The therapeutic potential of nTregs to prevent or cure multiple autoimmune diseases or GVHD in murine or xenogeneic models has been well documented (3
). Two critical obstacles to overcome before implementing this therapy in humans are generating sufficient cell numbers and demonstrating their in vivo safety and stability. Here we show that sort-purified nTregs could be expanded at least 50 million fold by repetitive stimulation with cell-based aAPCs while maintaining suppressive function in vitro and in vivo. Addition of rapamycin minimized contamination with Th1 inflammatory cytokine secreting cells, but not Th2 cells, which skew immunity away from inflammatory responses. Re-stimulated nTregs differentiated from a CD27+ memory phenotype to CD27− memory phenotype, but, importantly, did not adopt a senescent (CD57+) phenotype (33
). The lack of Vß skewing in the T cell receptor repertoire indicates that massively expanded nTregs retain a broad spectrum of reactivities and are not transformed.
Maximizing nTreg expansion, while minimizing loss of suppressive function and contamination with non-Tregs, is critical for establishing an nTreg cellular therapy. Three studies have shown that nTregs can be expanded >1000-fold if re-stimulated in the absence of rapamycin, but in each case cultures contained high numbers of IL-2- and IFNγ-secreting cells that were both Foxp3− and + (16
). We confirmed these data and found that nTreg cultures eventually lost Foxp3 and suppressive function in the absence of rapamycin. Loss of Foxp3 correlated with an increased ratio of cycling (i.e. Ki-67+) Foxp3− cells (Fig. S7
), suggesting loss of purity is due to the outgrowth of Foxp3− cells as opposed to conversion of Foxp3+ cells as suggested by one report (16
We previously demonstrated the increased stimulatory capacity of cell-based aAPCs allowed PB nTregs to be expanded 1000-fold with a single re-stimulation, even in the presence of rapamycin, and nTreg expanded with aAPCs were equal to anti-CD3/28 mAb-coated beads expanded cells at suppressing xenogeneic GVHD (Fig. S1K
)). For these initial studies, re-stimulation was performed at the growth plateau phase, but the high variability (day 8 to 12) and difficulty of determining this timepoint are not conducive to clinical production. Re-stimulation on a specific day is optimal for clinical trials. However, although studies without rapamycin showed re-stimulation on day 7 increased expansion, we observed no increase in expansion with day 7 re-stimulation (n=3) with bead-purified nTreg stimulated with anti-CD3/28 mAb-coated beads (25- vs. 18-fold for with or without day 7 re-stimulation, respectively). Although re-stimulation based upon cell size resulted in more variability in the day of optimal re-stimulation than would be the case at a single time point, such an approach identified a time range (day 13±1) more suitable for clinical re-stimulation.
All nTreg cultures contain some level of Foxp3− cells, which have the potential, especially after re-stimulation, to become effector T-cells and exacerbate disease. Although nTreg cultures re-stimulated in the absence of rapamycin contained high numbers of IL-2- and IFNγ-secreting cells, the number of these cells did not increase with re-stimulation in the presence of rapamycin. Furthermore, when transferred in vivo, Foxp3− cells present in nTreg cultures did not expand or persist long-term and, in contrast to cultures expanded from CD4+25− cells, did not exacerbate GVHD. In addition, studies show rapamycin temporally imparts Foxp3 expression and Treg-like activity to effector T-cells, which can re-acquire T-effector cell function if rapamycin is removed (35
). LAP expression differentiates activated nTregs from stimulated CD4+25− T-cells expressing Foxp3 spontaneously or after exposure to TGFß or rapamycin (17
). Even after four re-stimulations, most Foxp3+ cells expressed LAP even 7 days after re-stimulation, showing that the cultures remain primarily nTregs. Furthermore, cultures expanded over 1 million-fold maintained nTreg-specific demethylation in the Foxp3
gene. Murine T-cells expanded in rapamycin are Th2 skewed, secrete IL-4 and IL-10 and, after adoptive transfer, decrease allospecific IFNγ secretion and ameliorate disease in a murine model of GVHD (36
). Interestingly although rapamycin almost completely inhibited the differentiation of IL-2- and IFNγ-secreting cells in our cultures of human cells, the effect on IL-4 was not complete, and >50% of cells secreted IL-4 (both Foxp3+ and FoxP3– cells) after the fourth re-stimulation.
Murine and human nTregs are not terminally differentiated, and can be reprogrammed to secrete IL-17 in vitro or in vivo when activated in the presence of IL-6 (31
). Adoptive transfer of reprogrammed murine nTregs induced autoimmune diabetes but, unlike their human counterparts, these cells also produced IFNγ and TNFα. It is not known whether reprogrammed human nTregs will cause disease, since only ~5% of nTregs become IL-17+ in vitro (37
) and these retain suppressive function (31
). Several findings from this study suggest nTreg reprogramming may not be a grave issue in developing a cellular therapy for in vitro expanded nTregs. First, IL-17 was undetectable in the supernatants of all re-stimulation samples cultured with rapamycin (limit of detection 0.3pg/ml). Second, the number of expanded cells that were IL-17+ cells was very low (<2% total and ≤0.5% Foxp3+IL-17+) and, even more important, did not increase significantly over the 4 re-stimulation cycles (Fig. S1J
). Although the likelihood for in vivo reprogramming of nTregs and especially expanded nTregs may be context dependent, the high degree of TSDR demethylation of these cells may provide some degree of resistance to the reprogramming process.
In summary, the degree of nTreg expansion reported here could lead to the widespread application of nTreg cellular therapy for GVHD and graft rejection through the creation of an off-the-shelf therapy using nTreg banks generated from HLA-typed donors with known safety and potency records. The massive expansion observed with repetitive polyclonal stimulation should also allow relatively rare, auto-antigen-specific nTreg clones to be expanded to treat autoimmune diseases. Ultimately, this strategy could be applied to expansion of antigen-specific nTregs, which are more effective than polyclonal Tregs at suppressing disease. This strategy is potentially preferable to using Tregs induced in vitro by FoxP3
gene transfer or other conditions that favor FoxP3 expression. Furthermore, if increased purity and/or suppressive function is required, nTregs could be re-isolated after expansion using a protocol described recently by Shevach’s group based upon LAP expression (17
). Although GMP sorting can be challenging for many institutions, re-stimulation-driven expansion could produce sufficient numbers of cells in a small number of sorts to support the creation of a master cell bank that would contain matches for multiple patients. Finally, an nTreg master cell bank would be an effective treatment for multiple diseases because, as shown here, nTregs suppress third-party responses and ameliorate disease without long-term persistence and are also able to maintain suppressive function after freeze/thaw.