Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The title and abstract effectively cover the content and focus of the manuscript.
The introduction provides relevant theoretical background, a statement of the problem, and a proposed approach.

The paper is well written, clearly structured, and addresses an important and timely question: the mechanism explaining the connection between Regulatory T cells (Tregs, CD4⁺ CD25⁺ Foxp3⁺) and immune homeostasis in the ocular immune-privileged microenvironment. Accessible to readers, with references that strengthen the contextual framework, clearly presented.

The Introduction starts with Inflammatory eye diseases, indicating the necessity of changing traditional clinical treatments, which mainly rely on broad-spectrum anti-inflammatory drugs, to precise, low-toxicity targeted immunomodulatory therapies. What is the main significance of the fact that the eye has a unique immune privilege characteristic? The authors point out Tregs as a key set of cells maintaining immunotolerance in the eyes.

Treg cells are the core regulatory hub of ocular immune homeostasis. What are the other important factors contributing to immune privilege in the eye? Are they equally important as Tregs?

What kind of different Treg mechanisms could be expected during different pathological states? You explained some of them, precisely two types of them. Is there any kind of pathology where Tregs could have the most significant role, like autoimmune diseases?

The physiological distribution of Treg cells in the eye and the mechanism of maintaining immune homeostasis are very well written and explained. The table is detailed and precise, giving the readers important information on different Treg Cells in Major Ocular Tissues and Inflammatory Eye Diseases. And three other tables are precise and clear, which is very important to people interested in these themes.

The molecular mechanism of Treg Cells in Maintaining Ocular Immune Homeostasis is precisely given in the diagram.

Treg cells in inflammatory diseases are mainly included in two aspects: on the one hand, in the early stage of inflammation, the number of Treg cells increases in a compensatory manner to inhibit the excessive inflammatory response and prevent the spread of inflammation; on the other hand, in the middle and late stages of persistent inflammation, the pro-inflammatory microenvironment leads to the functional impairment or quantitative reduction of Treg cells. Could you please explain if there are any other aspects of including Treg cells in inflammatory diseases?

What is the mechanism of the CD25lo Treg subset in the cornea to transform into ex-Treg, secreting IFN-γ and promoting the progression of corneal stromal inflammation?

Is there some common mechanism that can explain all forms of uveitis or all forms of conjunctivitis, for example?

What is interesting is the MSC application in the healing of some ocular diseases. In your opinion, what eye inflammatory diseases could be treated in this way?

What is the difference in the results when using cell therapy or using the P2X7R antagonist? The same is the result: alleviating retinal inflammatory infiltration, but in different ways.

How do these ways differ mechanistically? What are the situations when we are using the first or the second, respectively?

The discussion is short but summarizes all the results the authors find in the literature.

The authors of the manuscript clearly and precisely defined limitations and future directions.

” In the future, it is necessary to further elucidate the disease-specific regulatory mechanisms of Treg cells, overcome technical bottlenecks, promote the translation of basic research into clinical applications, and ultimately develop safe and effective novel immunomodulatory therapies to improve the visual prognosis of patients with inflammatory eye diseases.”

This manuscript is very detailed and clear. The authors did an extraordinary job. My questions are for the future articles; I was just sincerely interested in them.

Kind regards

Author Response

Response to Reviewers' Comments

 

Dear Editor and Reviewers,

 

We sincerely thank you for your thorough review and valuable comments on our manuscript. After receiving your feedback, we carefully considered each point, meticulously reviewed the entire manuscript, and made revisions accordingly. All modifications are highlighted in red in the uploaded "Revised Manuscript." Below is our point-by-point response to the reviewers' comments.

 

Comment 1

Reviewer's Original Comment: The Introduction starts with Inflammatory eye diseases, indicating the necessity of changing traditional clinical treatments, which mainly rely on broad-spectrum anti-inflammatory drugs, to precise, low-toxicity targeted immunomodulatory therapies. What is the main significance of the fact that the eye has a unique immune privilege characteristic? The authors point out Tregs as a key set of cells maintaining immunotolerance in the eyes.

