PONE-D-24-54197Human cytomegalovirus-IE2 suppresses antigen presentation of macrophage through the IL10/STAT3 signalling pathway in transgenic mousePLOS ONE

Dear Dr. Xian juan,

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This research was funded by Shandong Provincial Science and technology Foundation (grant no. 2019JZZY011009), Qingdao Municipal Science and technology Foundation (grant no. 20-2-3-4-nsh), National Key Research and Development Program of China (grant no.2018YFA0900802), Shandong Provincial Natural Science Foundation (grant no. ZR2021QH254).

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Reviewers' comments:

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: 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

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Reviewer #1: No

Reviewer #2: Yes

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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:  In this study, Dr. Zhang and collaborators take advantage of a transgenic mouse model stably expressing the HCMV IE2 protein to prove that the long-term IE2 expression has a major negative impact on immune activation. Although some of the presented results are suggestive, the primary issue I can envisage is the lack of supporting experiments using human cells. These should be easily obtained with primary human macrophages transduced with a plasmid carrying the IE2 gene and with HCMV strains deleted for this gene. The author should explain why they did not pursue these approaches.

In general, the findings are poorly explained, and the majority of the experimental details are vague. Finally, the English language need accurate revision.

Specific details:

- the construct for transgene generation is poorly described;

- in Fig 2 and Fig 3: the authors should mention in the text or at least in the legend what T stands for (in WT+T and IE2+T)

- for Fig 3: a few details are provided in both the text and the legends. Please amend

- Fig 3C : please change “E Coil” to “E. coli”

- Fig 3E,F: how much MCP-1 was used for the transwell experiment? Please include details in the main text and/or in the M&M section

- In numerous places throughout the text, the MCP-1 acronym is wrongly reported as Mcp-1.

- M&M and legends are poorly detailed, with some inaccuracies (i.e., line 197: The Transwell® system WERE used)

Reviewer #2:  Summary:

The authors have developed a new transgenic mouse model that allows for investigation of the consequences of prolonged expression of the HCMV gene IE2 on immune activation in mice. Using this model, they found that prolonged expression of IE2 alters macrophage polarization and motility, the consequences of antigen-presenting to CD4 and CD8 T-cells, and that this might be due to IE2 expression leading to higher levels of IL-10 and STAT3 phosphorylation. The new model is interesting and could be potentially useful to others interested in studying functions of IE2. The data generally support the conclusions posited by the authors. The biggest concern I have is regarding the lack of discussion/presentation of any data regarding any other effects on the mice with prolonged expression of IE2. IE2 has many reported functions, not just immunomodulation, that could alter the biology of the mouse in important ways. Other minor concerns are enumerated below. Recommendation: minor revisions, focus on adding in more information about the mice.

Specific areas for improvement:

Major issues:

1. This is a new transgenic mouse model. While Figure 1 adequately shows where in the genome IE2 was knocked-in and identified mice with the knock-in by PCR, it’s important to provide more general information about the health of the mice – do they live as long as WT counterparts? Do they have any unexpected abnormalities? I am sure the authors have noted these things in the development of the transgenic mice – as a reader, I want to know these things to aid in my interpretation of the rest of the paper.

2. Line 52, p.3: “…mouse model simulates the expression process of viral proteins after HCMV infection in vivo to a certain extent.” – there is no data provided for this…? To WHAT extent? It is important to show more characterization of this mouse model, especially when claiming that it recapitulates the expression process. HCMV expresses genes in the typical cascade of IE->E->L, and this model relies on long-term sustained expression of IE2. How is this simulating the “normal” expression of IE2? Data on this would be extremely useful to aid readers in interpreting the reliability of extrapolating data from this study to what we think is going in in humans/human cells.

Minor issues:

1. Line 40 p.2: “macrophages are the first targets during HCMV infection.” – it is often said that epithelial, endothelial, or fibroblast cells are the first cells infected when a host encounters the virus, and that trafficking from these outer tissues to the endothelium allows the virus to encounter cells like macrophages. I definitely agree they are important for viral persistence and spread, but I would not definitively declare macrophages as the first target cells of HCMV.

2. Entire introduction – I would like more specific information included in the introduction, describing a bit more specifically what the cited studies showed. For example, line 43 “Previous studies have shown that HCMV infection mediates immune escape by regulating macrophage activity.” – how does it regulate it? What was shown? What part of macrophage activity was regulated? Was it IE2 or something else? What kinetic class of viral gene (if known) was implicated in regulating macrophage activity? What models were used? Etc

3. Fig. 1A – This is a nice map of the insertion, but it could use some more detail. Under what promoter was IE2 expression placed? The font is also quite small – readability could be improved by altering font sizes.

