CD103⁺ Treg cells potently ameliorated ongoing chronic GVHD. Sublethally irradiated BALB/c recipients were given 100 × 10⁶ DBA/2 donor spleen cells to induce chronic GVHD with proteinuria. Twenty days after HCT, the chronic GVHD recipients were infused with either CD103⁺CD4⁺ Treg cells from other recipients or freshly isolated or in vitro–activated CD25^hi natural Treg cells from DBA/2 donors (1-5 × 10⁶ each). The control recipients were given PBS only. The recipients were monitored daily for proteinuria and survival. (A) The percentage of recipients with reversal of proteinuria and percentage of survival. There were 6 to 12 recipients in each group combined from 2 or 3 replicate experiments. (B) The serum levels of anti-dsDNA IgG 20 days after infusion of CD103⁺ Treg cells (10⁶), in vitro–activated natural Treg cells (5 × 10⁶), or PBS. (C) Histopathology score (mean ± SE, n = 6). (D) Histology of skin, liver, and kidney of representative recipients before and after treatment with CD103⁺ Treg cells or in vitro–activated natural Treg cells. Tissues of BALB/c mice without HCT are shown as normal control. Slides were examined at 400× magnification. The samples were visualized with an Olympus BX51 fluorescent microscope equipped with Olympus 20×/0.70 NA and 40×/0.90 PlanApo objectives (Olympus America, Melville, NY) and a Pixera (600CL) cooled CCD camera (Pixera, Los Gatos, CA). Fluorescent images relative to each marker were collected using a corresponding filter set and Pixera Viewfinder acquisition software 3.0. Color composite images were generated using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA).