Dear Dr. Orange,

Thank you for submitting your manuscript entitled "Degranulation-enhanced presynaptic membrane packing protects NK cells from perforin-mediated autolysis" for consideration as a Research Article by PLOS Biology.

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Paula

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Paula Jauregui, PhD,

Associate Editor

PLOS Biology

Dear Dr. Orange,

Thank you very much for submitting your manuscript "Degranulation-enhanced presynaptic membrane packing protects NK cells from perforin-mediated autolysis" for consideration as a Initial Research Submission at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

In light of the reviews (below), we will not be able to accept the current version of the manuscript, but we would welcome re-submission of a much-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.

In particular, reviewer #1 wants to see the results from 2 cell lines in separate graphs and with a minimum of 10 data points/cell line, has several questions about figure 2, target cell lysis rate, NK cell death at 16 h time point, statistics, and presentation of 5 donors separately. Reviewer #1 also asks about the lipid composition of target cells, whether NK cells form conjugates with target cells in the presence of EDTA, and wants you to tone down the conclusions about tumor cell scape, saying that you should use a second resistant tumor cell line to validate your results. Reviewer #1 and #3 have concerns about the NK cell resistance to perforin in the absence of degranulation, and about the specificity of the 7KC treatment, suggesting to repeat the experiment with target cells. Reviewer #2 wants you to discuss more thoroughly previous similar work. Reviewer #2 and #3 have concerns due to 7KC not only impacting membrane order. Reviewer #3 has concerns with the small effect of 7KC on NK-cell autolysis, asks what happens with a flipped ratio of E:T, says that you should present data for PI uptake in CD107a-negative cells, and have further questions about 7KC-treated cells. Reviewer #3 also says that the data ruling out LAMP-1 is weak, and asks whether you can add a control to figure 5. Please address also the rest of the reviewers' comments.

We expect to receive your revised manuscript within 3 months.

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Paula

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Paula Jauregui, PhD,

Associate Editor,

pjaureguionieva@plos.org,

PLOS Biology

*****************************************************

REVIEWS:

Reviewer expertise:

Reviewer #1: Innate immunity and cancer.

Reviewer #2: Membrane structure/lipids.

Reviewer #3: Stephen Waggoner. NK cells.

Reviewer #1: This articles adresses the very important question of how NK cells (and possibly other cytotoxic lymphocytes) are protected against their own weapons, in particular perforin which, once released to the immunological synapse, promotes the formation of holes in the target cell membrane.

Overall this is a very well written and interestig article. The introduction is well documented and focused on the question adressed in the subsequent experimental work. The methodology is appropriate and the experimental work is of great quality. The conclusion (and title) that « Degranulation-enhanced presynaptic membrane packing protects NK cells from perforin-mediated autolysis » are supported by a significant amount of results. I listed below several concerns that need to be adressed before publication.

Figure 1.

a) The data obtained with YTS and NK-92 are depicted in the same graphs (panels C and F and possibly panel I). It would be nice to see the results of the two cell lines in separate graphs.

b) With this regard, a minimum of 10 data points/cell line for proper statistical testing would be appreciated.

c) Is the unpaired t-test appropriate here?

Figure 2.

a) Is a target cell lysis rate of 30% the max. cytolytic capacity of NK cells even at high E:T ratios?

b) Fig. 2D, the values for the 16 hours coculture are suprisingly lower than 4 or 8 hours and this is not reported in the result section. Could this be explained by the higher rate of YTS death shown for the same time point in Fig. 2E?. By the way, how do you explain the increase of NK cell death in the 16 hours cytotoxicity assay (while other incubation times are all similar)? Alternatively, did the authors account for the increase in NK cell death and adjust the target cell lysis accordingly?

c) Healthy donor derived NKs: because there is significant heterogeneity between donor NK cells, I suggest to show each set of data (i.e. for each donor) in addition to (as a supplementary Figure?) or instead of the averages values. Ideally, five donors should be used. Finally, it would be important to check and report target cell killing with eNKs as well (similar to what has been shown with NK cell lines, Figure 2A and B).

d) Statistical significance is sometimes missing. Not sure which test should be used.

Figure 3. Do NK cells form conjugates with target cells in the presence of EDTA?

Figure 4.

a) Panel D, NK cells were exposed to free perforin. Without degranulation events, the NK cell membrane shows no increase in lipid order (which was proposed to drive resistance). Thus, how the authors explain NK cell resistance to perforin (in the absence of degranulation)? Is it possible that 7KC treatment destroys the overall cell membrane structure of resting NK cells rendering them more vulnerable to perforin? Testing of membrane leakiness after 7KC treatment with cytoplasmic dyes could provide more insight into the specificity of this treatment on perforin susceptibility

b) A repetition of this experiment with target cells would give indications whether NK cells have intrinsically a cell membrane of higher lipid order than other cells, providing them with a resistance "bonus".

