Aligning cellular and molecular components in age-dependent tertiary lymphoid tissues of kidney and liver

Naoya Toriu; Yuki Sato; Hiroteru Kamimura; Takahisa Yoshikawa; Masaou Tanaka; Shinya Yamamoto; Shingo Fukuma; Masakazu Hattori; Shuji Terai; Motoko Yanagita

doi:10.1371/journal.pone.0311193

Peer Review History

Original SubmissionMay 16, 2024
16 May 2024 Author Response https://doi.org/10.1371/journal.pone.0311193.r001
12 Jul 2024 Decision Letter - Nebojsa Bacanin, Editor PONE-D-24-18840Aligning Cellular and Molecular Components in Age-Dependent Tertiary Lymphoid Tissues of Kidney and LiverPLOS ONE Dear Dr. Yanagita, 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. ============================== ACADEMIC EDITOR: Dear Authors, please revise proposed manuscript thoroughly according to all reviewers' comments. Additionally, please do the following: - Visualization of obtained results must be improved - Motivation behind proposed research should be more clearly explain. Please elaborate what is "beyond state-of-the-art" of proposed. study. - Make sure that the source code is available according to PLOS ONE publication policies. - Make sure that you have conducted rigid statistical analysis. All the best, Nebojsa Bacanin ============================== Please submit your revised manuscript by Aug 26 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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Additionally, please do the following: - Visualization of obtained results must be improved - Motivation behind proposed research should be more clearly explain. Please elaborate what is "beyond state-of-the-art" of proposed. study. - Make sure that the source code is available according to PLOS ONE publication policies. - Make sure that you have conducted rigid statistical analysis. All the best, Nebojsa Bacanin [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: No ****** 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: The authors have demonstrated that TLTs developed in the liver with aging, and that their cellular and molecular components were similar to those in the kidneys, such as senescence-associated T cells and age-associated B cells. Additionally, the authors have shown that the expression of TLT-related genes was elevated in the liver biopsy samples from HCV-infected patients compared with those without HCV infection. TLT formation was also associated with liver injury and fibrosis. The present study suggests that TLT formation is observed across the tissues over aging. Although the causal relationship between TLT formation, chronic inflammation and aging remains unclear, further understanding of this cross-organ phenomenon may lead to solutions to many diseases and health problems. HCV-related chronic hepatitis is now actively treated and curable disease. Is TLT formation associated with treatment response or liver function outcome? Is TLT formation associated with other liver dysfunction or disease such as non-C virus hepatitis and alcoholic/non-alcoholic/drug-induced/primary biliary cholangitis? P18 LL389 “liver fibrosis x” � “liver fibrosis” Reviewer #2: The present manuscript is about tertiary lymphoid tissues (TLT) in the liver. The authors found aging and HCV were associated with TLT formation in the liver, and might contribute to liver dysfunction and fibrosis. The topic is interesting, but some critical concerns should be addressed. 1. Which type of non-hematopoietic cells express CXCL13 in TLT in the liver tissue? 2. The interpretation of results and conclusion are too strong because statistical analyses have not been performed in Tables 1 and 2. Also, it seems to be difficult to describe TLT increases and develop to more advanced stages based on the data in Figure 4L-O. I understand that they are due to small sample size, but this point is critical because we should discuss based on data. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). 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Revision 1
