Gr1-expressing myeloid cells accumulate in the pancreatic lesions of CD101^+/+ NOD.B6 <i>Idd10</i> mice.

Representative FACS dot plots of Gr1- and/or CD11b-expressing myeloid cells from the pancreas (A) and the pancreatic lymph nodes (B) of 14-week-old CD101+/+ and CD101−/− NOD.B6 Idd10 mice are displayed (upper panels). The percentages of Gr1-positive and Gr1-negative cells among CD11b-expressing myeloid cells were compiled from 8 individual CD101−/− NOD.B6 Idd10 and CD101+/+ NOD.B6 Idd10 mice (A+B, lower panels). Statistical significance was determined using Mann-Whitney tests (*, p<0.05; **, p<0.01; ***, p<0.001). Error bars indicate the SD of the mean.</p

Deletion of <i>Cd101</i> exon 1 leads to a loss of protein expression.

Representation of the Idd10 and Idd18 regions and the NOD congenic strains used in this study (A). Lack of CD101 expression in CD101−/− NOD.B6 Idd10 (B) and CD101−/− NOD.B6 Idd10/18 mice (C). CD11b-positive cells in the spleens of CD101−/− NOD.B6 Idd10 (B) and CD101−/− NOD.B6 Idd10/18 mice (C) as well as CD101-expressing control littermates were evaluated for their CD101-expression by flow cytometry. Representative FACS dot plots are displayed.</p

Protection from T1D is associated with an accumulation of CD101-expressing myeloid cells.

Gr1-positive cells have reduced accumulation and CD101 expression (A-C) in the pancreatic lymph nodes of NOD scid recipients of CD101−/− as compared to CD101+/+ NOD.B6 Idd10 T cells. Representative FACS dot plots of Gr1-expressing myeloid cells in the pancreatic lymph nodes are displayed (A) as well as the percentages of Gr1-expressing cells (B) and the distribution of CD101 expression on them (C) compiled from 5 recipients in each cohort. Gr1-expressing myeloid cells from recipients of CD101−/− donor cells express significantly less TGF-β (D) and IL-10 (E) (N = 4 in each cohort for D and E). TGF-β and IL-10 mRNA copies were determined by RT-qPCR in purified Gr1-expressing cells. The ratios of the TGF-β and IL-10 mRNA copies relative to the HPRT copies were calculated. Statistically significant differences were determined using Student´s t-tests (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Error bars indicate the SD of the mean.</p

CD101-positive T cells accumulate in the pancreatic lymph nodes of NOD.B6 <i>Idd10</i> mice.

The expression of CD101 on TCRβ-positive T cells (A) and FoxP3-positive Tregs (B-E) was assessed by flow cytometry in the spleens (A, B), pancreatic (panc) (A-E) and popliteal (pop) (A, B) lymph nodes. The compiled data of five individual 4-8-week-old NOD.B6 Idd10 mice (A, B) are displayed. Representative FACS plots are shown in S2 Fig. The expression of FoxP3 and CD101 was assessed on T lymphocytes expressing the β-chain of the TCR and CD4 (C-E). Representative FACS plots for NOD.B6 Idd10 (upper panel) and NOD (lower panel) mice are displayed (C). The percentage of CD101-expressing Tregs (D) as well as the percentage of FoxP3-positive Tregs (E) was compiled of 4 (week 4, 10, 12 and 14 of age) or 5 (week 6 and 8) individual mice per time point. Comparisons between groups were performed using the Mann–Whitney nonparametric test (*, p<0.05; **, p<0.01; ***, p<0.001). Error bars indicate the SD of the mean.</p

CD101-expression protects NOD.B6 <i>Idd10</i> and NOD.B6 <i>Idd10/18</i> mice from T1D.

The frequency of T1D was assessed by the analysis of urinary glucose concentration (A-D) and histopathologic lesions (E) in the indicated number of females in parental NOD mice, CD101+/+ NOD.B6 Idd10 mice and CD101−/− NOD.B6 Idd10 mice (A-C) as well as in CD101+/+ NOD.B6 Idd10/18 mice and CD101−/− NOD.B6 Idd10/18 mice (D). Mice were derived from homozygous breeders except in the case of mice shown in (B) where progeny of heterozygous breeders were monitored for T1D. T1D incidence was assessed at Taconic (A+B, performed contemporaneously so the NOD control group shown in A is also the NOD control group for the groups of mice shown in B) and at CCHMC (C+D, performed contemporaneously so the NOD control group shown in C is also the NOD control group for the groups of mice shown in D). Histopathologic analysis of pancreata was performed in nondiabetic 8–10 week-old CD101−/− NOD.B6 Idd10 (n = 5), CD101+/+ NOD.B6 Idd10 (n = 6), CD101−/− NOD.B6 Idd10/Idd18 (n = 5) and CD101+/+ NOD.B6 Idd10/Idd18 (n = 4) female mice (E). For T1D frequency comparisons Kaplan–Meier survival curves were plotted for each mouse strain, and statistical significance was determined by log rank test (*, p<0.05; **, p<0.01; ****, p<0.0001). For insulitis comparisons a one-tailed t-text was used (*, p<0.05; **, p<0.01). Error bars indicate the SD of the mean.</p

Protection from T1D is associated with a restriction of T cell expansion and an accumulation of Tregs.

