Panel A shows flow cytometry plots of Foxp3 positive Tregs in the thymus and spleen of wild-type (WT) and knockout (KO) mice. The plots display CD8 and CD25 markers for thymus and CD3 and CD25 markers for spleen, with percentages of Foxp3 positive cells indicated. Panel B presents bar graphs comparing the percentage of Foxp3 positive cells among CD4 SP thymocytes, spleen, and pancreatic lymph node (PLN) between WT and KO mice. Panel C and D show bar graphs of soluble CD137 levels produced by cultured splenic Foxp3+ Tregs and circulating soluble CD137 in serum, respectively, with significant differences indicated. Panel E includes representative histology images of insulitis in prediabetic mice and a bar graph of mean insulitis scores, showing differences between WT and KO mice. Panels F, G, and H display line graphs of type 1 diabetes (T1D) incidence over time in female and male littermates with different genotypes, highlighting significant differences. Panel I shows a line graph of T1D incidence in NOD.Rag1 knockout mice receiving splenocytes from different donors. Panels J and K present histology images and bar graphs quantifying inflammation in lacrimal and salivary glands, respectively, with significant differences noted.
CD137 deletion in Foxp3 + Tregs leads to exacerbated diabetes development and Sjögren’s disease. (A and B) Percentages of Foxp3+ Tregs in the thymus, spleen, and PLN of 6- to 8-wk-old Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) female mice. (A) Representative flow cytometry plots. The plots of the KO spleen are also shown in Fig. S1 D. (B) Summarized results from three experiments. (C) Soluble CD137 produced by cultured Cre+-Tnfrsf9+/+ and Cre+-Tnfrsf9fl/fl splenic Foxp3+ Tregs (n = 3). *P < 0.05 by an unpaired t test. (D) Circulating soluble CD137 in 7- to 9-wk-old Cre+-Tnfrsf9+/+ and Cre+-Tnfrsf9fl/fl males. ****P < 0.0001 by an unpaired t test. (E) Insulitis in 8- to 10-wk-old prediabetic Cre+-Tnfrsf9fl/fl (KO) and Cre+-Tnfrsf9+/+ (WT) mice. Representative islet histology images and summarized mean insulitis scores are shown. The scale bar is 100 µm. *P < 0.05 by an unpaired t test. (F and G) T1D incidence of Cre+-Tnfrsf9+/+, Cre+-Tnfrsf9fl/+, Cre+-Tnfrsf9fl/fl, and Cre−-Tnfrsf9fl/fl female (F) and male (G) littermates. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001 by a log-rank test. (H) T1D incidence of CD137L-deficient (Tnfsf9−/−) Cre+-Tnfrsf9+/+, Cre+-Tnfrsf9fl/+, and Cre+-Tnfrsf9fl/fl female littermates. (I) T1D incidence of NOD.Rag1−/− females receiving CD137L-deficient (Tnfsf9−/−) Cre+-Tnfrsf9fl/fl or Cre−-Tnfrsf9fl/fl littermate splenocytes, combined from two transfer experiments. ***P < 0.0005 by a log-rank test. (J and K) Quantification of inflammation in lacrimal (J) and salivary (K) glands from 8- to 10-wk-old prediabetic Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) mice. Representative histology images of male lacrimal and female salivary glands and summarized focus scores are shown. The scale bar is 1 mm. *P < 0.05; **P < 0.005 by an unpaired t test.