31 Aug 2024 Author Response Point by point reply to the Editor and reviewers’ comments and suggestions We would like to thank the editors and the reviewers for their valuable and insightful comments and the useful suggestions that have helped us to improve our manuscript. - Visualization of obtained results must be improved Reply to the academic editor: Thank you for your important comments. We paid attention to the visualization quality of newly added data (Fig. 1R-S, S3_Fig) - Motivation behind proposed research should be more clearly explain. Please elaborate what is "beyond state-of-the-art" of proposed study. Reply to the academic editor: I appreciate your important comment. We have previously clarified the cellular and molecular components and clinical significance of TLTs in the kidney, and are curious whether our findings on renal TLTs could also be shared across the tissue. In this study, we therefore analyzed the cellular and molecular components of TLTs in the liver and their clinical significance, and found that there are similarities with TLTs in the kidney. We add the following sentence in the revised manuscript: “Although TLTs develop with aging in other organs as well, their cellular and molecular components, and clinical significance remain unclear.” (p.2, l.7-8) “we tried to characterize the cellular and molecular components of hepatic TLTs using superaged mouse and human livers, and to elucidate the clinical relevance of them.” (p.4, l.2-3) “demonstrate that the cellular and molecular components of hepatic TLTs are similar to those of renal TLTs and” (p.20, l.18-19) - Make sure that the source code is available according to PLOS ONE publication policies. Reply to the academic editor: Thank you for your suggestion. We have no source code for sharing. - Make sure that you have conducted rigid statistical analysis. Reply to the academic editor: Thank you for your suggestion. We performed statistical analysis in Table 1. In Table 2, Fig4 L, N-0, we could not perform statistical analysis because of the existence of a zero cell count. - Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Reply to the academic editor: Thank you for your suggestion. We followed the PLOS ONE’s style requirements. 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. Reply to the academic editor: Thank you for your comment. We added the following sentence in the “Material and methods” section in the revised manuscript: “Informed consent was obtained using an opt-out process on the website. Minors were not included among the patients.” (p.8, l.11-12) 3. Thank you for stating the following financial disclosure: [This research was supported by the Japan Agency for Medical Research and Development (AMED) under Grant Number AMED-CREST 19gm0610011, 22gm1210009, 22zf0127003h001, 22lm0203006, and 22ek0310020h0001; KAKENHI Grant-in-Aids for Scientific Research B (20H03697). This work was partly supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan.]. Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Reply to the academic editor: Thank you for your suggestion. We added the following statement in the revised manuscript and cover letter. "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." (p.21, l.10-11) 4. In the online submission form, you indicated that [The datasets of mice used and analyzed in the current study are available from the corresponding author upon reasonable request. The human datasets that support the findings of this study are available from the corresponding author, but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the ethics committee at Niigata University and the ethical committee at Kyoto University Hospital. Previously published RNA-seq data are available in GEO (GSE84346: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84346 and GSE171294: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171294).]. All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, 2. Within the manuscript itself, or 3. Uploaded as supplementary information. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval. Reply to the academic editor: Thank you for your suggestion. We included the mouse datasets in the supplement. However, as for human dataset, the ethics committees at Niigata University and Kyoto University Hospital have not approved uploading the human datasets because public availability would compromise patient privacy. Still, data are available from the authors upon reasonable request and with permission of the ethics committees at Niigata University and Kyoto University Hospital. We changed the data availability statement as follows: “The mouse datasets are available in the Supporting Information files (S2_Table).” (p.22, l.9) “The institutional contacts are: ethics@adm.niigata-u.ac.jp (the ethics committee at Niigata University) and ethcom@kuhp.kyoto-u.ac.jp (the ethical committee at Kyoto University Hospital).” (p.22, l.14-15) 5. We note that Figures 1, 2, and 4 in your submission contain [map/satellite] images which may be copyrighted. Reply to the academic editor: We apologize for not being clear for copyright. All figures included