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

RESEARCH ARTICLE Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta, Retinoic Acid, Rapamycin and Butyrate Angelika Schmidt1*, Matilda Eriksson1, Ming-Mei Shang1, Heiko Weyd2, Jesper Tegnér1 1 Unit of Computational Medicine, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet & Karolinska University Hospital, Stockholm, Sweden, 2 Division of Immunogenetics, Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany * Abstract OPEN ACCESS Citation: Schmidt A, Eriksson M, Shang M-M, Weyd H, Tegnér J (2016) Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta, Retinoic Acid, Rapamycin and Butyrate. PLoS ONE 11(2): e0148474. doi:10.1371/journal.pone.0148474 Editor: Luis Graca, University of Lisbon, PORTUGAL Received: August 27, 2014 Accepted: January 19, 2016 Published: February 17, 2016 Copyright: © 2016 Schmidt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme (FP7PEOPLE-2012-IEF; to AS), Dr. Åke Olssons Foundation (AS), KI Research Foundations (AS; JT), Swedish Research Council (JT), CERIC Linné Center (JT), AFA insurance (JT), Stockholm County Council (JT), and Torsten Söderberg Foundation (JT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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, IL10 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. Introduction CD4+CD25+Foxp3+ regulatory T cells (Tregs) play an indispensable role in the immune system as they are involved in the prevention of autoimmune disease, allergies and infection- PLOS ONE | DOI:10.1371/journal.pone.0148474 February 17, 2016 1 / 31 Comparative Analysis of Human iTreg Protocols Competing Interests: The authors have declared that no competing interests exist. induced organ pathology by suppression of other immune cells [1]. However, Tregs can also dampen immune responses against tumors in several settings [2]. Therefore, therapeutic manipulation of Treg number and function is subject to intense clinical investigations. Foxp3 was identified as a lineage-defining transcription factor (TF) for Tregs in mice and humans, and loss of Foxp3 leads to severe lethal autoimmune disease in mice and men [1]. Yet, Foxp3 cannot serve as a specific marker for Tregs but their suppressive function has to be determined, because human conventional CD4+CD25- T cells (Tcons) transiently express intermediate Foxp3 amounts upon activation [3,4]. Only recently, transient expression of low Foxp3 levels without commitment to the Treg lineage was also shown for murine T cells [5]. Contradictory reports leave it unclear whether Foxp3 expression is sufficient to confer suppressive abilities, whereas necessity of Foxp3 for Treg function is undisputed: Foxp3, in conjunction with several other TFs, activates or represses the expression of Treg signature genes [6,7]. Significant progress has been made in elucidating the regulation of Foxp3 expression. Activation of the Foxp3 gene is achieved by binding of several TFs to its promoter and intronic Conserved Non-coding DNA Sequences (CNSs) [8,9]. These TFs are activated via T cell receptor (TCR), IL-2 and TGF-β signaling and, depending on which CNS they act on, are implicated in either Foxp3 induction (CNS3), maintenance (CNS2) or TGF-β-enhanced expression (CNS1). It seems that a fine balance of TCR signal intensity, timing and quality defines the optimal conditions allowing for Foxp3 induction, and furthermore, TGF-β can decrease the sensitivity towards too strong TCR stimulation [10–12]. Foxp3 expression is negatively regulated by inactivation of the Foxp3-inducing TFs Foxo1 and Foxo3a through the Akt/mTOR pathway, which is activated largely by CD28 costimulatory signals, but also IL-2R and TCR signaling cross-talk with Akt via the kinase PI3K [10,13,14]. Along these lines, strong costimulation was suggested to inhibit Treg induction [10,15–17]. Hence, the clinically approved mTOR inhibitor rapamycin (Rapa) promotes Foxp3 expression as shown for murine Tregs [18–21]. Also for human Tregs, Rapa has been successfully used in expansion of Tregs, while at the same time it prevents growth of Tcons [22,23]. An additional layer of complexity is added by DNA methylation and histone modifications at the Foxp3 locus and, interestingly, an epigenetic “Treg signature” can be established independently of Foxp3 [24,25]. In particular, the CNS2 comprises the so-called Treg-specific demethylated region (TSDR), which includes several CpG motifs, demethylation of which is crucial for stable maintenance of Foxp3 expression: The TSDR is demethylated exclusively in stable Tregs while it is methylated in naïve and activated Tcons as well as in exTregs that have lost Foxp3 [26–28]. Peripheral tolerance is ensured not only by thymus-derived Tregs (tTregs, often called n (...truncated)