Higher Frequencies of T-Cells Expressing NK-Cell Markers and Chemokine Receptors in Parkinson’s Disease

Round 1

Reviewer 1 Report

The reviewer report

Jal 1976137

Higher frequencies of T-cells expressing NK-cell markers and chemokine receptors in Parkinson’s disease

The authors investigated the expression of receptors (CD161, NKG2A, NKG2C, and NKG2D) and chemokine receptors (CCR6, CXCR3, and CCR5) on peripheral T and NKT cells, respectively, for discovering and understanding their neurodegenerative effect in cross-sectional cohort focused on early PD patients compared with healthy subjects. The findings indicate the higher frequencies of peripheral CD8+ T-cells expressing NKG2A, NKG2C, and NKG2C+ CD8- cells (probably Th) and CD4-CD56+CCR5+ NKT-cells and suggest impaired T-cell activation, increased frequency of infiltrating NKT might be an inflammatory indicator for diagnosis of the early stage of PD.

Here are my contributions to the manuscript:

1. Row 160: The authors should add an explanation. On which cell type The chemokine receptors were investigated? 

2. Row 163 and 217: What does mean CCR5-expressing CD4+ T-cells? Are not they (CD56+CD4-CCR5+ T-cells) NKTs because of CD56 expression according to your gating strategy? The authors should revise the results and the discussion sections. (Ahmadi A et al. The role of NK and NKT cells in the pathogenesis and improvement of multiple sclerosis following disease-modifying therapies. Health Sci Rep. 2022 Jan 24;5(1):e489. DOI: 10.1002/hsr2.489.)

3. The authors determined the CMV serostatus but did not compare the expression of receptors CD161, NKG2A, NKG2C, and NKG2D, chemokine receptors CCR6, CXCR3, and CCR5 on T, NK, and NKT cells. (Vavilova JD et al. Reduced Immunosenescence of Peripheral Blood T Cells in Parkinson's Disease with CMV Infection Background. Int J Mol Sci. 2021 Dec 4;22(23):13119. doi: 10.3390/ijms222313119.)

4. References: There is one reference (No 50) dated the last five years. Here is an example from PubMed: Jun Tian et al. Specific immune status in Parkinson's disease at different ages of onset, NPJ Parkinsons Dis. 2022 Jan 10;8(1):5. doi: 10.1038/s41531-021-00271-x.

Here are some minor corrections:

5. S1 table:  The authors should add the meaning of abbreviations at the bottom of the table ( eg. m, f, UPDRS). The addition of a comma after years is better (in years, mean +/- SD)” and “± SD” instead of “+/- SD“.

6. S1 Fig:   cD3hiCD56

7. Figure 1. "CD8+/- T-cells." The authors should add NKT cells because of CD4-CD56+ T-cells.

8. Row 31 CD4-CD56+CCRR5+ T-cells

9. row 144 Figure 1A); row 147 Figure 1(b)); row148 Figure 1(c)); row 150 Figure 1(d)); row 163 Figure 1e); rows 154-157 (a)…. (b)….. (c)….. (d)…… (e). The uppercase should be better regarding the graphs in Fig1.

10. Row 149 mean fluorescence intensity; row155 Median fluorescence intensities; Fig1D median: Which is for MFI mean or median fluorescence intensity? 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The paper aims to characterise T cell expression of various Natural Killer (NK) cell markers and chemokine receptors in a Parkinson’s disease (PD) population that have been diagnosed in the last 3 years. To achieve this, they utilised multi-colour flow cytometry panels with patient and control PBMC samples. The study shows an increase in NKG2A, NKG2C and CCR5 in specific T cell subsets in the PD patients.

The study is well written with relatively good coverage of the PD literature around NK cell markers. One of the studies strengths is that the patient cohort is very well defined, other studies of PD fail to be specific with their cohort, which can distort the findings.

The main weaknesses of the paper include unusual phenotyping of T cells, for example gating for CD3+CD4-CD56+CCR5+, when it would be more appropriate to gate for CD3+CD8+CD56+CCR5+, as it is unclear whether the CD4- gating specify a legitimate T cell population.

Leaving negative results out of the main body of text as well as supplementary figures and discussing data in the main text that is not found in the main text or in supplementary figures. Moreover, it is unclear why in Figure 1 the Y axis changes from looking at population of interest compared to CD3+ as a percent (Figure 1A-C), to population of interest compared CD45+ as a percent (Figure 1E)? If the comparison to CD3 is used for Figure 1E does it remain significant? It is also unclear why the authors decided to only show MFI for NKG2D, it would be highly informative to see the MFI of the other markers investigated.

Furthermore, there are comparisons to Multiple Sclerosis throughout the paper, but it is unclear how relevant this is, as disease mechanism is very different, a comparison to Alzheimer’s or Huntingdon’s would be a much more apt comparison.

The authors conclude by suggesting that a CCR5 antagonist could be useful in the future for a therapeutic target for neuroinflammatory diseases but cite a paper (citation 49) that only suggests a CCR5 antagonist for autoimmune and infectious disease related CNS inflammation, not for neurodegenerative disorders like Alzheimer’s and Parkinson’s.