4. Lines 148-149: “indicating that the UL122 gene insertion does not affect the expression of neighboring genes.” – The assumption is the gene was inserted far enough away from neighboring genes to not alter expression. Including control RTqPCR or Western blots for Fam47c and Cfap47-205 in WT vs IE2 mice would strengthen this argument.

5. Line 158: “In the spleen, Mos were stained with CD11b+” – were cells isolated from the spleen specifically and then stained? Or is this some in vivo staining?

6. Throughout (example line 171) – specify that it is continued/sustained IE2 expression resulting in your phenotypes.

7. Figure 2B: the data are compelling on their own, but my first thought was to calculate in my head the fold changes relative to untreated for each mouse. It would be helpful to include in the figure the same data plotted relative to untreated (as a fold change)

8. Figure 3A-B: as a non-immunologist, it took awhile to parse out which data meant M1 vs M2. This could easily be fixed by adding labels to the figure, and strengthen the figure by making it more readable to people outside the field.

9. Lines 185-188: The experiment could use more explanation (again mainly for those readers who don’t frequently do phagocytosis assays) – where does the GFP come from? What do the images tell us specifically?

10. Lines 189-192: this simple inclusion of “the chemokine MCP-1 is known to induce migration” is a great example of the minor addition of explanation (needed for the phagocytosis assay) really making a difference for my understanding. This was great!

11. Fig 3E legend (line 197) – this could use more detail.

12. Effect of IE2 on T cells (lines 201-213): This section could use a bit more detailed explanation of the question you’re asking, what you measured by flow cytometry, and what it means for both CD4 helper T cells AND CD8 cytotoxic T cells (line 213 seems specifically focused on CD4 – what about CD8? What are the implications there?)

13. Figure 4: As mentioned for figure 2B (point 7 above), it would be useful to plot fold changes relative to the PBS control for each mouse in addition to the data presented. This would clearly show if there’s a specific change in activation as opposed to baseline changes overall in the IE2 mouse (this would also be interesting on its own!)

14. Figure 5: Again, this looks at the changes in IE2 vs WT in LPS-stimulated cells; however, it would be interesting to also include expression of these MHC markers or cytokines at baseline (without LPS) for IE2 and WT mice. The fold change compared to baseline would again be helpful. Same for the STAT3 Western blots.

15. Line 263: what is “E” gene product?

16. Line 293: This study revealed a correlation between IE2 expression and changes in IL-10/STAT3, but the mechanism feels lacking in data. This conclusion would be more fully supported if trans-complementing studies or something could be done – I am not sure whether that is meant to be in the scope of this paper or not. Thus, you could simply say “our study SUGGESTS a mechanism” instead of “our study REVEALS”

17. The last sentence regarding vaccine development is not necessary.

Final thoughts: these data suggest quite a cool link between IE2, STAT3/IL10, and macrophage activity. Looking forward to seeing more in depth follow up studies in the future.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-54197R1Human cytomegalovirus-IE2 suppresses antigen presentation of macrophage through the IL10/STAT3 signalling pathway in transgenic mousePLOS ONE

Dear Dr. Zhang ,

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 Apr 24 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,

Chunmei Cai

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: (No Response)

Reviewer #2: All comments have been addressed

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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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: No

Reviewer #2: Yes

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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: While the authors have adequately addressed the main concerns related to the first round of revisions, the following should still be amended:

- The authors have added some details about the generation of the transgene model, both in the M&M section and in the new Fig. S1. However, in the M&M the new paragraph sounds: “A pAV.Ex1d-CMV-IE2 vector containing a cDNA fragment of the IE2 gene was constructed, and the vector was injected into…”. No details are provided about the origin of the viral gene: where does this gene come from? What strategy did they employ for its amplification and cloning?

- Furthermore, the quality of figure S1 is poor, with some black squares partially covering the image and a scheme from the company's website that I'm not convinced can be used in this context.

- Finally, some more editing of the English language is required before acceptance.

Reviewer #2: The authors have adequately addressed reviewer concerns, which have strengthened the paper to be of higher quality for publication.

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Reviewer #1: No

Reviewer #2: No

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Human cytomegalovirus-IE2 suppresses antigen presentation of macrophage through the IL10/STAT3 signalling pathway in transgenic mouse

PONE-D-24-54197R2

Dear Dr. Xianjuan Zhang,

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,

Chunmei Cai

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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

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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

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

Reviewer #1: Yes

**********

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

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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

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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: (No Response)

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Reviewer #1: No

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