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. 2021 Aug 10;12:720133. doi: 10.3389/fimmu.2021.720133

Figure 1.

Figure 1

Rapid progression to overt diabetes after acute Treg cell ablation in adult NOD.DEREG females. Mice were either left untreated or injected with DT on two consecutive days, and subjected to further analysis on day 14, unless otherwise stated. (A) Efficiency of Treg cell ablation. Representative dot plots of CD4-gated cells from peripheral blood of 16-week-old NOD.DEREG females before (day 0) and after (day 3) injection with DT. Numbers in dots plots indicate mean percentage ± SD of 8 mice (unpaired t-test, p < 0.001) from a single experiment representative of 6 experiments performed (5-9 mice per experiment). (B–E) Pancreatic pathohistological changes. Histological sections were scored as described in Materials and Methods. (B) Insulitis scores of 4-5-week-old female (left, n = 10) and male (right, n = 9) NOD.DEREG mice. As all mice remained normoglycemic by day 7 after the first DT injection, two additional doses of DT were administered (day 7 and 8) prior to histology on day 14. (C) Representative histology and (D) insulitis score of 8-9-week-old NOD.DEREG females (8-10 mice per group). Untreated NOD.RAG (n = 4) and DT-injected DEREG– NOD (n = 5) females were included as controls. (E) Insulitis scores of 16-week-old DEREG females on different genetic backgrounds (4-7 mice per group). (F) Expression of mRNA encoding immune effector molecules with known function in autoimmune β cell destruction. Freshly isolated mRNA from pancreas and pLN (pooled from 4-5 mice per group) of 10-12-week-old NOD.DEREG females was subjected to real-time RT-PCR using β-Actin for normalization. Heat map (row normalized) shows mean values of triplicate samples from a single experiment representative of at least two independent experiments performed. Blue and red represent lowest and highest gene expression values, respectively. Pancreas, Unpaired t-test: p < 0.001: Gzma, Gzmb, Prf1, Tnf; p ≤ 0.01: Ifng. pLN, Unpaired t-test: p <0.001: Gzma, Gzmb; p ≤ 0.01: Prf1; not significant: Tnf, Ifng. (G, H) Manifestation of overt diabetes. (G) Cohorts of 16-week-old, initially non-diabetic NOD.DEREG mice were repeatedly injected with 2 doses of DT at 5-day-intervals, as indicated. Blood glucose levels (BGL) were determined at least three times per week. (H) Diabetes incidence of female NOD.DEREG mice. Mice were considered diabetic at blood glucose levels above 200 mg/dl on at least two consecutive measurements or with blood glucose levels once above 400 mg/dl. Data are from a single experiment (n = 10) representative of > 8 experiments performed. Note that the diabetes incidence of male NOD.DEREG mice is depicted in Supplementary Figure S3.