Figure 6.

- What is the lipid composition of target cells?

- As previously requested for figure 2: could the authors provide the data obtained for each primary NK cell donor. Ideally, please include 2 additional donors to better taker into account inter donor heterogeneity.

Figure 7

a) The legend is confusing. K562 and MB-231 cells are used in both panels E and F. Please check the figure legend.

b) The susceptibility of MB-231 is only minimaly increased following 7KC treatment. Accordingly the role of densely-packed post synaptic lipid membrane in tumor cell escape should not be overestimated. In the discussion section, the authors are invited to tone down their conlcusions and clearly state that although lipid density might contribute to tumor cell resistance against NK cells, it can likely not account for most of the resistance.

c) Do other (resistant) cancer cells respond to NK cell attack in the same way? Ideally a second resistant tumor cell line should be used to validate the results obtained with MD-231, in particular since the increase in susceptibility is not as high.

d) What is the fate of target cells that generate densely packed postsynaptic membranes? Do they survive?

e) Is there a higher autolysis of NK cells when cultured with MDA-MB-231 cells due to the denser postsynaptic membrane?

Additionnal points:

a) I suggest the authors to more explicitely explain how LAMP-1 has previously been suggested to provide NK cells with perforin protection (line 80). Would it be relevant to refer to #34 here?

b) I don't know what is PLOS Biology's policy oan siRNAs but most journals request to validate the results from gene KD with at least two different siRNAs. The "RNA interference" subsection in the Methods section only refers to the siRNA strategy for PRF1, while additional genes are knocked down using siRNAs.

c) Line 635: the authors should refer to the original study: https://pubmed.ncbi.nlm.nih.gov/30104240/ (not only to a review article).

d) Please check the statistical tests used throughout the article.

Reviewer #2: Overall, this manuscript describes what appear to be carefully done and extensive experiments probing (using membrane polarity sensitive dyes) and manipulating (using 7KC) the membrane order at the immune synapse, tying membrane order to the lytic activity of perforin in NK T-Cells.

I have 2 main comments/concerns:

The first being that the idea presented here is already present in the literature. This in itself is not a major problem, but I found it strange that it was not discussed more thoroughly. Namely, reference 36 of the manuscript is titled "Lipid order and charge

protect killer T cells from accidental death" and has a similar thesis to the present submission, although many of the experimental approaches differ.

My second main concern is the assertion that treatment with 7KC only impacts membrane order, which is certainly not the case. This lipid is added in a way that will also lead to the extraction of other lipids, especially cholesterol, and this could impact the observed processes in ways that do not depend exclusively on 'ordering'. This again is not necessarily a major problem as long as it is discussed appropriately. Ideally, experiments would also be done using other treatments that impact ordering in alternate ways, such as by adding bile acids or alcohols, feeding cells alternate fatty acids, or by altering lipid metabolism.

A possible way to address both concerns would be to narrow the scope of the current manuscript to more centrally focus on the specific inhibitory role of 7KC in the title and abstract.

Reviewer #3: This is a highly intriguing study from Li and Orange revealing formation of densely packed, highly ordered lipid membranes on NK cells at pre-synaptic sites of lytic granule fusion that prevent perforin binding and autolysis. They reveal that the unique lipid composition of lytic granules contributes to and/or reinforces this protection. Moreover, they show that an NK-cell resistant breast cancer cell line forms similar densely packed post-synaptic lipid membranes that contributes to evasion of NK-cell killing. The combination of fluorescent lipid packing sensors and microscopy is an asset that permits the additional strength of analysis across multiple NK-cell lines, primary NK cells, and tumor targets.

Concerns

1. While the effect of 7KC on high lipid order appears absolute in Figure 1, the effects on NK-cell autolysis in Figure 2 and elsewhere (notably Figure 7) are very small (this mechanism appears to protect 5% of NK cells). Intriguingly, these effects increase with effector cell density, potentially implicating fratricide over autolysis. What happens in an experimental setup with flipped ratio (1:10 E:T) that triggers greatest degranulation among large number of NK cells? While Figure 3G suggests degranulating cells appear to be most susceptible to death, data for PI uptake in CD107a-neagtive cells should be presented. In related queries, are the line colors mixed up in right graph in Figure 4A where NK92 survival would expected to be worst for 7KC-treated cells, which obfuscates the effect of CMA in this experiment. Finally, it is not clear why the measurement of 721.221 cell death in Figure 2D would decrease between 8 and 16 hours.

2. There is confusion about how Figure 4D fits with overall mechanism. If the synaptic formation of dense ordered lipid membranes that is enhanced by degranulation and is protective versus perforin, then what is the mechanism at play in resting NK cells treated with recombinant perforin? Is this different in activated NK cells?

