Article Text

  1. K. Pothoven,
  2. K. Tarbell,
  3. H. Yang,
  4. R. M. Steinman,
  5. M. Suthanthiran,
  6. X. Luo
  1. Northwestern University, Chicago, IL; New York, NY.


Background Thymic-derived CD25+CD4+ regulatory T (Treg) cells have been found to play an important role in the pathogenesis of autoimmune diabetes. Challenges for their application as a potent immunomodulatory therapy are (1) the small size of the naturally occurring CD25+CD4+ Treg population and (2) the polyclonal nature of the existing CD25+CD4+ Treg cells. Here we describe a novel system of using dendritic cell (DC)-stimulated expansion in the presence of TGF-β1 for in vitro generation of beta cell-specific CD25+CD4+ T cells that are potent suppressors of autoimmune diabetes.

Material and Methods Naive BDC2.5/NOD CD25CD4+ cells were obtained by cell sorting from pooled BDC2.5/NOD LNs. Splenic DCs from NOD mice were purified by CD11c-positive selection. Naive CD25CD4+ T cells were either cultured with irradiated CD11c+ DCs and BDC peptide (specific stimulation) or with anti-CD3 and anti-CD28 (nonspecific stimulation) for 7 days with or without TGF-β1, after which the CD25+ T-cell fraction was purified and analyzed.

Results Purity of the BDC2.5/NOD CD4+CD25 was routinely > 97%. At baseline, the CD4+CD25 BDC T cells express minimal Foxp3 measured by FACS analysis and real-time PCR. Stimulation in the presence of TGF-β1 with either specific or nonspecific conditions leads to marked induction of Foxp3 expression to a level comparable to that seen in naturally occurring CD25+CD4+ Treg cells. This induction was not seen in the absence of TGF-β1. The induced CD25+CD4+Foxp3+ BDC T cells generated with DCs plus BDC peptide (specific stimulation) maintained a high level of cell surface clonotype expression after stimulation and exert antigen-specific suppression in in vitro suppression assays. When cotransplanted with syngeneic islets in diabetic NOD mice, these cells significantly prolonged islet graft survival from a median of 12 to 46 days (p = .0008). When cotransferred with diabetogenic cells into NOD.scid recipients, theses cells significantly delayed the kinetics of diabetes onset (p < .0001). In contrast, CD25+CD4+Foxp3+ BDC T cells induced with anti-CD3 and anti-CD28 (nonspecific stimulation) show lower levels of clonotype expression on cell surface and were unable to suppress BDC peptide-stimulated proliferation in vitro or protect islet grafts in vivo.

Conclusion Beta cell-specific BDC2.5 CD25+CD4+ cells with high levels of Foxp3 can be induced from naive BDC2.5 CD4+CD25 cells by TGF-β1 in CD11c+ DC-stimulated expansions. These cells harbor potent suppressive activity in an islet antigen-specific manner and suppress autoimmune diabetes.

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.