Unraveling the Epigenetic Regulation of Regulatory T Cells in Cancer Immunity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review article by Subedi et al aims to comprehensively summarize the epigenetic mechanisms regulating regulatory T cell (Treg) development, stability, and suppressive function, with particular emphasis on cancer immunity. The authors provide an extensive overview of DNA methylation, histone modifications, non-coding RNAs, and Foxp3 post-translational modifications, as well as cancer-type–specific insights. The topic is highly relevant to immunology and oncology, and the manuscript is well referenced and addresses an important gap regarding how epigenetic programs shape Treg behaviour in the tumor microenvironment.

 

Strength:

1. Several recent reviews have covered the topic of Tregs in cancer immunology, but this manuscript adds value by organizing the epigenetic mechanisms systematically and by providing cancer-type–specific insights.
2. Comprehensive coverage of major epigenetic mechanisms (DNA methylation, histone modifications, noncoding RNAs, PTMs). And the explanation of Foxp3 regulation and lineage stability is clear
3. Strong collection of references, including recent publications. Most references are current and relevant.
4. Relevance to cancer immunology, potential therapeutic strategies against Tregs, especially the therapeutic potential of targeting epigenetic regulators in Treg provided translational insight in cancer immunotherapy.
5. Conclusions are generally consistent with the cited literature.

The review could be improved by making some minor correction in the following sections:

1. Lines 120-121 and 130-131 “These processes shape cellular identity and enable dynamic responses to environmental cues.” are replicated.
2. Lines 169–176: TSDR mechanisms are important; however, discussion should contrast natural Tregs vs induced Tregs more explicitly.
3. In section 4 Cancer type specific epigenetic regulation of Tregs, it would be good to clarify which epigenetic regulations occur within Tregs versus those occurring in tumor cells that indirectly affect Tregs.
4. Conclusions are generally consistent with the cited literature, but the final section could benefit from a more balanced discussion of limitations, therapeutic challenges, and emerging technologies.

Author Response

Reviewer 1

We thank the reviewer for the positive and encouraging evaluation of our manuscript and for recognizing its comprehensive and systematic overview of epigenetic mechanisms regulating Treg biology, the cancer-type specific insights, and its relevance to cancer immunotherapy. In response to the reviewer’s comments, we address each point individually as outlined below.

Comments and Suggestions for Authors:

This review article by Subedi et al aims to comprehensively summarize the epigenetic mechanisms regulating regulatory T cell (Treg) development, stability, and suppressive function, with particular emphasis on cancer immunity. The authors provide an extensive overview of DNA methylation, histone modifications, non-coding RNAs, and Foxp3 post-translational modifications, as well as cancer-type–specific insights. The topic is highly relevant to immunology and oncology, and the manuscript is well referenced and addresses an important gap regarding how epigenetic programs shape Treg behavior in the tumor microenvironment.

 Strength:

1. Several recent reviews have covered the topic of Tregs in cancer immunology, but this manuscript adds value by organizing the epigenetic mechanisms systematically and by providing cancer-type–specific insights.
2. Comprehensive coverage of major epigenetic mechanisms (DNA methylation, histone modifications, noncoding RNAs, PTMs). And the explanation of Foxp3 regulation and lineage stability is clear
3. Strong collection of references, including recent publications. Most references are current and relevant.
4. Relevance to cancer immunology, potential therapeutic strategies against Tregs, especially the therapeutic potential of targeting epigenetic regulators in Treg provided translational insight in cancer immunotherapy.
5. Conclusions are generally consistent with the cited literature.

Response: We sincerely appreciate the reviewer’s positive feedback and encouragement.

The review could be improved by making some minor correction in the following sections:

1. Lines 120-121 and 130-131 “These processes shape cellular identity and enable dynamic responses to environmental cues.” are replicated.

Response: We have removed this Lines 130-131 repetition from this section.

2. Lines 169–176: TSDR mechanisms are important; however, discussion should contrast natural Tregs vs induced Tregs more explicitly.

Response: We have updated these in lines 173-189.

3. In section 4 Cancer type specific epigenetic regulation of Tregs, it would be good to clarify which epigenetic regulations occur within Tregs versus those occurring in tumor cells that indirectly affect Tregs.

Response: We are grateful for reviewer feedback. We have modified these in Section 4.1 (Mechanism of Treg infiltration and suppressive function in cancer) with separation of epigenetic regulations occur within Tregs versus those occurring in tumor cells that indirectly affect Tregs in lines 575-626. It is also updated in melanoma section 4.2 in lines 643-680.