in our paper were taken by the authors and are not copyrighted by another organization. Reviewer: 1 Comments to the Author The authors have demonstrated that TLTs developed in the liver with aging, and that their cellular and molecular components were similar to those in the kidneys, such as senescence-associated T cells and age-associated B cells. Additionally, the authors have shown that the expression of TLT-related genes was elevated in the liver biopsy samples from HCV-infected patients compared with those without HCV infection. TLT formation was also associated with liver injury and fibrosis. The present study suggests that TLT formation is observed across the tissues over aging. Although the causal relationship between TLT formation, chronic inflammation and aging remains unclear, further understanding of this cross-organ phenomenon may lead to solutions to many diseases and health problems. HCV-related chronic hepatitis is now actively treated and curable disease. Is TLT formation associated with treatment response or liver function outcome? Reply to the reviewer 1: Thank you for your valuable and supportive comments and important questions. Unfortunately, this study did not collect the data on the usage of direct acting antivirals (DAAs). Combination therapy with DAA and interferon has been available in Japan since 2011. Among the patients analyzed, 37 patients received liver biopsy before 2011, and the remaining 10 patients received liver biopsy after 2011. Of these 10 patients, four patients on treated with interferon therapy, but the use of DAA is unknown. On the other hand, there are data about the treatment response to interferon therapy (newly added S3_Fig). The sustained virologic response at 24 weeks (SVR24) after interferon therapy was 100% for patients without TLTs, 73% for patients with stage I TLTs, and 78% for patients with stage II TLTs (S3_Fig A). While we could not perform Fisher’s exact test due to zero cell counts, there might be a tendency for TLTs in the liver to be associated with poor response to interferon therapy (S3_Fig A). On the other hand, TLT formation was not associated with the cumulative incidence of hepatocellular carcinoma (S3_Fig B). We add the following sentences and S3_Fig in the revised manuscript. “Kaplan-Meier method and log-rank test were used to depict and compare the cumulative incidence of hepatocellular carcinoma (HCC).” (p. 9, l.22-23) “We also investigated the relationship between hepatic TLTs and outcomes of 47 HCV patients. Among them, 25 patients were treated with interferon, and the ratio of patients who achieved negative for serum HCV RNA at 24 weeks after the end of interferon therapy (SVR24) was 73% for patients with stage I TLTs and 78% for patients with stage II TLT, compared with 100% for patients without TLTs (S3_Fig A). Among 47 HCV patients, we were able to follow the prognosis of 32 patients. Within five years after liver biopsy, TLT formation was not associated with the cumulative incidence of hepatocellular carcinoma (S3_Fig B).” (p. 17, l.8-14) Is TLT formation associated with other liver dysfunction or disease such as non-C virus hepatitis and alcoholic/non-alcoholic/drug-induced/primary biliary cholangitis? Reply to the reviewer 1: Thank you for your comment. As you suggested, TLT formation is associated with other liver disease such as HBV hepatitis (Link A et al. Am J Pathol. 2011;178(4):1662-75) autoimmune hepatitis, and primary biliary cholangitis (Pikarsky E et al. Gastroenterology. 2016;151(5):780-3, van Buuren N et al. JHEP Rep. 2022;4(1):100388). We added the following sentence in the revised manuscript: “Lymphoid aggregate, some of which are TLTs, can be found in patients not only with HCV but also with hepatitis B virus infection, autoimmune hepatitis, and primary biliary cirrhosis [41-43].” (p. 20, l.2-3) P18 LL389 “liver fibrosis x” � “liver fibrosis” Reply to the reviewer 1: Thank you for your kind comment and we are sorry for the typo. We corrected the sentence. Reviewer #2: Comments to the Author The present manuscript is about tertiary lymphoid tissues (TLT) in the liver. The authors found aging and HCV were associated with TLT formation in the liver, and might contribute to liver dysfunction and fibrosis. The topic is interesting, but some critical concerns should be addressed. 