Figure 5.

Depletion of Tregs stabilises the homoeostasis of CD4⁺ T cells in the lamina propria as well as of CD8α⁺ IELs in the duodenum. AP pancreatitis was induced in DEREG mice by partial duct ligation, T_regs were depleted by diphtheria toxin (DT) (n=11), controls receive PBS (n=7). (A) The efficiency of T_reg depletion with DT was verified by flow cytometry analysis of splenocytes. (B) Immunofluorescence detection of GFP-producing T_regs was performed in lymph nodes of DT-treated and PBS-treated mice. (C) Bar graphs show the ratios of CD25⁺ T-cells and GFP⁺/CD25⁺ T_regs analysed by flow cytometry of splenocytes. (D, E) Immunofluorescent labelling of CD3 and CD8α showed a significant persistence of the T-cell and IEL population in the absence of T_regs in DT-treated DEREG-mice compared with the PBS-treated group, (E). (F–N) Lymphocytes of the lamina propria and the epithelial layer were isolated from duodenum and analysed by flow cytometry. (F) Following T_reg-depletion, no GFP⁺/CD25⁺ T_regs were detected within the lamina propria. (G, H) Bar graph show the numbers of RORγt⁺/CD25⁺ Th17-cells (G) and TBET⁺/CD25⁺ Th1-cells (H). (I) The bar graph shows a higher ratio of CD4^hi expressing IELs in DT-treated mice. (J, K) In the CD4^hi IELs we observed a shift from GFP⁺ T_regs (J) to RORγt⁺ Th17-cells (K). (L) The ratio of CD8α⁺ IELs was not affected by T_reg-depletion. (M) Dot plot and bar graphs illustrate the ratio changes of TCRγδ⁺ IELs and TCRβ⁺ after DT-treatment. (N) Within the population of CD8α⁺ IELs we measured an increase in RORγt producing cells whereas TBET was not affected. Statistically significant differences were tested by unpaired student’s t-test for independent samples and significance levels of p<0.05 are marked by an asterisk. AP, acute pancreatitis; IELs, intraepithelial lymphocytes; PBS, phosphate-buffered saline.