Differential surface receptors, tissue migration, and suppressor function of CD103⁺ Treg and CD25^hi natural Treg cells. Naive CD25^hi natural Treg cells were freshly isolated from spleen of DBA/2 donor mice. The in vitro-activated and expanded CD25^hi natural Treg cells were from sorted naive CD25^hi natural Treg cells after being cultured with anti-CD3/CD28 beads and IL-2 for 7 days. The CD103⁺ Treg cells were from spleen of day 20 chronic GVHD recipients. (A) The viability as judged by 4,6 diamidino-2-phenylindole (DAPI) staining, the expression levels of FoxP3, CD25, CD103, CD62L, and CCR5 by freshly isolated CD25^hiCD4⁺ T cells, in vitro-activated CD25^hiCD4⁺ T cells, and in vivo–activated CD103⁺CD4⁺ T cells. One representative is shown of 4 replicated experiments. (B) CFSE-labeled in vitro–activated CD25^hi natural Treg cells and in vivo–activated CD103⁺ Treg cells (10⁶ each) were injected intravenously into chronic GVHD recipients, respectively. Twelve hours after injection, the percentage of CFSE⁺FoxP3⁺ cells among total FoxP3⁺ cells in the MLN, spleen, and liver was compared. One representative of 4 examined recipients in each group is shown. The means (± SE) of CD25^hi natural Treg cells versus CD103⁺ Treg cells in different tissues are as follows: 10.8% (± 1.2%) versus 4.9% (± 1.3%) in MLN, 0.3% (± 0.2%) versus 0.5% (± 0.3%) in spleen, and 0.4% (± 0.2%) versus 3.5% (± 0.8%) in liver. (C) Suppression of host DC-induced donor CD4⁺CD25⁻ T proliferation by freshly isolated and in vitro–activated CD25^hi natural Treg cells as well as CD103⁺ Treg cells. (D) CD103⁺ Treg cell suppression of donor CD4⁺CD25⁻ T proliferation induced by host, MHC-matched third-party DCs, or MHC-mismatched third-party DCs. (E) In vitro–activated CD25^hi natural Treg cell suppression of donor CD4⁺CD25⁻ T proliferation induced by host or MHC-mismatched third-party DCs. (C-E) Means (± SE) of 4 replicated experiments.