Dear Dr Coccia,

Thank you very much for submitting your manuscript "Differential plasmacytoid dendritic cell phenotype and type I IFN response in asymptomatic and severe COVID-19 infection" 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.

In particular, Reviewer #2 made comments about a need for additional controls and enhanced discussion of prior papers on pDCs and COVID-19. Reviewer #1 also made some experimental suggestions to consider.

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,

Michael S. Diamond

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

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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: The study by Martina Severa and colleagues shows that in vitro plasmacytoid DCs (pDCs) sense Covid-19 via a TLR7-dependent mechanism, leading to Type I IFN secretion. This Type I IFN will protect lung epithelial cells from viral replication and from releasing high levels of inflammatory cytokines, such as IL-6. Furthermore, pDCs stimulated with Covid-19 upregulate PD-L1 but not CD80, suggesting that they specialize in IFN-I production rather than in antigen presentation/costimulation. The authors also show that pDC percentages/numbers are reduced in Covid-19 patients in vivo, both in asymptomatic as well as in severe cases. However, in asymptomatic patients pDCs display a prevalent PD-L1+CD80- phenotype, while severe patients exhibit a PD-L1+CD80+ phenotype. Accordingly, asymptomatic patients display high levels of IFN-I induced genes and, in general, more IFN-a in sera, while severe patients have more inflammatory cytokines in sera. The authors conclude that Type I IFN is protective in Covid-19 infection, in agreement with previous studies showing that genetic defects in the IFN-I pathway predispose to severe Covid-19 pathology.

Overall, this is a well-conducted and informative study.

Reviewer #2: In this article entitled “Differential plasmacytoid dendritic cell phenotype and type I IFN response in asymptomatic and severe COVID-19 infection”, authors report interesting findings on the cellular source of type I IFN following Sars-CoV-2 challenge. They show a major role of pDC in this process. Interestingly, they were also able to analyse patient samples in the context of asymptomatic, or severe COVID-19. However, we have several concerns about the manuscript at this stage.

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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: There are a couple of points that should be experimentally addressed:

a) One of the most interesting aspects of the study is that UV-inactivated Covid-19 does not stimulate production of IFN-a by PBMCs. This begs the question what about isolated pDCs? If purified pDCs also do not respond to UV inactivated Covid-19 (differently from what observed for influenza virus and HSV), it would maybe suggest the requirement for a nonstructural viral protein for induction of IFN-I. A similar phenomenon was observed for Dengue type 2 virus (Pichyangkul et. Al. JI. 2003).

b) pDCs express neuropilin1 (BDCA-4), which was recently implicated in cell-entry of Covid-19. Does blockade of neuropilin1 reduce IFN-I production by pDCs?

Reviewer #2: 1. The introduction should be more comprehensive about published work on pDC in COVID-19. This is the main topic of the study. Several references are cited by the authors, but the associated work is not presented in the introduction: in particular Refs 39, 45, and 47-49. Authors should also introduce pDC response to other coronaviruses, which forms important background information: refs 42 and 44. On the contrary, other parts of the introduction appear less relevant to the present study, and could be moved to discussion.

2. In Figures 2, 3, and 6, important controls are missing:

The TLR7/8 inhibitor should be used in the presence of R848 in order to show efficient inhibition (positive control).

Control inhibitor (negative control: non-effective compound of similar chemical nature), should be used in comparison to the effective inhibitor for each viral titer.

The authors introduce Mock as medium only (Fig legend: “Mock medium only »). This is not the usual meaning for Mock. If the condition is Medium only, it should be marked as such on the figure and its legend.

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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: None

Reviewer #2: 1. Figure 1 presents descriptive negative results and should be shown as supplementary figure, unless the authors provide mechanistic insight.

2. Figure 4 legend mentions « in presence or absence of a 30 minute pre-treatment with a specific TLR7/8 inhibitor (I-TLR7/8, 1μM) ». However, this is not visible on the actual figure. There is no culture condition with the inhibitor.

3. Results presented in figure 5 are completely expected given the antiviral functions of type I interferons. It could be moved to supplementary.

4. In figure 6D, it is very surprising that the inhibitor induces TNF and IL-6 production by pDC. Could authors coment on that ? Is the figure just mislabled ?

5. In Figure 8 : authors should depict individual patient/donnor results in each graph (as in 8A), instead of histograms.

6. In Figure 8, IL-10 levels are actually very low in all conditions. Are these levels and differences biologically meaningful ? What is the sensitivity limit of the assay ? (should be shown on the graph)

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Reviewer #1: No

Reviewer #2: No

Figure Files:

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Dear Dr Coccia,

We are pleased to inform you that your manuscript 'Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection.' 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,

Michael S. Diamond

Section Editor

PLOS Pathogens

Michael Diamond

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: The authors have addressed my main concerns.

Reviewer #2: Manuscript has been adequately revised. Additional experiments were performed and important controls were added. Following the authors reply, we would agree to leave results of old Fig 5 presented in New Fig 3.

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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: No major issues were identified.

Reviewer #2: (No Response)

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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: No minor issues exist at this point.

Reviewer #2: (No Response)

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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