In Vivo Control of Diabetogenic T-Cells by Regulatory CD4+CD25+ T-Cells Expressing Foxp3

In Vivo Control of Diabetogenic T-Cells by Regulatory CD4 + CD25 + T-Cells Expressing Foxp3 Dorthe Lundsgaard , Thomas Lindebo Holm , Lars Hornum and Helle Markholst From the Hagedorn Research Institute, Gentofte, Denmark Address correspondence and reprint requests to Helle Markholst, MD, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820, Gentofte, Denmark. E-mail: hmar{at}hagedorn.dk Abstract To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitro−activated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3–4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4 + fraction, as purified CD4 + T-cells prevented the accelerated diabetes. When CD25 + cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. In contrast, two million CD4 + CD25 + cells (expressing Foxp3 ) prevented the accelerated diabetes when transferred both before and simultaneously with the diabetogenic T-cells. In addition, 2 million CD4 + CD25 + T-cells prevented spontaneous diabetes, even when given to rats age 42 days, whereas 20 million CD4 + CD25 − cells (with low Foxp3 expression) were far less effective. We thus demonstrated that CD4 + CD25 + cells exhibit powerful regulatory potential in rat diabetes. CFDA-SE carboxyfluorescein diacetate, succinimidyl ester cLN, cervical lymph node CTO, CellTracker Orange MFI, mean fluorescence intensity mLN, mesenteric lymph node panLN, pancreatic lymph node PE, phycoerythrin PMA, phorbol myristate acetate Footnotes D.L., T.L.H., L.H., and H.M. are employed by Novo Nordisk A/S; D.L., L.H., and H.M. hold stock in Novo Nordisk A/S; and Novo Nordisk A/S has contributed funds supporting the work of H.M.’s laboratory. Accepted January 5, 2005. Received June 21, 2004. DIABETES