PONE-D-24-38697Single-cell RNA-seq reveals disease-specific CD8+ T cell clonal expansion and a high frequency of transcriptionally distinct double-negative T cells in diabetic NOD micePLOS ONE

Dear Dr. Robben,

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.

We find that the manuscript requires increased precision in several areas, with certain data needing further completion and reinforcement. We invite you to submit a revised version that thoroughly addresses all points raised during the review process (refer to comments from the reviewer and academic editor below).

Please submit your revised manuscript by Dec 14 2024 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,

Prof. Pierre Bobé

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (a) methods of sacrifice, (b) methods of anesthesia and/or analgesia, and (c) efforts to alleviate suffering.

3. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, we expect all author-generated code to be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse.

4. Thank you for stating in your Funding Statement: 

This study was supported by grants from the Beatson Foundation (Grants ID 2800013). The salary of the author MZI was supported by Lundbeck Foundation, Copenhagen, Denmark (Grants ID R288-2018-1123).

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. 

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

5. Please note that your Data Availability Statement is currently missing the direct link to access each database. If your manuscript is accepted for publication, you will be asked to provide these details on a very short timeline. We therefore suggest that you provide this information now, though we will not hold up the peer review process if you are unable.

6. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

7. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Additional Editor Comments:

1) In the discussion section (line 372), it should be clarified that the lpr  mutation in the lupus-prone MRL genetic background (MRL/lpr ) or in the normal B6 genetic background (B6/lpr ) does not result from a null mutation in the fasL gene as indicated in the discussion. Instead, it arises due to the insertion of the retrotransposon ETn in the second intron of the fas   gene, leading to transcriptional repression. Therefore, lpr  mice, like NOD mice, possess functional copies of fasL genes . Additionally, DN T cells and CD8+ T cells from MRL/lpr  or B6/lpr  mice exhibit elevated expression of functional FasL on the plasma membrane. Furthermore, to the best of my knowledge, the current understanding is that pathogenic DN T cells in lpr  mice are mainly generated in the periphery as a result of dysregulated homeostasis of effector/memory T cells.

2) The authors should provide a detailed explanation of their methodology to demonstrate that the DN cells observed in NOD mice arise from their own proliferation, as stated line 403 (conclusion), rather than from the conversion of CD8+ cells into DN cells. Indeed, DN T cells could originate from CD8+ T cells, as shown in various lupus mouse models where chronic activation leads CD8+ T cells to downregulate their CD8 molecules. Therefore, have you quantified CD8 surface levels (MFI) in NOD CD8+ T cells using flow cytometry? Additionally, do DN T cells in NOD mice overexpress the phosphatase B220 (CD45RABC) along with Fas ligand, similar to DN T cells in lupus mouse models?

[Note: HTML markup is below. Please do not edit.]

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: Yes

**********

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

Reviewer #1: Yes

**********

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: Yes

**********

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

**********

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:  Dears,

I read with great interest and attention the manuscript “Single-cell RNA-seq reveals disease-specific CD8+ T cell clonal expansion and a high frequency of transcriptionally distinct double-negative T cells in diabetic NOD mice.” This study is original and relevant to the field, highlighting, through single-cell RNA sequencing, T cell subsets infiltrating the islets and potentially important T cell receptor sequences linked to the pathogenesis of type 1 diabetes.

Overall, the manuscript is well-constructed and fits within the journal's scope. The findings are interesting, properly interpreted, and well-discussed. Using robust and reproducible immunological methods, the authors demonstrate a high frequency of double-negative T cells throughout type 1 diabetes pathogenesis, as well as CD8+ T cells infiltrating with exhausted, cytotoxic, and inflammatory states during disease development

RECOMMENDATION: Publish after MINOR ADJUSTMENTS:

1) In the Materials and Methods section "Blood glucose measurement and diabetes diagnosis" (line 433), I suggest including a reference for the parameters used to diagnose diabetic mice (two consecutive glucose readings of ≥250 mg/dl considered diabetic).

2) Figure 1D, results: Is it possible to evaluate the frequency of T cells in the islets of non-diabetic mice using flow cytometry?

