Single-cell profiling reveals that dynamic lung immune responses distinguish protection from susceptibility to tuberculosis

PLOS Pathogens

Dear Dr. Duffy,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens'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 28 2026 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 plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Anne Jamet

Section Editor

PLOS Pathogens

Anne Jamet

Section Editor

PLOS Pathogens

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

1) We ask that a manuscript source file is provided at Revision. Please upload your manuscript file as a .doc, .docx, .rtf or .tex. If you are providing a .tex file, please upload it under the item type u2018LaTeX Source Fileu2019 and leave your .pdf version as the item type u2018Manuscriptu2019.

2) Please provide an Author Summary. This should appear in your manuscript between the Abstract (if applicable) and the Introduction, and should be 150-200 words long. The aim should be to make your findings accessible to a wide audience that includes both scientists and non-scientists. Sample summaries can be found on our website under Submission Guidelines:

https://journals.plos.org/plospathogens/s/submission-guidelines#loc-parts-of-a-submission

3) Please insert an Ethics Statement at the beginning of your Methods section, under a subheading 'Ethics Statement'. It must include:

i) The full name(s) of the Institutional Review Board(s) or Ethics Committee(s)

ii) The approval number(s), or a statement that approval was granted by the named board(s).

4) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines:

https://journals.plos.org/plospathogens/s/figures

5) Some material included in your submission may be copyrighted. According to PLOSu2019s copyright policy, authors who use figures or other material (e.g., graphics, clipart, maps) from another author or copyright holder must demonstrate or obtain permission to publish this material under the Creative Commons Attribution 4.0 International (CC BY 4.0) License used by PLOS journals. Please closely review the details of PLOSu2019s copyright requirements here: PLOS Licenses and Copyright. If you need to request permissions from a copyright holder, you may use PLOS's Copyright Content Permission form.

Please respond directly to this email and provide any known details concerning your material's license terms and permissions required for reuse, even if you have not yet obtained copyright permissions or are unsure of your material's copyright compatibility. Once you have responded and addressed all other outstanding technical requirements, you may resubmit your manuscript within Editorial Manager.

Potential Copyright Issues:

i) Figure 1A. Please confirm whether you drew the images / clip-art within the figure panels by hand. If you did not draw the images, please provide (a) a link to the source of the images or icons and their license / terms of use; or (b) written permission from the copyright holder to publish the images or icons under our CC BY 4.0 license. Alternatively, you may replace the images with open source alternatives. See these open source resources you may use to replace images / clip-art:

- https://commons.wikimedia.org

- https://openclipart.org/.

6) Kindly revise your competing statement in the online submission form to align with the journal's style guidelines: 'The authors declare that there are no competing interests.'

Reviewers' Comments:

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 lack of well-established surrogates of protection from Mtb hinders vaccine development. In this submission, Duffy et al. conducted a singe-cell RNAseq analysis of murine lungs following primary Mtb infection or following Mtb challenge in their previously established CoMtb model. Transcriptional responses are measured at three timepoints over 34 days, in response to two Mtb strains, and in both a “susceptible” and a “resistant” mouse strain. Importantly, by studying timepoints soon after infection, the confound of variable Mtb loads is removed. Notable, too, was the appropriate use of B6 sp140-/- mice as comparators with B/6 mice in the CoMtb study.

CoMtb mice exhibited higher expression of IFN-g response genes. Following Mtb challenge, CoMtb mice displayed early macrophage reprogramming as well as increased immune activation and migration signaling. These findings mirror some key features of IV BCG-elicited protection in NHP. Identifying such features associated with protection from Mtb across species helps the important quest to identify robust correlates of protection.

This study addresses an important issue and makes a valuable contribution to the field. This study was well designed and executed; in some cases, transcriptional signatures were verified by flow cytometry. The manuscript is well-written and the figures are appropriate and well laid-out. There are just a few concerns that should be addressed.

Reviewer #2: This is a comprehensive single cell analysis of the lungs of Mtb aerosol infected mice at early time points with a view to identifying correlates of protection. The experimental work is performed well with sufficient numbers of animals, a reproducible infection model and suitable hypotheses under test.

The approach is not novel - there are several studies already published in this arena - however there are elements that are important to the field of TB immunity. The comparison between mouse strains and genetically deficient strains as well as the comparison between naïve mice and mice with concomitant immunity are suitable comparisons and rationally based.

The strengths lie in the scope of the work and the in depth analysis - the data sets will be of use to the field as a whole for both model development and extension into human TB interventions.