4. Conclusions are generally consistent with the cited literature, but the final section could benefit from a more balanced discussion of limitations, therapeutic challenges, and emerging technologies.

Response: We are grateful for reviewer feedback. We have updated these in conclusion section in lines 873-904.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript addresses a highly relevant and timely topic at the intersection of tumor immunology and epigenetic regulation of Tregs. The authors survey a broad range of mechanisms, including DNA methylation, histone modifications, and noncoding RNAs, and attempt to integrate these with cancer-specific Treg biology across several tumor types. Foundational explanations of FOXP3 regulation and Treg epigenetic stability are well-cited, and the inclusion of ncRNA-based mechanisms is a notable strength. The manuscript demonstrates substantial effort, wide literature engagement, and an ambitious scope.

However, the manuscript often reads as a list of facts or disconnected mechanistic descriptions, rather than developing a clear conceptual narrative such as - how tumors epigenetically shape Treg identity? How Tregs suppress tumor immunity through epigenetic programs? How do these mechanisms differ by cancer type? what epigenetic “hallmarks” characterize tumor-infiltrating Tregs? What is the scope of epigenetic therapy for cancer?

Section-wise comments:

1. ABSTRACT:
2. Does not specify what gap this review fills.
3. Mentions textbook-like definitions; instead there’s a need to emphasize novelty, unresolved questions, and scope
4. Requires better rewording

 

1. INTRODUCTION:
2. Authors provide a redundant explanation of Treg biology and basic epigenetics, without sufficient conceptual framing.
3. There is no sufficient definition of tumor-specific Treg epigenetics, differences between nTreg vs pTreg vs TI-Treg, or how tumors remodel Treg chromatin.

 

• DNA MODULATION MECHANISMS:

1. Over-generalization: implies methylation globally shapes TI-Treg identity without citing tumor-specific data
2. Too much textbook material for each mechanism – almost all first paragraphs in this section are elaborate.
3. Legend for Figure-2 is too long and lists DNMTs and TETs without connecting to Treg-specific function.

 

1. HISTONE MODIFICATIONS & CHROMATIN REMODELING:
2. Excessive repetition of H3K4me3, H3K27ac, H3K27me3 definitions.
3. No definition or acronym of Tumor-infiltrating Tregs (TI Tregs).
4. No discussion of super-enhancer reprogramming in TI-Tregs (critical omission).
5. No mention of metabolic–epigenetic integration (acetate, lactate, hypoxia).

 

1. NON-CODING RNAs:
2. Tumor-derived and Treg-intrinsic miRNAs are discussed together without a clear distinction between the two.
3. Overstates role of miR-21 without proper citations.
4. No distinction between human and murine studies. Mostly, murine studies were included. Do the same miRNAs have similar function in mouse and human?
5. A table summarizing miRNA functions, target genes, associated Treg phenotypes, and animal models is recommended to avoid repetitive text.
6. Provide mechanistic diagrams showing how ncRNAs modulate Foxp3.
7. Incorrect citations/ no citations for role of Malat1. The citation reference #115 describes the role of a different lncRNA i.e., MIAT (not Malat1) in hepatocellular carcinoma which is not relevant to
8. For lncRNA Flatr, functional implication is listed but no insights into the mechanism was described, or relevant targets were listed.

 

1. FOXP3 POST-TRANSLATIONAL MODIFICATIONS:
2. Section is very descriptive. A table explaining all the post-translational modifications, with the modifiers and effect on Foxp3 expression, stability and Treg functions is ideal.
3. Multiple acronyms used without mentioning the full form.

 

• CANCER TYPE SPECIFIC EPIGENETIC REGULATION OF TREGS

Melanoma:

1. lncRNA discussion is superficial and incomplete and does not advance current understanding. Although authors list several lncRNAs, they do not discuss any mechanistic possibilities - Are these lncRNAs likely Treg-modulatory? Do any influence cytokines, chemokines, or immune checkpoints?
2. Weak integration of melanoma biology with Treg biology – how Melanoma epigenetics shape Treg recruitment or programming is not discussed.
3. HDAC6 inhibitors described without context. Why HDAC6 specifically matters in melanoma immune biology?

