Rapamycin is an immunosuppressive compound that is currently used to prevent acute graft rejection in humans. In addition, rapamycin has been shown to allow operational tolerance in murine models. However, a direct effect of rapamycin on T regulatory (Tr) cells, which play a key role in induction and maintenance of peripheral tolerance, has not been demonstrated so far. Here, we provide new evidence that rapamycin selectively expands the murine naturally occurring CD4⁺CD25⁺FoxP3⁺ Tr cells in vitro. These expanded Tr cells suppress proliferation of syngeneic T cells in vitro and prevent allograft rejection in vivo. Interestingly, rapamycin does not block activation-induced cell death and proliferation of CD4⁺ T cells in vitro. Based on this new mode of action, rapamycin can be used to expand CD4⁺CD25⁺FoxP3⁺ Tr cells for ex vivo cellular therapy in T-cell-mediated diseases. (Blood. 2005;105:4743-4748)

CD4⁺CD25⁺FOXP3⁺ T regulatory cells (Tregs) are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. Rapamycin has been shown to promote tolerance in experimental models and to favor CD4⁺CD25⁺ Treg-dependent suppression. We recently reported that rapamycin allows in vitro expansion of murine CD4⁺CD25⁺FoxP3⁺ Tregs, which preserve their suppressive function. In the current study, we show that activation of human CD4⁺ T cells from healthy subjects in the presence of rapamycin leads to growth of CD4⁺CD25⁺FOXP3⁺ Tregs and to selective depletion of CD4⁺CD25⁻ T effector cells, which are highly sensitive to the antiproliferative effect of the compound. The rapamycin-expanded Tregs suppress proliferation of both syngeneic and allogeneic CD4⁺ and CD8⁺ T cells. Interestingly, rapamycin promotes expansion of functional CD4⁺CD25⁺FOXP3⁺ Tregs also in type 1 diabetic patients, in whom a defect in freshly isolated CD4⁺CD25⁺ Tregs has been reported. The capacity of rapamycin to allow growth of functional CD4⁺CD25⁺FOXP3⁺ Tregs, but also to deplete T effector cells, can be exploited for the design of novel and safe in vitro protocols for cellular immunotherapy in T cell-mediated diseases.

Type 1 T regulatory (Tr1) cells suppress immune responses in vivo and in vitro and play a key role in maintaining tolerance to self- and non–self-antigens. Interleukin-10 (IL-10) is the crucial driving factor for Tr1 cell differentiation, but the molecular mechanisms underlying this induction remain unknown. We identified and characterized a subset of IL-10–producing human dendritic cells (DCs), termed DC-10, which are present in vivo and can be induced in vitro in the presence of IL-10. DC-10 are CD14+, CD16+, CD11c+, CD11b+, HLA-DR+, CD83+, CD1a−, CD1c−, express the Ig-like transcripts (ILTs) ILT2, ILT3, ILT4, and HLA-G antigen, display high levels of CD40 and CD86, and up-regulate CD80 after differentiation in vitro. DC-10 isolated from peripheral blood or generated in vitro are potent inducers of antigen-specific IL-10–producing Tr1 cells. Induction of Tr1 cells by DC-10 is IL-10–dependent and requires the ILT4/HLA-G signaling pathway. Our data indicate that DC-10 represents a novel subset of tolerogenic DCs, which secrete high levels of IL-10, express ILT4 and HLA-G, and have the specific function to induce Tr1 cells.