Forkhead Box Protein P3 in the Immune System

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments to the author

This paper provides an in-depth exploration of the role of FOXP3 in regulatory T cells (Tregs) and its implications across a broad spectrum of diseases. From autoimmune disorders like T1D and IBD to cancer and allergic diseases, the review covers many facets of immune regulation. Additionally, it delves into translational aspects, particularly gene and cell therapy applications, including the use of CRISPR/Cas9 and engineered Tregs (e.g., CAR-Tregs). While the review is comprehensive and well-structured, several strengths and weaknesses emerge upon further analysis.

Strengths:

 

The author provides a comprehensive Overview, detailing first, the multiple roles that FOXP3 plays in different diseases and immune processes. It covers not just autoimmune diseases and allergies but also cancer and transplantation, making it highly relevant to a wide range of researchers and clinicians. Second, the author incorporates Recent Technological Advances: The mention of CRISPR/Cas9 technology and engineered Tregs is highly timely and reflects ongoing innovations in immunology. Specifically, the focus on CAR-Tregs and engineered Tregs in autoimmune and alloimmune disorders demonstrates how modern therapeutic approaches are converging with our understanding of FOXP3 and Treg biology.

He provides the reader with detailed molecular and epigenetic insight, explaining the epigenetic regulation of FOXP3, including the role of Treg-specific demethylated region (TDRS) and CNS2 methylation in the development of Tregs and their functional stability. Linking FOXP3 Mutation to diease mechanisms.

Finally, the author provides the rider with Clinical Relevance. The sections on clinical applications, particularly the potential use of Treg-based therapies in conditions like autoimmunity, transplantation, and allergic diseases, are well-articulated. The paper bridges basic science with translational medicine, focusing on the promise and challenges of adoptive Treg transfer and engineered Tregs.

 

The tow figures are clear and helpful for the reader.

Weaknesses

1. Lack of In-Depth Analysis of Mechanisms in Diseases: While the paper covers a wide range of diseases and the role of FOXP3 in them, it tends to be more descriptive than analytical in terms of mechanistic insights. For example:
• In autoimmune diseases like T1D, IBD, and SLE, the author mentions Treg dysfunction but could have elaborated more on the underlying molecular mechanisms that cause Treg instability or failure in these diseases.
• The author mentions Treg imbalance in diseases like multiple sclerosis and myasthenia gravis, but does not delve into why this imbalance occurs or how it could be corrected at a mechanistic level. More emphasis on how specific genetic or epigenetic alterations in FOXP3 could lead to disease could have enhanced the scientific depth of the paper.
2. Controversial and Unresolved Issues are Underexplored: There are some controversial or unresolved issues in Treg biology that the review does not address, such as the heterogeneity of Tregs populations, Treg stability vs plasticity. For example, FOXP3+ Tregs can convert into effector T cells under certain inflammatory conditions, a topic that could have been explored in more depth.

 

3. Limited focus on the tumor microenvironment: Limited Focus on the Tumor Microenvironment: The role of Tregs in cancer is discussed, but the section could have been more expansive, especially with regard to the tumor microenvironment.
• Tregs are often recruited into tumors, where they suppress anti-tumor immunity, but this varies by cancer type. The review could have discussed the dual role of Tregs in cancer more thoroughly, noting how they can sometimes have tumor-promoting effects and other times help maintain tumor immune tolerance.
• Additionally, the role of FOXP3 in the tumor microenvironment could have been explored further. While it is mentioned that FOXP3 may function as an oncogene suppressor in breast cancer, the relationship of FOXP3 with immune checkpoint inhibitors (e.g., PD-1/PD-L1, CTLA-4) could have been discussed in greater detail.