General comments:

1. Several paragraphs in the introduction begin with textbook-style explanations that are wordy yet provide limited meaningful information. These should be more concise, precise, and focused on relevant content.
2. Incorrect grammar and sentence framing seen across multiple sections and paragraphs.
3. Structuring and nomenclature are inconsistent across the section ‘non-coding RNAs’. For e.g., subtitling miRNAs, a ‘:’ is used which is inconsistent for lncRNAs.
4. Full forms of acronyms are missing (e.g., TI-Tregs, PRMT5, NLK).
5. The authors did not cite some existing review articles (listed below) that are focused on epigenetic modifications in Tregs and did not mention what gaps in the literature this review fulfills.

• Khalilollah S, Kalantari Soltanieh S, Obaid Saleh R, Ali Alzahrani A, Ghaleb Maabreh H, Mazin Al-Hamdani M, Dehghani-Ghorbi M, Shafiei Khonachaei M, Akhavan-Sigari R. LncRNAs involvement in pathogenesis of immune-related disease via regulation of T regulatory cells, an updated review. Cytokine. 2024 Jul;179:156585. doi: 10.1016/j.cyto.2024.156585. Epub 2024 Apr 4. PMID: 38579428.
• Yue Y, Ren Y, Lu C, Li P, Zhang G. Epigenetic regulation of human FOXP3+ Tregs: from homeostasis maintenance to pathogen defense. Front Immunol. 2024 Jul 31;15:1444533. doi: 10.3389/fimmu.2024.1444533. PMID: 39144146; PMCID: PMC11323565.

Comments on the Quality of English Language

Needs writing and grammatical improvements

Author Response

Reviewer 2,

We thank you for your insightful and constructive critique. In response, we revised the manuscript to strengthen the conceptual narrative by explicitly integrating how tumors epigenetically shape Treg identity and function, how these programs differ across cancer types, and the emerging epigenetic hallmarks and therapeutic implications of tumor-infiltrating Tregs. We also incorporated discussion of super-enhancer reprogramming in tumor-infiltrating Tregs and metabolic-epigenetic integration.

To avoid redundancy, we summarized miRNAs and lncRNAs (Table 1) and Foxp3 post-translational modifications (Table 2) along with their roles in Treg biology. In addition, the sections on lncRNAs in melanoma and the integration of melanoma biology with Treg regulation were updated.

We are very grateful for these constructive comments, which have significantly improved the clarity and focus of our manuscript. A detailed, point-by-point response is provided below.

Comments and Suggestions for Authors

This manuscript addresses a highly relevant and timely topic at the intersection of tumor immunology and epigenetic regulation of Tregs. The authors survey a broad range of mechanisms, including DNA methylation, histone modifications, and noncoding RNAs, and attempt to integrate these with cancer-specific Treg biology across several tumor types. Foundational explanations of FOXP3 regulation and Treg epigenetic stability are well-cited, and the inclusion of ncRNA-based mechanisms is a notable strength. The manuscript demonstrates substantial effort, wide literature engagement, and an ambitious scope.

However, the manuscript often reads as a list of facts or disconnected mechanistic descriptions, rather than developing a clear conceptual narrative such as - how tumors epigenetically shape Treg identity? How Tregs suppress tumor immunity through epigenetic programs? How do these mechanisms differ by cancer type? what epigenetic “hallmarks” characterize tumor-infiltrating Tregs? What is the scope of epigenetic therapy for cancer?

Response: We thank the reviewer for this thoughtful critique. In response, we have revised the manuscript to strengthen the conceptual framework and improve narrative flow. Specifically, we now more clearly articulate how tumors epigenetically shape Treg identity and function, how Tregs suppress anti-tumor immunity through distinct epigenetic programs, and how these mechanisms vary across cancer types. We also highlight emerging epigenetic “hallmarks” of tumor-infiltrating Tregs and better define the scope and therapeutic potential of epigenetic interventions in cancer. These revisions aim to move beyond a descriptive presentation and provide a more integrated, concept-driven perspective. Thank you once again. We are very grateful for your constructive comments for this updated revised version.

Section-wise comments:

ABSTRACT:

Does not specify what gap this review fills.

Mentions textbook-like definitions; instead there’s a need to emphasize novelty, unresolved questions, and scope, requires better rewording

Response: We thank the reviewer for this constructive feedback. The abstract has been substantially revised to clearly define the knowledge gap addressed by this review, specifically the limited understanding of how tumor-specific epigenetic programs shape Treg identity and function across different cancer types. The revised wording improves clarity while highlighting the novelty and focus of the manuscript. (lines16-32).