 

Response: Thank you for this insightful question. We have revised the introduction to explicitly state the core significance of ocular immune privilege. As you rightly pointed out, its main significance lies in providing the essential supportive microenvironment for Treg cells to exert their function. The combination of physical barriers, anti-inflammatory humoral factors, and immunosuppressive resident cells collectively establishes a "low-inflammatory interference" setting. This environment protects Treg cells from excessive inflammatory pressure, allowing them to effectively maintain immune tolerance. Thus, immune privilege is the foundational basis that makes Treg cells the core regulators of ocular immune homeostasis. The specific modification content is in: line 38 - line 44

 

Comment 2

Reviewer's Original Comment: Treg cells are the core regulatory hub of ocular immune homeostasis. What are the other important factors contributing to immune privilege in the eye? Are they equally important as Tregs?

 

Response: We appreciate this critical perspective. We agree that describing Tregs as the sole "core hub" may be an oversimplification. In the revised manuscript, we have clarified that ocular immune privilege is a multi-factorial system. Tregs are a vital executive component, but they are supported by other equally important factors: the physical barrier of the blood-eye barrier, the humoral regulation of aqueous humor factors, and the active suppression by ocular resident cells. We now emphasize that these elements work synergistically in a network, each being indispensable for maintaining the overall immune-privileged state. The specific modification content is in: line 45 - line 50 and line 59 - line 62.

 

Comment 3

Reviewer's Original Comment: What kind of different Treg mechanisms could be expected during different pathological states? You explained some of them, precisely two types of them. Is there any kind of pathology where Tregs could have the most significant role, like autoimmune diseases?

 

Response: Thank you for these profound questions. We have expanded the description of Treg mechanisms in pathology to include a third crucial aspect: functional plasticity and pro-inflammatory conversion, as seen in HSV-1 keratitis where CD25lo Tregs become IFN-γ-secreting ex-Tregs. Regarding the pathology where Tregs are most significant, we fully agree with your suggestion: autoimmune diseases. We have added a clear statement in the Discussion (6.1) highlighting that in autoimmune uveitis and similar conditions, Treg dysfunction is a core pathogenic element, making Treg-targeted therapies particularly promising for these diseases. The specific modification content is in: line 215, line 221 - line 228 and line 620 - line 621.

 

Comment 4

Reviewer's Original Comment: Treg cells in inflammatory diseases are mainly included in two aspects: on the one hand, in the early stage of inflammation, the number of Treg cells increases in a compensatory manner to inhibit the excessive inflammatory response and prevent the spread of inflammation; on the other hand, in the middle and late stages of persistent inflammation, the pro-inflammatory microenvironment leads to the functional impairment or quantitative reduction of Treg cells. Could you please explain if there are any other aspects of including Treg cells in inflammatory diseases?

 

Response: This response has been merged with the revision for Comment 3. Following the originally described "two aspects," we have added a discussion on the "third aspect." The specific modification content is in: line 221 - line 228.

 

Comment 5

Reviewer's Original Comment: What is the mechanism of the CD25lo Treg subset in the cornea to transform into ex-Treg, secreting IFN-γ and promoting the progression of corneal stromal inflammation?

 

Response: This is an excellent question regarding a specific molecular mechanism. We have elaborated on the described process in section 3.1.2. The core mechanism involves the IL-12/STAT4/T-bet signaling axis. High local IL-12 levels during HSV-1 infection activate STAT4 in CD25lo Tregs, which induces T-bet. T-bet then acts as a master regulator, repressing Foxp3 and instigating a Th1-like, pro-inflammatory program characterized by IFN-γ secretion. This detailed pathway explains the molecular basis of Treg instability in viral keratitis. The specific modification content is in: line 253 - line 262.

 

Comment 6

Reviewer's Original Comment: Is there some common mechanism that can explain all forms of uveitis or all forms of conjunctivitis, for example?

 

Response: Thank you for this macro-level question. We have now explicitly stated the common mechanisms at the beginning of the respective sections. For uveitis, the shared mechanism is the breakdown of the Th17/Treg balance. For conjunctivitis, the commonality is altered Treg cell function, though the direction of change differs (loss of function in allergy vs. gain of immunosuppressive function in cancer). This clarification strengthens the logical flow between disease-specific discussions. The specific modification content is in: line 276 - line 278 and line 312 - line 317.