 

4. Insufficient Depth on Clinical Trials and Translational Applications: While clinical translation is mentioned, the paper could have been stronger by providing more detailed insights into the current state of clinical trials using Treg-based therapies. For example:
• How far have we progressed in adoptive Treg therapy for diseases like autoimmunity or transplantation? The review could have included data from recent trials, challenges faced, or efficacy outcomes that would provide readers with a clearer picture of what works and what needs improvement.
• The clinical trial data regarding CAR-Tregs or gene-edited Tregs could be expanded. There are some emerging clinical trials using CAR-Tregs for diseases like T1D, but this could have been elaborated on with more recent evidence.

 

5. There is a serious Lack of Discussion on Ethical and Safety Concerns: The review mentions CRISPR/Cas9-mediated gene editing and CAR-Tregs but does not discuss the ethical and safety concerns associated with these technologies. The potential for off-target effects, immune rejection, or long-term consequences of manipulating FOXP3 expression in humans remains a critical issue. A discussion on these topics would provide a more balanced view.

 

6. Visual aids could be added to this review, Figures or diagrams could have greatly enhanced the reader's understanding of the complex molecular mechanisms, Treg development, and interactions of FOXP3 in disease. This is especially true for complex topics like epigenetic regulation or Treg plasticity.

Opportunities for Improvement

1. Expanded Discussion on Treg Heterogeneity and Plasticity: As mentioned, Treg plasticity is an important concept that is not fully explored. The paper could include a discussion on how Tregs change their phenotype depending on environmental cues (e.g., inflammation, infections, cancer) and how this affects their functionality.
2. Clinical Data Integration: Incorporating more specific clinical data or case studies on Treg-based therapies, including details on patient outcomes and challenges faced in clinical trials, would strengthen the paper’s practical relevance. This could be done by including updated results from clinical trials investigating Treg transfer in autoimmune diseases, organ transplantation, and allergies.
3. Ethical Considerations in Treg Therapy: Including a discussion on the ethical implications and potential long-term risks of Treg-based therapies would add a critical layer to the review. This includes potential concerns about the stability and safety of engineered Tregs, as well as the social and ethical considerations of gene editing technologies like CRISPR/Cas9.
4. More Detailed Tumor Microenvironment Discussion: The tumor microenvironment and its interaction with FOXP3+ Tregs warrants deeper exploration, especially regarding how Tregs can both suppress anti-tumor immunity and potentially enhance tumor survival. The review could also include more details on how FOXP3 regulation in Tregs is targeted in immunotherapy.

 

Conclusion:

Overall, this paper provides a valuable and comprehensive summary of FOXP3’s role in immune regulation, its implications in disease, and the promise of Treg-based therapies. However, there is room for improvement in the depth of analysis, especially regarding mechanistic insights, clinical applications, and unresolved controversies in the field. More attention to the plasticity of Tregs, current clinical trials, and safety concerns related to emerging therapies would make this review more insightful and practical for researchers and clinicians working in immunology, gene therapy, and related fields.

Author Response

Comments to the author

This paper provides an in-depth exploration of the role of FOXP3 in regulatory T cells (Tregs) and its implications across a broad spectrum of diseases. From autoimmune disorders like T1D and IBD to cancer and allergic diseases, the review covers many facets of immune regulation. Additionally, it delves into translational aspects, particularly gene and cell therapy applications, including the use of CRISPR/Cas9 and engineered Tregs (e.g., CAR-Tregs). While the review is comprehensive and well-structured, several strengths and weaknesses emerge upon further analysis.

 

Strengths: 

The author provides a comprehensive Overview, detailing first, the multiple roles that FOXP3 plays in different diseases and immune processes. It covers not just autoimmune diseases and allergies but also cancer and transplantation, making it highly relevant to a wide range of researchers and clinicians. Second, the author incorporates Recent Technological Advances: The mention of CRISPR/Cas9 technology and engineered Tregs is highly timely and reflects ongoing innovations in immunology. Specifically, the focus on CAR-Tregs and engineered Tregs in autoimmune and alloimmune disorders demonstrates how modern therapeutic approaches are converging with our understanding of FOXP3 and Treg biology.