The frequency of T1D was assessed by the analysis of urinary glucose concentration (A) in NOD scid recipients upon transfer of 5x107 T cells from 6-8-week-old CD101+/+ NOD.B6 Idd10 or CD101−/− NOD.B6 Idd10 mice. 16 female recipients in each cohort were analyzed until day 100 after the adoptive T cell transfer. Kaplan–Meier survival curves were plotted for each mouse strain, and statistical significance was determined by log rank test (*, p+/+ NOD.B6 Idd10 and CD101−/− NOD.B6 Idd10 donors were CFSE-labelled and transferred into NOD scid recipients. 2 (upper panels), 3 (middle panels) and 4 weeks (lower panels) after transfer the division of T cells in the pancreatic lymph nodes was assessed by the analysis of CFSE dilution. The percentages of CFSE-negative cells reflecting the upper left gates in panel B were determined from all CD3-positive T lymphocytes and compared between recipient mice of CD101+/+ or CD101−/− donors (C). Representative FACS dot plots (B) and summaries (C) of donor T cells obtained from the pancreatic lymph nodes of 3–5 NOD scid recipients are displayed. All cells were pre-gated on CD45. Tregs are reduced in the pancreatic lymph nodes of NOD scid recipients of CD101−/− as compared to CD101+/+ NOD.B6 Idd10 T cells (D). The percentage of FoxP3+ cells in total CD4 T cells are shown (n = 5 in each cohort). To assess statistical significance a one-tailed t-text was used (*, p<0.05; **, p<0.01).</p

B6-derived <i>Idd10</i> region in myeloid cells as well as T cells provides the greatest degree of protection from T1D.

The frequency of T1D was assessed by the analysis of urinary glucose concentration in NOD Idd10 scid and NOD.B6 scid recipients upon transfer of 5x107 T cells from 6-8-week-old CD101+/+ NOD.B6 Idd10 (A) or CD101−/− NOD.B6 Idd10 mice (B). The indicated numbers of female recipients in each cohort were analyzed until day 100 after the adoptive T cell transfer. Kaplan–Meier survival curves were plotted for each mouse strain, and statistical significance was determined by log rank test (*, p<0.05).</p

Proliferation of adoptively transferred BDC2.5 Tg CD4 T and NY8.3 tg CD8⁺ T cells in pancreatic LN and spleen of anti PDL1 treated NOD and <i>Idd3/10/18</i> mice.

<p>a) Collective data from 3 out of 8 experiments of the percentage of CFSE-dividing cells (gated on CD4⁺ Vβ4⁺) are shown. Horizontal lines express mean value. For CD4⁺ T cells pLN, NOD, control vs. treated p = 0.0276; pLN, <i>Idd3/10/18</i>, control vs. treated p = 0.0002. b) A representative experiment from 3 performed is shown. For CD8 T cells pLN, <i>Idd3/10/18</i> control vs. treated p = 0.0321, spleen <i>Idd3/10/18</i> control vs. treated p = 0.0185.</p

Incidence of diabetes in NOD congenic mouse strains undergoing anti PDL1 treatment.

<p>Treatment was started at 10 weeks of age. a): Incidence of diabetes in <i>Idd5</i> and sub-congenics <i>Idd5.1</i>, <i>Idd5.2</i>, <i>Idd5.3</i> and <i>Idd5.2</i>/<i>5.3</i> until day 30 after anti PDL1 treatment. In <i>Idd5</i> mice (n = 16) 62.5% developed diabetes, in <i>Idd5.1</i> (n = 15) 66.6%, in <i>Idd5.2</i> (n = 20) 80%, in <i>Idd5.3</i> (n = 8) 87.5% and in <i>Idd5.2</i>/5.3 (n = 23) 66.5%. NOD mice (n = 28) had a 92.5% incidence of diabetes by day 30. All control treated mice did not develop diabetes. b): In <i>Idd9</i> (n = 26) 46.15% developed diabetes, in <i>Idd9.1</i> (n = 20) 95%, in <i>Idd9.2</i> (n = 30) 56.6% and in <i>Idd9.3</i> (n = 21) 90.5%. c): <i>Idd3</i> (n = 17) developed diabetes at a rate of 50%, <i>Idd10/18</i> (n = 18) at 94.1%, <i>Idd3/10/18</i> (n = 26) at 30.8%. d): In <i>Idd3/5</i> (n = 16) 0% of anti - PDL1 treated mice developed diabetes, in <i>Idd3/5.2</i>/5.3 (n = 13) 15.4%. Statistics and cumulative incidence for the strains are shown in separate Tables for Figure 1. P-values were calculated using Log-rank (Mantel-Cox test).</p

CD101-expression sustains CD8-positive T lymphocytes and Tregs.

Representative profiles of inflammatory infiltrates in pancreatic islets of CD101+/+ and CD101−/− NOD.B6 Idd10 mice show that they consist mainly of TCRβ+ T lymphocytes (A). The accumulation of CD8- over CD4-positive T lymphocytes is enhanced in CD101-expressing NOD.B6 Idd10 congenic mice (B). Representative FACS dot plots of T cells from pancreatic islets which were pre-gated on TCRβ and the summary of CD4- and CD8-positive T lymphocytes from five individual, 14 week-old female mice per strain are displayed (C+D). The expression of FoxP3 was assessed on lymphocytes expressing the β-chain of the TCR and CD4 in the pancreatic lymph nodes (E) and spleens (F) of the indicated mouse strains. The results of 13 individual, 10–13 week-old female mice were summarized from three independent experiments. Statistical significant differences were determined using Mann-Whitney tests (*, p<0.05; **, p<0.01; ***, p<0.001). Error bars indicate the SD of the mean.</p