Dear Dr Das Sarma,

Thank you very much for submitting your manuscript "CD40L protects against Mouse Hepatitis Virus-induced neuroinflammatory demyelination" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. Please note that a revised version needs to include at least some suggested experiments;  the proposed transfer experiments for example, would make the study much stronger.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Matthias Johannes Schnell, PhD

Associate Editor

PLOS Pathogens

Volker Thiel

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: in this study, Fareeha Saadi et al. show that mice deficient in CD40L are more susceptible than WT to neuroinflammatory demyelination induced by intracranial infection with mouse hepatitis virus. This is shown at 5, 10 and 30 days post infection after measuring a variety of parameters including virus loads, histopathology innate and adaptive cell infiltration in the brain, etc. Figure 2A requires more extended time of observation and statistical validation for the claim of difference in mortality. Most figures lack MI control (3, 4, 5, 6, 7, 8, 9, 10). and this is particularly important in Figure 7. In many micrographs, a greater magnification would help to see the differences. Some conclusions cannot be made. For example,CD4 T cell priming. More importantly, the data does not support that the increased disease in the C40L-/- mice is due to CD40L deficiency in CD4 T cells. This would require 1) Co-adaptive transfer experiment of CFSE (or similar stain) labeled WT and CD40L deficient CD4 T cells. 2) Reconstitute CD4 deficient mice with CD40L deficient or WT CD4 T cells. Some experiments

Reviewer #2: This is a very nicely done study by Saadi et al. examining the role of CD40L in the neurological damage following mouse hepatitis virus (RSA59) infection. The authors show that deficiency in CD40L results in increased neurological disease, which is associated with more severe pathology in early and late stages of disease as well as a decrease in CD4 T cells, microglia cells as well as monocyte/macrophages/neutrophil population (which was not sorted out). Long term damage was associated with prolonged virus in the brain. As the pathogenesis of virus-induced demyelination and damage to the CNS is still a strong area of interest and the authors study the interaction between the innate and adaptive immune response, this paper is of substantial interest to readers of PLOS Pathogens.

Reviewer #3: The Saadi et al. study describes the protective role of CD40L expressing CD4+T cells in reducing Mouse Hepatitis Virus-induced neuroinflammatory demyelination. The paper is well written and the experiments were well planned.

The authors performed a time course experiment assessing the brain and the cervical lymph node at different stages of MHV infection in wild-type and CD40L-/- mice, focussing on examining the molecular interaction between CD4+ T cells and microglia/macrophages. The lack of CD40L leads to increase in demyelination of axons, highlighting the protective role of this interaction.This result contrasts with the well-documented pathogenic role of the CD40-CD40L in an autoimmune disease model, thereby signifying this study.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: This paper is lacking in mechanism. It would be much improved with

1) Co-adoptive transfer of genetically marked and CFSE labeled WT and KO CD4 T cells and comparing their responses and migration in WT and CD40L -/- mice.

2) Show that the disease is recapitulated in CD4 deficient mice reconstituted with CD40L-/- mice while they are protected when given WT CD4 T cells.

In addition, the authors should see that all figures have MI control mice.

Reviewer #2: 1) One of the main questions of concern is what happens to the microglia population in the CNS of CD40L-/- mice. The number of resident brain microglia should not be changed dramatically unless the cells are undergoing apoptosis or are actively replicating. Do the numbers of microglia in WT mice increase compared to mock-infected animals? Alternatively, are the microglia in CD40L-/- mice dying, this could be determined by staining tissue sections for Iba1 and active Caspase 3.

2) the authors do not mention peripheral virus loads in the WT versus CD40L mice. It would be expected that they would be comparable based on the comparable loads in the brain at 5 and 7 dpi, but this should be addressed.

3) the experiment in Fig. 2 ends right when mice are developing clinical disease (day 10). The authors need to provide an explanation on why that time frame was not extended further. Was it due to the high level of weight loss in the CD40L-/- mice? Were any of the mice taken out past day 10?

Reviewer #3: None

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Line 46. Please define better the model. Introduce the virus, then explain the route of administration.

Lines 51-55. This is unclear. The model is about chronic infection but then talks about protective immunity. Please explain the model well and possible outcomes.

Line 110. Indicate route and pfu.

Figure 1A. Please describe in the introduction.

Line 132. From the data, it cannot be concluded that CD4 T cells are the primary population expressing CD40L. CD4 T cells could be involved in the recruitment of CD40L expressing cells.

Lane 145. Death of CD40L-/- mice does not seem to be less than 60%. Please indicate if there are significant differences or not in survival. Ideally, mice should be observed for a period of time, until death stabilizes.

Lane 171 should say Figure 3B.

Line 177. Indicate references demonstrating that CD45 intensity distinguishes cell origin.

Reviewer #2: 1) as the dose of inoculating virus changes between some experiments, it should be included in the figure legend.

2) lines 135-140 belong in the intro or discussion

3) the intro part of the abstract (lines 17-21) and should be rewritten

4) mock-infected controls in the flow data would be helpful to show increase/decrease in cell populations. Additionally, was a live/dead stain used to identify cells? What is the large negative cell population? Is that cellular debri?

5) the macrophage population was based on CD45 high, CD11b high - this population could also contain neutrophils. This should be mentioned.

6) magnification of axon blebbing (Fig. 8A) should be a higher resolution to observe

7) PLP staining was hard to discriminate from background stain in Fig. 8A

Reviewer #3: 1) Line 185: Please correct the figure number to figure 4 (CX3CR1)

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PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr Das Sarma,

Thank you very much for submitting your manuscript "CD40L protects against Mouse Hepatitis Virus-induced neuroinflammatory demyelination" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Matthias Johannes Schnell, PhD

Associate Editor

PLOS Pathogens

Volker Thiel

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: I think the experiments provide an excellent characterization of the response of CD40L deficient mice to IC infection with MHV-A59. The authors infer but do not explore the mechanisms involved.

Reviewer #2: the authors have addressed all of my concerns. Nice job on the manuscript.

Reviewer #3: In this revised manuscript, Saadi et al. have addressed most of the concerns raised by the reviewer. Despite these additions, the revised manuscript is not that well written and has too many main figures.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The manuscript contains too many main figures. Several figures can be moved to the supplementary section like Figs 2,4, 8,10 and 13.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Please check the manuscript for typos and be sure you define all the acronyms the first time.

Reviewer #2: (No Response)

Reviewer #3: Line 232. On day ?

Line 290: The paragraph should mention that this study assesses day 30 p.i. in the beginning of the paragraph.

Line 371 word spacing

Line 372: RSA59 induced (n missing)

Fig 1 C: There is less actin in the Mock infected control suggesting a loading issue. This figure should be repeated or removed.

Fg 12: A-C: At what point was the spinal cord of 5 week old mice assessed ? day 30 p.i?

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

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.

Dear Dr Das Sarma,

We are pleased to inform you that your manuscript 'CD40L protects against Mouse Hepatitis Virus-induced neuroinflammatory demyelination' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Matthias Johannes Schnell, PhD

Associate Editor

PLOS Pathogens

Volker Thiel

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):