Systemic delivery of miR-29b protects against adoptive transfer of T1D <i>in vivo</i>.

Abstract

<p>Ins-HA mice were treated intravenously with miR-29b, miR-127, HBS buffer or DOTAP alone, eighteen hours before receiving HA-specific CTLs from CL4-TCR mice. (A) Recipients were monitored for diabetes development for at least one month. The survival curves and table summarize the results of five independent experiments after transfer of 1 to 10×10⁵ cells, with miR-29b -injected mice as filled symbols, and HBS-injected mice as empty symbols. The table indicates, for each group, the percentage of final cumulative diabetes incidence and the number of diabetic mice among all mice in the group in brackets. A logrank test was performed for statistical significance of differences between Kaplan-Meier incidence curves. (B) Eighteen hours after miRNA injection, Ins-HA recipient mice received 5×10⁵ activated HA-specific CTLs, followed 48 h later by the intravenous administration of HA-pulsed «CFSE_high » and non-pulsed «CFSE_low » target cells mixed at a 1∶1 ratio. Splenocytes from recipient Ins-HA mice were analysed by flow cytometry, sixteen hours after target cell injection. The bar chart shows the compiled results of three independent experiments (n = 4–5 mice/group) as mean specific lysis ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 (Mann-Whitney). (C–E) Eighteen hours after miRNA injection, Ins-HA were transferred with 8×10⁵ activated HA-specific Thy1.1⁺ CTLs from CL4-TCR⁺Thy1.1⁺ mice. Four days later, spleens (C) and PLNs (D) were harvested from Ins-HA recipient mice and analysed by flow cytometry. Compiled results of two independent experiments are presented as the percentage of Thy1.1⁺ cells in individual mice gated on the CD3⁺ CD8⁺ T-cell population (n = 3–5 mice), and were confirmed in a third experiment. *<i>P</i><0.05 (Mann-Whitney). (E) Histological analysis of insulitis of pancreata: 0 = islet free of mononuclear cell infiltration (unfilled bars); 1 = peri-insular infiltration involving <10% of the islet area (punctuated bars); 2 = infiltration involving between 10% and 50% of the islet area (hatched bars); 3 = infiltration involving >50% of the islet area (black bars). The stacked vertical bar graph indicates the percentage of islets in each category described above. Results are presented as the mean percentage of n = 5 mice for miR-29b, n = 3 for miR-127, and n = 4 mice in the HBS group from three independent experiments. For each pancreas, an average insulitis score was calculated by adding up the score of each islet and dividing it by the total number of islets counted. Results show the individual insulitis scores for each group of recipient mice. *<i>P</i><0.05 (Kruskal-Wallis).</p