A Validation Study on Immunophenotypic Differences in T-lymphocyte Chromosomal Radiosensitivity between Newborns and Adults in South Africa

Round 1

Reviewer 1 Report

I have some specific comments for the authors to address regarding the re-written version of this manuscript.

1. While the authors have stated that this is a validation of the Vandevoorde study in a South African population in the simple summary, abstract and introduction. I think it would be useful for the authors to add some justification as to why they are repeating the studies in a different population. Were they expecting to find different results or the same results? It would also be useful to find other studies that have shown differences (or not) which would provide a rationale for repeating the studies.

2. Line 104 in the introduction is incomplete therefore the point of the sentence is unclear.

3. I’m not sure which of the two figures in Figure 5, the authors have chosen for the final figure (there is both a box and whisker plot as well as a bar graph) and the error bars are different between the two graphs. From the comments I received it appears as though I was correct in both previous versions of this manuscript that the authors had selected the wrong error bars. I stand by my original comments that this data is better represented as individual data points to show the true variation in the data which is not conveyed by a bar graph. Therefore, I suggest the authors present Figure 5 as individual data points, or if they are going to use a box and whisker plot, ensure that the error bars are correct.

4. In the discussion in Line 397-400 the authors make the statement: “No statistically significant difference was observed in radiosensitivity between the different racial groups (data not shown) and there is currently limited data on the effect of ethnicity on radiosensitivity which could give an indication of a possible difference.” This statement is ambiguous and it is not clear what the authors are trying to convey. Do they mean to say that they didn’t observe any differences, but there is limited data on the effects of ethnicity on radiosensitivity, so more similar studies are needed to support that there is no difference? Can the authors please consider re-wording to help clarify the point they are trying to make.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

All comments and questions have been answered and integrated in the new version by the authors.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Manuscript Number: Radiation_1392617

Title: Immunophenotypic Differences in Chromosomal Radiosensitivity Between Newborn and Adult T-lymphocytes (Original Paper)

Engelbrecht et al.

Overview and general recommendation:

The paper by Engelbrecht et al. investigated radiation sensitivity of umbilical cord blood (n=32) compared to peripheral blood of a group of adults (n=27) and possible underlying mechanisms. PBMCs were isolated and in vitro  exposed to ⁶⁰Co γ-rays ranging from 0.5 Gy to 4 Gy. Increased radiation sensitivity was determined by Cytokinesis-block micronuclei assay using a semi-automated detection system from Metasystems. Radiation induced micronuclei frequency was significantly increased for 0.5 Gy, 1 Gy, 2 Gy and 3 Gy in umbilical cord blood compared to peripheral adult blood. At 4 Gy micronuclei yield was also higher in umbilical cord blood but the difference was not significantly. Immunophenotyping was performed to analyse the fraction of naive T cells and memory subsets as detected by expression of the cell determinants CD45RA+ or CD45RO+ respectively by FACS analysis. As shown already in several studies before, umbilical cord blood is highly enriched by naive CD4 and CD8 Tcells expessing the CD45RA cell surface protein in comparison to adult peripheral blood cells in which the fraction of positive cells drops down from 90% in umbilical cord blood to about 45% in adult blood. Therefore the authors concluded that this difference is linked to the increased radiation sensitiviy of the umbilical cord blood cells.

The paper is nicely written, results are demonstrated clearly, techniques are well done, some minor changes can be recommended to improve the paper. However, the research results presented here have already been shown in several other studies, including the investigated micronuclei endpoint. T cells of umbilical cord blood are more radiation sensitive in comparison to T cells of peripheral adult blood. In addition it has long ago been shown that umbilical cord blood bears an increased fraction of naive Tcells. The identical hypothesis shown here have already been stated by a study performed by one of the authors before: C Vandevoorde, A Vral, B Vandekerckhove, J Philippé, H Thierens. Radiation Sensitivity of Human CD34(+) Cells Versus Peripheral Blood T Lymphocytes of Newborns and Adults: DNA Repair and Mutagenic Effects. Radiat Res. 2016 Jun;185(6):580-90. doi: 10.1667/RR14109.1. Epub 2016 May 19

Therefore my major concern is that the study does not provide new results as have already been shown and discussed by Floyed and Cassoni 1994, by Vandevoorde et al. 2016, by Bakhmutsky et al. 2014 or Gomolka et al. 2018. No new experiments were provided that do support the previous stated hypothesis from 2016 by Vandevoorde et al. In addition in the current study it has not been shown that the naive cell type do really show an increased radiosensitivty in respect to micronuclei induction. In the study here, the authors provide an indirect correlation.

Therefore I suggest to provide and show additional experiments in comparison to the studies mentioned before, e.g. to investigate and demonstrate the changes in different age groups, or to show mechanistically that radiosensitivity is linked to change in chromatin structure in the age group and that this change somehow is related to repair pathway choice. This would give a new impact to the study.

