Supplementary Figure S1: CD4, CD8 T Cells, M1 and M2 Macrophages Infiltrate the Injected

1Supplementary data

3Supplementary Figure S1: CD4, CD8 T cells, M1 and M2 macrophages infiltrate

4the injected muscles

5Immunohistological and FACS analyses of muscles of C57BL/6 and Sgca-/- mice, 14

6days after injection of 5x109 vg of either PBS or rAAV1_CMV_SGCA-HY vector. (a)

7Immunostaining of CD4 or CD8 (red) on cryosections of injected muscles with DAPI

8stained nuclei (blue). Images are representative of 4 mice per group. The bar scale is

9100 µm and the upper right insert shows a magnification of the selected area. (b)

10FACS analysis of the population of muscle-infiltrating CD11b+ cells enriched by

11magnetic cell sorting from the TA of C57/BL6 (left) or Sgca-/- (right) mice injected

12with PBS (top row) or vector (bottom row). Dot blots represent the expression of

13CD11b and F4/80 on live cells and the percentages of two distinct populations of

14F4/80low and F4/80high cells are indicated. Histograms represent the expression of

15CD206, iNOS and ARG-1 on F4/80low (red) and F4/80high (black) cells in the muscles

16that were injected with the rAAV. Isotype-matched negative controls (grey) are shown

17as overlays for intracellular stainings (iNOS and ARG-1).

19Supplementary Figure S2: Anti-transgene T cell responses and transgene

20expression in MHCII-/-, 2m-/-, µMT-/- and C57BL6 mice

21Muscle transgene expression and splenic T cell responses were measured following

22injection of PBS or of 5x109 vg of rAAV1_CMV_SGCA-HY in the left TA of different

23strains of mice. Data show values obtained for at least 6 mice per condition and per

24time point. (a) Expression of SGCA mRNA in the TA was measured at indicated time

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

2efficacy in this model. Results represent the average and SD of mice tested in the

3group. (b) At the time points indicated on the X axis (14 or 21 days post vector

4injection) the levels of Dby-specific CD4+T cell responses (left panel) and Uty-

5specific CD8+T cell responses (right panel) were measured by IFN-ELISPOT

6following peptide in vitro stimulation of spleen cells. Each dot represents data from

7individual mice after exclusion of aberrant data points, and horizontal bars indicate

8the average value.

9 10 11Supplementary Figure S3: Anti-transgene T cell responses, muscle integrity

12and cellular infiltration in CD4-/-Sgca-/- and CD8-/-Sgca-/- mice compared to

13CD4-/- and CD8-/- mice

14C57BL/6 mice, CD4-/- mice, CD8-/- mice, CD4-/-Sgca -/- mice, CD8-/-Sgca -/- mice,

15and Sgca-/- mice were injected IM into the TA with 5x109 vg of rAAV1_CMV_SGCA-

16HY vector, then anti-transgene immune responses and muscle histology were

17analyzed over time. (a) At the indicated times in the X axis (14, 21, and 42 days post

18vector injection) spleen CD4 T cell (left panels) and CD8 T cell (right panels)

19responses were measured by IFN-ELISPOT in the indicated strains of mice. Dots

20represent individual mouse data and horizontal bars indicate the average value. (b)

21At day 14, muscle histology was analyzed by HE staining in the indicated groups.

22Images are representative of 1 experiment with 4 mice analyzed per group. (c) At

23day 14, muscle cryosections of mice in the indicated groups were immunostained to

24detect CD3+ T cells (red) and CD11b+ myeloid cells (red). Nuclei were stained with

25DAPI (blue). The scale bar is 100 µm. The upper right inserts reflect a magnification

26of the selected areas.