PONE-D-22-09163

APRIL/BLyS Deficient Rats Prevent Donor Specific Antibody (DSA) Production and Cell Proliferation in Rodent Kidney Transplant Model

PLOS ONE

Dear Dr. Bath,

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PLOS ONE

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“RR- KL2 career development award (4KL2TR000428-10), https://ictr.wisc.edu/career-development-awards-2/; American Society of Transplant Surgeons Faculty Development Grant (MSN183242), https://asts.org/asts-foundation/grants-and-eligibility; American College of Surgeons Franklin Martin, MD, FACS Faculty Research Fellowship (MSN192116), https://www.facs.org/member-services/scholarships/research/acsfaculty.

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Reviewers' comments:

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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

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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 well performed study, with clear results and discussion. I would have a comment for the authors in the introduction - they state that: "despite advances in immunosuppression the ten-year survival rate remains stagnant due to lack of effective methods to target alloantibody". I do not completely agree with this statement because there is clear evidence from the CTS and other studies that long-term graft survival has significantly improved in comparison with graft survival in the 90 from the last century.

Reviewer #2: This study explores the effect of April and BlyS depletion in a rodent kidney transplant model. Using April -/- and BlyS-/- engineered rats, the authors described significant changes in antibody production, response to alloantigens, architecture of splenic germinal center and frequency of B cell and T cell subsets in various tissues, compared to WT animals. In sensitized transplanted April -/-/BlyS-/- rats, the authors found reduced alloantibody production, reduced early-stage and mature B lymphocytes, but not long-lived B lymphocytes, and less severe ACR and AMR, although the differences were not statistically significant. These results provide an insight into the effect of April and BlyS cytokines in a model of kidney transplantation and lay the ground for further studies exploring the value of these cytokines as therapeutic targets in transplantation.

I believe the following issues should be clarified:

1. Since depletion of early-stage and mature B lymphocytes has been observed in April -/-/BlyS-/- rats, is there any concern that B regulatory cell subsets may be among the depleted subsets? This has been a major concern in studies using B cell depleting agents, including studies using April/BlyS blockade (Bath et al, PLOS One, 2019).

2. The authors investigated the effect of April and BlyS depletion on alloantibody production by performing crossmatches with allogeneic T and B lymphocytes. Sera from non-sensitized and sensitized Lewis rats were used in these crossmatches. While testing sera from sensitized rats is meaningful, I am not clear why non-sensitized animals were also included. What level of alloantibodies was expected in these animals and what is the relevance of the crossmatch results?

3. Please provide representative flow cytometric data in the form of dot plots. That will help the reader to visualize and better understand the gating strategies, cell marker expression and the changes in cell subset frequencies.

4. The lack of data on transplant outcomes in animals who have not been previously sensitized should be listed as a limitation of the study.

Minor points:

1. Results section “April-/- and BlyS-/- lymphocyte proliferation significantly decreased when challenged with alloantigen” and Legend for Fig 4: For clarity, please specify that the responding lymphocytes were from Lewis rats.

2. The data presented in Figures 2 and 6 could be consolidated and presented in one figure.

3. The concluding remark of the Result section “Naïve and MZ B lymphocytes were significantly decreased in sensitized BlyS-/-“ is not clear. Please revise.

4. In the Result section “Splenic transitional zone (TZ) B lymphocytes significantly depleted by BlyS-/-“, the authors show that, in BlyS-/- animals, splenic TZ B cells were reduced while in other tissues these cells had similar frequency as observed in WT animals. Given these findings, it is not clear why the authors concluded that the absence of BlyS induced an accumulation of TZ B lymphocytes. Please clarify.

Reviewer #3: The article is well written and clearly demonstrates the findings based on the hypothesis and aims.

A few questions/comments

1. Please state what the abbreviation BCMA means.

2. How do you explain the difference in findings between the different lymphoid organs? eg lines 263-264?

3. The authors discuss their current findings and the use of belimumab in SLE. What study design would you employ to bridge the gap between the rodent model and clinical application? (Discuss next steps).

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Reviewer #1: Yes: Assoc. prof. Antonij Slavcev, PhD

Reviewer #2: No

Reviewer #3: No

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APRIL/BLyS Deficient Rats Prevent Donor Specific Antibody (DSA) Production and Cell Proliferation in Rodent Kidney Transplant Model

PONE-D-22-09163R1

Dear Dr. Bath,

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.

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Kind regards,

Hodaka Fujii, M.D., Ph.D.

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

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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

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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: Yes

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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

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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 comments, the authors have adequately corrected the manuscript, I would suggest acceptance. I had only one remark concerning the long-term survival rate of organ transplants which has significantly improved in the last decades.

Reviewer #2: My comments were appropriately addressed by the authors. I believe that the revised version of the manuscript is acceptable for publication.

Reviewer #3: The authors have addressed the comments and questions from the reviewers.

The article in its current form is suitable for publication.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Antonij Slavcev, Assoc. prof., MD, PhD

Reviewer #2: Yes: Adriana Colovai

Reviewer #3: No

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