PONE-D-24-38309Myosin 1f and Proline-rich 13 are transcriptionally upregulated yet functionally redundant in T helper cells during experimental malaria.PLOS ONE

Dear Dr. Haque,

Your manuscript was reviewed by two experts in the field and both of them were enthusiastic about the findings reported in your manuscript. There were, however, some concerns that were raised by one of the reviewers that need to be addressed prior to making a final decision on your manuscript. As such, my recommendation is minor revision. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Dec 07 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Dr. Joseph J Mattapallil

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: No

Reviewer #2: 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

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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: Positive feedback. I applaud the authors for moving forward with a publication of negative results. It is important to show that some suspected genes are not important in T cell responses (to malaria). The text is well written, figures are of a good quality (but see comments), and results seems solid (given performed experiments, see also below). I also appreciated authors showing the data from individual mice; this is important and should be the standard in the field.

Comments.

1. The rationalization of choosing these two genes to study is still poor. Ok, these genes are unregulated in scRNAseq data. So what? I predict hundreds of genes are unregulated - why these two? Are they unregulated to the highest level (mRNA)? Are they upregulated at the protein level? I found the data presented in Fig 1A being unclear. NOTE: because individual panels are not labeled, it is hard to refer to individual panels. But, in Fig 1A trajectories - how uncertain are these? In Fig 1A violin plots -> what are these (no labels given)? No stats are given either.

2. Measurements are mostly done at day 7. Why? Could it be too early to see a difference? Is there a positive control authors could present that would justify measurements at day 7 (or 28) as appropriate?

3. I could not understand the results shown in Fig 3D - the way I see these gels is that expression in WT vs. KO is the same, i.e., KO did not work. I think someone experienced in gels should look into these (e.g., Dr Elisabeth Bik @MicrobiomDigest on Twitter)

4. I am not sure about some statistical analyses showing no difference between WT and KO cells. For example, in Fig 4B and 4D (cannot point to panels!), there is a tendency of lower total T cell numbers in PbT-II KO cells. I wonder if experiments in which WT and KO cells are co-transferred to the mice and then look at the response magnitude. This way, the data will be paired and may allow for higher statistical power to detect difference.

Minor comments.

Improvements in figures must be made. Every panel in a figure should be "named", e.g., you can use Ai, Aii, Aiii, etc to label sub-panels in a figure with multiple elements (A/B/C etc).

I highly recommend generating pdf of the text with ALL figures and figure captions being on the same page, e.g., as done in published papers or grant applications. This makes reviewing of the paper easier. Also, number the lines so reference to specific lines in the text can be made. And obvious -> have page numbers!

The text in 2nd paragraph in Results is unclear. It reads as you put T cells into WT Cre-expressing mice but it does not seem to be correct as you generated KO mice first. Or perhaps I did not understand the text well -> in any case, please review and update if needed.

If KO T cells generate fewer Tfh cells, does this result in lower Ab (IgG?) levels to the parasites? Perhaps this needs to be shown.

The quality of the KO is stated in Methods to be shown in Fig 2B (cannot point more precisely because page and line numbers are missing). I cannot see this. Please make sure you refer to the right figures/panels.

Purity of CD4 T cells was exceeding approximately 80%. What does this mean? 81%? Or 99%. Please put specific numbers/range, e.g., 80-85%.

Statistical comparisons are not properly presented. In particular, it is not recommended anymore to use stars (**) but putting exact p values, the same for "ns" -> put exact value. Also, if possible, indicating fold change is highly recommended as showing effect size is as important as showing p values.

Given variability in some data (indicated by measurements in individual mice) - e.g., Fig 5A, how much does a gene deletion must impact Tfh (or something else) differentiation, so differences become statistically significant? Seems that with such variability these experiments may be simply underpowered to detect a difference. Experiments in which measurements are "paired" may be thus more powerful (see a suggestion above). This may need to be discussed.

Reviewer #2: This study carefully evaluates the potential role of Maf1, Myo1f and Prr13 on CD4 T cell maturation following Plasmodium chabaudi chabaudi AS (PcAS) infections in mice. All three genes had previously been shown to be upregulated during T cell differentiation in response to PcAS. They assessed both Plasmodium specific-TCR transgenic and polyclonal T cells and found the anticipated decrease in Tfh cell production following Maf1 deletion in Transgenic CD4+ cells, but neither Myo1f nor Prr13 deletion had a measurable effect. Myo1f or Prr13 have not been tested before for a role in T cell activity so this well-designed study advances the understanding of genes that although upregulated are not critical for CD4 T cell differentiation in response to malaria.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-38309R1Myosin 1f and Proline-rich 13 are transcriptionally upregulated yet functionally redundant in CD4+ T cells during blood-stage Plasmodium infection.PLOS ONE

Dear Dr. Haque,

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.

Please submit your revised manuscript by Mar 02 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Joseph J Mattapallil

Academic Editor

PLOS ONE

Journal Requirements:

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.

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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: (No Response)

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 #1: Partly

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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 #1: No

Reviewer #2: 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

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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: Comments.

1. The authors provided some justification of why these 2 genes (Myo1f and Prr13) were perhaps selected for further analysis but their abstract and most of text still did not explicitly say that prior knowledge was used. In particular, abstract ignores this completely. Please revised and put explicit statements that selection of these 2 genes was also driven by prior knowledge of importance of these genes in T cell responses and not just by scRNAseq data (which give hundreds of unregulated genes).

2. I understand your points of choosing day 7 to measure responses but I think you do not state that clearly in the main text. Please do.

3. The study could be underpowered - as authors agreed, so this should be listed in the Discussion, along with the point that you could not do experiments when co-transferring WT and KO cells due to cogenic marker identity.

4. I appreciate showing original gels but I still could not understand them. It could be my limitation, so editor may need to show these to the right expert to ensure they are accurate.

Minor comments

You did not format the figures/captions properly as we do in grants (perhaps you don't apply for funding). You MUST show the figure and caption together! (And preferably on the page near where that figure is first cited). I will not review another paper that is not formatted to help easier review process.

If Figure 1 has been published before (per disclaimer listed), why do we need it here? The authors anyway state that they focused on the genes found by mining previously published dataset (along with some prior knowledge), so do we need to see Figure 1? NOTE: not all panels in Figure 1 are labeled, e.g., Violin plots in A are not. And, you may want to mention that only about 50% of cells (based on shape of violin plots) express Myo1f (the middle panel in Figure 1A) -> is that important that 50% of cells do not upregulate this gene?

Please remove "ns" from all panels and put actual p values there. You did not interpret my comment correctly - do not use shortcuts but put actual p values, i.e., p=0.89 if difference is statistically non-significant.

The experiments with co-transfer of WT and KO cells could still perhaps be done (in the future) by using some type of cell label, e.g., CTV. The label does get diluted but CD4s are not expected to divide that much, so some level of labeling may be retained by day 7. Perhaps it is worth mentioning as future directions in the paper.

Line 113: do you mean "our data" or "our analysis"? Data had been published.

Line 139: "infected with PcAS infection" sounds weird. Buttery butter.

Line 145: you must include justification of why day 7 is a good day to measure this response.

Line 311: include justification of why these genes are good ones by citing relevant other studies.

340: "regulated" -> unregulated (?)

Reviewer #2: The rationale and figures have been clarified and the authors have responded to all the criticisms within the limits of the genetics of the experimental design.

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Reviewer #1: No

Reviewer #2: No

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Myosin 1f and Proline-rich 13 are transcriptionally upregulated yet functionally redundant in CD4+ T cells during blood-stage Plasmodium infection.

PONE-D-24-38309R2

Dear Dr. Haque,

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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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,

Joseph J Mattapallil

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

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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

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

Reviewer #1: Yes

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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 #1: No

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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

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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: I don't see that you fully addressed my comments but because they are becoming a mundane point, I give up. Why you chose those two genes remains a puzzle to me - but again, perhaps it could remain a puzzle.

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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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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

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