<p> 1Supplementary data</p><p>2</p><p>3Supplementary Figure S1: CD4, CD8 T cells, M1 and M2 macrophages infiltrate</p><p>4the injected muscles</p><p>5Immunohistological and FACS analyses of muscles of C57BL/6 and Sgca-/- mice, 14</p><p>6days after injection of 5x109 vg of either PBS or rAAV1_CMV_SGCA-HY vector. (a)</p><p>7Immunostaining of CD4 or CD8 (red) on cryosections of injected muscles with DAPI</p><p>8stained nuclei (blue). Images are representative of 4 mice per group. The bar scale is</p><p>9100 µm and the upper right insert shows a magnification of the selected area. (b)</p><p>10FACS analysis of the population of muscle-infiltrating CD11b+ cells enriched by</p><p>11magnetic cell sorting from the TA of C57/BL6 (left) or Sgca-/- (right) mice injected</p><p>12with PBS (top row) or vector (bottom row). Dot blots represent the expression of</p><p>13CD11b and F4/80 on live cells and the percentages of two distinct populations of</p><p>14F4/80low and F4/80high cells are indicated. Histograms represent the expression of</p><p>15CD206, iNOS and ARG-1 on F4/80low (red) and F4/80high (black) cells in the muscles</p><p>16that were injected with the rAAV. Isotype-matched negative controls (grey) are shown</p><p>17as overlays for intracellular stainings (iNOS and ARG-1). </p><p>18</p><p>19Supplementary Figure S2: Anti-transgene T cell responses and transgene</p><p>20expression in MHCII-/-, 2m-/-, µMT-/- and C57BL6 mice </p><p>21Muscle transgene expression and splenic T cell responses were measured following</p><p>22injection of PBS or of 5x109 vg of rAAV1_CMV_SGCA-HY in the left TA of different</p><p>23strains of mice. Data show values obtained for at least 6 mice per condition and per</p><p>24time point. (a) Expression of SGCA mRNA in the TA was measured at indicated time</p><p>25points by qRT-PCR and normalized to PO gene expression. Data were normalized to 1a reference of 1.0 corresponding to levels of SGCA known to provide therapeutic</p><p>2efficacy in this model. Results represent the average and SD of mice tested in the</p><p>3group. (b) At the time points indicated on the X axis (14 or 21 days post vector</p><p>4injection) the levels of Dby-specific CD4+T cell responses (left panel) and Uty-</p><p>5specific CD8+T cell responses (right panel) were measured by IFN-ELISPOT</p><p>6following peptide in vitro stimulation of spleen cells. Each dot represents data from</p><p>7individual mice after exclusion of aberrant data points, and horizontal bars indicate</p><p>8the average value. </p><p>9 10 11Supplementary Figure S3: Anti-transgene T cell responses, muscle integrity</p><p>12and cellular infiltration in CD4-/-Sgca-/- and CD8-/-Sgca-/- mice compared to</p><p>13CD4-/- and CD8-/- mice</p><p>14C57BL/6 mice, CD4-/- mice, CD8-/- mice, CD4-/-Sgca -/- mice, CD8-/-Sgca -/- mice,</p><p>15and Sgca-/- mice were injected IM into the TA with 5x109 vg of rAAV1_CMV_SGCA-</p><p>16HY vector, then anti-transgene immune responses and muscle histology were</p><p>17analyzed over time. (a) At the indicated times in the X axis (14, 21, and 42 days post</p><p>18vector injection) spleen CD4 T cell (left panels) and CD8 T cell (right panels)</p><p>19responses were measured by IFN-ELISPOT in the indicated strains of mice. Dots</p><p>20represent individual mouse data and horizontal bars indicate the average value. (b)</p><p>21At day 14, muscle histology was analyzed by HE staining in the indicated groups.</p><p>22Images are representative of 1 experiment with 4 mice analyzed per group. (c) At</p><p>23day 14, muscle cryosections of mice in the indicated groups were immunostained to</p><p>24detect CD3+ T cells (red) and CD11b+ myeloid cells (red). Nuclei were stained with</p><p>25DAPI (blue). The scale bar is 100 µm. The upper right inserts reflect a magnification</p><p>26of the selected areas.</p>