1. Which type of non-hematopoietic cells express CXCL13 in TLT in the liver tissue? Reply to the reviewer 2: Thank you for your important comment. Immunostaining analysis showed the colocalization of CXCL13 and Desmin and CD21, respectively (Fig. 1R, S), which suggest that Desmin+ fibroblasts and CD21+ follicular dendritic cells (FDCs) express CXCL13. We added the following sentence. “CXCL13 was expressed in Desmin+ fibroblasts and CD21+ FDCs (Fig 1R, S).” (p. 10, l.17-18) 2. The interpretation of results and conclusion are too strong because statistical analyses have not been performed in Tables 1 and 2. Also, it seems to be difficult to describe TLT increases and develop to more advanced stages based on the data in Figure 4L-O. I understand that they are due to small sample size, but this point is critical because we should discuss based on data. Reply to the reviewer 2: Thank you for your important suggestion, and we apologize for the too-strong interpretation. We performed statistical analysis in Table 1. Unfortunately, in Table 2, Figure 4L, and Figure 4N-O, we could not perform Fisher’s exact test due to the existence of zero cell counts. Furthermore, we modified the result and discussion sections to avoid strong statement on the data without statistical significance. We changed the result and discussion as follows. “The median age of patients in each group was 53 years for patients without TLTs, 62 years for patients with stage I TLTs, and 63 years for patients with stage II TLTs, respectively (Table 1). While none of the patients without TLTs have been previously treated with interferon, some of the patients with TLTs have been previously treated with interferon. In addition, ALBI scores were −3.03 for patients without TLTs, −2.90 for patients with stage I TLTs and −2.73 for patients with stage II TLTs. Furthermore, FIB-4 index was 1.59 for patients without TLTs, 2.61 for patients with stage I TLTs and 2.55 for patients with stage II TLTs.” (p. 15, l.15-21) “Overall, 100% of the aged patients (60 years of age or over) were positive for hepatic TLTs, while 72.7% of the young patients were positive for hepatic TLTs (Fig. 4L). The number of TLTs in the liver biopsy samples was not statistically different between two groups (Fig. 4M). The percentage of patients with stage II TLTs was 52% in aged patients and 27% in young patients (Fig. 4N).” (p. 16, l.12- p. 17 l.1) “The percentage of patients with severe fibrosis (F3) was 26.8% in patients with hepatic TLTs and 0% in patients without hepatic TLTs (Fig. 4O). All patients had no difference in activity grade (Table 2).” (p. 17, l.5-7) “With a clear definition of TLTs, we found that hepatic TLTs were observed more frequently than previously considered, especially in aged patients, in whom TLTs were present in 100% of patients.” (p. 19, l.15-17) “Our findings demonstrate that the cellular and molecular components of hepatic TLTs are similar to those of renal TLTs and suggest that hepatic TLTs might be associated with aging, liver injury and liver fibrosis.” (p. 20, l.18-19) Although the association between TLTs and liver fibrosis was not statistically supported in Figure 4, we reserved the sentence because of the association in Figure 3. To soften our statement, we changed "are" to "might be". Attachments Attachment Submitted filename: point_by_point 240824 FInal_NT.docx https://doi.org/10.1371/journal.pone.0311193.r003
16 Dec 2024 Decision Letter - Elsayed Seddek Ibrahem Mohammed, Editor PONE-D-24-18840R1Aligning Cellular and Molecular Components in Age-Dependent Tertiary Lymphoid Tissues of Kidney and LiverPLOS ONE Dear Dr. Yanagita, 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 Jan 30 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Elsayed Seddek Ibrahem Mohammed, 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. Additional Editor Comments: Dear Authors, On behalf of PLOS ONE, I would like to extend my gratitude for submitting your manuscript entitled “Aligning Cellular and Molecular Components in Age-Dependent Tertiary Lymphoid Tissues of Kidney and Liver” (Manuscript ID: PONE-D-24-18840R1). Your work represents a significant contribution to the understanding of the dynamic interplay between cellular and molecular components within tertiary lymphoid tissues and their age-related adaptations in the kidney and liver. I understand the limitations that you have faced in your study and the effort that you have put to get sufficient and informative data. After a thorough review process, I am pleased to inform you that your manuscript has been accepted for publication, pending the incorporation of minor corrections. These revisions will enhance the clarity and impact of your findings. Please see the attached document outlining the necessary modifications. We greatly appreciate your efforts and commend the scientific rigor and innovation reflected in your work. If you have any questions or require assistance during the revision process, please do not hesitate to reach out. Thank you once again for contributing your research to PLOS ONE. We look forward to the final version of your manuscript and its forthcoming publication. Editor comments to the authors: 1- Page 5 line 96 : the Formaline used in fixation was 4% paraformaldehyde or 4% NBF,, Please describe 2- Page 5 line 97: the model and manufacturer of the microtome used in this study. 