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. 2018 Dec 4;26(1):40–56. doi: 10.1038/s41434-018-0052-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — AAV2/8-insulin-based therapy elicits a cellular immune response against the vector and the transgene in NOD diabetic mice. Livers of NOD mice treated with AAV2/8-HLP-hINSco (indicated with AAV2/8) show signs of T cell infiltration observed in pre-diabetic NOD mouse pancreata. a Pancreas sections from a 8-week-old control NOD female pancreas (a, top panel), a 30-day AAV2/8-insulin-treated C57BL/6 (a, middle panel) and a 30-day AAV2/8-insulin-treated NOD (a, bottom panel) were stained for insulin (red) and glucagon (green), and CD3 (yellow) to show β-cell destruction in treated mice. b Livers from AAV2/8-HLP-hINSco -injected C57BL/6 (b, left) and NOD (b, right) mice were stained for insulin and CD3 to test for T cell infiltration. c Livers from AAV2/8-HLP-hINSco-injected NOD mice were stained for insulin and CD8. Blue shows nuclear DAPI staining. Scale bar 50 μm. d, e Ex vivo stimulated splenocytes from AAV2/8-HLP-hINSco -treated NOD mice and C57BL/6 controls for IFN-γ ELISPOT assay. Cells from NOD and C57BL/6 mice were harvested after 30 days (d) or more than 200 days (e) from the day of the injection of the AAV2/8-insulin vector. Cells were stimulated in vitro for 40 h with CD8⁺ immunodominant peptides (either InsB_15–23 peptide for NOD or insulin K^b-restricted epitope A_12–21 containing peptide for C57BL/6) or the AAV8 capsid-specific CD8⁺ T cell peptide NSLANPGIA and the number of resulting IFN-γ spots was counted with an ELISPOT reader. f Levels of anti-inflammatory cytokines in NOD mice compared to C57BL/6. Splenocytes from C57BL/6 and NOD mice were harvested at the end of a 30-day treatment with AAV2/8-HLP-hINSco 5 × 10⁹ vg and stimulated with PMA/Iono for 48 h. Supernatants were then collected and tested for IL-10 production. g Mouse anti-AAV8 ELISA performed on blood plasma of diabetic, healthy and 5 × 10⁹ vg AAV2/8-HLP-hINSco-treated NOD and C57BL/6 mice. Treated mice were tested for anti-AAV8 antibodies 30 days after the beginning of the therapy. The absorbances at 405 nm correlate with the concentration of the antibody. *(p ≤ 0.05), **(p ≤ 0.01), ***(p ≤ 0.001), unpaired Student’s t-test. Data shown are expressed as mean ± SE and are representative of two independent experiments

Fig. 4 — AAV2/8-insulin-based therapy elicits a cellular immune response against the vector and the transgene in NOD diabetic mice. Livers of NOD mice treated with AAV2/8-HLP-hINSco (indicated with AAV2/8) show signs of T cell infiltration observed in pre-diabetic NOD mouse pancreata. a Pancreas sections from a 8-week-old control NOD female pancreas (a, top panel), a 30-day AAV2/8-insulin-treated C57BL/6 (a, middle panel) and a 30-day AAV2/8-insulin-treated NOD (a, bottom panel) were stained for insulin (red) and glucagon (green), and CD3 (yellow) to show β-cell destruction in treated mice. b Livers from AAV2/8-HLP-hINSco -injected C57BL/6 (b, left) and NOD (b, right) mice were stained for insulin and CD3 to test for T cell infiltration. c Livers from AAV2/8-HLP-hINSco-injected NOD mice were stained for insulin and CD8. Blue shows nuclear DAPI staining. Scale bar 50 μm. d, e Ex vivo stimulated splenocytes from AAV2/8-HLP-hINSco -treated NOD mice and C57BL/6 controls for IFN-γ ELISPOT assay. Cells from NOD and C57BL/6 mice were harvested after 30 days (d) or more than 200 days (e) from the day of the injection of the AAV2/8-insulin vector. Cells were stimulated in vitro for 40 h with CD8⁺ immunodominant peptides (either InsB_15–23 peptide for NOD or insulin K^b-restricted epitope A_12–21 containing peptide for C57BL/6) or the AAV8 capsid-specific CD8⁺ T cell peptide NSLANPGIA and the number of resulting IFN-γ spots was counted with an ELISPOT reader. f Levels of anti-inflammatory cytokines in NOD mice compared to C57BL/6. Splenocytes from C57BL/6 and NOD mice were harvested at the end of a 30-day treatment with AAV2/8-HLP-hINSco 5 × 10⁹ vg and stimulated with PMA/Iono for 48 h. Supernatants were then collected and tested for IL-10 production. g Mouse anti-AAV8 ELISA performed on blood plasma of diabetic, healthy and 5 × 10⁹ vg AAV2/8-HLP-hINSco-treated NOD and C57BL/6 mice. Treated mice were tested for anti-AAV8 antibodies 30 days after the beginning of the therapy. The absorbances at 405 nm correlate with the concentration of the antibody. *(p ≤ 0.05), **(p ≤ 0.01), ***(p ≤ 0.001), unpaired Student’s t-test. Data shown are expressed as mean ± SE and are representative of two independent experiments