PONE-D-21-02602

Semaphorin 3E Deficiency Dysregulates Dendritic Cell Functions: In Vitro and In Vivo Evidence

PLOS ONE

Dear Dr. Gounni,

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Amarjit Mishra, PhD

Academic Editor

PLOS ONE

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: 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: Comments for the Author:

In the present manuscript authors intends to elucidate the non-redundant role of Semaphorin 3E (Sema3E) in Dendritic cell migration and antigen uptake during allergen sensitization in mice. They have shown increased migratory phenotype of Sema3E deficient BMDCs with increased CCR7 expression compared to Sema3E sufficient BMDCs after culture with ligand CCL21. Further, author have shown increased Rac1 GTPase activity and F-actin expression in absence of Sema3E. Further, author have indicated increased accumulation of lung CD11b+ cDCs in Sema3E knock out mice and enhanced expression of PD-L2 and IRF-4 compared to wild type controls.

Author have tried to elucidate functional importance Sema3E in the inflammation, specifically during sensitization phase which is very interesting and relevant to the field. However, there are few concerns regarding experiments, figures and data which need to be answered before publication of this manuscript. My specific comments are:

Comments:

1. Author have used in vitro BMDCs model to show the migratory defect in Sema3E deficient BMDCs compared to Sema3E sufficient one. The data showing increased CCR7 expression in Sema3E deficient BMDCs in presence of CCL21 looks promising. However, flow data presented is not convincing as basal CD11c expression in Sema3E deficient BMDCs seems high while after adding CCL21 it comes to normal. Author should also include the data with alternative ligand CCL19 which could add to conclusion that CCL21 and CCL19 both lead to the CCR7 pathway activation controlling the migration of DCs.

2. After sensitization with HDM-APC it is clearly shown the increased uptake of allergen by lung CD11b+cDCs of Sema3E deficient mice. It would better idea to add the HDM-APC+ CD11b+cDCs data from mediastinal lymph node with same experiment. Also, it is not mentioned how many mice were used to perform this experiment.

3. Figure 3, the representative flow plot for gating strategy do not add to results, it could be moved to supplemental data. What is FITC in SSC vs FITC? Is that representing SiglecF?

4. Increased expression level of PD-L2 and IRF-4 in lung cDCs seems very promising. What are the numbers representing? Is the data from a whole lung?

5. Apart from CCR7, what was the expression level of other activation markers in the DCs during the sensitization phase? Did author check for activation status of maturation markers in the DCs from Sema3E deficient mice?

6. Figure1B, flow plots are not uniform that needs to be replaced.

7. Figure2A, HDM amount (100 ug) contradicts the materials methods (page7, line 126, 1 ug).

8. Page7, line 116, clone 418 should be N418.

9. Page2: Line 29, replace ‘time’ with ‘type’.

10. Page5: Line 80, replace ‘BMDC’ with ‘BMDCs’.

11. Page14: Line 273, OF should be corrected.

Reviewer #2: In this study, Gounni et al, have explored the role of semaphoring 3E in dendritic cell differentiation and migration along with, the contributory role of CCR7 expression, elevated rac1 GTPase activity, and F-actin polymerization. The authors have successfully demonstrated that, Sema3E deficiency leads to an enhanced expression of PD-L2 and IRF-4, which results in enhanced antigen uptake by differentiated dendritic cells.

This research has merit as it will help in development of novel immunotherapeutic strategies for the treatment of allergen based diseases in which immune regulation is impaired.

Authors, have presented the data clearly and have conducted all the necessary assays and statistical analysis.

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Reviewer #1: Yes: Anil Kumar Jaiswal

Reviewer #2: No

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Semaphorin 3E Deficiency Dysregulates Dendritic Cell Functions: In Vitro and In Vivo Evidence

PONE-D-21-02602R1

Dear Dr. Gounni,

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,

Amarjit Mishra, PhD

Academic Editor

PLOS ONE

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

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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: Partly

**********

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

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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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

**********

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: Comments for the Author:

In the present manuscript Movassagh et. al intends to show role of Semaphorin 3E (Sema3E) in Dendritic cell migration and antigen uptake during allergen sensitization in mice. During, first review author has address most of the issue. However, few technical issues remain the same.

Comments:

1. Figure1B: This figure is not impressive, and author failed to address the question. The flow contour plot shows that addition of CCL21 to Sema3e-/- BMDC leads to increased number of CD11c-low expressing cells while it is reverse with WT BMDCs.

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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: Anil Kumar Jaiswal