3) Considering the complementary determining region (CDR3) of autoreactive T cell clones, it could be interesting to determine the antigen fragments potentially recognized using the BLAST tool. This could reinforce that, in particular, Trav16n, Trbv29, and Trav8d-2 exhibit CDR3 regions that may potentially recognize insulin peptides.

**********

6. 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

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-24-38697R1Single-cell RNA-seq reveals disease-specific CD8+ T cell clonal expansion and a high frequency of transcriptionally distinct double-negative T cells in diabetic NOD mice

PLOS ONE

Dear Dr. Robben,

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.

==============================

The authors have addressed many of the issues that emerged during the review process.

• However, some minor points persist in the text, such as the inconsistent spelling of "pseudo," which is incorrectly written as "psuedo" at least six times.
• Furthermore, I recommend that the authors include the FSC-A vs. SSC-A dot plot in their gating strategy (Supplementary Figure 5) to demonstrate that only single cells are included in the flow cytometry analysis, while doublets or multiplets are excluded.
• I found the statements regarding Fas-deficient lupus mice in the Discussion section on page 20 to be ambiguous in the first instance and inaccurate in the second: “Studies in the MRL-lpr mice ….. have connected lymphoproliferation of DN T cells to a reduced apoptotic signal of negatively selected T cells in the thymus [50], as well as “ …….would suggest peripheral development of DN T cells not seen in lpr mice. There is substantial evidence indicating that peripheral DN T cells are generated in the periphery of lpr mice and patients with autoimmune lymphoproliferative syndrome (ALPS), rather than representing primitive αβ T cells that escape late-stage thymocyte development before migrating to peripheral tissues. Specifically, multiple reports have shown that B220⁺ DN T cells originate from CD8⁺  T cells at the effector/memory stage following repeated TCR engagement in Fas-deficient lupus mice. Similar findings have also been reported in human studies.
• To avoid any confusion for the readers, I recommend adding "SP" when referring to the CD4+ and CD8+ phenotype, for instance, CD4/DN and CD4+/CD8+ could be written CD4+ SP/DN and CD4+/CD8+ SP T cells.
• Additionally, when mentioning the MRL/lpr mouse strain, I suggest including the term "lupus-prone" (e.g., lupus-prone MRL/lpr mice) for those who may not be familiar with this mouse model of systemic lupus erythematosus (page 9).

==============================

Please submit your revised manuscript by Feb 09 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,

Prof. Pierre Bobé

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments:

• The spelling of "pseudo," appears incorrect at least six times as "psuedo."
• Furthermore, I recommend that the authors include the FSC-A vs. SSC-A dot plot in their gating strategy (Supplementary Figure 5) to demonstrate that only single cells are included in the flow cytometry analysis, while doublets or multiplets are excluded.
• I found the statements regarding Fas-deficient lupus mice in the Discussion section on page 20 to be ambiguous in the first instance and inaccurate in the second: “Studies in the MRL-lpr mice ….. have connected lymphoproliferation of DN T cells to a reduced apoptotic signal of negatively selected T cells in the thymus [50], as well as “ …….would suggest peripheral development of DN T cells not seen in lpr mice. There is substantial evidence indicating that peripheral DN T cells are generated in the periphery of lpr mice and patients with autoimmune lymphoproliferative syndrome (ALPS), rather than representing primitive αβ T cells that escape late-stage thymocyte development before migrating to peripheral tissues. Specifically, multiple reports have shown that B220⁺ DN T cells originate from CD8⁺  T cells at the effector/memory stage following repeated TCR engagement in Fas-deficient lupus mice. Similar findings have also been reported in human studies.
• To avoid any confusion for the readers, I recommend adding "SP" when referring to the CD4+ and CD8+ phenotype, for instance, CD4/DN and CD4+/CD8+ could be written CD4+ SP/DN and CD4+/CD8+ SP T cells.
• Additionally, when mentioning the MRL/lpr mouse strain, I suggest including the term "lupus-prone" (e.g., lupus-prone MRL/lpr mice) for those who may not be familiar with this mouse model of systemic lupus erythematosus (page 9).

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Single-cell RNA-seq reveals disease-specific CD8+ T cell clonal expansion and a high frequency of transcriptionally distinct double-negative T cells in diabetic NOD mice

PONE-D-24-38697R2

Dear Dr. Robben,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Prof. Pierre Bobé

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: