PONE-D-19-23862

Bypassing ubiquitination enables LAT recycling to the cell surface and enhanced signaling in T cells.

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?

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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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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: The underlying molecular interactions at the initiation of the T cell response has been widely studied. One of the molecules that is implicated in the T cell response is Linker of Activation of T cells (LAT). The mechanism that underlies the turnover of LAT is the focus of the studies in this manuscript. LAT levels at the plasma membrane, its endocytosis after phosphorylation and its recycling back to the cell surface are all points in LAT turnover that can affect the T cell response. There are 2 pools of LAT; at the cell surface and in vesicular compartments. The mobilization of LAT may be regulated by its ubiquitination. In this manuscript, the authors assess the role of ubiquitination on LAT turnover.

To assess ubiquitination, a chimeric molecule was constructed that contained the extracellular domain of CD4 and the cytoplasmic domain of LAT with or without mutated lysines. Following CD4 ligation with anti CD4 antibody or cell activation with anti-CD3, they monitored the redistribution of the chimeric CD4/LAT molecule within the cell. The results of the experiments are clearly presented.

Within the context of these experiments, as an additional evaluation of the ubiquitination of LAT, experiments in the presence of cell permeable ubiquitin inhibitors that target the ubiquitin proteasome system at an early point (an E1 inhibitor) or later in the process (proteasome inhibitor) would be informative. The expectation is that the CD4WTLAT and 2KRLAT will be differentially affected. Those differences should complement the studies reported here.

Reviewer #2: This manuscript needs some revision as there are mistakes /inaccurate statements. Below is what I found. Authors need to proof-read it carefully.

Line 57: Why capitalized Cell

Line 58: Phosphorylation of the TCR on cytosolic tyrosine residues ...... Really ?

Line 61: Wrong reference. The first author seems to love to cite her own papers.

Line 66: leading to activation of the downstream kinases and transcription factors......what kinases down of LAT? why only kinases and transcription factors? Please rewrite this sentence.

Line 67: effector functions as T cell proliferation and cytokine expression. Is T cell proliferation an effector function?

Line 68: wrong reference

Line 81: a defect in protein turnover rate. How a turnover rate could be defective? Should be either decreased or increased!

Line 84: enhance T-cell potency. why we are talking potency here? potent in doing what?

Line 92: move some cited references that reported essential role of surface LAT.

Line 107: LAT endocytosis in these cells. What are these cells? Did you talk about any cells in this paragraph?

Line 110: Molecular switch? a fancy word. perhaps simply targets LAT for degradation.

Line 272: significantly effecting. should be affecting.

Line 276-277: make it clearer for " dose dependent and TCR dose"

Line 277: ubiquitin is a signal for the loss of cell surface LAT. Weird statement

Line 300: labeling of the construct at the cell surface. Construct?

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Reviewer #1: No

Reviewer #2: No

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Bypassing ubiquitination enables LAT recycling to the cell surface and enhanced signaling in T cells.

PONE-D-19-23862R1

Dear Dr. Balagopalan,

We are pleased to inform you that 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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With kind regards,

Mariola J Edelmann, 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

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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

**********

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

Reviewer #1: Yes

Reviewer #2: 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

**********

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

**********

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 Response)

Reviewer #2: This study demonstrated that LAT ubiquitination plays an important role in LAT half-life and its function in TCR-mediated signaling. My concerns were all addressed.

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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 #1: No

Reviewer #2: No