Abstract
FOXP3+ regulatory T cell (Treg) based cellular therapies represent promising therapeutic options in autoimmunity, allergy, transplantation and prevention of Graft Versus Host (GVH) Disease. Among human FOXP3-expressing CD4+T cells, only the CD45RA+ naïve Treg (nTreg) subset is suitable for in vitro expansion. However, FoxP3 expression decays in cells using currently described culture protocols.Rapamycin alone was not able to prevent FOXP3 loss in nTregs cells, as only a half of them maintained FOXP3 expression after 14 days of culture. In contrast we report a novel combined drug regimen that can drastically stabilize FOXP3 expression in cultured Tregs. IL-2, rapamycin, histone deacetylase and DNA methyltransferase inhibitors act in synergy to allow expansion of human regulatory T cells with sustained high expression of FOXP3 and CD15s with potent suppressive capacities in vitro and control of murine xeno-GVH reactions. Of note, an additional subsequent infusion of expanded nTreg cells did not improve survival of mice.Combination of IL-2, rapamycin, histone deacetylase and DNA methyltransferase inhibitors is optimal for the expansion in vitro of pure effective nTreg maintaining high levels of FOXP3 for therapeutic purposes.
Highlights
Thymus derived naturally occurring FOXP3 expressing CD4+ regulatory T cells (Treg cells) are indispensable for the maintenance of self tolerance and immune homeostasis [1]
In the presence of rapamycin in addition to IL-2, the proportion of expanding FOXP3lowCD45RAnon Treg cells maintaining FOXP3 expression was higher, which is consistent with the finding that rapamycin promotes the expansion of genuine Treg cells at the expense of non Treg cells [22]
Significant proportion of expanding non Treg cells still lost FOXP3 expression after 7 days of culture, even in the presence of rapamycin (Figure 2A). This finding in addition to the poor capacity of eTreg cells to survive even in the presence of high dose IL-2 indicates that FOXP3+ cells with a CD45RA- phenotype are improper for expansion
Summary
Thymus derived naturally occurring FOXP3 expressing CD4+ regulatory T cells (Treg cells) are indispensable for the maintenance of self tolerance and immune homeostasis [1]. Because FOXP3 expressing Treg cells represent 1 to 6 % of CD4+ T cells in humans [9], in vitro expansion strategies are required to enable the infusion of significant number of Treg cells, given that 2 million Treg cells are usually required for infusion in mice (105 Treg cells per gram) to prevent autoimmunity [10]. Most strategies aiming at the expansion of human Treg cells have been mainly based on the isolation of CD25+CD4+ T cells, giving rise to expanding cells that contain significant proportions of cells that do not express FOXP3 [17, 18]. Because FOXP3 is a key molecule in the development and function of Treg cells, and because high levels of FOXP3 are more correlated with potent suppression than low levels of FOXP3 [19], Treg expansion protocols should incorporate means to maintain high levels of FOXP3 expression
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