Abstract
Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) has been successfully utilized to treat graft versus host disease and represents a promising strategy for the treatment of autoimmune diseases and transplant rejection. The aim of this study was to evaluate the effects of all-trans retinoic acid (atRA) and rapamycin (RAPA) on the number, phenotype, homing markers expression, DNA methylation, and function of induced human Treg cells in short-term cultures. Naive T cells were polyclonally stimulated and cultured for five days in the presence of different combinations of IL-2, TGF-β1, atRA and RAPA. The resulting cells were characterized by the expression of FOXP3, activation, surface and homing markers. Methylation of the Conserved Non-coding Sequence 2 was also evaluated. Functional comparison of the different culture conditions was performed by suppression assays in vitro. Culturing naive human T cells with IL-2/TGFβ1 resulted in the generation of 54.2% of Treg cells (CD4+CD25+FOXP3+) whereas the addition of 100 nM atRA increased the yield of Treg cells to 66% (p = 0.0088). The addition of RAPA did not increase the number of Treg cells in any of these settings. Treg cells generated in the presence of atRA had an increased expression of the β7 integrin to nearly 100% of the generated Treg cells, while RAPA treated cells showed enhanced expression of CXCR4. The differential expression of homing molecules highlights the possibility of inducing Treg cells with differential organ-specific homing properties. Neither atRA nor RAPA had an effect on the highly methylated CNS2 sites, supporting reports that their contribution to the lineage stability of Treg cells is not mediated by methylation changes in this locus. Treg cells generated in the presence of RAPA show the most potent suppression effect on the proliferation of effector cells.
Highlights
The discovery, isolation, and generation of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) represent a remarkable breakthrough in modern immunology[1,2]
The results shown are consistent with a higher yield of Treg cells in cultures supplemented with all-trans retinoic acid (atRA) 100nM, which is related to a higher percentage of FOXP3 expressing cells
One of the main limitations to an effective adoptive therapy with Treg cells is the high number of cells needed and the difficulties related to achieving this goal
Summary
The discovery, isolation, and generation of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) represent a remarkable breakthrough in modern immunology[1,2]. In the context of organ and bone marrow transplantation, Treg cells contribute critically to the acceptance of allogeneic implants [8]. In this regard, the adoptive transfer of Treg cells in animal models of transplantation has shown to improve graft survival [9,10]. The manufacture and use of Treg cells for the advance of solid organ transplantation is being evaluated in a large cooperative project, the “ONE Study” [14,15]
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