https://orcid.org/0000-0003-1521-4557
Peer Review History
| Original SubmissionMay 25, 2021 |
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Dear Dr Cutler, Thank you very much for submitting your manuscript "EBV+ tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22" 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. 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, Christian Munz Associate Editor PLOS Pathogens Blossom Damania Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens *********************** 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: Here the authors show that LMP1 fosters CCL17 and CCL22 expression in EBV+ tumor cell lines supporting the hypothesis that CCL17 and CCL22 expression induced by LMP1 contributes to EBV+ HL and NPC tumor biology. The chemokines CCL17 and CCL22 are usually secreted by APCs to attract T cells. CCR4, the receptor for CCL17 and CCL22, is expressed on tumor-infiltrating FOXP3+CD4+ Tregs, which play an important role in suppressing anti-tumor immunity. The authors suggest that, by secreting the two chemokines, EBV/LMP1+ tumors attract Tregs which suppress anti-tumor immunity and thereby foster immune escape of EBV/LMP1+ tumors. Accordingly, EBV+ HL tumor biopsies show significant coincidence of EBER, CCL17 and FOXP3 staining. Also in NPC biopsies chemokine expression and tumor-infiltrating Tregs were detected, although no clear coincidence with the presence of EBV was observed. EBV+ NPC xenografts showed tumor-intrinsic and -extrinsic expression of CCL22, which was not dependent on the presence of LMP1. Thus, no clear link between LMP1 expression, chemokine expression and Treg infiltration exists in NPC, in contrast to HL. To clarify a potential contribution of LMP1 to epthelial tumor biology and the attraction of infiltrating Tregs, the authors make use of an elegant colon tumor xenograft model. CT26 cells were engineered to express LMP1 (or OVA as control) and transplanted into mice. GFP-marked mouse iTregs were co-transferred and iTreg migration into the tumors was analysed. Notably, LMP1 induced a marked increase in Treg infiltration. In the CT26-LMP1 model, LMP1 enhanced chemokine expression induced by IFN gamma. Most notably, Treg migration into these tumors was blocked by the CCR4 small molecule inhibitor CCR4-351. The authors conclude that the CCR4 pathway is a novel target for pharmacological inhibition to restore anti-tumor immunity of EBV+ tumors. This claim is based on the following data: the CCR4 inhibitor CCR4-351 interfered with migration of CCRF-CEM T-lymphoblasts and iTregs induced by Raji supernatants in culture, the inhibitor was able to block iTreg migration into Raji or Daudi xenograft tumors in NOD-SCID mice, and, finally, CCR4-351 inhibited Treg migration into tumors formed by CT26-LMP1 cells. Taken together, this is a very interesting manuscript on the biology of EBV/LMP1+ tumors highlighting the relevance of tumor-intrinsic, and in the case of NPC also -extrinsic, CCL17 and CCL22 expression for Treg attraction. However, the observation that LMP1 augments CCL17 and CCL22 expression in tumor cells is not entirely new. This mechanism likely plays an important role in suppressing anti-tumor immunity in EBV+ tumors, although the latter has not been proven formally. The highlight of this manuscript is clearly the identification of the CCR4 pathway as novel potential target for pharmacological immune therapy of EBV+ tumors. Reviewer #2: Jorapur and colleagues use multiple lines of evidence (in vitro, in vivo. public databases, cell lines, patient samples, siRNA and small molecule blockade) to examine the basis for inflammation in EBV+ epithelial (gastric and NPC and LMP1 gene inserted) and B cell (Hodgkin and atypical Burkitt) malignancies. They conclude that via intrinsic (B cell) and epithelial (intrinsic and cell extrinsic) mechanisms, these tumours produce CCL17 and CCL22, which result in induction and migration of TREGs and migration of DCs. The data indicates that LMP1, possibly influenced by IFNg, are responsible for this induction, via activation of CCR4. The precise mechanism by which LMP1 upregulates these chemokines, and how EBV might induce cell extrinsic effects, is not explored. Interestingly, a novel CCR4 inhibitor reduced TREG migration, suggesting a potential therapeutic avenue to reduce immune evasion via the tumour microenvironment. The study is well conducted and extremely (particularly the results) well written. The data is novel, and provides an important counter-point to the paper of Edwards et al. As an interesting footnote, the gastric ca tumour NCI-N87, believed to be EBV-negative, was found to be EBV-pos. ********** 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: 1. sFig1 and lane 115 to 117) In contrast to previous reports the gastric carcinoma line NCI-N87 is now found to be EBV+. Are NCI-N87 cells used in this work derived from a certified source? This cell line should be verified. 