Acute GVHD is associated with lymphangiogenesis during experimental GVHD. (A) Representative images of increased lymphangiogenesis in the colon during GVHD (right) versus no GVHD (left) on day +15 after HSCT. Colon sections of HSCT recipients with GVHD and without GVHD were stained with Lyve1 antibody (green) and counterstained with the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI). (B) Quantification of the Lyve1 positive area in the colon on day +15 after HSCT (n = 5 per group). (C) Lymph vessels in the mesenteric window during GVHD (right) versus no GVHD (left) on day +15 after HSCT. Isolated mesenteric windows of HSCT recipients with GVHD versus no GVHD were stained with Lyve1 antibody (green) and counterstained with DAPI. (D) Quantification of the Lyve1 positive area in the mesenteric window on day +15 after HSCT (n = 4, no GVHD; n = 5, GVHD). (E-F) Quantification of lymphatic endothelial cells (gp38+ ECs) via fluorescence-activated cell sorter (FACS). Endothelial cells were isolated from mesenteric (n = 6 per group) (E) and peripheral (n = 3 per group) (F) lymph nodes of HSCT recipients with GVHD versus no GVHD on day +7 after HSCT. (G) Quantification of peripheral lymph node addressin (PNAd)–positive endothelial cells in peripheral lymph nodes of HSCT recipients with GVHD versus no GVHD (n = 3 per group). Error bars indicate mean ± SEM; significance was tested with an unpaired Student t test. (A-E) LP/J → C57BL/6 (1.5 × 107 BM cells, 2 × 106 splenic T cells). (F-G) C57BL/6 → BALB/c (5 × 106 BM cells, 1 × 106 splenic T cells).