The research area the authors are investigating is highly relevant, as the past 10 years has seen a significant increase in publications examining the role the immune system plays in the development and progression of PD.

Below are specific comments with line number references.

Specific comments –

Abstract

Line 23: “activated immune cells could interesting for understanding” doesn’t make any sense

Line 31: typos throughout “CCRR5+” should be “CCR5+”

Line 29: Unclear why they state NKG2C+ CD8- frequency was higher, do they mean CD4+ NKG2C+? Is NKG2C+ CD8- a relevant phenotype?

Line 31: Again, unclear why they say CD4-CD56+CCRR5+, do they mean CD8+CD56+CCR5+ T cells? Unclear if CD4-CD56+CCRR5+ is a relevant phenotype?

Introduction

No mention of T cells infiltration into substantia nigra in post-mortem PD brains, the relevant paper must be cited.

Line 58: “NK-cells are increased in PD patients controls” what is meant by PD patients controls?

Line 68: It should be mentioned that another study has found no difference in NKG2D expression, see PMID: 17702627

Line 69: How relatable is the pathology in MS to the pathology in PD?

Line 69: Very different mechanisms of disease between MS and PD, so I’m not sure how useful it is referring to any similarities between MS and PD. Furthermore, MS is considered an autoimmune disease, not a neurodegenerative disorder. Unclear if this would justify the inclusion of NKG2C and CD161 in the study.

Line 68: The study that found a decrease in NKG2A (PMID: 17702627), also found no difference in NKG2D levels, this should be mentioned

Other changes in NK cell phenotype should be stated i.e. increased MFI of CD16 on CD56dim cells in early PD compared to HC, but also late PD in males PMID: 35026420 (2022).

Methods

Line 138: Unclear why only the MFI of NKG2D was investigated and not the other markers? Why not be consistent and examine proportion of markers as well as MFI of markers for all markers investigated?

Results

Line 148: Again, why only show MFI of NKG2D and not the other markers? This has not been explained. My suspicion is that the other markers were not significant, MFI of NKG2D is only just (p=0.04). The results that weren’t significant should be shown in supplementary figures if not shown in main text.

Line 150: If CD161 was investigated, then why is it not shown?

Figure 1: Why does the Y axis change from looking at population of interest compared to CD3+ as a percent (Figure 1A-C), to population of interest compared CD45+ as a percent (Figure 1E)?

Line 161: Why CD4-CD56+? Is this a legitimate T cell population, should this be defined as CD8+CD56+?

Line 163-167: Multiple data sets discussed in text, but no reference to a graph or table in results section or supplementary figures. This should be shown.

Table 1 is unclear, e.g., states “PD patients % (median (range))”, percent of what exactly? Percent of total lymphocytes? Percent of white blood cells in general?

Table 1: Why does Table 1 not show CD3-CD56highCD16lowCCR5+ frequencies?

In summary there are multiple graphs that I’m unclear about and require major revisions to.

Discussion

Line 177: Should state “higher frequency of CD8+ T cells expressing NKG2C relative to CD3+”

Line 179: Again, should say “higher frequencies of CD8+ T-cells expressing the inhibitory receptor NKG2A relative to CD3+”

A lot of papers look at “percentage of lymphocytes” therefore any change from this should be clearly stated

Line 184: Such a low percentage of T cells have NK markers on them, I don’t think you can you really suggest that these are the T cells found in the substantia nigra? As there is such a small number of T cells found in the substantia nigra in the first place.

Line 189: Mentions that in MS, CD4+ NKG2C+ T cells can contribute to tissue damage when they migrate into the CNS, you can’t relate that to your data as you found a difference in CD3+CD8-NKG2C+ populations, not CD4+. For this comparison to be legitimate, the data for CD4+CD56+NKG2C+ should be shown and is dependent on whether there is significance.

Line 212: Compares CCR5 expression in MS lesions, unclear if this is a relevant comparison as discussed above

Line 213: An increase of CCR5 after stimulation of cells with Aβ is not comparable to PD, should cite a study that shows an increase of CCR5 when stimulated with α-synuclein

Line: 217: CD4- is not a legitimate T cell type, it should be CD8+ if you want to claim an increase in CCR5

Line 238: The results stand in contrast to 3 studies, not 2. See PMID: 28791571.

References

References are inconsistent, some using all capital letters and others not.

Supplementary figures

Supplementary figures require FMO’s for more smeary stains to justify placement of gate e.g. CXCR3, CCR6, CCR5 and CD161

Supplementary figure 1 page 4, top right gating: Unclear how you made the distinction between CD3+CD56+ and CD3highCD56+?

Supplementary figure 1 page 4, bottom left gating: What is the population going off the scale to the right of CD16highCD56low?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I thank the authors for the elaborate corrections. I invite them kindly read the attachment for some minor improvements.

Comments for author File: Comments.pdf

Author Response

We appologise for the typos in the supplement and have corrected them in tables, figures and legends.

In S1 Fig. we deleted the duplicate plot and for figures in general we adjusted the description and added arrows for a better understanding of the gating strategy.

Reviewer 2 Report

My concerns with the manuscript have been answered.

Author Response

Thanks again for your support to improve the manuscript.

Latest review is an update to correct some typos in the supplement.