VSTM1-v2 does not drive human Th17 cell differentiation: A replication study

Helen J. von Richthofen; Florianne M. J. Hafkamp; Anouk van Haperen; Esther C. de Jong; Linde Meyaard

doi:10.1371/journal.pone.0284404

Peer Review History

Original SubmissionOctober 5, 2022
10 Nov 2022 Decision Letter - Sumit Kumar Hira, Editor PONE-D-22-25685VSTM1-v2 does not drive human Th17 cell differentiation: a replication study.PLOS ONE Dear Dr. Meyaard, 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. Please submit your revised manuscript by December 2 2022 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. 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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. Please update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by Amsterdam UMC, University of Amsterdam, and by a Vici grant from the Netherlands Organization for Scientific Research (NWO, grant no. 91815608)" We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "This work was supported by Amsterdam UMC, University of Amsterdam, and by a Vici grant from the Netherlands Organization for Scientific Research (NWO, grant no. 91815608, www.nwo.nl). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [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: Partly ******** 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: For figure 1B, n=7 seems too low for providing conclusive results. few more samples should be included ( n=20 fir example). Representative Dot plot for IL17+ cells should be included at least as supporting figures. ELISA of IL17 from Naive and memory T cells after treatment ( for cytokine induced Th17 differentiation ) could be considered Reviewer #2: The manuscript provides a controversial opinion on the role of VSTM1-v2 in human Th17 cell differentiation against a previous study. To draw a clear conclusion, the author should supply additional data to answer the following questions: 1) The VSTM1-v2 expression construct has not be verified by sequencing therefore, the protein generated using this construct may not be correct. 2) It is not clear whether VSTM1-v2 interacts with a ligand used by the full length SIRL-1. 3) Since purified VSTM1-v2 may lose function, overexpression of this protein may be more feasible. ****** 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: Sankar Bhattacharyya, Ph. D, Associate Professor, Department of Zoology, Sidho Kanho Birsha University, India. 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.] 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. https://doi.org/10.1371/journal.pone.0284404.r001
Revision 1
17 Jan 2023 Author Response Manuscript reference number: PONE-D-22-25685 Dear Editor, We thank you for the evaluation of our manuscript entitled “VSTM1-v2 does not drive human Th17 cell differentiation: a replication study”. We have addressed the reviewers’ comments and performed the requested experiment to increase the number of replicates. You can find our point-by-point reply to the reviewers’ comments below. Changes in the original manuscript are indicated as tracked changes. We hope that, with these changes, you will find the manuscript acceptable for publication in PLOS ONE. Our funding statement is as follows: “This work was supported by Amsterdam UMC, University of Amsterdam, and by a Vici grant from the Netherlands Organization for Scientific Research (NWO, grant no. 91815608, www.nwo.nl ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Yours sincerely, Helen von Richthofen Linde Meyaard Reviewer #1: For figure 1B, n=7 seems too low for providing conclusive results. few more samples should be included ( n=20 fir example). Representative Dot plot for IL17+ cells should be included at least as supporting figures. ELISA of IL17 from Naive and memory T cells after treatment ( for cytokine induced Th17 differentiation ) could be considered Reply: As requested by reviewer #1, we increased the number of replicates to strengthen the conclusion that VSTM1-v2 does not enhance DC-driven Th17 cell development from human naive T cells. Addition up to n=20 was not feasible as per experiment this takes approximately three weeks, but we did add n=3 to the figure with now a total of n=10. Statistical analysis still shows that no significant induction of Th17 cells occurs by VSTM1-v2. Furthermore, we included representative dot plots for IL-17+ cells as panel B in Figure 1. We thank reviewer #1 for the suggestion to consider an ELISA to measure IL-17 in supernatant as a measure for Th17 cell differentiation. In our previous publication by Souwer et al. [1], we also assessed DC-driven Th17 differentiation with or without neutrophils, and we measured a similar fold induction in % IL-17+ cells versus IL-17 concentration in supernatant. Thus, these methods seem equally sensitive. However, measuring the % of IL-17+ cells also gives single cell information as opposed to using ELISA, which is why we chose this method. Reviewer #2: The manuscript provides a controversial opinion on the role of VSTM1-v2 in human Th17 cell differentiation against a previous study. To draw a clear conclusion, the author should supply additional data to answer the following questions: 1) The VSTM1-v2 expression construct has not be verified by sequencing therefore, the protein generated using this construct may not be correct. Reply: We did sequence the VSTM1-v2 construct as part of our standard cloning procedure, and confirmed that the sequence is correct. The sequence can be found in our recent publication in J Immunol (Supplemental Figure 1) [2]. 2) It is not clear whether VSTM1-v2 interacts with a ligand used by the full length SIRL-1. Reply: We did not detect direct binding between SIRL-1 and its ligands (S100 proteins, LL-37 and phenol soluble modulins) [3, 4], possibly due to low affinity interactions or the requirement of an additional binding partner. As a consequence, we were unfortunately not able to technically assess whether VSTM1-v2 competes with the binding between SIRL-1 and its ligands. We now clarified this in sentence 168-170 of the manuscript. 3) Since purified VSTM1-v2 may lose function, overexpression of this protein may be more feasible. Reply: We appreciate the suggestion of the reviewer, but we purposefully chose to use purified recombinant VSTM1-v2 for several reasons; 1) we aimed to replicate the study by Guo et al., in which purified VSTM1-v2 was used; 2) purification of recombinant proteins by His-tag is an approved and commonly used method to obtain functional recombinant proteins [5], and 3) we made use of primary cells in this system, which are technically challenging to transfect for overexpression. References 1. Souwer Y, Groot Kormelink T, Taanman-Kueter EW, Muller FJ, van Capel TMM, Varga DV, et al. Human TH17 cell development requires processing of dendritic cell-derived CXCL8 by neutrophil elastase. The Journal of allergy and clinical immunology. 