Stability of human rapamycin-expanded CD4+CD25+ T regulatory cells

Abstract

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

Comparison of Regulatory T Cells in Hemodialysis Patients and Healthy Controls

“…Freshly isolated Tregs from HD patients stained marginally more positively for IL-17 and after activation in IL-17-inducing cytokines (IL-2, IL-1b, IL-6, and TGF-b [32]), produced more IL-17 than those Tregs from HCs, but these differences did not reach statistical significance (Figure 4, A-C). After expansion ex vivo, low-level staining for IL-17 was observed in Tregs from both HCs and HD patients, with HD Tregs, as before, being slightly more positive for IL-17 ( Figure 4, D and E).…”

“…Nevertheless, this finding is the clearest indication yet that Treg-based cell therapy for renal allograft transplantation should use a Rapamycin-based protocol. Rapamycin is an ideal choice for Treg-based therapy in solid organ transplantation, because it preferentially expands the most stable Treg subpopulation (population I) and inhibits IL-17 production from other subpopulations (32,44). Rapamycin interferes with early T cell signaling events (Akt/mammalian target of rapamycin), which are critical in T cell fate decisions (45).…”

“…Rapamycin promotes the expansion of phenotypically and functionally stable Tregs in GMP-compatible systems while impairing the proliferation of CD4 + CD25 2 T effectors (13,32,42). Thus, in the absence of Rapamycin treatment, the elevated IL-17 production by HD Tregs may be attributed to increased contaminating Th17 cells or a greater propensity for these cells to differentiate into Th17 cells (consistent with the chronic inflammation seen in HD and/or with vitamin D insufficiency/deficiency in ESKD, because this vitamin prevents Th17 differentiation) (43).…”

“…Having established that HD patients have the same numbers of Tregs as HCs with normal expression of maturation and activation markers within subsets, albeit with slight reduction in population II, we next sought to determine whether they could be expanded in vitro under the GMP-compatible conditions (13,32) as required for a program of cell therapy.…”

Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of human CD4+CD25+FOXP3+ T regulatory cell subpopulations

“…To address this concern, Treg lines were cultured in the presence of pro-inflammatory cytokines previously reported to favor Th17 conversion. 8,28 Two cytokine cocktails were selected: cocktail A (IL-1β, IL-2, IL-6 and TGF-β) and cocktail B (IL-2, IL-21, IL-23 and TGF-β). Analysis of supernatants from freshly isolated or 28-day untreated Tregs revealed that IL-17 production was increased by the addition of each cytokine cocktail.…”