-regulated vector induced a potent CD4+ T-cell-mediated immune response reaching after 14 days the same levels of response as the non-regulated vector (Figure 5b). The levels of transgene-specific T cells in the animal can be tracked over time following the injection of congenic (CD45.1+) transgene-specific CD4+ T cells before gene transfer vector administration. With the non-regulated vector, the kinetics of transgene-specific blood CD4+ T-cell levels were similar in C57BL/6 (Figure 6a) and Sgca-/- mice (Figure 6b), starting day 6, peaking around 8 days even though the retraction phase was slightly different. Administration of the Mir142T-regulated vector in C57BL/6 mice did not result in a significant amplification of T cells as compared with PBS. On the contrary, in Sgca-/- mice T-cell responses were not controlled by the Mir142.3p-regulated vector, which induced a striking expansion of CD4+ T cells that peaked at day 11, beyond the peak time of the non-regulated immune response.

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