PONE-D-19-24723

Contribution of ROS and metabolic status to  neonatal and adult CD8+T cell activation

PLOS ONE

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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?

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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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: While interesting major elements are missing from the paper and need to be added.

1. Important details are missing in the methods, particularly the number of cord blood and adult samples obtained. Were these from particular sexes? What was the time from the obtaining of the cord blood samples to their processing? Was this variable and did this affect the result. What were the age and sex of the adult donors? Was there a similar interval between taking of blood from adult donors and its processing? Was the cord blood from natural births or caesarians. Had the mothers received any drugs during labour that might have affected the T cell responses. Was any attempt made to obtain parallel blood from the mothers to compare to the cord blood as this would exclude any drug or hormonal confounders that might be affecting the cord blood T cells.

2. Fig 2 seems to suggest there were only 3 cord blood and 3 adult samples obtained? How do the authors know these responses are representative? As cord blood may not be representative of blood of young children due to prevailing maternal and birth stress factors, was any attempt made to obtain blood from young babies to show that these exhibited the same pattern as the cord blood samples?

3. In view of the above the authors need to add a paragraph at the end of the study describing shortcomings in the study design and cautions in interpretation of the data.

Reviewer #2: The manuscript by Sanchez-Villanueva et al sought to determine the correlation of reactive oxygen species (cytosol and mitochondria) against the known lower T cell activation profile of neonates. Specifically, they obtained cord blood or adult blood and stimulated with a polyclonal activator and then determined mROS and cROS in CD8 T cells. They found neonates consistently had high levels of mROS while adults had a mix of high and low mROS. They then built a logic model to depict the effects of activation on neonatal CD8 T cells.

The overall results of the study are quite interesting with respect to the mROS differences. There are a few issues that should be addressed to improve the manuscript:

1. A through reading of the manuscript to improve the wording would help the reader. There are few word choices and sentence mechanics all throughout the manuscript that make it a bit difficult to read.

2. In figure 2, the authors found a large difference in HV-1 expression between infants and adults. However, it appears that GADPH was used as the normalization control which is no longer appropriate. GADPH is so widely impacted by immunological processes (i.e. TNFa or IFNg mRNA transcripts bind to it) that it is not an appropriate housekeeping control. Their data may still be relevant, but they should ensure the rigor of their findings with another calibrator, especially when comparing neonates to adults where transcripts such as IFNg are widely different during activation

3. Comparisons of neonates to adults naïve T cells is not an apples to apples comparison. Neonates are loaded with recent thymic emigrants in blood that express lower T cell function that may enter the competitive pool for activation. Since the authors did not gate on these or remove them, they should at least bring up the topic of RTEs in the discussion.

4. The authors should at least discuss the high mROS they observed in the context of NFAT and IL-2. Both of these are lower in neonates, affect the activation profile even at 6 hours, and are inhibited by high mROS. Contrary, IL-4 which is supposedly higher in neonates is also inhibited by high mROS and thus this should all be in the discussion section.

5. Calcium influx increases mROS in T cells as determined in a number of prior studies. The authors cite reference 20 to indicate that influx may be high in infants. There are a number of studies that indicate that the influx may also be lower in neonates and that the increase is only in CD4 T cells (and non-Th1 cells at that). They authors should at least discuss this.

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Reviewer #1: No

Reviewer #2: No

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Contribution of ROS and metabolic status to neonatal and adult CD8+ T cell activation

PONE-D-19-24723R1

Dear Dr. Santana,

We are pleased to inform you that the revised version of your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Yours Sincerely,

Lucienne Chatenoud

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 #2: 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 #2: (No Response)

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

Reviewer #2: (No Response)

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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 #2: (No Response)

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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 #2: (No Response)

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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 #2: (No Response)

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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.

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Reviewer #2: No