https://orcid.org/0000-0002-6973-9474
Peer Review History
| Original SubmissionJuly 22, 2021 |
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Dear Dr Pampusch, Thank you very much for submitting your manuscript "CAR/CXCR5-T cell immunotherapy is safe and potentially efficacious in promoting sustained remission of SIV infection" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation. When you are ready to resubmit, please upload the following: [1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. [2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments. Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts. Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments. Sincerely, Daniel C. Douek Associate Editor PLOS Pathogens Thomas Hope Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens *********************** Reviewer's Responses to Questions Part I - Summary Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship. Reviewer #1: Pampusch et al present a manuscript entitled “CAR/CXCR5-T cell immunotherapy is safe and potentially efficacious in promoting sustained remission of SIV infection” for publication in PLOS Pathogens. The authors hypothesize that increased trafficking of virus-specific CD8 T cells to B cell follicles, i.e. via modification with a CD4-MBL CAR and forced expression of CXCR5, will lead to ART-free virus control in their NHP model of suppressed HIV-1 infection. Tissue analyses in a pilot animal necropsied 2 days post-CAR infusion show predominant trafficking of the infused cells to B cell follicles; 4% of SIV+ cells in these sites were in direct contact with CAR+ cells by duplex RNAscope. A cohort of animals that received SIV-negative T cell products (T2) showed more promising impacts on post-ATI plasma viral load than animals for which infused cells were SIV+ in origin (T1). These T2 animals’ infused cells expressed Ki67 and showed dimmer CTV within B cell follicles, consistent with a proliferative phenotype at a single time point. In line with the time frame in which CAR+ cells were detected in peripheral blood (the first 2 weeks post-infusion), the vast majority of assayed follicles in T2 animals contained CAR+ signal, but CAR+ levels in blood and tissues dropped precipitously thereafter. Nevertheless, the infused cells led to particularly promising reductions in SIV viremia in the T2 cohort vs. controls. This is a well-designed and important study that applies cutting-edge imaging assays to characterize a promising approach to HIV cure. As with the results in most preclinical HIV cure experiments, the key measure of success (ART-free virus control) is not clear-cut, although the infused CAR T cells are having an appreciable impact on viral replication in the absence of ART. The data presented here are valuable, and open numerous doors and pose important key questions for the field moving forward. Reviewer #2: The manuscript by Pampush et al. details the use of a SIV-specific chimeric antigen receptor that is coexpressed with CXCR5 to attempt to target persistently infected cells in lymphoid follicles in SIV infected rhesus macaques. They attempt to address their central hypothesis that the “infusion of T cells engineered to express a potent SIV-specific CAR along with a B cell homing molecule, CXCR5, will control SIV-infection by reducing viral replication in follicles”. Targeting persistently infected reservoirs, and in particular, lymphoid follicles, is a key issue with many different therapeutic approaches to HIV/SIV and this study addresses a potentially unique strategy to permit virus specific CAR expressing cells to attack infected cells at this site. The rationale, strategy, and model system in this study can potentially provide unique insight into a CAR based approach to specifically target a persistently infected reservoir. This study does demonstrate, at a reasonable level for the short term, that infusion of CAR/CXCR5 expressing cells is relatively safe, as the animals did not appear to experience treatment-related toxicities. However, the data, as it is presented, falls short of justifying their conclusions and addressing their hypothesis (see below). In sum, this is a potentially interesting study attempting to target SIV/HIV persistence in lymphoid follicles that is limited by the lack of inclusion of controls demonstrating that SIV-specific CAR/CXCR5 modified cells can target this site better than unmodified cells or cells expressing the CAR alone. ********** Part II – Major Issues: Key Experiments Required for Acceptance Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions. Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject". Reviewer #1: -In Figures 1A, 4A-B, and elsewhere, the difference between CTV and CAR/CXCR5 RNAscope labeling should be clarified. CTV will label the entire infused cell product, not just the 55.0-79.4% of cells that have been modified with the CAR/CXCR5 vector. It would be more accurate to refer to data using CTV as “the infused T cell product,” rather than CAR/CXCR5-T cells, and CTV and CAR/CXCR5-T RNAscope labeling should preferably be shown in different colors, to avoid confusion. -What was the frequency of CAR+ (and CTV+) cells in the blood in the pilot animal in Figure 1? -The authors state from their previous work that “the average ratio of in vivo effector (virus-specific CD8+ T cells) to target (virus-infected cells) cells is over 40-fold lower in follicular (F) compartments compared to extrafollicular (EF) compartments of secondary lymphoid tissues” (lines 68-70). Can this ratio be quantified in the current manuscript? How does it compare to the percentage of follicles with CAR+ T cells (Figure 5D) and the percent of SIV vRNA+ cells in direct contact with CAR/CXCR5-T cells (Figure 1B)? -Are data from all 9 animals shown in each panel of Figure S1? Panel F appears to only show cohort T2, whereas panels A-C appear