3. On line 135, the results in Figure S2 are said to be similar to non-synaptic regions in Figure 1, but this is not readily apparent from the visible spectra representing general polarization values (bright yellow in unconjugated cells and dark orange to red in non-synaptic regions). Minimally this could be clarified with presentation of mean/SD values of GP for unconjugated cells. Is it feasible to capture images of lipid order in synapse and 7KC disruption (Figure 1) in primary NK cells (only tested in killing)? This would be a major plus but understandable if primary cells are less amenable to the imaging techniques employed.

4. The data ruling out LAMP-1 is weak. How do we know CD107a neutralizing antibodies worked as advertised? What is a positive control for this reagent? Likewise, in LAMP-1 knock-down cells, the LAMP-1 expression levels within granules (highly enriched) should be assessed as well as cell surface levels of CD107a after degranulation to ensure the knockdown was effective where it matters.

5. The approach to dissecting the role of dense ordered lipid membranes is restricted to use of 7-ketocholesterol. This is somewhat problematic given the toxicity of this drug in cells and the difficulty of determining that the only relevant effect of 7KC is the desired disruption of dense lipid formations rather than anti-mitotic or effects on mitochondrial membrane potential. The authors make several attempts to rule out direct toxicity of 7KC, which are good but could be strengthened. For example, not clear what cell density is used in Figure 3A (incubation of NK cells without targets), but would be important to use NK cells at same density as used in 10:1 E:T wells and to include controls of activated NK cells (without targets) to determine if a combination of 7KC and activation signaling is problematic for NK cells. Ideally, a second non-7KC method for disrupting these dense lipid formations if possible would be a huge improvement. A related question is whether the smaller size of 7KC-treated cells in Figure 1D/E is a consistent feature and how this might impact other measurements in this study?

6. Is there any potential control (i.e. loading control) for Figure 5? Disconcerting to see the shrinking numerator without any denominator.

Minor points

1. There is a lot of interpretation and discussion contained within the results section that should ideally be restricted to the discussion.

2. Typo in Figure 1F legend (p=12, presumably meant to be 0.12). This value should be presented in place of ns on the figure image itself. All ns should be replaced with actual values.

3. If p<0.0001 is used a 'lower limit', then p<0.001 (Fig 1C), p<0.01 (Fig 4C), and p<0.05 (multiple spots) should be replaced with actual p values.

4. Colors in line graphs a bit hard to discern, not to mention for any readers who are colorblind.

5. Please put a label distinguishing the two pie charts in Figure 6C.

6. Statement on line 539 about GM3 should be cited to reference Table S1 since this information is not clear in Figure 6.

7. Control I and II labels in Figure 6E should be better defined (these are two non-synaptic regions apparently?)

8. Are bars switched in right bar graph in Figure S4?

9. Several images in Figure S5C and E are not discernable (appear teal)

10. Does green NK cell label on images in Figure 7 refer to YTS? B and C should be quantified like A and D.

Dear Dr. Orange,

Thank you for submitting your revised Research Article entitled "Degranulation-enhanced presynaptic membrane packing protects NK cells from perforin-mediated autolysis" for publication in PLOS Biology. I have now obtained advice from the original reviewers and have discussed their comments with the Academic Editor. 

Based on the reviews, we will probably accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests.

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Since you are using cells from donors, please include information about the form of consent (written/oral) given for research involving human participants. All research involving human participants must have been approved by the authors' Institutional Review Board (IRB) or an equivalent committee, and all clinical investigation must have been conducted according to the principles expressed in the Declaration of Helsinki.

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Please do not hesitate to contact me should you have any questions.

Sincerely,

Paula

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Paula Jauregui, PhD,

Associate Editor,

pjaureguionieva@plos.org,

PLOS Biology

 ------------------------------------------------------------------------

Reviewer remarks:

Reviewer #1: Clément Thomas

Reviewer #3: Stephen Waggoner

Reviewer #1: Most of my concerns have been properly addressed and I am in favor of publication of the revised version of the manuscript.

Reviewer #3: The authors were thorough and highly responsive in addressing the previous concerns of the referees. The revised manuscript is greatly improved.

Dear Dr. Orange,

On behalf of my colleagues and the Academic Editor, Hans-Uwe Simon, I am pleased to say that we can in principle offer to publish your Research Article "Degranulation enhances presynaptic membrane packing, which protects NK cells from perforin-mediated autolysis" in PLOS Biology, provided you address any remaining formatting and reporting issues. These will be detailed in an email that will follow this letter and that you will usually receive within 2-3 business days, during which time no action is required from you. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have made the required changes.

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Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Paula

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Paula Jauregui, PhD 

Associate Editor 

PLOS Biology

pjaureguionieva@plos.org