 INTRODUCTION:

1. Authors provide a redundant explanation of Treg biology and basic epigenetics, without sufficient conceptual framing.
2. There is no sufficient definition of tumor-specific Treg epigenetics, differences between nTreg vs pTreg vs TI-Treg, or how tumors remodel Treg chromatin. (do I add in Introduction)

Response: We thank the reviewer for this constructive feedback. In response, we have revised the Introduction to reduce redundant textbook-style descriptions of general Treg biology and basic epigenetic mechanisms and to improve conceptual framing. We now explicitly define tumor-specific Treg epigenetics in the section 4. Cancer type specific epigenetic regulation of Tregs in lines 594-626 and clearly distinguish between thymus-derived Tregs (nTregs), peripherally induced Tregs (pTregs) (Lines 70-83), and tumor-infiltrating Tregs (TI-Tregs) in lines 87-89. In addition, we have expanded the tumor microenvironment, metabolic constraints, hypoxia, and tumor-derived signals, thereby reinforcing Foxp3 expression and suppressive function in lines 606-626. These revisions provide a clearer framework for the review and better contextualize subsequent sections.

 

DNA MODULATION MECHANISMS:

1. Over-generalization implies methylation globally shapes TI-Treg identity without citing tumor-specific data
2. Too much textbook material for each mechanism – almost all first paragraphs in this section are elaborate.
3. Legend for Figure-2 is too long and lists DNMTs and TETs without connecting to Treg-specific function.

Response: We thank the reviewer for these insightful comments regarding the DNA Modulation Mechanisms section. First, we agree that the original text overgeneralized the role of DNA methylation in shaping tumor-infiltrating Treg (TI-Treg) identity. In the revised manuscript, we have tempered these statements and now restrict conclusions to findings supported by tumor-specific or Treg-focused studies, explicitly noting where evidence is derived from non-tumor or non-Treg contexts. Second, to improve focus and reduce textbook-style content, we have substantially condensed the introductory paragraphs for each DNA modulation mechanism, retaining only information directly relevant to Tregs and Foxp3 regulation. Third, we have revised the legend of Figure 2 to shorten (Line 145-150). These changes enhance clarity and strengthen the relevance of this section to TI-Treg-specific epigenetic regulation.

 

HISTONE MODIFICATIONS & CHROMATIN REMODELING:

1. Excessive repetition of H3K4me3, H3K27ac, H3K27me3 definitions.
2. No definition or acronym of Tumor-infiltrating Tregs (TI Tregs).
3. No discussion of super-enhancer reprogramming in TI-Tregs (critical omission).
4. No mention of metabolic–epigenetic integration (acetate, lactate, hypoxia).

Response: We thank the reviewer for these detailed and constructive comments. In response, we have carefully revised the Histone Modifications & Chromatin Remodeling section to improve clarity, focus, and completeness. First, to reduce redundancy, we have removed repeated definitions of common histone marks (H3K4me3, H3K27ac, and H3K27me3) and now provide a single concise description at their first mention, using abbreviated references thereafter. Second, we have defined and introduced the acronym “tumor-infiltrating regulatory T cells (TI-Tregs)” at its first occurrence to improve readability. Third, we have added a dedicated subsection of 2.4 Chromatin Organization and Accessibility in Tregs. Under this subheading 2.4.1 Super-enhancer reprogramming in Tregs (Lines 424-453), we have highlighted its role in reshaping Foxp3-associated transcriptional networks and reinforcing Treg identity within the tumor microenvironment. Finally, to address the critical link between metabolism and epigenetic regulation, we have added a new subheading 2.4.3 Metabolic-Epigenetic integration in Tregs, (Lines 470-496) which discusses how metabolic cues such as acetate availability, lactate accumulation, and hypoxia influence chromatin modifications and transcriptional programs in Tregs. These revisions substantially strengthen the conceptual framework and address the reviewer’s concerns.

 

 

NON-CODING RNAs:

1. Tumor-derived and Treg-intrinsic miRNAs are discussed together without a clear distinction between the two.

Response: We thank the reviewer for highlighting this important point. We agree that tumor-derived miRNAs and Treg-intrinsic miRNAs were not sufficiently distinguished in the initial version. In the revised manuscript, we have clearly separated these two categories.

2. Overstates role of miR-21 without proper citations.

Response:  We thank the reviewer for this important clarification regarding the role of miR-21. We agree that the original text overstated the functional importance of miR-21 in regulatory T cell differentiation and function. In the revised manuscript, we have substantially revised this section to accurately reflect the current literature (Lines 333-344).

