Epigenetic Profile of Treg-like Cells Induced By Mesenchymal Stem Cells in Vitro Resembles That of Natural Treg
Authors: Azevedo Rita I , Minskaia Ekaterina , Lobato da Silva Cláudia , Fernandes Platzgummer Ana , Vieira Ana I.S. , Cabral Joaquim M.S. , Lacerda Joao F 
Published: October 2019

Abstract
Regulatory T cells (Treg) are a key population in immune tolerance and their potential use in the treatment of chronic inflammatory diseases has been increasingly investigated. A major hurdle to adoptive Treg therapy in humans is the difficulty in obtaining sufficient numbers of clinical grade Treg.
We and others have investigated the potential of mesenchymal stem cells (MSC) to induce and/or expand Treg in vitro. Our goal is to ascertain whether MSC co-culture supports the recruitment and expansion of Treg from a non-Treg starting population, which would allow for clinically relevant numbers to be reached, whilst bypassing the costly isolation of Treg under GMP conditions prior to expansion.
We found that co-cultures of human peripheral blood mononuclear cells (PBMC) from healthy donors with allogeneic bone marrow-derived MSC specifically increase the absolute counts and frequency (4 and 6-fold, respectively) of Treg-like cells with a CD4+ CD25high Foxp3+ CD127low phenotype. We further observed that this increase in Treg numbers after MSC co-culture is mainly due to the induction of conventional CD4 T cells (Tcon) to acquire a Treg-like phenotype, rather than to Treg expansion.
These data led us to investigate if these Treg-like cells induced by MSC also resemble Treg in terms of suppressive ability and epigenetic profile, which would inform on their functional potential and stability, two key features for their applicability in a clinical setting.
We performed suppression assays using FACSorted Treg-like cells after co-culture of purified Tcon with MSC as suppressor cells and autologous fresh Tcon labelled with CFSE as responder cells. We compared the suppressive ability of Treg-like cells to that of Treg co-cultured with MSC, as well as of fresh Treg. We found that induced Treg-like cells isolated from Tcon:MSC co-cultures (P=0.0121), natural Treg isolated from Treg:MSC co-cultures (P=0.0025) and fresh Treg (P=0.0006) all significantly suppress Tcon proliferation (% divided cells at 1:1 Tcon:Treg ratio compared to Tcon alone, Mann Whitney test).
We observed significantly higher levels of TGF-β in PBMS:MSC co-culture supernatants compared to that of PBMC cultured alone (P<0.05 on Day 10, P<0.01 on Day 14, two-way ANOVA), indicating a role for TGF-β in the induction of these Treg-like cells. TGF-β-induced Treg have been suggested to be less stable than natural Treg, likely due to epigenetic regulation. In particular, DNA demethylation has been shown to be a key factor in Treg stability. The potential therapeutic application of MSC-induced Treg-like cells would hinge on these cells being epigenetically stable. Hence, we investigated whether MSC-induced Treg-like cells are more similar to the original Tcon that they arose from or to natural Treg in terms of their DNA methylation profile.
Most Treg epigenetic studies focus on the Treg-specific demethylated region (TSDR) of the FOXP3 gene and, given that FOXP3 is encoded on the X chromosome, opt to use male donors to bypass artefacts of X-chromosome inactivation. In order to avoid a gender biased analysis, we sought an alternative to TSDR. CAMTA1 (calmodulin-binding transciption activator 1) is a Ca2+-dependent calmodulin-binding transcription factor encoded on chromosome 1q3.6, which has been proposed as a potential molecular marker that distinguishes Treg from Tcon in both male and female donors. We assessed the methylation status of 13 CpG sites within the 470bp CAMTA1 intronic region 3 based on the coding DNA strand in Treg-like cells purified after Tcon:MSC co-cultures, as well as in fresh Treg and Tcon from the same donor. Fresh Tcon cells from one of the donors showed 100% methylation of CpGs 1-13, whereas fresh Treg and Treg-like cells from the same donor showed much lower levels of methylation (33.8% and 0%, respectively), with the highest differences in methylation observed in CpGs 2 and 11 (15% in fresh Treg; 0% in Treg-like cells; 100% in fresh Tcon). These two CpG positions, which have been previously demonstrated to be the ones distinguishing Treg from Tcon, were consistently more demethylated in fresh Treg and Treg-like cells than in fresh Tcon, with an average methylation levels of 5.8% for fresh Treg, 17% for Treg-like cells and 73% for fresh Tcon.
Overall, our data demonstrate that MSC induce a subset of Tcon to acquire a Treg-like phenotype, which is accompanied by a suppressive ability and an epigenetic profile that resemble that of natural Treg.

Disclosures
No relevant conflicts of interest to declare.