Synergistic effects of anti-PDL-1 with ablative radiation comparing to other regimens with same biological effect dose based on different immunogenic response

Maedeh Alinezhad; Mohsen Bakhshandeh; Elham Rostami; Reza Alimohamadi; Nariman Mosaffa; Seyed Amir Jalali

doi:10.1371/journal.pone.0231507

Peer Review History

Original SubmissionDecember 15, 2019
21 Jan 2020 Decision Letter - Jianxin Xue, Editor PONE-D-19-33249 Synergistic Effects of Anti-PDL-1 with Ablative Radiation Comparing to other Regimens with Same Biological Effect Dose based on Different Immunogenic Response PLOS ONE Dear Dr Jalali, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it is not fully meet PLOS ONE’s publication criteria. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. In addition, during our internal assessments, we noted that 6 animals died due to lowering temperature during the irradiation procedures.Some editors raised the great concerns about the animal welfare. We expect your response about this matter. We would appreciate receiving your revised manuscript by Mar 06 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled '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. We look forward to receiving your revised manuscript. Kind regards, Jianxin Xue Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. As part of your revision, please complete and submit a copy of the ARRIVE Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://www.nc3rs.org.uk/arrive-guidelines. Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 4. We note that Figures in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: a) You may seek permission from the original copyright holder of the Figures to publish the content specifically under the CC BY 4.0 license. We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.” Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].” b) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. [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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: Reviewer’s comments The current study has evaluated the synergistic effect of RT combined with immunotherapy using the αPDL-1 antibody in three different RT regimens in a xenograft tumor model in BALB/c mice. The synergistic effect of the αPDL-1 antibody in RT regimens is interesting. The findings would be helpful to develop the new immunomodulator and to design the more efficient radiation regimens to control the tumor outgrowth. Comments to the author 1. In the Introduction section, the author should favorably explain the motivation of the research to differentiate the current study from the other applied papers in this field. 2. The author should explain more about the expression of CD8+ and CD4+ cells after radiation in the first paragraph of the introduction. Methods: 3. The author should mention the fractionation schedules. How many fractions of RT were given per day and for how many days/ weeks? 4. The mostly used hyper fractionation RT regimens in clinics are 2Gy fraction per day. Is there any specific reason to use 3Gy fractions? 5. Radiation with or without causes nephrotoxicity. The author should evaluate the nephrotoxicity after RT ± anti-PD-L1 and could cite the following paper (PMID: 27836988) Results and discussion 6. The quality of the figures is unsatisfactory, please improve the quality of the figures. 7. The findings have been discussed to some extent to support the claims made in the hypothesis. The manuscript adds some critical information to advance the field. The results presented in the paper are substantial enough for publication in Plosone after minor revision. Reviewer #2: Excellent paper. Please add a paragraph after the discussion: Conclusion and discuss clinical implications of your findings and how your findings could be translated into the clinical studies. Lung SBRT is a good example where we deliver ablative dose of radiation. ****** 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: Yes: ANIS AHMAD Reviewer #2: 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0231507.r001
Revision 1
9 Mar 2020 Author Response February 1, 2020 Prof. Jianxin Xue Academic Editor PLOS ONE Dear Professor Jianxin Xue: Thank you very much for your letter regarding our manuscript (PONE-D-19-33249) entitled" Synergistic Effects of Anti-PDL-1 with Ablative Radiation Comparing to other Regimens with Same Biological Effect Dose based on Different Immunogenic Response”. The reviewers’ comments were very helpful, and we made revisions to the manuscript according to their criticisms. I think described point together with the other minor points that we have addressed made the manuscript much improved. Please find attached a detailed reply to the referees’ comments and description of other changes to the manuscript. We would like to thank the reviewers again for their important comments. Our responses to the comments are as follows: #Academic editor: In addition, during our internal assessments, we noted that 6 animals died due to lowering temperature during the irradiation procedures. Some editors raised the great concerns about the animal welfare. We expect your response about this matter. Response: We appreciate you taking the time and the valuable suggestions offered. The mice were adapted to the conditions after two weeks in the animal research room. Then, after tumor induction, eight groups were randomly selected. At the stage of tumor induction and irradiation, they were anesthetized to reduce pain tolerance. Symptoms of pain were monitored for up to 4 hours after irradiation, and if any symptoms of pain such as paleness, anorexia, weight loss and back flexion were observed, they were given pain relief. After reaching the final stage and finding endpoint criteria, they were anesthetized by isoflurane and sacrificed ten minutes later. #Reviwer 1: 1. In the Introduction section, the author should favorably explain the motivation of the research to differentiate the current study from the other applied papers in this field. Response: We thank the reviewer for their close attention. The motivations for the study were written in the form