Sustained store-operated calcium entry utilizes activated chromatin state leads to instability in iTregs

Reviewer #1 (Public Review):

This study revealed that one of the mechanisms for iTreg (induced-Treg) lineage instability upon restimulation is through sustained store-operated calcium entry (SOCE), which activates transcription factor NFAT and promotes changes in chromatin accessibility to activated T cell-related genes. The authors revealed that, unlike thymus-derived Tregs (tTreg) with blunted calcium signaling and NFAT activation, iTregs respond to TCR restimulation with fully activated SOCE and NFAT similar to activated conventional T cells. Activated NFAT binds to open chromatin regions in genes related to T helper cells, increases their expression, and leads to the instability of iTreg cells. On the other hand, inhibition of the SOCE/NFAT pathway by chemical inhibitors could partially rescue the loss of Foxp3 expression in iTreg upon restimulation. The conclusion of the study is unexpected since previous studies showed that NFAT is required for Foxp3 induction and iTreg differentiation (Tone Y et al, Nat Immunol. 2008, PMID: 18157133; Vaeth M et al, PNAS, 2012, PMID: 22991461). Additionally, Foxp3 interacts with NFAT to control Treg function (Wu Y et al, Cell, 2006, PMID: 16873067). The data presented in this study demonstrated the complex role NFAT plays in the generation and stability of iTreg cells.

Several concerns are raised from the current study.
1. Previous studies showed that iTregs generated in vitro from culturing naïve T cells with TGF-b are intrinsically unstable and prone to losing Foxp3 expression due to lack of DNA demethylation in the enhancer region of the Foxp3 locus (Polansky JK et al, Eur J Immunol., 2008, PMID: 18493985). It is known that removing TGF-b from the culture media leads to rapid loss of Foxp3 expression. In the current study, TGF-b was not added to the media during iTreg restimulation, therefore, the primary cause for iTreg instability should be the lack of the positive signal provided by TGF-b. NFAT signal is secondary at best in this culturing condition.

2. It is not clear whether the NFAT pathway is unique in accelerating the loss of Foxp3 expression upon iTreg restimulation. It is also possible that enhancing T cell activation in general could promote iTreg instability. The authors could explore blocking T cell activation by inhibiting other critical pathways, such as NF-kb and c-Jun/c-Fos, to see if a similar effect could be achieved compared to CsA treatment.

3. The authors linked chromatin accessibility and increased expression of T helper cell genes to the loss of Foxp3 expression and iTreg instability. However, it is not clear how the former can lead to the latter. It is also not clear whether NFAT binds directly to the Foxp3 locus in the restimulated iTregs and inhibits Foxp3 expression.

Reviewer #2 (Public Review):

The phenotypic instability of in vitro-induced Treg cells (iTregs) has been discussed for a long time, mainly in the context of the epigenetic landscape of Treg-signature genes; e.g. Treg-specifically CpG-hypomethylated Foxp3 CNS2 enhancer region. However, it has been insufficiently understood the upstream molecular mechanisms, the particularity of intracellular signaling of natural Treg cells, and how they connect to stable/unstable suppressive function.

Huiyun Lv et al. addressed the issue of phenotypic instability of in vitro-induced regulatory T cells (iTregs), which is a different point from the physiological natural Treg cells and an obstacle to the therapeutic use of iTreg cells. The authors focused on the difference between iTreg and nTreg cells from the perspective of their control of store-operated calcium entry (SOCE)-mediated cellular signaling, and they clearly showed that the sustained SOCE signaling in iTreg and nTreg cells led to phenotypic instability. Moreover, the authors pointed the correlation between the incomplete conversion of chromatin configuration and the NFAT-mediated control of effector-type gene expression profile in iTreg cells. These findings potentially cultivate our understanding of the cellular identity of regulatory T cells and may shed light on the therapeutic use of Treg cells in many clinical contexts.

The authors demonstrated the biological contribution of Ca2+ signaling with the variable methods, which ensure the reliability of the results and the claims of the authors. iTreg cells sustained SOCE-signaling upon stimulation while natural Treg cells had lower strength and shorter duration of SOCE-signaling. The result was consistent with the previously-proposed concept; a certain range of optimal strength and duration of TCR-signaling shape the Treg generation and maintenance, and it provides us with further in-depth mechanistic understanding.

In the later section, authors found the incomplete installment of Treg-type open chromatin landscape in some effector/helper function-related gene loci in iTreg cells. These findings propose the significance to focus on not only the "Treg"-associated gene loci but also "Teffector-ness"-associated regions to determine the Treg conversion at epigenetic level.

Limitations and weaknesses;
(1) Some concerns about data processing and statistic analysis.
The authors did not provide sufficient information on statistical data analysis; e.g. lack of detailed descriptions about
-the precise numbers of technical/biological replicates of each experiment
-the method of how the authors analyze data of multiple comparisons... Student t-test alone is generally insufficient to compare multiple groups; e.g. figure 1.
These inappropriate data handlings are ruining the evidence level of the precious findings.

(2) Untransparent data production; e.g. the method of Motif enrichment analysis was not provided.
Thus, we should wait for the author's correction to fully evaluate the significance and reliability of the present study.

(3) Lack of evidence in human cells.
I wonder whether human PBMC-derived iTreg cells are similarly regulated.

(4) NFAT regulation did not explain all of the differences between iTregs and nTregs, as the authors mentioned as a limitation.
Also, it is still an open question whether NFAT can directly modulate the chromatin configuration on the effector-type gene loci, or whether NFAT exploits pre-existing open chromatin due to the incomplete conversion of Treg-type chromatin landscape in iTreg cells. The authors did not fully demonstrate that the distinct pattern of chromatin regional accessibility found in iTreg cells is the direct cause of an effector-type gene expression.