A proportion of lymph node CD4⁺CD8α⁺ double-positive (dp) T cells comprises FoxP3⁺ regulatory T cells.

(A) Representative results of FoxP3 expression in CD4+CD8α+ dp T cells of secondary lymphatic and non-lymphatic organs, and of CD4+CD8α+ dp thymocytes are shown. Two independent experiments with n = 4–10 dogs in total were performed. The numbers in the flow cytometry plots indicate percentages. Appropriate gating was confirmed by internal negative (CD8α+ single-positive (sp) T cells) and positive (CD4+ sp T cells) controls (B). Presented is the FoxP3 expression of CD4+ sp and CD8α+ sp T cells in canine lung being representative for all analyzed organs. (C) Proportions of FoxP3 expression of CD4+ sp (green hexagons) and CD4+CD8α+ dp (red triangles) T cells were analyzed in indicated organs (tLN: tracheobronchial lymph node, mLN: mesenteric lymph node, PP: Peyer’s patches, IEL: intraepithelial lymphocytes of the small intestine). Each symbol represents one individual dog, the horizontal bars indicate median values. The Mann-Whitney U test was performed to check for statistical significance between CD4+ sp and CD4+CD8α+ dp T cells (two-tailed; * p < 0.05, ** p < 0.01, *** p < 0.001).