Review Reports - Tumor Acidosis Boosts Antibody-Driven Cytotoxic Potential of Neutrophils

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Formatting: The manuscript should be thoroughly revised for formatting consistency before resubmission:

Ensure proper spacing between words.
Define each abbreviation in its first apparition.
The citation formatting should be standardized.
Please review the names in italics (e.g in vivo, in vitro…).

The authors should state the method they used to determinate the percentage purity of neutrophils they obtained.

The quality of the figures and Western blot images should be improved.

Adjusting extracellular pH experimentally may not fully reproduce chronic tumor acidosis. In tumors, acidity is accompanied by changes in lactate, glucose availability, oxygen tension, bicarbonate concentrations, and other metabolites that may independently affect neutrophil function.

Increased LTB4 production and ERK signaling correlate with enhanced neutrophil activity, but this does not by itself demonstrate that the LTB4-ERK pathway is responsible for the increased antibody-dependent tumor killing. Pharmacological inhibition, receptor blockade, or genetic approaches would strengthen the causal interpretation

Another point, PBMC-mediated ADCC is attributed largely to NK cells, but PBMCs also contain monocytes and lymphocyte subsets. Without purified NK-cell experiments or detailed immune-subset analyses, it may be difficult to determine precisely which cells account for the variable IgG-mediated effects.

Immune cells obtained from healthy individuals may behave differently from neutrophils isolated from patients with cancer, which can display altered phenotypes and functional states within the tumor environment.

The observation that acidity suppresses ROS and NET formation while enhancing antibody-dependent killing is interesting, but whether this balance improves therapeutic efficacy without producing other unwanted inflammatory effects needs further investigation.

Author Response

1. General Formatting: The manuscript should be thoroughly revised for formatting consistency before resubmission:

Ensure proper spacing between words.
Define each abbreviation in its first apparition.
The citation formatting should be standardized.
Please review the names in italics (e.g in vivo, in vitro…).

We thank the reviewer for pointing this out and carefully checked and edited the things mentioned above.

2. The authors should state the method they used to determinate the percentage purity of neutrophils they obtained.

We thank the reviewer for this comment. We assessed the cellular composition of the isolated PBMC and neutrophil fractions by flow cytometry using the staining panel as described in the Methods. The relative proportions of the identified immune cell populations were already shown in Supplemental Figure 2B-C. We have now clarified the flow cytometry approach and the markers used to identify each population in the Methods section.

3. The quality of the figures and Western blot images should be improved.

We appreciate the reviewer’s comment. We have carefully reviewed the figures and Western blot images and confirmed that the figures are provided at a resolution of at least 300 dpi. We have therefore reviewed the overall figure presentation, including labeling, sizing, and consistency across panels, and made adjustments where appropriate. As we are not sure what was specifically meant by the reviewer about the quality of the figures needed to be improved, we would greatly appreciate more specific suggestions if still needed. In case inserting the figures in the word document resulted in loss of resolution, we also supplied the figures as high quality PDF files.

4. Adjusting extracellular pH experimentally may not fully reproduce chronic tumor acidosis. In tumors, acidity is accompanied by changes in lactate, glucose availability, oxygen tension, bicarbonate concentrations, and other metabolites that may independently affect neutrophil function.

We thank the reviewer for raising this important consideration. We agree that experimentally adjusting extracellular pH does not fully reproduce the complex metabolic environment associated with tumor acidosis. In vivo, alterations in extracellular pH occur alongside changes in lactate and glucose availability, oxygen tension, bicarbonate concentrations, and other metabolites, which may independently affect neutrophil function. We have now incorporated this limitation into the Discussion and clarified that our approach was designed to specifically assess the effects of extracellular pH and therefore does not capture the full complexity of the tumor microenvironment (lines 516-520).

5. Increased LTB4 production and ERK signaling correlate with enhanced neutrophil activity, but this does not by itself demonstrate that the LTB4-ERK pathway is responsible for the increased antibody-dependent tumor killing. Pharmacological inhibition, receptor blockade, or genetic approaches would strengthen the causal interpretation

The reply to this comment is provided in the “Non-published Material” document.

6. Another point, PBMC-mediated ADCC is attributed largely to NK cells, but PBMCs also contain monocytes and lymphocyte subsets. Without purified NK-cell experiments or detailed immune-subset analyses, it may be difficult to determine precisely which cells account for the variable IgG-mediated effects.