3. No distinction between human and murine studies. Mostly, murine studies were included.

Response:  We thank the reviewer for this important comment. We agree that the initial version mostly included murine studies. In the revised manuscript, we have explicitly indicated the species for each study discussed in Table 1 and clarified that the majority of functional evidence is derived from murine models. Where available, we now highlight human data and note instances in which similar miRNAs have been reported to play comparable roles in mouse and human Tregs.

4. A table summarizing miRNA functions, target genes, associated Treg phenotypes, and animal models is recommended to avoid repetitive text.

Response:  We thank the reviewer for this helpful suggestion. To address this comment and reduce repetitive text, we have now added a table summarizing key miRNAs and long noncoding RNAs, their impact on Tregs, and their Targets with species involved in Table 1. This Table 1 provides a concise overview of the available evidence and improves clarity and readability of the manuscript.

5. Provide mechanistic diagrams showing how ncRNAs modulate Foxp3.

Response:  We appreciate the reviewer’s suggestion. While mechanistic diagrams of ncRNA-mediated Foxp3 regulation would be informative, the manuscript is intended to provide an integrated overview of Foxp3 regulation and is therefore not focused exclusively on ncRNAs. Accordingly, we have revised the text and provided Table 1 (Lines 1732-1734) to more explicitly describe established ncRNA–Foxp3 regulatory mechanisms without adding a standalone mechanistic diagram.

6. Incorrect citations/ no citations for role of Malat1. The citation reference #115 describes the role of a different lncRNA i.e., MIAT (not Malat1) in hepatocellular carcinoma which is not relevant to

Response:  We thank the reviewer for pointing out the incorrect citation regarding the role of lncRNA MALAT1. We agree that reference #115 describes the function of a different lncRNA, MIAT, in hepatocellular carcinoma and is not relevant to MALAT1. In the revised manuscript, we have corrected this error by removing the inappropriate citation and replacing it with relevant and accurate references that specifically describe the role of MALAT1 in immune regulation and/or disease contexts discussed in our study. All citations related to MALAT1 have been carefully re-checked to ensure accuracy and relevance. Please see lines 391-401.

7. For lncRNA Flatr, functional implication is listed but no insights into the mechanism was described, or relevant targets were listed.

Response:  We thank the reviewer for raising this important point. In the revised manuscript, we have rewritten the description of lncRNA Flatr to better clarify its potential function. Literatures support regulatory role of Flatr in the upstream cascade of Treg differentiation. However, its molecular mechanism and direct targets remain fully characterized.

 

FOXP3 POST-TRANSLATIONAL MODIFICATIONS: Section is very descriptive. A table explaining all the post-translational modifications, with the modifiers and effect on Foxp3 expression, stability and Treg functions is ideal.

Response:  We thank the reviewer for this helpful suggestion. To improve clarity and accessibility, we have added a new Table 2 (Lines 1737-1739)  summarizing the reported post-translational modifications of Foxp3, including the locations, corresponding modifiers and their effects on Foxp3 expression and species involved, protein stability, and Treg function. We believe this table provides a concise overview that complements the detailed discussion in the text.

1. Multiple acronyms used without mentioning the full form.

Response:  We thank the reviewer for pointing this out. All acronyms have now been defined in full at their first mention in the manuscript to improve clarity and ensure accessibility for a broad readership.

 

        CANCER TYPE SPECIFIC EPIGENETIC REGULATION OF TREGS

        Melanoma:

1. lncRNA discussion is superficial and incomplete and does not advance current understanding. Although authors list several lncRNAs, they do not discuss any mechanistic possibilities - Are these lncRNAs likely Treg-modulatory? Do any influence cytokines, chemokines, or immune checkpoints?

Response: We agree with the reviewer and have significantly expanded the lncRNA section to move beyond descriptive listing toward mechanistic interpretation. In the revised manuscript, we now discuss how melanoma-associated lncRNAs such as MALAT1, BANCR, HOTAIR and SNHG5 may participate in melanoma growth that may indirectly foster a TME that regulate Tregs (please see lines 643-662). However direct connection is not known. There is limited information on cytokines, chemokines or immune checkpoint involved on these lncRNAs.

2. Weak integration of melanoma biology with Treg biology – how Melanoma epigenetics shape Treg recruitment or programming is not discussed.

