The role of Foxp3+CD4+CD25+ regulatory T (Treg) cells has increasingly been demonstrated in modulating allograft rejection and mediating allograft tolerance in rodents. In humans, a favorable transplantation outcome is frequently associated with increased frequencies of Foxp3+CD4+CD25+ Treg cells. Over the past several years, we have been interested in using natural CD4+CD25+ Treg (nTreg) as cellular therapy to prevent heart allograft rejection in mice. Due to low frequencies in fresh tissues, ex vivo expansion is essential for generating sufficient numbers of CD4+CD25+ nTreg cells to target the large repertoire of alloreactive effector T (Teff) cells in transplant recipients. Strategies for ex vivo expansion have evolved from alloantigen (alloAg)-nonspecific (e.g., anti-CD3/CD28 mAb-coated beads) approaches to alloAg-specific (e.g., live donor bone marrow-derived dendritic cells [BM-DC]) methods, with reasonable cell expansion after 20 days of culture. An unexpected observation was a gradual decrease of the transcription factor Foxp3, a critical regulator for suppressive function during ex vivo expansion, when in vitro cultures were supplemented with only interleukin (IL)-2. We systemically examined the kinetics of Foxp3 expression in CD4+CD25+ and CD4+Foxp3+ nTreg cells (FACS-sorted from Foxp3-green fluorescence protein [GFP] knock-in mice) during ex vivo expansion, and confirmed the activation of T cell receptors (TCR), leading to the loss of Foxp3 from the original Foxp3+ precursors. We have made significant progress towards preserving Foxp3 expression by modifying expansion protocols to include transforming growth factor (TGF)-β, all-trans retinoic acid (RA), histone deacetylase inhibitor trichostatin A (TSA), or combinations of these reagents. Importantly, fresh CD4+CD25+ nTreg cells expanded by the "modified" culture conditions (e.g., RA/TSA/TGF-β/IL-2) not only expressed high Foxp3 in vitro (∼45%) but also retained high Foxp3 expression in vivo (∼91%) during prevention of heart allograft rejection. As a result, long-term survival of fully major histocompatibility complex (MHC)-mismatched heart allografts was successfully achieved in wild-type mice without using conventional immunosuppressants. Further optimization of protocols for expanding CD4+CD25+ nTreg cells ex vivo may be essential for the successful use of these cells in preventing heart allograft rejection in immune competent mice.
|Original language||English (US)|
|Title of host publication||Heart Transplantation|
|Subtitle of host publication||Indications and Contradictions, Procedures and Complications|
|Publisher||Nova Science Publishers, Inc.|
|Number of pages||25|
|State||Published - Jan 1 2009|
ASJC Scopus subject areas