We thank the reviewer for raising this point. While PBMCs contain multiple immune cell subsets, our complementary experiments with purified NK cells (Supplemental Figure 1) indicate that NK cells are the main contributors to the IgG-mediated tumor-cell killing observed in the PBMC assays. Additionally, given the short duration of the assay, we consider contributions from other PBMC subsets, including monocytes and other lymphocyte populations, to be less likely to account substantially for the observed killing. Nevertheless, we acknowledge that contributions from other subsets cannot be completely excluded. We have revised the Results section to more clearly emphasize the relevance of the purified NK-cell experiments presented in Supplemental Figure 1 to the PBMC findings (lines 313-315).

7. Immune cells obtained from healthy individuals may behave differently from neutrophils isolated from patients with cancer, which can display altered phenotypes and functional states within the tumor environment.

We thank the reviewer for raising this important consideration. We agree that neutrophils present within the tumor microenvironment may differ phenotypically and functionally from neutrophils isolated from healthy peripheral blood. In other studies from our group, however, neutrophils isolated from the peripheral blood of patients with cancer exhibited comparable tumor-killing capacity to neutrophils from healthy donors (Stip et al., JITC, 2023; van der Peet et al., manuscript in preparation). Nevertheless, these circulating patient-derived neutrophils do not necessarily reflect neutrophils exposed to the TME, and local tumor-derived signals may alter neutrophil phenotype and function in ways not captured by our experimental system. We have added this consideration to the Discussion and clarified that the extent to which such TME-associated changes influence neutrophil-mediated ADCC under acidic conditions remains to be determined (lines 543-550).

8. The observation that acidity suppresses ROS and NET formation while enhancing antibody-dependent killing is interesting, but whether this balance improves therapeutic efficacy without producing other unwanted inflammatory effects needs further investigation.

We agree with the reviewer that the balance between reduced ROS/NET formation and enhanced antibody-dependent tumor killing under acidic conditions warrants further investigation, particularly regarding its potential impact on therapeutic efficacy and inflammatory responses. We have added the following statement to the Discussion: “Although our study shows that acidic conditions suppressed ROS production and NET formation while enhancing antibody-dependent tumor cell killing, the broader consequences of this functional shift for therapeutic efficacy and inflammatory responses remain to be determined.”

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Holtrop et al present results of their studies to test effects of an acidic environment on antibody-dependent cellular toxicity induced by neutrophils, much like that produced in the tumor microenvironment. The authors utilize peripheral blood neutrophils vs. mononuclear cells to test for cytotoxicity effects on multiple antibody-treated tumor cell lines, each tested in neutral vs. acidic conditions (pH 7.4 vs. 6.3, respectively). The authors convincingly demonstrate tumor cell lysis by both IgG- and IgA-opsinized tumor cells, with consistently higher levels provided by IgA, but both showing increased lysis at pH 6.3. The neutrophils also show increased trogocytosis, an interesting response by neutrophils to attack tumor cell membranes. In contrast, multiple neutrophil functional responses were inhibited by the acidic environment including ROS production and NET formation, indicating that along with the increase cytotoxicity by neutrophils, their pro-tumor properties are inhibited. Further studies indicated effects of low pH on neutrophil morphologies, directional movements and migration velocities, which are enhanced at low pH, particularly in response to IgA. This antibody response also led to increase LTB4 secretion and increased ERK1/2 phosphorylation, further demonstrating enhanced neutrophil activities. Overall, the results are interesting and add to our knowledge of how the TME can affect neutrophil functions, which may help in designing new ways to control tumor cell growth. The data are well described, the figures are for the most part convincing, and the text is easily navigated with just a few noted grammatical issues (although a careful review is suggested). There are some minor issues to consider as follows.

Lines 118-119, there is a grammatical error with "exposing effector cells being to acidic pH", which might be simply changed to "exposing effector cells to acidic pH...."

Line 126, might simply define ADCC as "antibody-dependent cell-mediate cytotoxicity".

Line 281, might simply add "by ADCC" after "was assessed" to help guide the reader through Figs. 1A and B.

Lines 290-292, the sentence is grammatically incorrect with the opening of "Especially", so suggest slight adjustment (e.g., add "was" after "an effect").

Lines 319-321, the first sentence in Figure 1 legend is confusing - assume the first part is meant as the title sentence, in which case this should end at "neutrophils or PBMCs at pH 7.4 and 6.3." The second half should then start with "(A, C, E, G) in bold, following by specifics for neutrophils, then (B, D, F, H) for PBMCs.