3- Page 5 line 97: the reference of the stain H and E and staining protocol (for example Bancroft et al., or Savana et al., 2018) 4- Page 5 line 103:the cryostat model and manufacturer is missing. 5- Page 6 line 117: the used formaldehyde for fixation is it the same in all the experiments / if so Please unify in all the manuscript as mentioned in the first comment. Reviewer comments to the author: Dr. Yanagita and colleagues submitted their manuscript, "Aligning Cellular and Molecular Components in Age-Dependent Tertiary Lymphoid Tissues of Kidney and Liver," for review. The study addresses a significant topic in immunology and aging, offering novel insights into the systemic formation of tertiary lymphoid tissues (TLTs). The manuscript is scientifically sound and presents an important advancement in the understanding of TLTs in aging and liver pathology. Recommendations are provided to refine and enhance the manuscript for publication. 1. Figures and Visual Representation (1) Figure 1: • Use uniform scale bars across sub-panels, positioned consistently (e.g., bottom-right). • Clarify differences between TLT stages (I, II, III) in panel (V) with added markers or legends. (2) Figure 4: • Improve legends for panels (L–N) to explain the relationship between age and TLT stages. (3) Supplementary Figures: • Ensure all axes in the figures (e.g., S3) are clearly labeled, with legends comprehensively describing the content. 2. Interpretation of Results • While the manuscript successfully tempers conclusions due to small sample sizes in some analyses, consider further nuanced discussions of statistical limitations. For instance: Clarify the significance of TLT presence in fibrosis-related outcomes when statistical significance is not achieved (e.g., Figure 4). 3. Clinical Applications and Generalizability • Strengthen the discussion on the broader implications of TLT findings in conditions beyond HCV, such as non-alcoholic steatohepatitis or autoimmune hepatitis. Once these minor issues are addressed, I am confident that the manuscript will meet the high standards of PLOS ONE and greatly benefit its readership. Thank you for the opportunity to review this excellent work. Please do not hesitate to contact me if further clarification is needed. Best Regard's, [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: All comments have been addressed Reviewer #2: All comments have been addressed Reviewer #3: 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: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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 Reviewer #3: No ****** 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 Reviewer #3: 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: (No Response) Reviewer #3: Drs. Yanagita and colleagues submitted their manuscript, "Aligning Cellular and Molecular Components in Age-Dependent Tertiary Lymphoid Tissues of Kidney and Liver," for review. The study addresses a significant topic in immunology and aging, offering novel insights into the systemic formation of tertiary lymphoid tissues (TLTs). The manuscript is scientifically sound and presents an important advancement in the understanding of TLTs in aging and liver pathology. Recommendations are provided to refine and enhance the manuscript for publication. 1. Figures and Visual Representation (1) Figure 1: • Use uniform scale bars across sub-panels, positioned consistently (e.g., bottom-right). • Clarify differences between TLT stages (I, II, III) in panel (V) with added markers or legends. (2) Figure 4: • Improve legends for panels (L–N) to explain the relationship between age and TLT stages. (3) Supplementary Figures: • Ensure all axes in the figures (e.g., S3) are clearly labeled, with legends comprehensively describing the content. 2. Interpretation of Results • While the manuscript successfully tempers conclusions due to small sample sizes in some analyses, consider further nuanced discussions of statistical limitations. For instance: Clarify the significance of TLT presence in fibrosis-related outcomes when statistical significance is not achieved (e.g., Figure 4). 3. Clinical Applications and Generalizability • Strengthen the discussion on the broader implications of TLT findings in conditions beyond HCV, such as non-alcoholic steatohepatitis or autoimmune hepatitis. ****** 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 Reviewer #2: Yes: Satoshi Hara Reviewer #3: 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.0311193.r004
