Associations of lymphocyte subpopulations with clinical phenotypes and long-term outcomes in juvenile-onset systemic lupus erythematosus

Butsabong Lerkvaleekul; Nopporn Apiwattanakul; Kanchana Tangnararatchakit; Nisa Jirapattananon; Supanart Srisala; Soamarat Vilaiyuk

doi:10.1371/journal.pone.0263536

Peer Review History

Original SubmissionNovember 5, 2021
6 Dec 2021 Decision Letter - Masataka Kuwana, Editor PONE-D-21-35318Associations of lymphocyte subpopulations with clinical phenotypes and long-term outcomes in juvenile-onset systemic lupus erythematosusPLOS ONE Dear Dr. Vilaiyuk, 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. Our reviewers found some interests in this manuscript, but one of them pointed out a number of comments that require improvement. I ask the authors to fully respond to all comments made by the reviewer in the revised manuscript. Please submit your revised manuscript by Jan 20 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. 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, Masataka Kuwana, MD, PhD 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 https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and " ext-link-type="uri" xlink:type="simple">https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf" 2. Thank you for stating the following in the Funding Section of your manuscript: SV received funding support from the Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand (RF_59002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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: SV received funding support from the Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand (RF_59002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. (URL funder website https://www.rama.mahidol.ac.th/en) Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 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. [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: Lerkvaleekul B et al. evaluated the association of lymphocytes subsets with clinical manifestations in patients with JSLE. The manuscript sounds interesting, but I have some concerns which authors need to answer. 1. Previous studies have demonstrated quite contradictory results regarding with frequency of Treg cells. Some reported a reduced frequency of circulating Foxp3+ Treg cells, but others found an increased or comparable frequency of circulating Foxp3+ Treg cells. I suggest authors to discuss this issue in discussion section. 2. Since the subsets of peripheral lymphocytes were dramatically changed by the background treatment, including glucocorticoid dose, and immunosuppressive agents. I suggest author to add background treatment in Table 1 and 2. 3. Authors compared the clinical manifestations between high and low frequency of each subset in Figure 3. I wonder how did authors select the manifestations, only mucosal ulcer, arthritis, AIHA, vasculitis and LN? Please show all manifestations listed in BILAG and compare them between high and low frequency of each lymphocyte subset. 4. Before showing Table 4, additional Table needs to be created comparing clinical characteristics depending on achievement of remission on therapy and select covariates for multivariate analysis using items having a P-value 0.05 in univariate analysis. There was no data supporting the items were properly selected as covariates for multivariate analysis in Table 4. 5. In Figure 4 and Figure 5 showed the serial change of each subset and Treg proportion and Figure 6 showed the probability of clinical remission depending on the frequency of Treg cells. These results were influenced by background treatment, and I suggest authors to show these results were independent of background treatment. Reviewer #2: There are no reports of lymphocyte subsets in Asian children with SLE, and this is an important finding that correlates with the phenotype. Of particular importance is the significantly lower proportion of regulatory T cells (Tregs) found only in patients with lupus nephritis (LN), a major cause of refractory disease. ****** 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 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.0263536.r001
Revision 1
19 Jan 2022 Author Response Response to reviewers 19 January 2022 Dear Editor, Thank you for allowing us to revise this manuscript. We have responded and revised this manuscript as reviewers' suggestions. We highlighted the revised sentences in yellow color throughout the entire manuscript. We also revised the format of the manuscript and added minimal data set as the journal's recommendation (2 supplement figures and 4 supplement tables). We removed the funding information from the manuscript and did not amend the previous funding statement given to the journal. With Regards, Soamarat Vilaiyuk Comments from reviewers Reviewer #1: Lerkvaleekul B et al. evaluated the association of lymphocytes subsets with clinical manifestations in patients with JSLE. The manuscript sounds interesting, but I have some concerns which authors need to answer. 1. Previous studies have demonstrated quite contradictory results regarding with frequency of Treg cells. Some reported a reduced frequency of circulating Foxp3+ Treg cells, but others found an increased or comparable frequency of circulating Foxp3+ Treg cells. I suggest authors to discuss this issue in discussion section. Response: We would like to thank the reviewer for your valuable comments on this manuscript. We have discussed this issue and added it in the discussion section as follows. The contradictory results of Tregs in various studies are from multiple factors. First, since there are several markers of Tregs with multiple phenotypic features, the variable Treg markers in each study cause the difference of Tregs results. The earlier studies found that the percentage of Tregs was significantly lower in active SLE compared to controls when CD25+ or CD25high cells were used as Treg markers. In contrast, other studies that used FoxP3+ or CD127low staining showed a comparable percentage of Tregs between active SLE and controls [20,22]. A previous study suggested that CD25 alone should not be classified as Tregs because many of these cells were FoxP3 negative, and other activated T cells can express CD25 [20]. Regarding extracellular staining CD4+CD25+CD127- cells, a previous study reported that CD127 is also downregulated during early activation of effector T cells, and around one-third of CD127low cells did not express FoxP3 [24]. Therefore, low expression of CD127 might not be a good marker that represents the Treg population. Unlike an important regulator in Treg development, a transcription factor FoxP3 is a more specific marker and remains the best marker of Tregs up to this point [20,22]. Second, each study had a different definition of active SLE disease, and the studies with higher cut-off SLEDAI scores tended to have a lower percentage of Tregs [22]. Our study results also supported this finding that the percentage of Tregs had increased while the disease activity had decreased. Third, since SLE is a heterogeneous disease, it is difficult for all studies to have the same patients' baseline