PONE-D-24-17797Naïve intestinal T cells in food-allergic model mice, expanded with anti-CD3 and anti-CD28 antibodies while under regulatory T cell-polarization culture condition, can exhibit suppressive activityPLOS ONE

Dear Dr. Nakajima-Adachi,

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Masanori A. Murayama

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PLOS ONE

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 [This work was supported by grants from the Kieikai Research Foundation (HNA, Grant number; 2017S063, https://www.nakashima-foundation.org/kieikai/) and 

Grant-in-Aid for Scientific Research (B) from

the Japan Society for the Promotion of Science (JSPS)(SH, Grant number; 26292065, https://www.jsps.go.jp/j-grantsinaid/index.html) .

The analysis for Fig. 5 and S5 Fig was funded by Meiji Holdings Co., Ltd (HNA and SH). ].  

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Additional Editor Comments:

Thank you for submitting manuscript. This research is very interesting, however, this manuscript is open to discussion in this time.

As reviewer indicated, title is too difficult to understand the contents of this manuscript. To be honest, this manuscript is hard to understand in whole. So this manuscript need to help with English language editing.

As major point, this study just focused on the difference of R23-3 and RagD10, and the experiments and discussion was lack for the development of clinical application. Thus, this manuscript requires significant revision.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General comments

This study tried to find the difference in T cell characteristics between food allergic and non-allergic animal models. The article includes several interesting findings although there are some points which are requested to be improved to publish in PLOS ONE.

Specific comments

1. The title is difficult to understand the aim of this study.

2. I do not understand the reason why the authors stimulated T cells by anti-CD3 and anti-CD28 antibodies. What does the stimulation with the antibodies physiologically mean?

3. Related to above comment, I do not understand the reason why the responses to anti-CD3 and anti-CD28 were different from that to antigen stimulation.

4. The authors mentioned the Tregs from the mice as ‘allergen-specific Tregs”. I was wondering if the Tregs inhibited allergy in an allergen-specific manner. The authors should confirm the allergen specificity of the suppressive activity.

5. The authors wrote “ Naïve CD4+ T cells isolated from CN-fed Rag23-3 mice and stimulated with anti-CD3 and anti-CD28 mAbs differentiate into Tregs” (line 369). However, the authors used not only CD-fed mice but also EW-fed mice in the section. I do not understand the reason why title of the section was different from the content.

6. The position of markers in the graph right bottom of Fig. 2a seems out of alignment.

7. The authors wrote “Fig. 2c (data not shown).” (line 434). The authors should provide all data you used in this manuscript.

8. Fig. 3b shows that each of the anti-IL-4 or anti-IFN-g antibody almost completely inhibited the activity of the Sup by itself. I was wondering why IFN-g in anti-IL-4 condition or IL-4 in anti-IFN-g condition did not affect Treg induction.

9. I was wondering why the characteristics of naïve T cells of EW-fed mice differed from those of CN-fed mice in Fig. 5c. Were the naïve T cells of EW-fed mice really naïve?

10. It was very difficult to understand the purpose of each experiment throughout the manuscript. Please give it a clear title easy to understand the purpose of each section of results and explain the reason why the experiments were adopted for the purpose of each section.

Reviewer #2: <major comment="">

This manuscript showed a potential of ex-vivo polarization of functional Treg cells from the naïve intestinal T cells of on-going allergic enteropathy model mouse. Furthermore, overexpression of IL-4 and IFN-� from activated T cells suppressed the induction of Treg cells in the allergic mouse model. The experiments were conducted appropriately, and the detail of methods and results are clearly shown.

Although the length of manuscript is not limited in PONE journal, the text in method, result and figure legends is overlapping, that makes the redundant impression to the readers. Especially, the text in the figure legends may be shorter, because they use almost an equal volume as the text.

<minor points="">

1. Reference

The Ref. 1,2,4 should be updated to represent the recent clinical situations regarding food allergy and oral immunotherapy.

2. L. 371-373

Immobilized anti-CD3 stimulation should be TCR-mediated, although not antigen-specific stimulation.

3. L. 434

Fig 2c might be Fig 2b?

4. Statement of S3 Fig is missing in the text.

5. L.439-442

Blocking either IL-4 or IFN-� alone almost completely restored the suppressive effect of the Rag23-3_EW_Sup, suggesting these two cytokines are affecting to one lymphocyte simultaneously. How do authors explain this mechanism? Does a naïve T cell express both IL-4 receptor and IFN-� receptor on the surface? I’m wondering if direct supplementation of IL-4 and/or IFN-� shows the same effect?

