Panel A and B: Transplanted hearts were harvested from (A) rejecting mice or from (B) tolerant C3H mice at POD 100 and subsequently immunostained for Foxp3 (red) and FGL2 (green) (magnification 200×). Nuclei were visualized with DAPI (blue). Tolerant mice had significantly increased numbers of Foxp3⁺ Treg (white arrow). Whereas Foxp3⁺ Treg from tolerant mice largely expressed FGL2, Foxp3⁺, Treg in rejecting mice did not express FGL2. Inset shows a FGL2⁻ Treg in a rejecting allograft and a FGL2⁺ Treg in a tolerant allograft (magnification 1000×). Panel C–E: Morphometric analysis of the immunostained sections was performed using a Definiens analysis assessing the (C) number of Foxp3⁺/μm²,(D) FGL2⁺/μm², and (E) Foxp3⁺FGL2⁺/μm². Cardiac myocytes were excluded from analysis using size exclusion. Lymphocytes were defined based on size of 10 microns or less. The morphometric analysis of heart allografts is from 6 rejecting mice, 7 tolerant mice, and 3 syngeneic mice, with 4 serial sections taken at multiple levels of the heart. Data are expressed as the mean±SEM. Statistical significance was assessed using Student’s t test. Copyright (2014) John Wiley and Sons. Used with permission from Urbanellis P, Shyu W, Khattar R, et al.⁴⁹