Immunophenotyping and Functional Characterization of NK Cells in SARS-CoV-2 Infection

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article is very interesting and presents several strengths. However, there are multiple aspects that the authors need to review and correct:

• The reference for the first paragraph of the introduction must be included.
• The overall writing format should be improved; for example, the abbreviation for natural killer is defined three times and is inconsistently used throughout the text.
• The authors should clarify why the patient group in P1 included only 5 individuals, while P2 included 15. It is unclear if it was a follow-up study. It must be clarified if the patients were the same or if the groups were different. Additionally, it should be specified whether age and gender matching were performed. This must be clearly explained.
• A section of the introduction is based on the process of NK cell exhaustion and reduced cytotoxic potential, where the upregulation of inhibitory receptors is relevant. However, it is not clear why three activating receptors (NKG2D, NKp46, NKG2C) were included along with only one inhibitory receptor (NKG2A), which forms a complex with NKG2C. The authors should justify why other inhibitory receptors, such as KIRL (CD158), were not considered.
• While it is true that activated NK cells express CD25, it should be clarified that CD25 is a subunit of the IL-2 receptor and should not be classified as an activating receptor, as it is mainly associated with processes such as proliferation and the production of lytic molecules like perforin and granzyme B, rather than degranulation. This should be corrected.
• The authors should indicate whether the patients in P1 were in the acute phase of the disease and specify the duration of illness at the time of sampling. Additional demographic and clinical data, such as age, gender, and disease severity, should be reported. These factors could influence NK cell frequency in P1.
• It must be specified whether the SARS-CoV-2 variants were identified. Various variants circulated during 2023 and 2024, mainly from the Omicron family, which show differences in immunogenicity and may have affected immune responses and cell populations due to immune evasion mechanisms. If the variant was not identified, this should be included as a limitation.
• The authors should include the frequency of CD56bright cells in Table 1, as Figure 1A shows they represent a significant percentage.
• It should be clarified whether the analysis of CD16bright and CD16dim subpopulations was considered. This analysis should be included, ideally in Table 1.
• Section 2.4 needs improvement; it is unclear whether any normality tests were performed or how the format for data presentation was decided. For example, in Figures 1 through 5, it is not specified whether means and standard deviations are shown, or what values are represented. This should be clarified.
• Some sentences from the results section should be moved to the discussion, as they are not direct results and should be supported by references—for example, lines 156–158, 170–172, 279–281, among others.
• Figure 7 is missing the “-1” label on one of its bars.
• Figures 8 and 9 raise multiple concerns and need to be restructured. Please consider the following:

1) The p-values must be added to confirm whether the correlations are statistically significant.

2) The data used for the correlation analyses must be clarified. Each parameter shows only 10 values, which do not match any of the study groups. If the data are from P2, there should be 20 values. If GraphPad was used, it is important to note that this software does not allow individual plots per variable; it uses a single symbol to indicate correlation.

3) Figure 9 should be reviewed and restructured. If the correlation is negative (r = - 0.951), the bar should be oriented in the opposite direction.

4) In terms of formatting, it is recommended to merge Figures 8 and 9 into a single figure (e.g., 8A and 8B), remove the titles (they are implied in the figure legend) or adapt them to an appropriate size, label the axis titles and units, and clarify whether percentages or MFI values were correlated.

• The errors identified in the correlation analyses of Figures 8 and 9 also cast doubt on the presentation of Figure 7. This figure should be described more precisely. Several relevant correlations are mentioned, but it is unclear whether they are statistically significant; if so, the corresponding p values must be reported.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper of Petrov et al. describes phenotypical changes in circulating blood NK cells in course of SARS-CoV-2 infection. The study, in general, is not original and doesn’t add too much to the present knowledge in the field.

I have several important concerns.

The number of patients enrolled in the study is far too low to draw any reliable conclusion, especially that NK cell activity displays a huge variability between different subjects. What was the result of statistical power calculation?

The study should be designed as a follow-up, otherwise differences between groups may accidentally be a result of patients’ variability.

The patients must be described in detail: severity, duration of symptoms, etc. Did the results correlated with e.g. a severity of disease. What was the gender of the patients?

Gating strategy should be presented (as supplemental data) for each analyzed marker.

Percent cytotoxicity results have by definition a not normal distribution. The very high SD value confirms this, so the results must be presented as median and interquartile range. A scatterplot presentation would be encouraged.

There is something wrong with the content and statistical evaluation shown in Fig. 8 and 9.

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The clinical and sociodemographic characteristics of the patients (Section 2.1) should be moved to the Results section; they are not part of the Materials and Methods section.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I uphold my previous opinion that the number of patients is not sufficient to draw any reliable conclusions. In the present shape the paper doesn't meet criteria for being published in a reputable journal.

Author Response

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Author Response File: Author Response.pdf