of a few questions at the end of the last paragraph, which we completed by adding a few sentences. However, studies have not yet determined whether different regimens with the same BED (when the biological effects of the regimens are equal) have different immunogenic effects, whether they affect tumor recurrence associated with immunological responses. In addition, can these regimens respond differently to combination therapy? Because it can improve the quality of immune responses into TME by use of modification in the dose and number of fractions radiation to the extent that the radiation clinic limitations and technology of the radiation devices allow us. Also it can lead to memory responses, which prevents tumor recurrence that is a common problem after treatment. To address these issues, we aimed to evaluate the synergistic effect of RT combined with immunotherapy using αPDL-1 in three different RT regimens with the help of modeled tumor BALB/c mice. #Reviwer 1: 2. The author should explain more about the expression of CD8+ and CD4+ cells after radiation in the first paragraph of the introduction. Response: Yes, Sure, in the first paragraph, we noted the effect of radiation on CD4 and CD8 cells. Irradiation has multiple inhibitory and stimulatory effects on the immune system such as increased CD4+, CD8+ and Treg Cells infiltration into TME (3, 4) Some regimens of radiation like ablative RT was associated with increased infiltration of exhausted CD8+ T cells into the tumor, which induced radiation resistance. (5, 6). The combination of radiotherapy and Immune checkpoint blockers therapy can reduce these inhibitory effects by stimulating immune cells and enhancing the response to radiation. Preclinical studies showed that RT increased PDL-1 expression on tumor cells (7, 8), and anti-PDL1 (αPDL-1) mAb combined with radiation had a synergistic effect on immune response induction dependent IFN-γ producing CD8 T cells activations. (9, 10). #Reviwer 1: 3. The author should mention the fractionation schedules. How many fractions of RT were given per day and for how many days/ weeks? Response: Thank you for pointing this out. In Methods, Treatment section adds these sentences: The experimental schedule of ablative radiation therapy was only one fraction of 15 Gy in 18 days’ post-initial inoculation. In Hypofraction RT regimens, two fractions of 10 Gy were performed on days 18 and 28 after inoculation. In Conventional RT, ten fractions of 3 Gy were given from day 18 to day 31 for 14 days at a time interval of one day or at most two days. (Figure 1A). #Reviwer 1: 4. The mostly used hyper fractionation RT regimens in clinics are 2Gy fraction per day. Is there any specific reason to use 3Gy fractions? Response: We thank the reviewer for their close attention. Given that the clinic conventional RT has a range of 1.8 to 2.5 Gy1, we wanted to assay immunological responses when deliver a 40 Gy equivalent total BED in these regimens. But on the other hand, we were going to have to choose the 2g dose for conventional regimen, we would have to give 17 fractions of radiation, due to the limitation of the number of irradiations in the pre-clinical studies, 3Gy fractions were selected for 10 fractions. Of course, further studies are ongoing for lower dose of BED regimens closer to the clinic regimens. #Reviwer 1: 5. Radiation with or without causes nephrotoxicity. The author should evaluate the nephrotoxicity after RT ± anti-PD-L1 and could cite the following paper (PMID: 27836988) Response: The main purpose of this study was to evaluate the immunological responses in irradiated regimens with the same biological effects. Further studies on radiation induced nephropathy should be considered after identifying the most appropriate regimen in any body tissue according to the immunological response and combination of radiation therapy with immunotherapy. #Reviwer 1: 6. The quality of the figures is unsatisfactory, please improve the quality of the figures. Response: The correction was made in the revised manuscript. The quality of figures are improved. #Reviwer 1:7. The findings have been discussed to some extent to support the claims made in the hypothesis. The manuscript adds some critical information to advance the field. The results presented in the paper are substantial enough for publication in Plosone after minor revision. Response: We greatly appreciate the reviewer’s efforts to carefully review the paper. #Reviwer 2: Excellent paper. Please add a paragraph after the discussion: Conclusion and discuss clinical implications of your findings and how your findings could be translated into the clinical studies. Lung SBRT is a good example where we deliver ablative dose of radiation. Response: We greatly appreciate the reviewer’s efforts to carefully review the paper. References: 1. John. L. Meyer. IMRT, IGRT, SBRT: Advances in the Treatment Planning and Delivery of radiotherapy. Vol. 43. Page 396 Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0231507.r002
25 Mar 2020 Decision Letter - Jianxin Xue, Editor Synergistic Effects of Anti-PDL-1 with Ablative Radiation Comparing to other Regimens with Same Biological Effect Dose based on Different Immunogenic Response PONE-D-19-33249R1 Dear Dr. Jalali, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Jianxin Xue Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. 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 ****** 5. 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 ****** 6. 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: (No Response) ****** 7. 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: Yes: ANIS AHMAD https://doi.org/10.1371/journal.pone.0231507.r003
Formally Accepted
30 Mar 2020 Acceptance Letter - Jianxin Xue, Editor PONE-D-19-33249R1 Synergistic Effects of Anti-PDL-1 with Ablative Radiation Comparing to other Regimens with Same Biological Effect Dose based on Different Immunogenic Response Dear Dr. Jalali: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Jianxin Xue Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0231507.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .