Distorted TCR repertoires define multisystem inflammatory syndrome in children

Amna Malik; Eszter N. Tóth; Michelle S. Teng; Jacob Hurst; Eleanor Watt; Lauren Wise; Natalie Kent; Jack Bartram; Louis Grandjean; Margarita Dominguez-Villar; Stuart Adams; Nichola Cooper

doi:10.1371/journal.pone.0274289

Peer Review History

Original SubmissionMarch 22, 2022
16 Jun 2022 Decision Letter - Sebastian D. Fugmann, Editor PONE-D-22-08475Distorted TCR repertoires define multisystem inflammatory syndrome in childrenPLOS ONE Dear Dr. Cooper, 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. 1) After receiving the comments of one reviewer one big issue needs to be addressed in order to make this manuscript suitable for consideration in PLoS ONE. 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Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information [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: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? 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Reviewer #1: 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: This is a new manuscript on the TCR repertoire analysis in patients with MIS-C. A total of 20 children with COVID-19 have been included. In the manuscript the authors state that “whether the specific T cell clones contribute to the hyperinflammatory state or if there is a difference in the T cell repertoire composition, antigen specificity is unknown”. This is not correct, and several studies have covered the repertoire of MIS-C patients on largest cohorts (Moreews et al. Science Immunol 2021, Hoste et al. JEM 2022, Sacco et al Nat Med 2022). The omission to refer to these papers in the manuscript and this statement are very surprising. -The group of severity are poorly described and the “mild disease” comprise asymptomatic patients (that can thus not be defined as mild) and the severe group only comprise MIS-C patients (no other subset of ICU and pediatric COVID-19). This should be corrected, and the authors should clarify that they compare MIS-C to SARS-CoV2-positive children. The severe group is actually restricted to MIS-C. I suggest to avoid to name this group Severe COVID-19 (MIS-C instead). There are two severe manifestations of COVID-19 in children: -Severe / fatal pneumonia, occurring in immunocompromised children (IFN-I pathway, in particular) and MIS-C. Here the authors only highlight the MIS-C phenotype. -The use of DNA instead of RNA for the TCR sequencing is more challenging and associate a -greater number of biases. -The junctional diversity of TRBV11-2 was also previously reported. In addition, it is present in both CD4/CD8 T cells and some functional studies have also tested T cell activation to various SARS-CoV2 antigens in MIS-C patients (Moreews et al. Hoste et al…). -Actually the polyclonal expansion of T cells is a feature of superantigen immune reaction that was highlight in previous (omitted)studies. The other patients that experience an acute infection present an antigen-specific immunity that is also reported and physiological. -The authors state that the TCR bias is responsible for the hyperinflammatory syndrome but this causal link is not supported by any data provided here. -The timing to infection should be indicated. The so-called group “mild “ was possibly sampled at the acute phasis of the infection whereas the MIS-C are sampled at the time of the episode which is post-infectious (about 4 weeks after the virus encounter). This information is important. -The clinical data on the MIS-C are missing (vasoplegia, blood pressure, shock, erythema…). MIS-C usually occurs in patients with no comorbidities. How MIS-C was confirmed in the patient with hepatoblastoma ? The TRBV11-2/ Vb21.3 expansion is now considered as a diagnosis marker. How was individual TRBV11-2 expansion (especially in the patient with hepatoblastoma)? Is there any pediatric controls in the repertoire assessment? In total, this is a replication study of numerous previous work on MIS-C (not severe pediatric COVID) and use a more challenging technique (DNA seq) to address this question on a lowest number of patients. The add-value of this work is limited compared to previous studies and the authors may provide information on how this report replicate previous work and what is new. ****** 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. 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Revision 1
4 Aug 2022 Author Response Comments from Reviewer #1: 1. In the manuscript the authors state that “whether the specific T cell clones contribute to the hyperinflammatory state or if there is a difference in the T cell repertoire composition, antigen specificity is unknown”. This is not correct, and several studies have covered the repertoire of MIS-C patients on largest cohorts (Moreews et al. Science Immunol 2021, Hoste et al. JEM 2022, Sacco et al Nat Med 2022). Response: All these papers and other relevant papers have now been cited. 2. The group of severity are poorly described and the “mild disease” comprise asymptomatic patients (that can thus not be defined as mild) and the severe group only comprise MIS-C patients (no other subset of ICU and pediatric COVID-19). This should be corrected, and the authors should clarify that they compare MIS-C to SARS-CoV2-positive children. The severe group is actually restricted to MIS-C. I suggest to avoid to name this group Severe COVID-19 (MIS-C instead). There are two severe manifestations of COVID-19 in children: -Severe / fatal pneumonia, occurring in immunocompromised children (IFN-I pathway, in particular) and MIS-C. Here the authors only highlight the MIS-C phenotype. Response: We have now changed the text in the manuscript to represent this. 3. The use of DNA instead of RNA for the TCR sequencing is more challenging and associate a -greater number of biases. Response: Although using DNA is more challenging and can lead to greater number of biases, the protocol we employed addresses these issues. The protocol used in this study has been standardised and used routinely in clinical setting for minimal residual disease (MRD) in cancer (Bartram et al.). Furthermore, analysis of the raw TCR sequences was performed using MiXCR which removes PCR errors before assembling clonotypes. Using DNA for bulk sequencing is also more quantitative, because there is one copy/cell, so the result will look more similar to e.g. single-cell sequencing, as opposed to RNA sequencing where you have varying number of RNA copies/cell. 4. The junctional diversity of TRBV11-2 was also previously reported. In addition, it is present in both CD4/CD8 T cells and some functional studies have also tested T cell activation to various SARS-CoV2 antigens in MIS-C patients (Moreews et al. Hoste et al…). Response: We have now added these references to the manuscript. 5. Actually the polyclonal expansion of T cells is a feature of superantigen immune reaction that was highlight in previous (omitted)studies. The other patients that experience an acute infection present an antigen-specific immunity that is also reported and physiological. Response: We have added the relevant references in the manuscript. 6. The authors state that the TCR bias is responsible for the hyperinflammatory syndrome but this causal link is not supported by any data provided here. Response: We agree with the reviewer and have changed the wording in the manuscript to represent that this is a speculation. The timing to infection should be indicated. The so-called group “mild “ was possibly sampled at the acute phasis of the infection whereas the MIS-C are sampled at the time of the episode which is post-infectious (about 4 weeks after the virus encounter). This information is important. Response: There was no significant difference in sampling between the two groups. The mild group was sampled at a median of 8 days post COVID-19 diagnosis and the MIS-C group was sampled at a median of 10 days post COVID-19 diagnosis. 7. The clinical data on the MIS-C are missing (vasoplegia, blood pressure, shock, erythema…). MIS-C usually occurs in patients with no comorbidities. How MIS-C was confirmed in the patient with hepatoblastoma ? Response: Patient with hepatoblastoma was diagnosed based on having central nervous system disease, a clinical manifestation of MIS-C. We don’t have access to any further clinical data on the patients. 8. The TRBV11-2/ Vb21.3 expansion is now considered as a diagnosis marker. How was individual TRBV11-2 expansion (especially in the patient with hepatoblastoma)? Response: Patient with hepatoblastoma was diagnosed based on having central nervous system disease, a clinical manifestation of MIS-C. We don’t have access to any further clinical data on the patients. The spread of TRBV-12 expansion is shown on the box plot. The level of TRBV11-2 is significantly higher in MIS-C compared to controls; therefore, it is possible to say that it can be used as a biomarker. The spread in the MIS-C patients was quite even. 9. Is there any pediatric controls in the repertoire assessment? Response: There are paediatric control included in the repertoire analysis and referred to as ‘pre-COVID-19’ or ‘unexposed’ in the figures. 10. In total, this is a replication study of numerous previous work on MIS-C (not severe pediatric COVID) and use a more challenging technique (DNA seq) to address this question on a lowest number of patients. The add-value of this work is limited compared to previous studies and the authors may provide information on how this report replicate previous work and what is new. Response: Although the expansion and the polyclonality of TRBV11-2 have been shown before in MIS-C, we however, report the use of two metrics to define MIS-C in children infected with SARS-CoV-2: 1) CDR3-independent expansion of TRBV11-2+ T cells, 2) a lack of SARS-CoV-2 specificity in TRBV11-2+ T cells, measured by distance to Sars-Cov2-specific MIRA clones. These two metrics can serve as biomarkers for early detection of MIS-C guiding physicians to start precision immunotherapeutics that can prevent the development of severe, life-threatening complications and lasting disability in children. More specifically, the use of network embedding and the distance to MIRA analysis is the added value of our study. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0274289.r002
25 Aug 2022 Decision Letter - Sebastian D. Fugmann, Editor Distorted TCR repertoires define multisystem inflammatory syndrome in children PONE-D-22-08475R1 Dear Dr. Cooper, 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, Sebastian D. Fugmann, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0274289.r003
Formally Accepted
30 Sep 2022 Acceptance Letter - Sebastian D. Fugmann, Editor PONE-D-22-08475R1 Distorted TCR repertoires define multisystem inflammatory syndrome in children Dear Dr. Cooper: 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. Sebastian D. Fugmann Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0274289.r004

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