6. L. 658-660

In this TCR-transgenic mouse model, how authors consider the difference between OVA+APC stimulation and anti-CD3+anti-CD28 stimulation? The difference of TCR signal transduction, or the other signals through co-stimulatory molecules on the APC? In other words, does OVA+APC stimulation with Treg-polarization condition on EW-fed Rag23-3 cells have a potential to induce Tregs?</minor></major>

Reviewer #3: In their manuscript "Naïve intestinal T cells in food-allergic model mice, expanded with anti-CD3 and anti-CD28 antibodies while under regulatory T cell-polarization culture conditions, can exhibit suppressive activity," Haruyo Nakajima-Adachi et al. compared two strains of OVA-specific TCR transgenic mice and found the distinct induction of Tregs and effector memory T cells in food allergen-mediated enteropathy.

1. This manuscript is difficult to follow and understand the emphasized points in its current version. The manuscript could benefit from English language editing to enhance clarity and readability. I strongly suggest this is necessary in its current format.

2. The study begins with the introduction of potential therapy using Treg administration for food allergy patients. Is there any clinical perspective on that? To avoid confusion for the reader, the authors should cite adequate references for Treg therapy in food allergies. Compared to other inflammatory diseases, cellular interventions such as cell therapy for food allergies represent an ultimate approach. The importance of how this research can be substantiated is in question.

3. Two strains of OVA-specific TCR tg mice are used in the manuscript, and they published the differential function of Tregs between these mouse strains in a 2017 PLOS One paper. In that paper, the amount of IL-4 was shown to be a causative factor in the regulation of Treg function. In this manuscript, their aim is to understand the stability of Tregs, but the study lacks analysis of how the cells are “stabilized”, for instance, how epigenetic regulations differ in the two mice lines of iTregs. How about RORgt+ Treg which is important cell population regulating allergic disorder. Is this caused by intrinsic control based on the strength of TCR signaling? How are Tregs differently induced in the two lines? The authors can experimentally define this by measuring the strength of TCR signaling.

4. Counter gating should be shown in Figure 1a of effector memory T cells with adequate control.

5. Upon activation, CD4+ T cells transiently express FOXP3, especially in activated CD4+CD25– T cells, thus CD25 expression should be shown in the panel throughout the manuscript.

6. I don’t understand why IL-2 is significantly induced in the control of Rag23-3 mice. Fully discuss this.

7. TCR is specific for OVA; however, EMT cells are significantly induced in the mice without OVA. What is the underlying mechanism of these phenotypic discrepancies?

8. The author indicated the importance of IL-4 for the induction of Treg. Does IL-4 administration induce Tregs in Rag23-3 CN mice?

9. TGF-beta should be referred to as “TGF-beta1” throughout the manuscript.

10. In terms of Treg induction via non-TCR mediated stimulation, I do not understand why the authors stimulated with an anti-CD3 antibody, which mimics TCR stimulation. The authors should reconsider the fundamental concept of this study and rearrange the experimental design.

11. The authors stop explaining the details of the results obtained about the different induction rates of Tregs between spleen and MLN by citing the Mucosal Immunology paper from Traxinger BR. The authors should fully explain and conclude the results based on their data.

12. IL-4 and IFNg are produced at higher levels in the Rag23-3 mice, thus Th1 and Th2 cells may be induced in the Treg-induction experiments. The authors should show if IL-4 and IFNg suppresses or skews Th1/Th2 cells by analyzing intracellular FACS of Th1/2 in mice.

13. Line 403: “The finding that OVA did not fully induce Treg differentiation, but stimulation with antibodies induced Treg differentiation in CN-fed Rag23-3 mice suggests the presence of factors” – however, they mimic TCR signaling by anti-CD3 and anti-CD28. I don’t understand this part.

14. The authors indicate Tregs derived from Rag23-3 mice have sufficient regulatory activity; however, it is important to examine the functional differences in the suppressive roles of RagDO10 and Rag23-3 derived Tregs in vivo and in vitro. Cytokine productions are different?

15. Lines 552 to 557 are hard to understand. Why do naïve T cells ameliorate differentiation to Tregs? This part is complicate to the reader. Please reorganize this part.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-24-17797R1Ovalbumin-specific regulatory T cells with the naïve phenotype (CD62LloCD44hi) from mesenteric lymph nodes suppress food-allergic enteropathy in mice, when expanded by anti-CD3/CD28 antibodies but not by ovalbumin plus antigen-presenting cellsPLOS ONE

Dear Dr. Nakajima-Adachi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 20 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Masanori A. Murayama, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

Thank you for re-submission of your manuscript. This revised manuscript is interesting, but it has open issues. Please check comments from reviewer2. And I have some comments; In introduction section, please explain why authors use different OVA-TCR Tg mice. At least, authors should explain what is the difference OVA23-3 and DO11.10 mice at basic levels.