Response: We appreciate this comment and have revised the manuscript to more explicitly integrate melanoma epigenetic regulation with Treg biology. Our main purpose was to focus on Treg biology in melanoma. However, the revised text now emphasizes how melanoma-specific epigenetic programs, including lncRNA expression, miRNA expression, immune check point actively shapes the immune landscape rather than passively reflecting tumor progression (Lines 643-680).

 

General comments:

1. Several paragraphs in the introduction begin with textbook-style explanations that are wordy yet provide limited meaningful information. These should be more concise, precise, and focused on relevant content.

Response: We agree with the reviewer and have revised the Introduction to remove overly general, textbook-style descriptions. These sections have been condensed and refocused to emphasize information directly relevant to Treg biology, epigenetic regulation, and cancer immunity, thereby improving precision and readability while maintaining necessary context for the review.

2. Incorrect grammar and sentence framing seen across multiple sections and paragraphs.

Response: We appreciate this observation and have thoroughly edited the manuscript to correct grammatical errors, improve sentence structure, and enhance overall readability. All sections were carefully reviewed to ensure clarity, consistency, and adherence to formal scientific writing standards.

3. Structuring and nomenclature are inconsistent across the section ‘non-coding RNAs’. For e.g., subtitling miRNAs, a ‘:’ is used which is inconsistent for lncRNAs.

Response: We thank the reviewer for highlighting this inconsistency. The “Non-coding RNAs” section has been restructured to ensure uniform formatting and nomenclature. Subheadings for miRNAs and lncRNAs now follow a consistent style throughout the manuscript, improving coherence and professional presentation.

3. Full forms of acronyms are missing (e.g., TI-Tregs, PRMT5, NLK).

Response: We have revised the manuscript to ensure that all acronyms are defined at first mention. Specifically, we now include the full forms for tumor-infiltrating regulatory T cells (TI-Tregs), protein arginine methyltransferase 5 (PRMT5), and Nemo-like kinase (NLK), as well as other abbreviations used throughout the text.

4.The authors did not cite some existing review articles (listed below) that are focused on epigenetic modifications in Tregs and did not mention what gaps in the literature this review fulfills.

Response: We agree and have now incorporated the suggested review articles and other relevant literature into the revised manuscript. In addition, we have clarified the distinct contribution of our review, emphasizing that while previous reviews have focused broadly on epigenetic regulation in Tregs, our manuscript specifically integrates non-coding RNA regulation, chromatin-modifying enzymes, and melanoma immune biology, highlighting unresolved mechanistic gaps and emerging therapeutic opportunities. We have updated the references in Line 1222 (Khalilollah S  et al., 2024) and Line 1382 (Yue et al., 2024).

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have reviewed the literature relevant to the role of epigenetic regulation in T regs.  Epigenetic coverage is relatively complete with the exception of any comments on nucleosome location.  Nucleosome location has been studied in many systems most recently by ATAC-Seq and i am surprised that it was not included.  At a minimum i think the authors should at least indicate that nucleosome phasing/positioning is important and the reason why they chose not to include it.  I also notice that there is an "of"  missing in line 498.  

Author Response

Reviewer 3

Thank you for providing us thoughtful insight to the nucleosome phasing/positioning. Emerging chromatin accessibility data strongly suggest that nucleosome organization represents a key regulatory layer in Treg biology and warrants deeper investigation in future studies. In response to your suggestion, we have briefly discussed this aspect in the revised manuscript, as outlined in the paragraph below.

Comments and Suggestions for Authors

The authors have reviewed the literature relevant to the role of epigenetic regulation in T regs.  Epigenetic coverage is relatively complete with the exception of any comments on nucleosome location.  Nucleosome location has been studied in many systems most recently by ATAC-Seq and i am surprised that it was not included.  At a minimum i think the authors should at least indicate that nucleosome phasing/positioning is important and the reason why they chose not to include it.  I also notice that there is an "of" missing in line 498.  

Response: We thank the reviewer for this insightful comment. Emerging chromatin accessibility data strongly suggest that nucleosome organization is a key regulatory layer in Treg biology and warrants deeper investigation in future studies. Nucleosome positioning in Tregs has been briefly alluded in subtitled 2.4.2 Nucleosome Positioning and Chromatin Accessibility in Tregs under 2.4 Chromatin Organization and Accessibility in Tregs; in the revised manuscript, and its importance in regulating chromatin accessibility and Treg-specific transcriptional programs (Lines 455-468).

We have also updated line 498.