characteristics, especially the frequency of lupus nephritis, which might have an influence on the percentage of Tregs the most. 2. Since the subsets of peripheral lymphocytes were dramatically changed by the background treatment, including glucocorticoid dose, and immunosuppressive agents. I suggest author to add background treatment in Table 1 and 2. Response: We have added more details about medications in Table 1 and Table 2 as your suggestion. (Highlight in yellow color) 3. Authors compared the clinical manifestations between high and low frequency of each subset in Figure 3. I wonder how did authors select the manifestations, only mucosal ulcer, arthritis, AIHA, vasculitis and LN? Please show all manifestations listed in BILAG and compare them between high and low frequency of each lymphocyte subset. Response: We analyzed all clinical manifestations but showed only the clinical manifestations that were significantly different as stacked bar chart. More details about lymphocyte subsets in each clinical manifestation are in supplement table (S1 Table). We also highlighted the yellow color in the clinical presentations and cells that were statistically different between the high and low frequency of each subset. 4. Before showing Table 4, additional Table needs to be created comparing clinical characteristics depending on achievement of remission on therapy and select covariates for multivariate analysis using items having a P-value 0.05 in univariate analysis. There was no data supporting the items were properly selected as covariates for multivariate analysis in Table 4. Response: We have added the supplement table (S4 Table) that showed the comparison of clinical characteristics between patients who had achievement and non-achievement of clinical remission on therapy. We found that absolute lymphocyte count and lupus nephritis were significantly different between both groups (yellow highlight). The presence of the direct Coombs test also tended to have a significant difference. Therefore, we selected these three factors with our interesting factor, high Tregs, and performed univariate analysis. All of them showed significance in univariate analysis. Furthermore, we performed the multivariate analysis using these four factors as covariates, which showed significance. We also added other factors that readers might be interested in and showed in the univariate analysis. However, those factors were not significant. We also added more details as your suggestion in the result section of the manuscript, as shown below. Furthermore, multivariate analysis was performed by selecting four significant covariates in univariate analysis, and it showed that predictors of remission on therapy were high Tregs, high ALC (1.5×106/L), positive DCT, and non-LN JSLE at enrollment (Table 4). 5. In Figure 4 and Figure 5 showed the serial change of each subset and Treg proportion and Figure 6 showed the probability of clinical remission depending on the frequency of Treg cells. These results were influenced by background treatment, and I suggest authors to show these results were independent of background treatment. Response: We have changed the presentation of Figure 4 and Figure 5 per your suggestion. Instead of presenting all JSLE patients, we classified patients into treatment-naïve and treated patients at enrollment. Overall results corresponded with the initial one (all JSLE patients), and it showed a clearer pattern, especially a significant increase of Treg percentage at 6 months in treatment-naïve patients with lupus nephritis. We also added more details in the result and discussion sections below (yellow highlight). In the result section. The percentage of Tregs between treatment-naïve patients and treated patients at enrollment is shown in Fig 5A. There was a significant increase of Treg percentage at 6 months in treatment-naïve patients with LN (Table in S3 Table). During the 6-month follow-up, SLEDAI-2K decreased over time in all JSLE patients, particularly in non-LN JSLE patients (Fig 5B). In the discussion section. In a previous study, SLE patients under immunosuppressive medications had significantly increased Tregs levels compared with untreated patients [55]. We demonstrated percentage of Tregs between treatment-naïve and treated JSLE with LN during the 6-month follow-up period and showed a clearer alteration pattern of Treg percentages in treatment-naïve JSLE patients. LN patients with naïve treatment also had a lower percentage of Tregs than treated patients at baseline. In addition, they showed significantly increased Treg percentage at 6 months, implying that lower Treg percentage at baseline derived from the active disease more than immunosuppressive medications. However, further study with a larger sample size regarding the treatment affecting Tregs should be performed. Regarding Fig 6, we have added another graph of Kaplan Meier analysis, which was performed only in treatment-naïve patients (Fig 6B). The graph of treatment-naïve patients demonstrated the same pattern with all JSLE patients (Fig 6A). However, the graph in treatment-naïve patients showed a more significant difference. We have added more details in the result section as below. Moreover, we also performed the Kaplan-Meier analysis only on the treatment-naïve patients (Fig 6B). This result corresponded with the finding from all JSLE patients (Fig 6A) and showed the difference between Tregs low and high groups more distinct. In the treatment-naïve patients, the median time to achieve remission on therapy was longer in the Tregs low group (58.40 [CI 20.51-96.29] months) compared with the Tregs high group (14.70 [CI14.16-15.24] months) with more statistical significance (p0.001) as shown in Fig 6B. In addition, 50% of treatment-naïve patients in the Tregs low group and 100% of Tregs high group achieved remission on therapy. Reviewer #2: There are no reports of lymphocyte subsets in Asian children with SLE, and this is an important finding that correlates with the phenotype. Of particular importance is the significantly lower proportion of regulatory T cells (Tregs) found only in patients with lupus nephritis (LN), a major cause of refractory disease. Response We would like to thank the reviewer for your valuable comments on this manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0263536.r002
21 Jan 2022 Decision Letter - Masataka Kuwana, Editor Associations of lymphocyte subpopulations with clinical phenotypes and long-term outcomes in juvenile-onset systemic lupus erythematosus PONE-D-21-35318R1 Dear Dr. Vilaiyuk, 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, Masataka Kuwana, MD, PhD 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: No https://doi.org/10.1371/journal.pone.0263536.r003
Formally Accepted
28 Jan 2022 Acceptance Letter - Masataka Kuwana, Editor PONE-D-21-35318R1 Associations of lymphocyte subpopulations with clinical phenotypes and long-term outcomes in juvenile-onset systemic lupus erythematosus Dear Dr. Vilaiyuk: 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 Prof. Masataka Kuwana Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263536.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 .