Revision 2
17 Jan 2025 Author Response Point by point reply to the Editor and reviewers’ comments and suggestions We would like to thank the editors and the reviewers for their valuable and insightful comments and the useful suggestions that have helped us to improve our manuscript. 1- Page 5 line 96 : the Formaline used in fixation was 4% paraformaldehyde or 4% NBF,, Please describe Reply to the editor: Thank you for your comment. We used 10% NBF and described the fixation method in the Methods. 2- Page 5 line 97: the model and manufacturer of the microtome used in this study. Reply to the editor: Thank you for your comment. We described the model and manufacturer of the microtome. 3- Page 5 line 97: the reference of the stain H and E and staining protocol (for example Bancroft et al., or Savana et al., 2018) Reply to the editor: Thank you for your comment. We cited the reference for the staining protocol. 4- Page 5 line 103:the cryostat model and manufacturer is missing. Reply to the editor: Thank you for your comment. We described the model and manufacturer of the cryostat. 5- Page 6 line 117: the used formaldehyde for fixation is it the same in all the experiments / if so Please unify in all the manuscript as mentioned in the first comment. Reply to the editor: Thank you for your comments. The used formaldehyde for fixation is the same in all the experiments. We described the fixation method. 1. Figures and Visual Representation (1) Figure 1: • Use uniform scale bars across sub-panels, positioned consistently (e.g., bottom-right). Reply to the reviewer 3: Thank you for your suggestion. We unified the scale bars, and positioned at the bottom-left consistently. • Clarify differences between TLT stages (I, II, III) in panel (V) with added markers or legends. Reply to the reviewer 3: Thank you for your valuable comments. We added the following sentences to the figure legends. “Stage I TLTs are the aggregation of lymphocytes with the sign of proliferation, and do not include FDCs or germinal centers; Stage II TLTs include FDCs, but not germinal centers; Stage III TLTs include FDCs and germinal centers.” (p.11, l.13-15) “Stage I TLTs are the aggregation of lymphocytes with the sign of proliferation, and do not include FDCs or germinal centers; Stage II TLTs include FDCs, but not germinal centers.” (p.14, l.26-28) (2) Figure 4: • Improve legends for panels (L–N) to explain the relationship between age and TLT stages. Reply to the reviewer 3: Thank you for your valuable comments. We changed the legend for panels (L-N) as follows: “(L) TLT frequencies in young and aged patients are 72.7% and 100%, respectively. (M) The relative numbers of TLTs per portal tract number. The medians of those in young and aged patients are 0.125 and 0.138, respectively. (N) The proportion of patients in each TLT stages. The proportion of patients with stage I TLTs in young and aged patients are 45% and 48%, respectively, while that of patients with stage II TLTs are 27% and 52%, respectively.” (p.15, l.2-6) (3) Supplementary Figures: • Ensure all axes in the figures (e.g., S3) are clearly labeled, with legends comprehensively describing the content. Reply to the reviewer 3: Thank you for your valuable comments. We changed the axis of S3 Fig A to “% of patients with SVR24 (%)”. We also corrected the typo of the y axis of S3 Fig B. In addition, we added the sentence in the legend. “The proportion of patients with SVR24 in patients without TLTs, with Stage I TLTs and with Stage II TLTs are 100%, 73% and 78%, respectively.” (p.27, l.33-34) “The cumulative incidences of HCC in patients with TLTs are 4.17, 4.17, and 8.73% at 1, 3, and 5 years after the liver biopsy, respectively. Those without TLTs are 0, 0, and 0% at 1, 3, and 5 years after the liver biopsy, respectively. Log-rank test is used to compare the cumulative incidence of HCC.” (p.27, l.36-39) Furthermore, we changed the label of Fig1W and S1 Fig as follows. “% of each stage among all TLTs” (Fig 1W) “CD90.2 CD45 DAPI” (S1 Fig) 2. Interpretation of