He provides the reader with detailed molecular and epigenetic insight, explaining the epigenetic regulation of FOXP3, including the role of Treg-specific demethylated region (TDRS) and CNS2 methylation in the development of Tregs and their functional stability. Linking FOXP3 Mutation to diease mechanisms.

Finally, the author provides the rider with Clinical Relevance. The sections on clinical applications, particularly the potential use of Treg-based therapies in conditions like autoimmunity, transplantation, and allergic diseases, are well-articulated. The paper bridges basic science with translational medicine, focusing on the promise and challenges of adoptive Treg transfer and engineered Tregs.

 

The tow figures are clear and helpful for the reader.

 

Weaknesses

1. Lack of In-Depth Analysis of Mechanisms in Diseases: While the paper covers a wide range of diseases and the role of FOXP3 in them, it tends to be more descriptive than analytical in terms of mechanistic insights. For example:
• In autoimmune diseases like T1D, IBD, and SLE, the author mentions Treg dysfunction but could have elaborated more on the underlying molecular mechanisms that cause Treg instability or failure in these diseases.
• The author mentions Treg imbalance in diseases like multiple sclerosis and myasthenia gravis, but does not delve into why this imbalance occurs or how it could be corrected at a mechanistic level. More emphasis on how specific genetic or epigenetic alterations in FOXP3 could lead to disease could have enhanced the scientific depth of the paper.

(Response) While Treg biology is extensively studied in healthy donors, the clinical research especially on the molecular mechanism of allergy and immune mediated disease is not sufficient. We provided the limitations in the dedicated section.

 

1. Controversial and Unresolved Issues are Underexplored: There are some controversial or unresolved issues in Treg biology that the review does not address, such as the heterogeneity of Tregs populations, Treg stability vs plasticity. For example, FOXP3+ Tregs can convert into effector T cells under certain inflammatory conditions, a topic that could have been explored in more depth.

 

(Response) Thank you so much for pointing this out. We fully agreed with the heterogeneity and plasticity of Treg. We have added dedicated section describing Treg heterogeneity and plasticity. The section will provide more insights on Treg biology and clinical translation.

 

1. Limited focus on the tumor microenvironment: Limited Focus on the Tumor Microenvironment: The role of Tregs in cancer is discussed, but the section could have been more expansive, especially with regard to the tumor microenvironment.
• Tregs are often recruited into tumors, where they suppress anti-tumor immunity, but this varies by cancer type. The review could have discussed the dual role of Tregs in cancer more thoroughly, noting how they can sometimes have tumor-promoting effects and other times help maintain tumor immune tolerance.
• Additionally, the role of FOXP3 in the tumor microenvironment could have been explored further. While it is mentioned that FOXP3 may function as an oncogene suppressor in breast cancer, the relationship of FOXP3 with immune checkpoint inhibitors (e.g., PD-1/PD-L1, CTLA-4) could have been discussed in greater detail.

 

(Response) We provided the figure and added the discussion on tumor metastasis in the dedicated section.

 

1. Insufficient Depth on Clinical Trials and Translational Applications: While clinical translation is mentioned, the paper could have been stronger by providing more detailed insights into the current state of clinical trials using Treg-based therapies. For example:
• How far have we progressed in adoptive Treg therapy for diseases like autoimmunity or transplantation? The review could have included data from recent trials, challenges faced, or efficacy outcomes that would provide readers with a clearer picture of what works and what needs improvement.
• The clinical trial data regarding CAR-Tregs or gene-edited Tregs could be expanded. There are some emerging clinical trials using CAR-Tregs for diseases like T1D, but this could have been elaborated on with more recent evidence.

 

(Response) The aim of this review is to provide current knowledge on the role of Treg in immune and allergic conditions. I fully understand it would be important to provide more description of Treg-based therapy. However, we try just to introduce Treg cell products and the in detailed information would be provided in the future review.