The weaknesses lie in the presentation and the failure to deliver causal connections between the observations and the outcomes. The mouse model is useful because it allows direct mechanistic assessment of the immune response to infection over time, dose and route; these definitive outcomes can then be integrated into models wherein these causal connections cannot be made.

1. The presentation of the paper indicates rushed presentation. There are numerous grammatical errors and the text of the results section has the feel of a laboratory group presentation. A thorough re-edit of the results section with removal of the technical discussions - i.e. sort out how to account for the differences in baseline between the mouse strains and naïve/concomitant groups rather than stating it as problem for analysis.

2. Some of the data is discussed as being in the figures but is not shown - also the figures are not referenced correctly i.e. Figure 1 C does not show day 10 counts for all conditions and it is referred to as Fig 1B in places. Figure 3 has 3C before 3B in the figure layout. These are just things that should have been addressed in the editing process by the senior author.

3. Overall the presentation of the results suffers from having too many variables lumped together in presentation. This makes it exceptionally difficult to present the outcomes clearly, and importantly, the quality and impact of the data sets is lost to the reader. Pick a subject, explain it from start to finish and then move to the next subject. I would address the differences between the primary response of the strains first B6 to C3H then sp140, then move to the impact of concomitant immunity. Trying to bring all these together to generate a single (or simple) correlate of protection would have worked if there was one - but there isn't one common theme, and therefore just a straightforward description of the data is more helpful to the field and to our understanding of TB immunity.

4. Most of the outcomes noted are as expected but there are some novel observations such as the transcriptional differences in the myeloid cells. It would be important however to make some causal connection between any one of the observations reported and the altered immunity. i.e. removal of a cell type, disruption of a communication channel etc.

5. The data sets are largely transcriptional in nature - it would be very helpful for the figure legends to be explicit about the technical elements of the data shown - what is flow data, what is transcriptional data, what are the n numbers? how many experiments were performed and how much of the data is shown

6. One technical issue is that of the potential differences in the inoculum between the SA161 and the H37Rv used for aerosol. Can the group be certain the presentation of bacteria to the innate immune response is the same between the two during the first 10 days - should be addressed in the text just to show it has been considered.

Overall good source of data but presentation leaves the field more complex rather than clarified.

**********

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.required to validate study conclusions.required to validate study conclusions.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: None

Reviewer #2: The data sets are of interest but there is no causal connection made between the observations and the outcomes. Is there any data set that could be supplied that would show the importance of any one of the new pathways identified to protection?

While a computational tool could be used to make quasi-causal connections, this would not be the best option.

**********

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: Fig 1C shows “Mtb lung CFU post infection over time, stratified by mouse strain, Mtb strain, and CoMtb status”. However, the figure appears to differentiate only Mtb strain and mouse status (Primary v CoMtb). Which dots represent B6 and which represent C3H? Why is there no CFU data at 10 days following infection with H37Rv, which is important to support the contention that bacterial burden is similar at this time point.

Line 43: The term “symptomatic” implies self-reported feelings of illness. The more correct term is “signs of disease”.

Mice were aerogenically infected with 50-100 CFU, which is often used in the field, but is supraphysiologic.

Reviewer #2: This is not really a minor issue but the presentation of the data and the results section compromises the clarity and impact of the data. However as this can be addressed by effective senior editing then it is a minor issue. Pleaser ensure the data is presented rationally in sequence with single variables addressed clearly. This can be followed by integration of the data - and even some novel computational analysis to bring the observations together would be helpful.

**********

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 published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy....

Reviewer #1: No

Reviewer #2: No

Figure resubmission:-->While revising your submission, we strongly recommend that you use PLOS’s NAAS tool (https://ngplosjournals.pagemajik.ai/artanalysis) to test your figure files. NAAS can convert your figure files to the TIFF file type and meet basic requirements (such as print size, resolution), or provide you with a report on issues that do not meet our requirements and that NAAS cannot fix.-->-->

Reproducibility:

?>

Dear Dr. Duffy,

We are pleased to inform you that your manuscript 'Single-cell profiling reveals that dynamic lung immune responses distinguish protection from susceptibility to tuberculosis' 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,

Helena Ingrid Boshoff

Section Editor

PLOS Pathogens

Anne Jamet

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

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 #2: (No Response)

**********

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.required to validate study conclusions.required to validate study conclusions.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 #2: (No Response)

**********

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 #2: (No Response)

**********

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 published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy....

Reviewer #2: No