 

Comment 7

Reviewer's Original Comment: What is interesting is the MSC application in the healing of some ocular diseases. In your opinion, what eye inflammatory diseases could be treated in this way?

 

Response: We appreciate the interest in this promising therapeutic avenue. In the revised manuscript (Section 5.1.1), we have added our perspective on the most promising applications for MSC-based therapy in ophthalmology. We highlight autoimmune diseases, high-risk corneal transplantation, retinal ischemic injury, and severe immune-mediated dry eye as prime candidates, given the strong preclinical evidence supporting MSC-mediated immunomodulation and tissue protection in these conditions. The specific modification content is in: line 510 - line 518.

 

Comments 8 & 9

Reviewer's Original Comment: 8. What is the difference in the results when using cell therapy or using the P2X7R antagonist? The same is the result: alleviating retinal inflammatory infiltration, but in different ways. 9. How do these ways differ mechanistically? What are the situations when we are using the first or the second, respectively?

 

Response: We have combined our response to these two interrelated questions. We have added a new comparative analysis section (5.2.3) that explicitly contrasts the two strategies.

Mechanistic Difference: We clarify that Treg therapy is an "active addition" of suppression, while P2X7R blockade is an "indirect de-repression" of endogenous Tregs by calming innate immunity.

Application Context: We propose that cell therapy is suited for acute, severe cases requiring potent intervention, whereas P2X7R antagonists may be ideal for chronic management or early intervention where fine-tuning the microenvironment is the goal. This provides a clearer rationale for choosing between these approaches.

The specific modification content is in: line 560 - line 575.

 

The authors would like to thank the reviewers' kind comments and constructive suggestions to improve our manuscript!

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction

Abbreviations need to be as full when mentioned first time example in introduction line 70 PD-1, but also all others.

-lines 79-80, there is mention as follows, “the regulatory mechanisms of Treg cells in different inflammatory eye diseases show significant heterogeneity”

Please specify few examples.

 

Main text

-Table 1 is not easy to read. It needs to be clarified. It looks like text is added just into table. Table should be fast information souce, thus id need to be simplified. Simply, introduce tissue Treg Cells found, characteristic, associated disease, regulatory mechanism, detection method and species, and references. Key experimental evidence may no need to include into Table, to make is more informative for key findings. Shortly. Try to fit into one page (short amount of text, table layout/structuring, font etc.). Also, include additional information in the text that is not needed in the Table. Text should also introduce the key thing from the Table.

 

-2.3.1 to 2.3.7, All these headings with included texts are quite short. Is it possible to add a little bit more information to paragraphs? Or, for example, combine some of those or all into a single subheading? Or add more text to each of them.

- 2.3.1 to 2.3.7 are also under the subheading of 2.2. The specific distribution of Treg cells in ocular tissues. But the text in 2.3.1 to 2.3.7 paragraphs is not related to the location. Instead, those are more related signaling pathways, the act of Treg cells etc. Please clarify headings correctly.

-subheading 2.3.5 Please, add something related to Tregg cells, for example, concrete examples, how they affect the promotion of polarization of macrophages to the M2 anti-inflammatory phenotype.

-Table 2 is better than only being included in the text, but since it is also possible to make also figure. For example complementary figures under each subtitle (4.2, 4.3 etc). Table 2 is also too large, and it should be simplified. Also, abbreviations should be introduced as full text below the table. Specific factor and related mechanism of action are quite hard to read when the table is in its current format.

-Table 3 also need to be simplified and make clearer and shorter. Single page included tables are best chose. Less text and more informative as single read. Additional information is added into text. Simply and collective tables for fast basic short text/sentences and most informative. Also, divide text by lines into spots etc. It makes it easier to read.

- Add some text between Table 3 and 4. Table 4 starts confusing just after Table 3. Similar kind of comments to make clearer and easier to read Table 4 as mentioned related to Tables 2 and 3.