 

1. There is a serious Lack of Discussion on Ethical and Safety Concerns: The review mentions CRISPR/Cas9-mediated gene editing and CAR-Tregs but does not discuss the ethical and safety concerns associated with these technologies. The potential for off-target effects, immune rejection, or long-term consequences of manipulating FOXP3 expression in humans remains a critical issue. A discussion on these topics would provide a more balanced view.

 

(Response) It is very important to describe the limitation and problems in the Treg-based therapy including ehical and possibly technical consderations. During my previous experience, the safety and rationale of the study is always asked during clinical development. I am very happy to share and provide the discussions through my past experience.

 

1. Visual aids could be added to this review, Figures or diagrams could have greatly enhanced the reader's understanding of the complex molecular mechanisms, Treg development, and interactions of FOXP3 in disease. This is especially true for complex topics like epigenetic regulation or Treg plasticity.

 

(Response) We have extensively generated figures to help understanding of the readers. Thank you so much for valuable comments.

 

Opportunities for Improvement

1. Expanded Discussion on Treg Heterogeneity and Plasticity: As mentioned, Treg plasticity is an important concept that is not fully explored. The paper could include a discussion on how Tregs change their phenotype depending on environmental cues (e.g., inflammation, infections, cancer) and how this affects their functionality.

 

(Response) Thank you so much for pointing this out. We fully agreed with the heterogeneity and plasticity of Treg. We have added dedicated section describing Treg heterogeneity and plasticity. The section will provide more insights on Treg biology and clinical translation.

 

1. Clinical Data Integration: Incorporating more specific clinical data or case studies on Treg-based therapies, including details on patient outcomes and challenges faced in clinical trials, would strengthen the paper’s practical relevance. This could be done by including updated results from clinical trials investigating Treg transfer in autoimmune diseases, organ transplantation, and allergies.

 

(Response) Thank you so much for the suggestions. It is wonderful idea to add the current status of clinical trials. There are few reviews on clinical trial of Treg cell therapy, but it is important to update the ongoing clinical trial. This could strengthen the impact of the review.

We have provided dedicated section to address current status of Treg based cell therapy.

 

1. Ethical Considerations in Treg Therapy: Including a discussion on the ethical implications and potential long-term risks of Treg-based therapies would add a critical layer to the review. This includes potential concerns about the stability and safety of engineered Tregs, as well as the social and ethical considerations of gene editing technologies like CRISPR/Cas9.

 

(Response) It is very important to describe the limitation and problems in the Treg-based therapy including ehical and possibly technical consderations. During my previous experience, the safety and rationale of the study is always asked during clinical development. I am very happy to share and provide the discussions through my past experience.

 

1. More Detailed Tumor Microenvironment Discussion: The tumor microenvironment and its interaction with FOXP3+ Tregs warrants deeper exploration, especially regarding how Tregs can both suppress anti-tumor immunity and potentially enhance tumor survival. The review could also include more details on how FOXP3 regulation in Tregs is targeted in immunotherapy.

 

(Response) The study on tumor microenvironment is topic of the field. We have included more refenreces and discussion in the corrensponding section.

 

 

Conclusion:

Overall, this paper provides a valuable and comprehensive summary of FOXP3’s role in immune regulation, its implications in disease, and the promise of Treg-based therapies. However, there is room for improvement in the depth of analysis, especially regarding mechanistic insights, clinical applications, and unresolved controversies in the field. More attention to the plasticity of Tregs, current clinical trials, and safety concerns related to emerging therapies would make this review more insightful and practical for researchers and clinicians working in immunology, gene therapy, and related fields.

 

(Response) I would like to appreciate again for the thoughtful and insightful suggestions and comments from the reivewer. The reviewer provided the strength of the review by including important topic on current knowledge in the field.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “FoxP3 in the immune system” By Sato is an interesting review of FOXP3 and its role as a master regulator of Regulatory cells. Although the information provided is very compelling, it lacks a lot of the depth that would be needed for a solid review. The structure of the paper is great and the topics are of great interest, however often in the sections are a bit to space, most only being two to five sentences long. One aspect of this is that the sections are often divided into the role of FOXP3 expression and the role of Tregs in that particular topic. I agree that covering both topics are critical to a comprehensive review of the subject, as they go hand in hand. Significant expansion of all the topics would greatly improve the impact of the review. Covering a broader swath of what is know and published to convey a greater understanding of the role of FOXP3 expression and T-Regulatory cells.

