LSel^hi Tregs inhibit effectors in secondary lymphoid organs and GVHD target tissues. Lethally irradiated MHC class II–disparate bm12 and syngeneic B6 mice were infused with B6 BM. Cohorts of mice received 2 × 10⁶ B6 GFP⁺ CD25-depleted CD4⁺ effector T cells alone or with ex vivo activated and expanded B6 (non-GFP) LSel^hi or LSel^lo Tregs in a ratio of 1 to 3 Tregs. Mice were imaged 1, 2, and 3 weeks after BMT. Representative images from 1 of 3 mice per group at 1 and 2 weeks after BMT are shown. Data were replicated in a second experiment. (A) Images of lymphoid organs including inguinal and mesenteric LNs, Peyer patch, and spleen. (B) Images of GVHD target tissues including skin, gingiva, liver, lung, ileum, and colon. Stereomicroscope was set to × 3.2 zoom factor for inguinal LNs and skin; × 4.0 for mouth and mesenteric LNs; × 4.5 for colon; × 7.0 for Peyer patch, ileum, liver, and spleen; and × 10.0 for lung. Exposure times were optimized for GVHD control mice for each organ and identical times were used for all other groups. The following exposure times were used for organs shown: 154 msec, inguinal LN; 80 msec, mesenteric LN; 110 msec, Peyer patch; 870 msec, spleen; 1.0 sec, skin; 490 msec, gingiva; 910 msec, liver; 540 msec, lung; 110 msec, ileum; 120 msec, colon. Negative controls of mice not receiving GFP⁺ effectors to verify lack of autofluorescence resulted in dark images at indicated exposure times (not shown).