Figures 3F and G, the images used to indicate NET formation are problematic in that multiple arrows do not appear to show anti-MPO staining indicative of extracellular nets. Given that the results in 3H and I indicate that NETs are indeed being detected in response to the IgA and IgG (at least at ph7.4), perhaps the authors can adjust the arrows or have other convincing pictures to show. It would also be helpful to compare their results to a positive control in order to be convinced of significant NET formation.

For lines 404-406, do the authors have any quantitative measurements of IgA-mediated lysis, at least based on color changes shown in the images? This would help support the images shown in understanding the percentage changes (despite the comment on half of the cells were dead by 120 min).

Lines 412-414 and Figures 4B and C, the authors show data derived from their studies of neutrophil roundness, but the protocol for this is lacking other than the use of SAMJ software - how were the cells prepared prior to scans, and do they have any images to indicate the differences between round vs. non-round cells (or "spread-out" morphologies)?

Line 469, the authors need to mention in the figure legend for 5D that the results from the different cell lines are from exposure to neutrophils.

Lines 477-478, while the low levels of ERK1/2 phosphorylation at pH 7.4, in particular at 2 min, significantly contrasts that exhibited by cells at pH6.3, a negative control with no stimulant would be good to show for either set of blots.

Line 487, the sentence has a grammatical error with "binding at in acidic pH".

Author Response

1. Lines 290-292, the sentence is grammatically incorrect with the opening of "Especially", so suggest slight adjustment (e.g., add "was" after "an effect").

We thank the reviewer for the critical reading, and the sentence has been re-written in the revised manuscript with the aim to improve clarity: “This was of particular interest given previous reports that NK cells lose their spontaneous tumor-killing ability after 20 hours, an effect that is not reversible even upon subsequent culture at pH 7.4 [9]”

2. Lines 319-321, the first sentence in Figure 1 legend is confusing - assume the first part is meant as the title sentence, in which case this should end at "neutrophils or PBMCs at pH 7.4 and 6.3." The second half should then start with "(A, C, E, G) in bold, following by specifics for neutrophils, then (B, D, F, H) for PBMCs.

We thank the reviewer for the suggested correction. We have now adapted the sentence in the figure legend accordingly: “Figure 1. Antibody-mediated lysis of four different tumor cell lines with neutrophils or PBMCs at pH 7.4 and pH 6.3. (A, C, E, G) Specific tumor lysis by freshly isolated neutrophils (E:T = 40:1) or (B, D, F, H) PBMCs (E:T = 100:1) using a 4-hour ⁵¹Cr release assay at pH 7.4 or 6.3. The IgG- and IgA-formats of (A-B) Trastuzumab (HER2) to target SKBR3 cells,”

3. Figures 3F and G, the images used to indicate NET formation are problematic in that multiple arrows do not appear to show anti-MPO staining indicative of extracellular nets. Given that the results in 3H and I indicate that NETs are indeed being detected in response to the IgA and IgG (at least at ph7.4), perhaps the authors can adjust the arrows or have other convincing pictures to show. It would also be helpful to compare their results to a positive control in order to be convinced of significant NET formation.

We thank the reviewer for their helpful suggestions. In response, we have added magnifications from the microscopy images to better highlight the regions where NETs can be observed. Unfortunately, no positive control samples were available for the microscopy experiments. For the ELISA assays, however, we did include samples from neutrophils stimulated with PMA as a positive control. The corresponding data have been added below for the reviewer's reference. We chose not to include these data in the manuscript because the PMA-treated samples show substantial variability, which could distract from the main message and interpretation of the figure. Nevertheless, these results support the validity of the assay and are provided here for completeness.

Figure 1: NET formation was quantified by ELISA in supernatants collected from neutrophil-tumor cell co-cultures following treatment with IgA, IgG, or PMA as a positive control. NETs were quantified with anti-MPO antibody and anti-DNA-POD antibody.

4. For lines 404-406, do the authors have any quantitative measurements of IgA-mediated lysis, at least based on color changes shown in the images? This would help support the images shown in understanding the percentage changes (despite the comment on half of the cells were dead by 120 min).

We thank the reviewer for the comment. We believe that killing is not the main outcome of this experiment, since this is better quantified with our ADCC assay. We have adjusted the text, so it lays less focus on killing and more on the effect of IgA on the neutrophils.

