PONE-D-20-08786

The NKG2D ligand ULBP4 is not expressed by human monocytes

PLOS ONE

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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?

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Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

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

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: N/A

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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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: I have suggested minor revision as I feel the publication satifys the criteria for publication in PLOS ONE

The paper is well written, easily understood, concise and provides a good search of the literature. The paper highlights the need to properly test and validate antibodies even from commercial retailers to avoid incorrect conclusions. This you have successfully highlighted in Sharma and Markiewicz 2019. This highlights the need for additional testing not reliant on specificity of an antibody, in particular when results are not as expected as in the case of Sharma and Markiewicz 2019. The examination of both ULBP4 transcript levels in addition to the use of an in-house produced monoclonal antibody and the use of a positive control further re enforces the validity of your finding that monocytes do not express ULBP4, in contrast to the findings of Sharma and Markiewicz 2019. Further the investigation of 3 different databases in regards to ULBP4 transcripts in immune cells further supports your conclusion.

The minor revisions are in regards to the presented figure.

1) Figure 1A, part of the flow cytometry gating strategy is missing- in particular the initial cell gate and the live dead stain.

2) Figure 1 and 2 ideally should be edited to be lighter, this is in particular regards to Figure 2C where datapoints are difficult to visualize.

3) Figure 1B shows a change in the color of the presented histograms (blue, all except one) than what is described in the text (gray). This should be corrected.

4) Figures 1D and 2C need to state what the bars represent (means or medians, IQR?)

5) Figure 1D and 2B should state what n,d respresents (assuming not detected) in figure text.

6) Flow cytometry data should be uploaded to the flow respository or similar to be made publically avaliable.

Reviewer #2: The manuscript from Lazarova and colleagues reports the lack of expression of the NKG2D ligand ULBP4 in the major immune cell subsets within the PBMC fraction, at least in the tested conditions. They highlight the divergent results obtained with two antibody clones reported to bind ULBP4, and suggested one of them to be non-specific. In general, I consider reporting on negative results a very important issue, especially in a period of “reproducibility crisis”. Therefore, I would recommend this paper for publication, upon revision of some minor points, listed below.

- A major limitation of this study is the lack of data on the CD14- monocyte fraction. This applies to every figure provided, and is particularly relevant as the current title refers to (total) “human monocytes”. In order to support with data their general statement, I would here recommend to perform experiments on the total monocyte population. Another reason for this is that, as the authors report in Figure 3B, different monocyte subsets can express different levels of NKG2D ligands, at least for what concerns MICA RNA. Where applicable, it would be useful to know whether ULBP4 expression changes across subsets.

-A related point is on the qPCR experiments (Figure 2B), where, again, there are no stimulated CD16+ CD14- monocytes. Also, since they perform qPCR on an “enriched” cellular fraction, the authors should provide data on the purity of isolated cells. Otherwise, FACS-sorting would be appreciated.

- Figure 1: A relevant point here is the lack of any type of positive control for the DUMO1 antibody binding (even on non-immune cells/on cell lines/transfectants). If possible I think the authors should provide it. As a minor technical comment, the staining for NK cells is suboptimal, and it would be nice to see a quantification on CD56+ CD3- cells.

- The use of public RNAseq datasets is convincing and the MICA vs MICB difference in absolute counts is striking. To complement these data, I would encourage the authors to include data relative not only to steady state conditions, but also to inflammatory conditions where, theoretically, ULBP4 might be involved (e.g. cancer). There is a number of repositories human of bulk/single cell sequencing data from immune/stromal cells where this information can easily be collected. This would nicely pair up with the in vitro stimulation data presented in Figure 2.

- Minor text point:

Page 4, line 12 “which are in part not ULBP4-specific”, can it be made clearer?

Reviewer #3: In the current study by Lazarova M. et al the authors report the unspecific binding of the anti-ULBP4 mAb 709116 (R&D systems).The effort made by the author to advise the scientific community about the careful usage of such reagents is considerable. However their statements would be better supported by showing unspecific binding of the mAb 709116 in a cell line that do not express the endogenous protein and that is transfected with a plasmid encoding ULBP4 or a control vector. Finally it will be interesting to understand in the future the specific role and tissue-restricted expression of ULBP4 compared to the other members of the same family.

Comments for figure 1:

□ The authors should also include the forward and side scatter plot to show proper gating of monocytes and lymphocytes that are characterized by different size

□ In figure 1B the author should correct the color on the histogram for the monocytes stained with the 709116 ab to much the other histograms

□ In figure 1B and C the authors should include legends to help undertanding which is the isotype and which is the stained sample

□ The authors should specify the stimulation conditions for the monocytes in the text, in the figure and in the figure legend

□ Is the 709116 ab detecting ULBP4 expression also after TLR3 and TLR4 stimulation?

Comments for figure 3:

□ The authors should include expression levels of other proteins (ULBPs) of the same family from the same dataset

□ The authors should include proteomic data from the ProteinAtlas dataset to corroborate their conclusions

Statistical analysis must be provided

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Reviewer #1: No

Reviewer #2: Yes: Andrea Ponzetta

Reviewer #3: No

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The NKG2D ligand ULBP4 is not expressed by human monocytes

PONE-D-20-08786R1

Dear Dr. Steinle,

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,

Silke Appel, PhD (Dr. rer. nat.)

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: The authors have addressed all my questions satisfactory and have updated figures and text accordingly. Due to the importance of investigations into specificity of antibodies used in research and their impact on reproducibility and interpretability of research, I recommend this paper for publication.

Reviewer #2: The authors have now addressed the major issues raised in the former manuscript version, and their work has now improved significantly in terms of content and clarity of the message.

Although the technical point relative to NK cell identification strategy (thus relevant for ULBP4 quantification on NK cells shown in Fig 1B rather than quantification of NK cells as a population) was not addressed, I agree with the authors on the focus on the paper being on monocytes rather than on other immune subsets.

Nice work and congratulations to the authors

Reviewer #3: The authors have addressed the questions from the reviewers and the quality of the paper is significantly improved.

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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: No

Reviewer #2: No

Reviewer #3: No