Results • While the manuscript successfully tempers conclusions due to small sample sizes in some analyses, consider further nuanced discussions of statistical limitations. For instance: Clarify the significance of TLT presence in fibrosis-related outcomes when statistical significance is not achieved (e.g., Figure 4). Reply to the reviewer 3: Thank you for your valuable and supportive comments We added the following sentence. In addition, 26.8% in patients with hepatic TLTs had severe fibrosis, whereas 0% in patients without hepatic TLTs had severe fibrosis. Due to the presence of a zero cell, statistical testing could not be performed.” (p.19, l.20-22) 3. Clinical Applications and Generalizability • Strengthen the discussion on the broader implications of TLT findings in conditions beyond HCV, such as non-alcoholic steatohepatitis or autoimmune hepatitis. Reply to the reviewer 3: Thank you for your valuable and supportive comments. It is reported that non-alcoholic steatohepatitis is not associated with hepatic TLTs in human patients and mouse models (Pikarsky E, et al. Gastroenterology. 2016;151(5):780-3.). Therefore, we discussed about autoimmune hepatitis. We added the following sentence. “however, as for autoimmune liver diseases, it is speculated that TLTs might maintain and propagate autoimmunity by providing an environment where self-antigens can be presented, and antigen-specific T cells can be activated [43]. Analysis of the role of hepatic TLTs in autoimmune hepatitis may contribute to an enhanced understanding of the relationship between liver disease and hepatic TLT.” (p.20, l.15-19) Attachments Attachment Submitted filename: Response to Reviewers_FINAL.docx https://doi.org/10.1371/journal.pone.0311193.r005
30 Jan 2025 Decision Letter - Elsayed Seddek Ibrahem Mohammed, Editor Aligning Cellular and Molecular Components in Age-Dependent Tertiary Lymphoid Tissues of Kidney and Liver PONE-D-24-18840R2 Dear Dr. Yanagita, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Elsayed Seddek Ibrahem Mohammed, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Subject: Acceptance of Manuscript PONE-D-24-18840R2 Dear editor , We are pleased to inform you that your manuscript titled "Aligning Cellular and Molecular Components in Age-Dependent Tertiary Lymphoid Tissues of Kidney and Liver" (Manuscript ID: PONE-D-24-18840R2) has been accepted for publication in PLOS ONE. Your study provides a significant contribution to our understanding of the cellular and molecular dynamics of tertiary lymphoid tissues, especially in the context of age-dependent changes. The thorough analysis and presentation of findings in this manuscript exemplify the high standard of research we strive to publish. Before finalizing the publication, we would like to request a minor revision to Reference 31: Current: Kim SS, Layton C, Bancroft JD (2018) Bancroft’s theory and practice of histological techniques, 8th edn. Elsevier, Amsterdam. Revised: Suvarna KS, Layton C, Bancroft JD (2018). Bancroft's theory and practice of histological techniques. Elsevier health sciences. Please ensure this revision is incorporated in your reference list before publication. Additionally, kindly review the references thoroughly to ensure accuracy and compliance with the journal’s formatting guidelines. Congratulations on your successful submission! We appreciate your contribution to the field and look forward to showcasing your work to the broader scientific community. Sincerely, Elsayed Mohammed PLOS ONE Editorial Team 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 #3: 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 #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: 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 #3: 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 #3: 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 #3: Dr. Yanagita and his team revised and resubmitted the manuscript, 'Aligning Cellular and Molecular Components in Age-Dependent Tertiary Lymphoid Tissues of Kidney and Liver.' I have no further concerns about this paper and recommend it for publication. ****** 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 #3: No ******** https://doi.org/10.1371/journal.pone.0311193.r006
Formally Accepted
Acceptance Letter - Elsayed Seddek Ibrahem Mohammed, Editor PONE-D-24-18840R2 PLOS ONE Dear Dr. Yanagita, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Elsayed Seddek Ibrahem Mohammed Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0311193.r007

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