to only show 1-2 animals from each of the 3 cohorts. Where average values form multiple animals are shown, this should be made clearer, i.e. with a different symbol. Showing data from the control cohort in Figure S1 E-F (and/or in Figure 4E-F) would be helpful to establish the background for each assay. -Is there direct evidence supporting “initial immediate spike in viral loads due to the presence of virus in the infused SIV-infected transduced cells” (lines 164-165)? Are data available that quantify the amount of SIV that was present in the infused cell products from cohort T1? -Plasma viral loads at a single time point (27-30 days post-transplant, Figure 3D) are consistent with SIV RNA levels in lymph nodes at the same time point (28 days post-transplant, Figure 6), but do not assess virological trends over time. Are other data available to corroborate this finding across a broader time frame following ART interruption, e.g. plasma viral load area under the curve, PBMC-associated viral RNA, viral DNA, or SIV Gag+ cells? -CM9 tetramer+ assays on their own seem insufficient to conclude that “the differences in viral loads between groups was not likely driven by differences in the endogenous response” (lines 181-182, Figure S3). Were other virus-specific immune response parameters measured? -The authors highlight that their study is “the first to visually confirm the in vivo expansion of autologous SIV-specific CAR T cells” (lines 301-302), presumably referring to their imaging data. Figure S4 convincingly shows that flow and imaging readouts are tightly correlated, lending credence to their imaging approach. However, the Ki67 data in Figure 4C-D is only a single time point, which makes it difficult to draw conclusions about a kinetic property like cell expansion. Figure 5C-D does show kinetic measurements of the authors’ imaging data, but calling this “expansion” is somewhat subjective. The authors’ key assertion that they are seeing expansion without addition of exogenous antigen (line 303) necessitates a clearer rationale and/or supporting data. Reviewer #2: There are a few critical issues that should be addressed in this manuscript: 1. A key issue with every aspect of the study in addressing their hypothesis is the lack of proper controls, ideally with animals that received non-transduced or control vector modified cells. The control animals, from all appearances, were not treated with an infused cell product. Due to this, and the reported fact that approximately 40% of the cells that were injected into the treated animals were not expressing, or were not transduced, with the CAR/CXCR5 vector, it is difficult to determine if greater levels of CAR expressing cells were infiltrating the follicles due to CXCR5 co-expression. Since the entire cell product was labeled with the fluorescent Cell Trace Violet (CTV), it is not clear whether the CAR/CXCXR5 vector modified cells were behaving in any way different than unmodified cells or infused cells in a control animal. It also is not clear, particularly in animals that received CAR/CXCR5 modification after SIV infection, whether the cells found in the follicles are endogenous SIV-specific T cells from the infused CTV-labeled fraction or are other cells that migrated there. There is some RNAscope in situ hybridization presented that shows localization of CAR/CXCR5 modified cells in and around the follicle, but it is not clear that the presence of vector expressing CXCR5 increases this. In addition, the levels of CAR/CXCR5 modified cells versus unmodified cell populations at these anatomical sites are not clear. The inclusion of a control, optimally animals receiving cells modified with a control vector, or another manner of analyzing the present data would strengthen their conclusions that the CXCR5 component on the cells was facilitating greater follicular localization. 2. The inclusion of a control along with or in the “initial” animal data shown in Figure 1 would strengthen these results and make them more meaningful. It is not clear if unmodified cells would anatomically distribute this way as well. 3. The viral load data described in Figure 3 appear to indicate possible effects of CAR/CXCR5 modified infused cells in 4 of 6 animals. However, there is natural decrease in the untreated control animals to low levels at the final time points as well. Since this control did not receive infused cells, the significance of this is not clear (other groups have demonstrated that untreated cell infusions can affect viral loads). In addition, without matched controls for the extended time period in the T2 animals shown in Figure 3c, the significance of the presence of low viral loads at these later time points is not clear. In the absence of detection of CAR/CXCR5 expressing cells, is this a natural phenomenon? ********** Part III – Minor Issues: Editorial and Data Presentation Modifications Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. Reviewer #1: -Were all animals A*01+? If not, could MHC haplotype contribute to virus control in any of the animals? -Despite statements in lines 119-120, the animals in cohorts C, T1, and T2 do not appear to be particularly well-matched in terms of age, sex, or weight. -It would be helpful to expand Figure 2 to include schematics of what tissues were collected from each animal at which time points. - Figure 3 appears to show plasma viral loads, which makes the reference in the Figure 3 legend to a beta actin housekeeping gene and use of cell pellets confusing. -What does the staining pattern shown in Figure S2 look like in animal(s) from the control cohort, i.e. is this flow cytometry assay specific for CAR+ cells? -In Figures 4-5, are data available from cohort T1? -The statement in the discussion, “Interestingly, we detected no SIV vRNA in CAR/CXCR5 cells in the examined sections.” (lines 325-326) should be tempered, indicating that only imaging-based assays were applied. Alternatively, flow- and PCR-based assays to confirm that CAR+ cells are not infected in vivo would significantly strengthen this assertion. Reviewer #2: 1. The methodology involved in the overall approach is not clear. A better description of key elements of this, such as greater details of the CAR/CXCR5 vector, cell processing methodologies/cell activation techniques, and the inclusion or absence of conditioning (such as lymphodepletion) regimens would strengthen this manuscript. Some reference to these is provided, but a clearer description and inclusion of these in the text would strengthen this manuscript. ********** 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 Figure 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. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Data Requirements: Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5. Reproducibility: To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols |
| Revision 1 |
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Dear Dr Pampusch, Thank you very much for submitting your manuscript "CAR/CXCR5-T cell immunotherapy is safe and potentially efficacious in promoting sustained remission of SIV infection" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the minor recommendations of reviewer #1. Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. When you are ready to resubmit, please upload the following: [1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out [2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments. Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments. Sincerely, Daniel C. Douek Associate Editor PLOS Pathogens Thomas Hope Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens *********************** Reviewer Comments (if any, and for reference): Reviewer's Responses to Questions Part I - Summary Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship. Reviewer #1: Pampusch et al present a revised manuscript containing important data updates. Included are new images of CAR staining in lung (Fig. 1), updated study schematics (Fig. 3), updated plasma viral load (Fig. 4) and CAR expression data (Fig. 5), imaging of CAR T cells and virus in ileum tissue (new Fig. S1), clarification of detection limits in cytokine data (Fig. S2), increased information for flow cytometry gating (Fig. S3), SIV measurements in CAR infusion products from cohort T1 (new Fig. S4), and additional quantification of imaging of CAR vs. viral RNA (new Table 3). Updated language in reference to total (CTV-labeled) vs. CAR+ T cells and clarification of important details regarding MHC-I typing, lack of comparability in animals’ demographic info, etc are appreciated. The revised interpretation of their results in the discussion section (lines 411-422) seems reasonable and is tempered. Reviewer #2: The authors have presented a significantly revised manuscript that sufficiently addresses previous concerns. While the lack of an optimal control is not presented, they do provide additional data that strengthens their conclusions. ********** Part II – Major Issues: Key Experiments Required for Acceptance Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions. Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject". Reviewer #1: N/A Reviewer #2: (No Response) ********** Part III – Minor Issues: Editorial and Data Presentation Modifications Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. Reviewer #1: Additional imaging-based follicular E:T ratio data is a key aspect of the revised manuscript, but no methodology is presented. Important details to include in the manuscript include i) how these values were calculated, ii) the maximum distance between a given CAR RNA+ effector and viral RNA+ target, iii) whether this distance would be expected to facilitate direct target killing, and iv) possible limitations to calculating E:T ratios using RNA signal, e.g. due to CAR RNA+ and/or viral RNA+ cells that aren’t expressing CAR/virus proteins. Reviewer #2: (No Response) ********** 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 Figure 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. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Data Requirements: Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5. Reproducibility: To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols References: 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. |
| Revision 2 |
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Dear Dr Pampusch, We are pleased to inform you that your manuscript 'CAR/CXCR5-T cell immunotherapy is safe and potentially efficacious in promoting sustained remission of SIV infection' has been provisionally accepted for publication in PLOS Pathogens. Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests. Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated. IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS. Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards, Daniel C. Douek Associate Editor PLOS Pathogens Thomas Hope Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens *********************************************************** Reviewer Comments (if any, and for reference): |
| Formally Accepted |
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Dear Dr Pampusch, We are delighted to inform you that your manuscript, "CAR/CXCR5-T cell immunotherapy is safe and potentially efficacious in promoting sustained remission of SIV infection," has been formally accepted for publication in PLOS Pathogens. We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication. The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly. Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers. Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards, Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens |
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