The online version of this article has a Supplementary Appendix. BackgroundThe clinical use of ex vivo-expanded T-regulatory cells for the treatment of T-cell-mediated diseases has gained increasing momentum. However, the recent demonstration that FOXP3 + Tregulatory cells may contain interleukin-17-producing cells and that they can convert into effector cells once transferred in vivo raises significant doubts about their safety. We previously showed that rapamycin permits the ex vivo expansion of FOXP3 + T-regulatory cells while impairing the proliferation of non-T-regulatory cells. Here we investigated the Th17-cell content and the in vivo stability of rapamycin-expanded T-regulatory cells as pertinent aspects of cell-based therapy. Design and MethodsT-regulatory-enriched cells were isolated from healthy volunteers and were expanded ex vivo with rapamycin with a pre-clinical applicable protocol. T-regulatory cells cultured with and without rapamycin were compared for their regulatory activity, content of pro-inflammatory cells and stability. ResultsWe found that CD4+ T cells (i.e., precursor/committed Th17 cells) contaminate the T-regulatory cells cultured ex vivo in the absence of rapamycin. In addition, Th17 cells do not expand when rapamycin-treated T-regulatory cells are exposed to a "Th17-favorable" environment. Rapamycin-expanded T-regulatory cells maintain their in vitro regulatory phenotype even after in vivo transfer into immunodeficient NOD-SCID mice despite being exposed to the irradiation-induced pro-inflammatory environment. Importantly, no additional rapamycin treatment, either in vitro or in vivo, is required to keep their phenotype fixed. ConclusionsThese data demonstrate that rapamycin secures ex vivo-expanded human T-regulatory cells and provide additional justification for their clinical use in future cell therapy-based trials.Key words: T-regulatory cells, ex vivo expansion, rapamycin, cell therapy, T-regulatory-cell stability. + T regulatory cells. Haematologica 2011;96(9):1357-1365
+ CD25+ T-regulatory cells" Haematologica 2011;96(9):1357-65.We have read the letter by He and Lv 1 and we think the issues raised by the authors are not pertinent to our published work. Specifically, He and Lv raise three issues. 1) They claim that we cannot conclude from our study that rapamycin has an intrinsic role in the stability of Treg cells. We never asserted or even suggested that, based on our data, rapamycin has a direct effect on the stability of Treg cells. Our experiments show that a mixed population of Treg and non-Treg cells cultured for ten days in the presence of rapamycin is stable both in vitro and in vivo. We have never hypothesized a direct effect of rapamycin on Treg cells. Indeed, it was our group who first showed that rapamycin selectively inhibits the proliferation of effector T cells while sparing that of Treg cells 2 and not Strauss et al.,3,4 as wrongly stated by the authors in their comment. Thus, it was us who had demonstrated that rapamycin does not have a direct role on the Treg cells themselves, and these findings were further confirmed in our recent paper in Haematologica.2) He and Lv assert that we cannot claim that rapamycin-expanded Treg cells are stable in vivo since we only showed the frequency of FOXP3
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