5. Lines 412-414 and Figures 4B and C, the authors show data derived from their studies of neutrophil roundness, but the protocol for this is lacking other than the use of SAMJ software - how were the cells prepared prior to scans, and do they have any images to indicate the differences between round vs. non-round cells (or "spread-out" morphologies)?

We thank the reviewer for pointing out this unclarity. We added some sentences to the Methods section of the manuscript (lines 251-254): “In short, the EfficientViTSAM-l2 model of the SAMJ plugin was loaded in FIJI, after which neutrophils were manually selected using the selection tool. When all regions of interest were selected, shape descriptors, including roundness, were determined by clicking analyze”. In addition, we have added zoomed-in images of neutrophils to Figure 4 zoom-ins to better illustrate the differences in their morphology (round vs non-round).

6. Line 469, the authors need to mention in the figure legend for 5D that the results from the different cell lines are from exposure to neutrophils.

We agree with the reviewer’s comment, and we have now adapted the sentence in the revised manuscript as follows: “(B) 4 hours and (C) 24 hours. (D) LTB4 ELISAs of SKBR3, A431, IMR32, and UM9 cells, co-cultured with neutrophils in the presence or absence of antibodies in normal or acidic pH.”

7. Lines 477-478, while the low levels of ERK1/2 phosphorylation at pH 7.4, in particular at 2 min, significantly contrasts that exhibited by cells at pH6.3, a negative control with no stimulant would be good to show for either set of blots.

We agree that an unstimulated control is useful to assess basal ERK1/2 phosphorylation. For the IgA experiment, we have an unstimulated (time 0) condition available, which is shown below and provides a baseline for comparison with the stimulated conditions. Although equal protein loading was performed, technical difficulties during subsequent staining resulted in loss of the corresponding loading control signals in some wells. Additionally, as an equivalent time 0 sample was not available for the IgG immunoblot, we had not included this condition in the main figure, as we aimed to present the IgA and IgG immunoblots consistently to allow comparison between the datasets.

Ideally, we would like to have repeated this experiment so this time point could be added in both blots. However, this was not possible in the short revision period we were given. Thus, we have now incorporated the time point 0 for the IgA blot in the revised manuscript (Figure 5).

8. Line 487, the sentence has a grammatical error with "binding at in acidic pH".

We thank the reviewer for noticing this grammatical error, which has now been corrected in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors of this manuscript entitled “Tumor Acidosis Boosts Antibody-Driven Cytotoxic Potential Of Neutrophils", investigated the effects of extracellular acidity on immune cell function and how it modulates anti-body-dependent cytotoxicity by neutrophils and peripheral blood mononuclear cells in vitro. Although this manuscript had an interesting ideal there are methodological limitations that require attention.

Major comments:

1) The authors need to demonstrate baseline viability/apoptotic levels of their tumor cell cultures at pH 6.3 for 4 hours. The fluorescent lipophilic probe that they observe in neutrophils via flow cytometry might be a result of phagocytosis of apoptotic material rather than specific trogocytosis. They should also try to demonstrate their findings with high precision confocal microscopy apart from flow cytometry in order to observe the size of these fragments.

2) Why are they observing NETs after 24 hours incubation? This is inappropriate for in vitro neutrophil cultures since more than 40% of neutrophils undergo a necrotic death by that time point in vitro. They should demonstrate what happens within the standard timeframe of 4 hours that NETs are usually studied. They should also stain specifically for citrullinated H3 so that they make sure what they see are actual NETs and not necrotic bodies that happen to bear also MPO.

3) In the 51Cr release ADCC assay, neutrophils are co-cultured with target cells at a 40:1 E:T ratio. However, for the live-cell imaging assay, the E:T ratio drops significantly to 10:1. Although, this decision is based probably to prevent overcrowding under the microscope, doesn’t it alter the dynamics of the swarming behavior and the killing kinetics? How do the authors comment on this?

4) Primary human neutrophils exhibit donor-to-donor variability. In the experiments for the trogocytosis and NET release assays, they rely on n=3. The authors should consider increasing the biological replicates to 5-6 for better reliability.

5) Follow Ficoll density gradient isolation the authors perform RBC lysing. Although there are several publications demonstrating RBC lysing as a suitable procedure for functional assays for neutrophils, many of these protocols induce baseline NET release due to the transient presence of hemoglobin which activates neutrophils. Have the authors tried to see the discrepancies between RBC lysing and neutrophils without RBC lysing?

