Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta, Retinoic Acid, Rapamycin and Butyrate

Abstract

Regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance. Therapeutic manipulation of Tregs is subject to numerous clinical investigations including trials for adoptive Treg transfer. Since the number of naturally occurring Tregs (nTregs) is minute, it is highly desirable to develop a complementary approach of inducing Tregs (iTregs) from naïve T cells. Mouse studies exemplify the importance of peripherally induced Tregs as well as the applicability of iTreg transfer in different disease models. Yet, procedures to generate iTregs are currently controversial, particularly for human cells. Here we therefore comprehensively compare different established and define novel protocols of human iTreg generation using TGF-β in combination with other compounds. We found that human iTregs expressed several Treg signature molecules, such as Foxp3, CTLA-4 and EOS, while exhibiting low expression of the cytokines Interferon-γ, IL-10 and IL-17. Importantly, we identified a novel combination of TGF-β, retinoic acid and rapamycin as a robust protocol to induce human iTregs with superior suppressive activity in vitro compared to currently established induction protocols. However, iTregs generated by these protocols did not stably retain Foxp3 expression and did not suppress in vivo in a humanized graft-versus-host-disease mouse model, highlighting the need for further research to attain stable, suppressive iTregs. These results advance our understanding of the conditions enabling human iTreg generation and may have important implications for the development of adoptive transfer strategies targeting autoimmune and inflammatory diseases.

TGF-β Affects the Differentiation of Human GM-CSF+ CD4+ T Cells in an Activation- and Sodium-Dependent Manner

“…Activation-induced low and transient FOXP3 expression in human activated conventional T cells further complicates such studies, which however mostly concur in the conclusion that (low) FOXP3 expression is insufficient to confer suppressive function. In a setup similar to the studies in the present work, we determined that despite high FOXP3 expression, iTreg suppressive function in vitro did not exceed unspecific “suppression” of FOXP3-negative control stimulated cells, except when iTregs were induced in the presence of TGF-β + retinoic acid + rapamycin (34). Nevertheless these TGF-β + retinoic acid + rapamycin iTregs did not suppress in a, though artificial, xenogeneic in vivo suppression assay (34), highlighting the complexity of assays for determining iTreg suppressive function.…”

“…In a setup similar to the studies in the present work, we determined that despite high FOXP3 expression, iTreg suppressive function in vitro did not exceed unspecific “suppression” of FOXP3-negative control stimulated cells, except when iTregs were induced in the presence of TGF-β + retinoic acid + rapamycin (34). Nevertheless these TGF-β + retinoic acid + rapamycin iTregs did not suppress in a, though artificial, xenogeneic in vivo suppression assay (34), highlighting the complexity of assays for determining iTreg suppressive function. A major complication with assays using human iTregs is that these cells lack epigenetic demethylation in the so-called Treg-specific demethylated region (TSDR) in the FOXP3 locus (56, 57) and hence lose FOXP3 upon restimulation (34, 58).…”

“…Notably, we observed that TGF-β increased FOXP3 [a well-known effect (31)] irrespective of NaCl addition to the medium (Figure 5D). Correspondingly, a slight inhibition of IFN-γ by TGF-β, which is a known effect (32–34), seemed to occur under low sodium conditions; however in the presence of physiological sodium concentrations, the fractions of IFN-γ + cells were generally much higher and an effect of TGF-β was not apparent (Figure 5E). Together, these results suggest that the effect of TGF-β specifically on GM-CSF expression is affected by sodium concentrations in the medium.…”

“…We and others have extensively studied the immune-suppressive function of human in vitro TGF-β-induced FOXP3 + Treg cells (iTregs) (53), with conflicting results possibly due to differences in experimental setup during Treg induction or setup and controls used in suppression assays (34, 54, 55). Activation-induced low and transient FOXP3 expression in human activated conventional T cells further complicates such studies, which however mostly concur in the conclusion that (low) FOXP3 expression is insufficient to confer suppressive function.…”

IL-33 improves the suppressive capacity of human regulatory T cells

“…Thus, we decided to evaluate the expression of certain genes involved in Tregs activity/stability such as Foxp3, ST2 and Amphiregulin (Areg) using quantitative PCR (qPCR), Figure 3C. As mentioned before, Foxp3 has been established as the master transcription factor for Tregs and is involved in the maintenance of the regulatory phenotype and suppressive function [14,15]; ST2 is the receptor for IL-33 and it has been linked with Tregs function in gut mucosa [11,16]; Areg, on the other hand, is an epidermal growth factor that improves Tregs function in vitro and in-vivo mouse models of colitis and tumor, favoring Tregs stability, avoiding conversion toward an inflammatory phenotype [17,18]. For these three genes studied, we observed a clear trend in the up-regulation of their mRNA, although the variations were not significantly different.…”