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. 2019 Aug 1;68(10):1990–2003. doi: 10.2337/db19-0239

Figure 2.

Figure 2

CCL21 local secretion by β-cells does not prevent insulitis. A: Light and confocal microscope images of pancreatic sections of 4-week-old nontransgenic and Ins2-CCL21 NOD mice stained with hematoxylin and eosin (left) or with insulin (red) and glucagon (green) (right) antibodies. Nuclei were counterstained with DAPI (white). Scale bar, 50 μm. B: Insulitis quantification (grade 1–5) of pancreatic sections of 4- (n = 6), 8- (n = 5), and 12-week-old (n = 5) nontransgenic and age-matched Ins2-CCL21 NOD mice. Four-week-old nontransgenic NOD mice: 86% grade 1, 2% grade 2, 11% grade 3, 1% grade 4, and 1% grade 5. Four-week-old Ins2-CCL21 NOD mice: 62% grade 1, 9% grade 2, 22% grade 3, 6% grade 4, and 1% grade 5. C: Quantification of pancreatic infiltrates of 4-week-old nontransgenic (n = 5) and Ins2-CCL21 NOD (n = 5) mice by flow cytometry. Data are indicated as % CD4+ live cells. Tn: naive T cells (CD44−CD127−CD62L+) transgenic: 38.05 ± 0.08, nontransgenic: 19.45 ± 4.62, P < 0.001; Tef (effector T cells): CD44+CD127−; Tm (memory T cells): CD44+CD127+. D: Quantification of blood leukocytes of 4-week-old nontransgenic (n = 5) and Ins2-CCL21 NOD (n = 5) mice by flow cytometry. Data are indicated as % CD45+ live cells. CCR7+: transgenic, 13 ± 11; nontransgenic, 3 ± 3 (P < 0.05). E: Confocal images of pancreatic sections of 4-week-old nontransgenic and Ins2-CCL21 NOD mice stained with insulin (cyan), B cells (B220, red), and T cells (CD3, green) antibodies; insulin (red) and CD4 (green); insulin (red) and CD8 (green); insulin (cyan), T cells (CD3, green), and Tregs (CD3+FoxP3+, red) antibodies. Scale bar, 50 μm. F: Confocal images of skin-draining LN sections of 4-week-old Ins2-CCL21 NOD mice immunostained with B220 (red) and CD3 (green) at two different magnifications. Scale bar, 50 μm. G: Quantification of Treg density in islets from pancreatic sections of 4-week-old nontransgenic (n = 4) and age-matched Ins2-CCL21 (n = 4) NOD mice. H: Quantification of pancreatic infiltrates of 12-week-old nontransgenic littermate (n = 6) and Ins2-CCL21 NOD (n = 6) mice by flow cytometry. Data are indicated as total number of live cells. wks, weeks.