Author Response

(Response) Thank you so much for the insightful comments. We would like to apologize for the quality of the manuscript. Now, we have added figures (Figure3-8) explaining each topic to provide more information and help understanding of the readers. In addition, we have added 3 additional sections according to the other reviewer's suggestions which will also increase the quality of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The review manuscript is well written. This review also emphasizes on the role and function of regulatory T cell and the related marker, FOXP3 in immune regulation and tolerance in different pathophysiological diseased condition including autoimmune disease, cancer and allergies. Although there are few reviews are also available on the same topic as a whole but the uniqueness of this review is to describe the role of the cell type marker and Treg cell into each individual pathophysiological conditions categorically. This helps to pinpoint the role of this particular subtype of cells related to different conditions individually. Although there are not enough evidences showed in each condition but that has been also stated as a caveat for not having enough references available.

Overall the manuscript is good but there are few minor comments that author may consider to enhance the concept and the write up for this review.

In some cases, the lines can be changed and re-written to make the statements more clear to the reader but it is not an absolute necessity. Like, author may think to rewrite the section in paragraph 3.2.

The role and function of FOXP3 can be elaborated further as this is an important aspect in the metastasis. As this review is concentrated only on the role of these cells and their role in corresponding diseases, the write up should incorporate few underlying mechanism by which it proves the  modulation of different pathophysiological condition. For example, in the section of paragraph 7, the write up concludes the role underlying mechanism to develop allergic reactions. But this lacks in other sections.

Treg cell therapy and engineered Tregs are very recent concepts. So, it would have been better if the author could discuss these deeply rather describing this superficially for the interest to the readers i.e., the differences and the usefulness using CAR-Tregs over polyclonal Tregs.

Author Response

Comments and Suggestions for Authors

The review manuscript is well written. This review also emphasizes on the role and function of regulatory T cell and the related marker, FOXP3 in immune regulation and tolerance in different pathophysiological diseased condition including autoimmune disease, cancer and allergies. Although there are few reviews are also available on the same topic as a whole but the uniqueness of this review is to describe the role of the cell type marker and Treg cell into each individual pathophysiological conditions categorically. This helps to pinpoint the role of this particular subtype of cells related to different conditions individually. Although there are not enough evidences showed in each condition but that has been also stated as a caveat for not having enough references available.

 

(Response) We addressed the limitation of current literatures.

 

Overall the manuscript is good but there are few minor comments that author may consider to enhance the concept and the write up for this review.

 

In some cases, the lines can be changed and re-written to make the statements more clear to the reader but it is not an absolute necessity. Like, author may think to rewrite the section in paragraph 3.2.

 

(Response) We have revised throughout paragraph 3.2.

 

The role and function of FOXP3 can be elaborated further as this is an important aspect in the metastasis. As this review is concentrated only on the role of these cells and their role in corresponding diseases, the write up should incorporate few underlying mechanism by which it proves the  modulation of different pathophysiological condition. For example, in the section of paragraph 7, the write up concludes the role underlying mechanism to develop allergic reactions. But this lacks in other sections.

 

(Response) Thank you so much for your insightful comments. We have provided the role of Treg in tumor metastasis.

 

Treg cell therapy and engineered Tregs are very recent concepts. So, it would have been better if the author could discuss these deeply rather describing this superficially for the interest to the readers i.e., the differences and the usefulness using CAR-Tregs over polyclonal Tregs.

 

(Response) Thank you so much for your insightful comments. We have provided in depth description of Treg based cell therapy.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks to the author considering my comments and suggestions to revise the review. I agree, that added a significant improvement the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The updated manuscript looks great! Love the additional content and the figures really help convey the message. Great job!