(A–C) Gross image (n = 6) (A), H&E staining (n = 6) (B), and CCSP immunofluorescence staining (n = 2) (C) of left lung transplant from BALB/c donor into CSB-treated B6 recipient, at least 30 days after engraftment. Scale bars: 100 μm. (D) Two-photon intravital microscopy of BALB/c lung at least 30 days after transplantation into CSB-treated B6 CD11c-EYFP/Foxp3-GFP recipient (n = 3). CD11c⁺ cells are yellow, Foxp3⁺ cells are green, collagen appears blue owing to second-harmonic generation (SHG), and vessels are red following i.v. injection of quantum dots. Scale bar: 20 μm. (E–I) Representative flow cytometry plots and quantification of abundance of Foxp3-expressing CD45⁺CD90.2⁺CD4⁺CD8^– T cells (E), CD25-expressing CD45⁺CD90.2⁺CD4⁺CD8^–Foxp3⁺ T cells (CD25 expression determined based on isotype control staining) (F), effector memory (CD44^hiCD62L^lo), central memory (CD44^hiCD62L^hi), and naive (CD44^loCD62L^hi) CD45⁺CD90.2⁺CD4⁺CD8^–Foxp3⁺ T cells (G), CD69-expressing CD45⁺CD90.2⁺CD4⁺CD8^–Foxp3⁺ T cells (H), and (I) PD-1–expressing CD45⁺CD90.2⁺CD4⁺CD8^–Foxp3⁺ T cells in BALB/c lung and recipient spleen at least 30 days after transplantation into CSB-treated B6 recipients (n = 4). At least 30 days after transplantation of BALB/c lungs into CSB-treated B6 Foxp3-GFP mice, CD45⁺CD90.2⁺CD4⁺CD8^–GFP⁺ cells were sorted and processed for TCR and genome sequencing (n = 2). (J) Gini coefficient (0 represents maximal diversity) comparison of clonal expansion (left) and number of cells in the top 11 shared clonotypes between Foxp3⁺ cells in tolerant lung allografts and recipient spleens (right) (triangles and circles denote individual mice). (K) Heatmap of most highly differentially expressed genes in Foxp3⁺ cells between tolerant lung allografts and recipient spleens (2 pooled lungs and spleens). (L–N) Gross image (n = 4) (L), H&E staining (n = 4) (M), and CCSP immunofluorescence staining (n = 2) (N) of BALB/c lungs 30 days after transplantation into CSB-treated B6 Foxp3-YFP-Cre Areg^fl/fl mice. Scale bars: 100 μm. Results expressed as mean ± SEM. **P < 0.01, ***P < 0.001. d30, day 30; q-dot, quantum dot; Tcm, T central memory; Tem, T effector memory; Treg, regulatory Foxp3⁺ T cell; TX, transplanted lung.