6) The immunoblots only track ERK1/2 activation up to 5 minutes post-stimulation. and then they argue this signaling contributes to functional resilience seen at 4 and 24 hours in the discussion. Have they tried to investigate what happens in later time points? Also the sample size is not mentioned in the respective figure legend.

Minor Comments

Incomplete Sentence

Line 460–461.

The paragraph ends abruptly mid-sentence: "In contrast, PBMCs showed a much more limited LTB4 response (Supplemental".

Typographical Errors

Line 42: ...immunotherapies therapies." (Remove the second "therapies").
Line 314: ...lysis was significantly reduced in under acidic conditions." (Remove the word "in").
Line 561: "the LBT4 receptor LTB4R1" should be corrected to "LTB4 receptor".

Author Response

Major comments:

We thank the reviewer for raising this important point. We agree that tumor cell viability and apoptosis are relevant when interpreting the fluorescent signal detected in neutrophils, as apoptotic tumor-cell material could potentially contribute to the observed fluorescence. We cultured tumor cells in both normal and acidic conditions for 4 hours and performed an Annexin V staining to check their apoptosis status. Only a very small percentage of cells underwent apoptosis (~10%), and this was similar in both pH conditions, indicating that the increase in fluorescence observed via flow cytometry upon acidosis cannot be attributed to the phagocytosis of tumor apoptotic material. We have now incorporated these data in Supplemental Figure 2 and added a sentence to the Results section of the revised manuscript.

Additionally, in our live cell imaging experiments performed without antibody, tumor cells remained PI-negative after 2 hours at both pH 7.4 and pH 6.3, indicating minimal tumor cell death during the assay period. We have now included these data as Supplemental videos 5 and 6. Furthermore, we evaluated the minimal release in the ADCC assays, which represents the baseline level of tumor cell death in the absence of antibodies or PMNs. The graphs below show the Normal/Low pH ratio, demonstrating that all cell lines have ratios close to 1. Taken all these data together, we can conclude that that there are no significant differences in baseline tumor cell death between normal and low pH conditions.

Figure 1: Minimal release in the ADCC assay represents baseline tumor cell death in the absence of antibody or PMNs. Data shown as the normal/low pH ratio for each tumor cell line.

The trogocytosis assay used in our study follows the approach described in the study already cited in the manuscript (Matlung et al., Cell Reports, 2018), in which membrane transfer was quantified by flow cytometry and further visualized by live cell confocal microscopy. Thus, our flow cytometry-based approach is consistent with a previously established method for assessing trogocytosis. Although confocal imaging would provide additional visualization of the membrane transfer process, the minimal tumor cell death observed in our live cell imaging, together with the established assay methodology, supports our interpretation of the fluorescent signal as membrane transfer consistent with trogocytosis.

We appreciate the reviewer’s concern regarding neutrophil viability during the extended incubation period. To support neutrophil viability during the extended culture period, G-CSF was added overnight. As shown in Supplemental Figure 2, approximately 30% of neutrophils underwent apoptosis at normal pH after prolonged incubation, compared with approximately 10% under acidic conditions. Thus, although some loss of viability occurs during the extended incubation, most neutrophils remained viable under both conditions.

We also examined NET formation both at 4 and 24 hours using both microscopy and our MPO-DNA ELISA assay, as shown in Figure 3F-I. At 4 hours, NET formation was detectable, but we did not observe significant differences between conditions (Figure 3H). In contrast, differences between the pH conditions became apparent after 24 hours (Figure 3I). This suggests that the effect of extracellular acidity on NET formation becomes more pronounced with prolonged exposure.

The structures shown in Figure 3 have a thread-like extracellular morphology, and the MPO-DNA ELISA further supports the presence of MPO-DNA complexes associated with NET formation. We will provide higher-magnification images to better illustrate these structures. We agree that citrullinated histone H3 staining would provide additional specificity for NETosis, however, this additional analysis was not feasible within the current revision period.

As the reviewer points out, the different E:T ratios were indeed chosen to accommodate the requirements of the respective assays, with the lower ratio used for live cell imaging to allow for clear visualization of neutrophil-tumor cell interactions and avoid excessive cell density. We agree that reducing the number of neutrophils could potentially influence collective behaviors such as LTB4-mediated recruitment and swarming. Within our laboratory, we do most of the 51Cr-release assays in a ratio of 40:1, and to keep our experiments reproducible and comparable, this is our preference. However, we have previously performed dedicated experiments to test lower E:T ratios (n=2), as shown below. We did notice that at a 10:1 ratio the killing capacity of neutrophils tends to go down, which we think also reflects their high abundance in nature. However, for the purpose of imaging the killing behavior of neutrophils, we indeed choose this lower E:T ratio to prevent overcrowding under the microscope, exactly as suggested by the reviewer.

