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. 2025 Jun 24;152(8):537–554. doi: 10.1161/CIRCULATIONAHA.125.073691

Figure 2.

Figure 2.

Graft CD34-lineage T cells mainly originate from the thymus and are drained by a lymphatic network. A, The proportion of early T cell progenitors (CD4−CD8−CD25−CD44+CD117+) among total thymocytes in the indicated groups via FC. B, The proportion of early T cell progenitors among total lymph node (LN) cells via FC. C, Schematic of the experiment design. Cd34-CreERT2;Rosa26-tdTomato mice were administered tamoxifen for 3 weeks, followed by transplantation of allogeneic grafts and thymectomy (allogeneic graft + thymectomy) or sham surgery (allogeneic graft + sham). Samples were collected 4 weeks after surgery for FC analysis (n=6 per group). D, Quantification of T cell subsets within an allograft artery in the indicated groups (n=3 per group). E, Representative immunofluorescence (IF) images of allografted arteries in the indicated groups stained for CD3 (green), B220 (grey), tdTomato (red), and 4’,6-diamidino-2-phenylindole (DAPI; blue) to assess T cell localization in tertiary lymphoid organs. F, Quantification of tdTomato+CD3+ T cells (left) in total arteries via IF staining (n≥30 fields per group from 6 mice, each spot represents the average of one mouse), quantification of tertiary lymphoid organ counts (middle) in allograft arteries via IF staining, and intima-to-media ratio (right) in allograft arteries in the indicated groups. G, Schematic illustrating the study design for LN dissection with or without lymphangiogenesis blockage in the allograft model. I, C57BL/6J mice underwent LN dissection and served as allograft recipients for vascular transplantation. The VEGFR-3 inhibitor was continuously administered, and tissues were harvested 4 weeks after vascular transplantation. II, C57BL/6J mice underwent LN dissection and served as allograft recipients for vascular transplantation, with tissues harvested 4 weeks after vascular transplantation. III, C57BL/6J mice served as allograft recipients for vascular transplantation, with tissues harvested 4 weeks after vascular transplantation. H, Quantification of the tertiary lymphoid organ counts in the indicated groups (n=6). I, Quantification of the ratio of CD3+ cells among total cells of a remodeled artery via IF staining in the indicated groups (n≥30 fields per group from 6 mice, each spot represents the average of one mouse). J, Schematic illustrating that Cd34-CreERT2;Rosa26-tdTomato mice with tdTomato fluorescence-labeled cells were used as receptors for bone marrow transplantation; bone marrow cells were cleared by irradiation, and C57BL/6J mice served as donors. The chimeric mice served as recipients for vascular transplantation. Throughout the procedure, tamoxifen was maintained to fluorescently label CD34-lineage cells, and the VEGFR-3 inhibitor was administrated to suppress lymphangiogenesis, with tissue harvested 4 weeks after transplantation for analysis. K, The proportion of early T cell progenitors (CD4−CD8−CD25−CD44+CD117+) among total thymocytes in the indicated groups via FC. L, Quantification of the ratio of tdTomato+ cells among total cells of a remodeled artery via FC in the indicated groups (n=6 per group). M, Representative images showing the distinct populations of CD34-lineage cells (tdTomato+), T cells (CD3+), and B cells (B220+) via IF staining in the indicated groups. N, Quantification of the T cells (top) or B cells (bottom) among total cells of a remodeled artery via IF staining in the indicated groups (n≥30 fields per group from 6 mice, each spot represents the average of one mouse). O, Schematic illustrating that CD34-lineage cells from LNs, transported by lymph vessels, contributed to vascular remodeling. Data shown are mean±SD (A, B, D, F, H, I, K, L, and N). For A, B, D, F, and K, normality of the data was analyzed by D’Agostino-Pearson omnibus test, and data that passed the normality tests were tested with standard unpaired Student t test with Welch’s correction. For H, I, L, and N, data were first analyzed for normality test by a Shapiro-Wilk test, followed by 1-way ANOVA with Tukey test. P<0.05 was considered to be statistically significant. A indicates adventitia; AG, allogeneic graft; BM, bone marrow; BMT, bone marrow transplantation; ETP, early T cell progenitor; FC, flow cytometry; I, intima; LN, lymph node; LND, lymph node dissection; M, media; PBMC, peripheral blood mononuclear cell; Tam, tamoxifen; Thy, thymocyte; TLO, tertiary lymphoid organ; and VEGFR, vascular endothelial growth factor receptor.