2. Fig 1C) The mechanism of chemokine induction by LMP1/LMP2A is not sufficiently adressed. LMP2A seems to considerably contribute to CCL17 expression in Raji. Jijoye and NC-37 express low LMP2A levels. Is LMP1 sufficient to induce chemokines in these cells or may other mechanisms contribute? The authors should express LMP1, LMP2A and a combination of both in EBV-negative cells such as Ramos or NPC cells and analyse CCL17 levels. Also LMP1 with mutations in CTAR1 and/or CTAR2 could then be easily tested. Reviewer #2: 1. It is not clear to this reviewer if the authors are stating that TREG tumour infiltration was induced or due to migration. If this cannot be inferred, please clarify this in the discussion. 2. Please discuss the mechanisms by which EBV might influence tumour extrinsic chemokine production. 3. Figure 4 and 5 are difficult to understand and view. Images (particularly on the right hand) are blurred, and colours are indistinct from each other, and the legends do not explain what we are meant to be observing. This needs considerable attention and re-working. ********** 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: 1. Statistics: Which T-Test was used? 2. 1A) Technical or biological replicates? Where do the LMP1/LMP2A expression data come from? Statistics is required. 3. sFig1A) Are all panels shown for a specific antibody derived from the same blot? Are the protein levels comparable? 4. Fig 1B) Does control LMP2A siRNA affect LMP1 expression? 5. Fig 4) What is known about the status of LMP1/LMP2A expression in the HL and NPC biopsies? 6. Fig 8) Statistics is required. Reviewer #2: Was CCL17 proportional to the density of Raji cells in culture (as per CCL22). Legend to Fig 2: explain CCRE CEM cells as CD4 lymphoblasts. ********** 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 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 |
| Revision 1 |
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Dear Dr Cutler, Thank you very much for submitting your manuscript "EBV+ tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22" 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, Christian Munz Associate Editor PLOS Pathogens Blossom Damania Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens *********************** 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 significanty improved the manuscript. I am now supportive for publication of the manuscript in PLoS Pathogens after may last minor specific points regarding statistics have been addressed (see below). My previous second major point regarding the mechansim of CCL17 induction by LMP1 (and LMP2A) has, unfortunately, not been addressed by the authors. Such data would have added substantially to the manuscript, especially since CCL17 and CCL22 induction by LMP1 has been reported previously. However, I follow the authors' argument that the focus of the present manuscript is rather the dissection of immune evasion by EBV+ tumors and, especially, its pharmacological inhibition. The latter is a very important finding with regard to the potential treatment of EBV+ tumors and warrants publication in PLoS Pathogens. Reviewer #2: Following revision, the study is sufficiently strong to recommend acceptance. ********** 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: I reviewed the authors responses to reviewers comments, primarily focussing on my requested revisions. These have all been sufficiently addressed. ********** 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: 1. Statistics of Figure 1A): Significance of the claimed major differences should be verified by T-Tests and p-values should be given. 2. Statistics of Figure 8: Standard deviations should be shown. Is there a significant difference between CT26, OVA and LMP1? From this reviewer's point of view, this could be tested by ANOVA comparing the LMP1 with the CT26, OVA curves, or by T-Tests at a distinct IFNg concentration. In case of doubt, a statistician should be consulted. Reviewer #2: Nil. ********** 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 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. |
| Revision 2 |
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Dear Dr Cutler, We are pleased to inform you that your manuscript 'EBV+ tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22' 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, Christian Munz Associate Editor PLOS Pathogens Blossom Damania Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens *********************************************************** Reviewer Comments (if any, and for reference): |
| Formally Accepted |
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Dear Dr. Kassner, We are delighted to inform you that your manuscript, "EBV<sup>+<sup> tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22," has been formally accepted for publication in PLOS Pathogens. We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication. The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly. Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers. Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards, Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens |
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