Figure 2: Specific tumor cell lysis by freshly isolated neutrophils with varying effector to target ratios (40:1, 20:1, 10:1) using a 4-hour 51Cr release assay. N=2 different donors performed in technical triplicates.

We thank the reviewer for this consideration. While we recognize the inherent donor-to-donor variability of primary human neutrophils, the three independent donors in these experiments showed consistent overall responses, with the observed variation falling within the expected range for primary neutrophils, as illustrated in the graph shown below. As n=3 (independent donors) was used consistently across our neutrophil experiments, we have retained the current number of biological replicates.

Figure 3: Increased trogocytosis observed in acidic pH for all cell lines tested. (A) A431, (B) SKBR3 and (C) IMR32 cells were labeled with DiO, a fluorescent lipophilic probe that gets incorporated into the plasma membrane. Antibody-mediated trogocytosis was quantified by measuring the percentage of DiO+ neutrophils overtime. (D) Bar graph of mean antibody-mediated trogocytosis per cell type. Two-way ANOVA, with Tukey multiple comparison test. N=3, in duplicate. Individual donors are indicated by different symbols. * p=0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

We thank the reviewer for raising this consideration. We acknowledge that the RBC-lysis procedure may contribute to some degree of baseline neutrophil activation, as we indeed observe a baseline level of NET formation in the no-antibody control (Figure 3). However, NET formation was substantially increased in the presence of antibody, particularly after 24 hours, indicating that the antibody-dependent response is clearly distinguishable from the baseline signal observed in the absence of antibody. A direct comparison of neutrophils prepared with and without RBC lysis could be valuable to further assess the potential contribution of the isolation procedure, but this was not performed in the current study.

We thank the reviewer for the comment. The immunoblots were performed after antibody stimulation and therefore captured the early ERK1/2 phosphorylation response following antibody addition. Importantly, the functional assays were performed after 4 hours of antibody exposure following the 20-hour culture period, rather than after 24 hours of continuous stimulation. ERK1/2 phosphorylation is a rapid response to stimulation, consistent with other reports (Martínez, et al., Journal of Immunology, 2006;176: 1163-7; Lokuta & Huttenlocher, JLB, 2005;78(1):210-219). We agree, however, that our experiments do not address whether ERK1/2 phosphorylation is maintained at later time points or establish a direct link between the early signaling response and the functional effects observed at later time points. We have clarified this point in the Discussion (lines 605-610).

The effect of acidic pH on phospho-ERK1/2 was observed in six independent donors. Although equal protein loading was performed, technical difficulties during subsequent staining resulted in loss of the corresponding loading control signals in some experiments. We subsequently optimized the protocol, and two independent experiments were obtained with complete blot sets, including the loading controls. The figure therefore shows representative results from these two independent experiments, and the number of independent experiments is now specified in the figure legend.

Minor Comments

Incomplete Sentence

Line 460–461.

The paragraph ends abruptly mid-sentence: "In contrast, PBMCs showed a much more limited LTB4 response (Supplemental".

We thank the reviewer for the critical reading, and we understand that the sentence seems to be ending abruptly. The remaining words of this sentence existed at line 476 after Figure 5, but it has now been adapted and moved to line 461.

Typographical Errors

Line 42: ...immunotherapies therapies." (Remove the second "therapies").
Line 314: ...lysis was significantly reduced in under acidic conditions." (Remove the word "in").
Line 561: "the LBT4 receptor LTB4R1" should be corrected to "LTB4 receptor".

We thank the reviewer for noticing these typographical errors which have all now been corrected in the revised manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

None

Reviewer 3 Report

Comments and Suggestions for Authors

Although the authors indeed demonstrate differences in NET release between 2 different conditions of RBC lysed preparation, there are several cases that this procedure primes neutrophils to respond to stimuli that they wouldn't if they were in actual resting conditions. And the fact that the neutrophils require so long to respond to NET release is rather surprising considering the time frame that they usually release NETs according to other physiological stimuli.

Apart from these considerations the manuscript has been significantly improved.