In Results section, authors should delete the supplementary explanation about fundamental knowledge, and some sentence should move discussion section. To promote understanding the contents, authors need concise result section. I think the result section is too long. As pointed out by reviewer 2, conclusions are unclear.

At all figures, it was very difficult to understand what is the significantly difference. Please use * or #, instead of a, b, c, and d, and use lines between graphs. And please change small to large alphabet, Fig1a -> Fig1A. And please change IFN-gamma -> IFNγ, TGF beta1 - TGFβ1.

In result section, authors investigated some cytokine from helper T cells, but not IL-10 and IL-17. Why authors did not investigate IL-10 expression?

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors appropriately answered to my questions and comments, and revised the manuscript according to the answers.

Reviewer #2: The manuscript has been appropriately edited, and the methods and results sound much clearer. The role of excessive amount of IL-4 and IFN-gamma, either autologous production or supplementation, on the suppression of Treg differentiation has now been clearly shown.

Reviewer #3: In their manuscript, "Naïve intestinal T cells in food-allergic model mice, expanded with anti-CD3 and anti-CD28 antibodies under regulatory T cell-polarization culture conditions, can exhibit suppressive activity," the authors currently describe, "Ovalbumin-specific regulatory T cells with the naïve phenotype (CD62L^lo^CD44^hi^) from mesenteric lymph nodes suppress food-allergic enteropathy in mice, when expanded by anti-CD3/CD28 antibodies but not by ovalbumin plus antigen-presenting cells." Haruyo Nakajima-Adachi et al. compared two strains of OVA-specific TCR transgenic mice and found distinct differences in the induction of regulatory T cells (Tregs) and effector memory T cells during food allergen-mediated enteropathy.

The comparison between anti-CD3/CD28 antibodies and ovalbumin plus antigen-presenting cells is a critical aspect of the paper. I suggest a revised title for the manuscript: "Stable and suppressive regulatory T cells from mesenteric lymph nodes suppress food-allergic enteropathy in mice" as the current title is long and somewhat complex.

Extracting Tregs from mesenteric lymph nodes (MLN) for therapeutic use in food allergy patients seems impractical. Would it not be possible to use Tregs derived from peripheral blood instead as an experimental setting which strength the requirement of their study? The manuscript appears to lack appropriate control experiments to address this question. The authors mention in line 79 that “it is difficult to separate allergen-specific Tregs from the total population of peripheral blood mononuclear cells.” However, obtaining Tregs from MLN seems even more challenging than isolation from peripheral blood mononuclear cells (PBMCs).

The authors highlight that iTregs derived from naïve CD4+ T cells of OVA-transgenic mice strongly suppress allergies. However, their explanation about how antigen-specific naïve T cell-derived Tregs could be obtained in non-transgenic models is overly complex for a general understanding of immunological mechanisms.

There are several established mouse models for food allergy, such as OVA-Alum. It would be more robust to test the expansion of Tregs using these models, comparing the two methods described in the manuscript. Demonstrating the inhibition of allergic symptoms with the authors’ method would strengthen the study’s findings.

Additionally, the manuscript cites a new reference (Reference 24). However, the cited paper’s results seem contradictory to the authors’ findings, stating that “iTregs generated by activation with antigen/APC are more suppressive than iTregs generated by anti-CD3/CD28 antibodies.” The cited manuscript attributes these functional differences to chemokine-related gene expression. If the authors choose to reference this paper, they should address this apparent discrepancy in their discussion.

Overall, the manuscript leaves its conclusions unclear. It does not effectively explain what was discovered regarding the differences between the two OVA-TCR mouse strains or the mechanisms of Treg induction by different antigen stimulation methods. While the stated aim is to evaluate the stability of Tregs, the study lacks critical analysis, such as how epigenetic regulation might contribute to stabilization in the two strains of iTregs. To provide generalizable insights, I recommend additional experimental comparisons of Tregs derived from these strains to clarify their cellular properties.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Ovalbumin-specific regulatory T cells differentiated from the naïve phenotype (CD44loCD62Lhi) in mesenteric lymph nodes stably suppress enteropathy even in severe food-allergic mice

PONE-D-24-17797R2

Dear Dr. Haruyo Nakajima-Adachi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Masanori A. Murayama, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for submitting revised manuscript. I am pleased for sending this mail. This manuscript is completely revised according to reviewer's comments. Thus, my decision is accept in this revision. Congratulations.

Reviewers' comments: