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. 2020 Apr 9;10:6156. doi: 10.1038/s41598-020-62881-4

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© The Author(s) 2020

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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Optimization of therapy with LL-CFA/I. Four wk-old NOD females were dosed with LL-CFA/I or PBS in varying doses or frequencies. At 11 wks of age, 1 wk after the final dose, mice were euthanized and insulitis levels examined. (A) Frequencies of observed insulitis scores of mice dosed with 5 × 10⁷ CFUs every 1 (n = 17), 2 (n = 17), or 3 wks (n = 7); ***p < 0.00005 as indicated between PBS and LL-CFA/I treated every 2 wks and between mice treated weekly and every 2 wks with LL-CFA/I. (B) Frequencies of observed insulitis scores of mice dosed with 1 × 10⁸ (n = 7), 5 × 10⁷ (n = 17), or 1.25 × 10⁷ (n = 7) CFUs of LL-CFA/I every 2 wks; ***p < 0.00005 as indicated between PBS and 5 × 10⁷ CFUs LL-CFA/I. Insulin-specific (C) reduction in diabetogenic CD4⁺ and (D) CD8⁺ T cells from females treated with the various doses of LL-CFA/I were observed using tetramers specific to InsB:9-23 and InsB:15-23 (n = 5/group), respectively; ^*p < 0.05 between PBS and LL-CFA/I (5 × 10⁷ CFUs). (E) Frequencies of observed insulitis scores (n = 8/group), left, and summary of insulin-specific CD4⁺ and CD8⁺ T cells from mice dosed with 5 × 10⁷ CFUs of LL-CFA/I, LL vector, or PBS every 2 wks (n = 7/group), right; ⁺p < 0.05 and ⁺⁺⁺p < 0.00005 between LL-CFA/I and PBS; ^#p < 0.05 and ^###p < 0.00005 between LL-CFA/I and LL vector^. Data are represented as means ± SEM.