 

 

 

 

 

Author Response

Response to Reviewers' Comments

 

Dear Editor and Reviewers,

 

We sincerely thank you for your thorough review and valuable comments on our manuscript. After receiving your feedback, we carefully considered each point, meticulously reviewed the entire manuscript, and made revisions accordingly. All modifications are highlighted in red in the uploaded "Revised Manuscript." Below is our point-by-point response to the reviewers' comments.

 

Comment 1

Reviewer's Original Comment: Introduction: Abbreviations need to be as full when mentioned first time example in introduction line 70 PD-1, but also all others. -lines 79-80, there is mention as follows, "the regulatory mechanisms of Treg cells in different inflammatory eye diseases show significant heterogeneity" Please specify few examples.

 

Response: We have addressed both points meticulously.

Abbreviations: We have thoroughly reviewed the manuscript and ensured that all abbreviations are defined in full upon first use in the text and in the footnotes of the tables. The specific modification content is in : line 50, line 59, line 60, line 96, line 102, line 108, line 109, et al.

Heterogeneity Examples: We have added specific examples in the Introduction (as shown above) to illustrate the heterogeneity. We contrast the roles of Tregs in corneal transplant rejection vs. viral keratitis, and the differing regulatory pathways involved in uveitis vs. dry eye, making the claim of "significant heterogeneity" concrete and evidence-based. The specific modification content is in: line 73 - line 78.

 

Comments 2, 6, 7, 8

Reviewer's Original Comment: 11. Table 1 is not easy to read... Simply, introduce tissue Treg Cells found, characteristic, associated disease, regulatory mechanism, detection method and species, and references. 15. Table 2 is too large... should be simplified. 16. Table 3 also need to be simplified... 17. Add some text between Table 3 and 4.

 

Response: We sincerely thank the reviewer for the critical and constructive feedback on the tables. We modified the title of Table 1 and Table 2 by removing the column "Key Experimental Evidence" in Table 1. We have checked and discussed the contents of Table 2, Table 3 and Table 4 in depth. We believe that the current Table Table 2 shows the contents corresponding to the pathway and mechanism clearly and intuitively, and there are not many specific experimental data. In order to ensure the readability of the table content, we still keep the contents of Table 2. Figure 1- mechanistic diagram of the molecular network that regulates regulatory T cell (Treg) functional homeostasis and plasticity in the eye (marked in the first paragraph of item 4 of the text; details of the figure are provided after the table at the end of the text). The contents of Table 3 and Table 4 respectively listed different treatment methods. We tried to merge them and found that the contents were too long after merging and the readability of the Table became worse, so we continued to keep the current Table 3 and Table 4. The specific modification content is in: line 403, line 746 - line 749, line 750, line 759 - line 762.

 

Comments 3 & 4

Original reviewer comments: 12. 2.3.1 to 2.3.7, All these headings with included texts are quite short... add a little bit more information... Or... combine... 13. 2.3.1 to 2.3.7 are also under the subheading of 2.2... But the text... is not related to the location. Please clarify headings correctly.

Response to reviewer comments: We carefully discussed this comment. In sections 2.3.1-2.3.7, we listed "The Molecular Mechanism of Treg Cells in Maintaining Ocular Immune Homeostasis". As this involves multiple mechanisms, and the discussion of mechanisms is a core part of this article, we considered retaining the current structure without merging, in order to make the content in this section appear more logically clear and allow readers to quickly understand the framework of the mechanisms.

 

Comment 5

Original reviewer comment: subheading 2.3.5 Please, add something related to Treg cells, for example, concrete examples, how they affect the promotion of polarization of macrophages to the M2 anti-inflammatory phenotype.

Response to reviewer comment: We have enriched the relevant section (2.3.5) with the requested concrete details. We now describe the molecular mechanisms (TGF-β/IL-10 secretion) by which Tregs drive M2 polarization and provide specific disease model examples (autoimmune dacryoadenitis, wet AMD) where this Treg-macrophage axis has been experimentally demonstrated. This moves the description from a general statement to a biologically grounded process. The specific modification content is in: lines 184-189.

 

The authors would like to thank the reviewers' kind comments and constructive suggestions to improve our manuscript!