2018;141(6):2286-9 e5. 2. von Richthofen HJ, Westerlaken GHA, Gollnast D, Besteman S, Delemarre EM, Rodenburg K, et al. Soluble Signal Inhibitory Receptor on Leukocytes-1 Is Released from Activated Neutrophils by Proteinase 3 Cleavage. J Immunol. 2023. 3. Rumpret M, von Richthofen HJ, van der Linden M, Westerlaken GHA, Talavera Ormeno C, Low TY, et al. Recognition of S100 proteins by Signal Inhibitory Receptor on Leukocytes-1 negatively regulates human neutrophils. Eur J Immunol. 2021;51(9):2210-7. 4. Rumpret M, von Richthofen HJ, van der Linden M, Westerlaken GHA, Talavera Ormeno C, van Strijp JAG, et al. Signal inhibitory receptor on leukocytes-1 recognizes bacterial and endogenous amphipathic alpha-helical peptides. FASEB J. 2021;35(10):e21875. 5. Spriestersbach A, Kubicek J, Schafer F, Block H, Maertens B. Purification of His-Tagged Proteins. Methods Enzymol. 2015;559:1-15. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0284404.r002
16 Feb 2023 Decision Letter - Sumit Kumar Hira, Editor PONE-D-22-25685R1 VSTM1-v2 does not drive human Th17 cell differentiation: a replication study. PLOS ONE Dear Dr. Meyaard, 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. Please submit your revised manuscript by Apr 02 2023 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, Sumit Kumar Hira, Ph.D. 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. [Note: HTML markup is below. Please do not edit.] 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: (No Response) Reviewer #2: 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 Reviewer #2: No ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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) Reviewer #2: Although purified proteins usually have activity that was stated in literature, it is not necessary true in every lab and for every protein. The authors produced the protein in primary cells, the supernatants or the primary cells themselves carrying the transfected expression construct would be a useful resource containing the protein that more likely has activity. ****** 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: Sankar Bhattacharyya 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.] 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. https://doi.org/10.1371/journal.pone.0284404.r003
Revision 2
27 Feb 2023 Author Response Manuscript reference number: PONE-D-22-25685 Dear Dr Sumit Kumar Hira, Thank you for your evaluation of the revision of our manuscript entitled “VSTM1-v2 does not drive human Th17 cell differentiation: a replication study” for PLOS ONE. Both reviewers now indicate that all their comments were addressed in the revised manuscript, yet your recommendation is to revise the manuscript again. We assume this is because of reviewer #2’s comment to question 6, suggesting to use supernatants of VSTM1-v2-transfected cells instead of purified VSTM1-v2 for our experiments. In the first round of review, reviewer #2 also raised this concern. We replied to this comment in the following way: “… we purposefully chose to use purified recombinant VSTM1-v2 for several reasons; 1) we aimed to replicate the study by Guo et al., in which purified VSTM1-v2 was used; 2) purification of recombinant proteins by His-tag is an approved and commonly used method to obtain functional recombinant proteins, and 3) we made use of primary cells in this system, which are technically challenging to transfect for overexpression.” These arguments still stand, we purposely chose to use purification of His-tagged recombinant VSTM1-v2 with a Ni-Sepharose column in order to replicate the method used by Guo et al. Additionally, cell culture supernatant contains a plethora of compounds which may have indirect effects on the co-culture system that we use. We characterized VSTM1-v2 by SDS-PAGE and Western blot after purification (von Richthofen et al., 2023). VSTM1-v2 had a molecular weight of approximately 50 kDa, and deglycosylated VSTM1-v2 had a molecular weight of 37 kDa. This is in accordance with the findings by Guo et al.. Moreover, we could detect native VSTM1-v2 with anti-SIRL-1 antibodies (two clones), which is to be expected since VSTM1-v2 is a splice isoform of SIRL-1. This suggests that purification of VSTM1-v2 resulted in a viable protein. We did not describe the characterization of VSTM1-v2 protein in the current manuscript, since we already described it in our earlier publication. However, to avoid confusion, we now added the following sentence to the discussion, after sentence 175: “Purified VSTM1-v2 had a similar molecular weight as in the study by Guo et al., as assessed by SDS-PAGE (von Richthofen et al., 2023). “ Taken together, we conclude that using supernatants of VSTM1-v2 transfected cells rather than purified VSTM1-v2 will not improve the quality of the manuscript. We therefore hope that with this addition the manuscript is acceptable for publication in PLOS ONE . Yours sincerely, Helen von Richthofen Linde Meyaard von Richthofen, H. J., Westerlaken, G. H. A., Gollnast, D., Besteman, S., Delemarre, E. M., Rodenburg, K., . . . Meyaard, L. (2023). Soluble Signal Inhibitory Receptor on Leukocytes-1 Is Released from Activated Neutrophils by Proteinase 3 Cleavage. Journal of Immunology, 210(4), 389-397. doi:10.4049/jimmunol.2200169 Attachments Attachment Submitted filename: Rebuttal.docx https://doi.org/10.1371/journal.pone.0284404.r004
30 Mar 2023 Decision Letter - Sumit Kumar Hira, Editor VSTM1-v2 does not drive human Th17 cell differentiation: a replication study. PONE-D-22-25685R2 Dear Dr. Meyaard, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 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, Sumit Kumar Hira, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0284404.r005
Formally Accepted
6 Apr 2023 Acceptance Letter - Sumit Kumar Hira, Editor PONE-D-22-25685R2 VSTM1-v2 does not drive human Th17 cell differentiation: a replication study. Dear Dr. Meyaard: I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Sumit Kumar Hira Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0284404.r006

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