Minor Comments

A 4 Gy exposure is possibly to high and the results of semiautomated analysis are in this dose range not really reliable, because of the fraction of dead cells.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper analysed radiation induced damage in mononuclear cells derived from umbilical cord blood and adult peripheral blood. The analysis used a single assay (cytokinesis-block micronucleus assay) to evaluate chromosomal damage following exposure to increasing doses of ⁶⁰Co γ-radiation. The authors also analysed the immunophenotype of T-lymphocytes from umbilical cord blood and adult peripheral blood.

The introduction was well written and gave a good introduction to the body of work. The methods were very detailed and could be easily replicated from the information provided. The authors obtained ethical consent for the use of the samples in this study.

I have some concerns regarding the results section as well as the conclusions drawn from this study. I have outlined my concerns below.

1. In the abstract the authors mention an increase in radiosensitivity of 34%, 42%, 29%, 26% and 16% was observed for newborns compared to adults at 0.5, 1, 2, 3 and 4 Gy. They make no further mention of this except in the discussion, so I was wondering how they calculated these percentages and why there is no reference to this result in the results section.
2. Is the data in Figure 2 and Figure 3 the same data as that represented in Figure 1, but just broken down into Male vs Female? In the methods section and in the figure legends the authors indicate that all data is presented with standard error of the mean (SEM). However, the error bars are very different between Figure 1 and Figure 2/3. Can the authors please check that they have correctly represented the error bars.
3. Can the authors clarify why they have chosen a non-parametric test (Kruskal Wallis) to analyse statistical significance.
4. In Figure 5 the y-axis title is confusing and needs to clearly state what is being measured.
5. In Figure 5, was the immunophenotyping carried out on different samples than the CBMN assay (n=33 for UCB in immunophenotyping and n=32 in UCB for CBMN assay, n=18 for APB in immunophenotyping and n=27 in APB for CBMN assay). Why is there a discrepancy in the n numbers between the two assays?
6. In Figure 5, the error bars (SEM) are overlapping, so I am not sure if the results are statistically significant as indicated. It might be useful to plot individual data points for all of the graphs, rather then box plots so that the reader can better interpret the results.
7. Why did the authors switch from presenting the data as mean percentage of lymphocytes in Figure 5 to median in Table 2, as I believe they are derived from the same flow cytometry data. Also, in Section 3.2 the authors refer to the data in Table 2 as median in line 277, then as mean in line 286. It would be useful if all the data was presented consistently to allow better interpretation of the results.
8. In the discussion the authors state that “The results indicate that the immunophenotypic profile of the T- lymphocytes influences its radiosensitivity” line 306-307. I don’t believe this can be concluded from these results. The authors show that newborn UCB have increased sensitivity to radiation compared to adult PB and that newborns have a different T-cell immunophenotype then adults, however they have not shown a causative relationship. To do this they would need to sort cells into CD45RA+ and CD45RO+ populations, perform the CBMN assay and show that there is a difference in the radiosensitivity between naïve and memory T-cells. Then they could suggest that the finding may be causative.
9. In the conclusions the authors state “This study showed that the IR-induced MN yields in the newborn T-lymphocytes were higher compared to the MN yield in adult T-lymphocytes” line 484-485. This is not necessarily true as the authors performed the CBMN assay on bulk umbilical cord mononuclear cells and adult peripheral blood mononuclear cells. While the majority of mononuclear cell populations prepared by density gradient centrifugation contain T-cells, there are also B-cells, NK cells, monocytes and dendritic cells present, as well as stem/progenitor cells in the UCB. Therefore, conclusions on the T-lymphocytes alone cannot be drawn from this data. The authors would need to sort the T-lymphocytes from the other cells types and repeat the study to draw this conclusion.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Review: radiation-1392617 (Report 2)

General Comments:

Also in the resubmitted version there is no add on to already published results. The authors have extensively described in their response that originally they intended to investigate ethnical differences and do agree that the presented study is just a validation study. I do understand that the authors have no possibility to investigate their original research question: radiosenstivity among different ethnical groups. But even if they had been allowed to publish the differences, there is no sound statistics from what was shown in the authors response. In addition is it really to be expected that fundamental differences will be observed?

In summary there are no new facts therefore it has to be really stressed that this study is a validation study of already published results in another population.

Specific Comments:

Short summary

Line 21: „The aim of this study was to gain more insight in the intrinsic higher radiosensitivity of newborns (0 days old) compared to adults.“

Focus on what was really investigated here, since it is a validation study in another population of previous results, please state this clearly in the summary:

The aim of this study was to validate published results on immunophenotypic differences underlyinging the increased radiosensitivity in newborns in an South African population.

Abstract

Line 34-36: In this study, the potential age dependency in chromosomal radiosensitivity was evaluated by means of the cytokinesis-block micronucleus (CBMN) assay in T-lymphocytes isolated from umbilical cord and adult peripheral blood.

Please also state clearly:

In this study, previous results on the potential age depencency…  was validated again …. And adult peripheral blood in an South African population.

Line 43-45„This demonstrates that the observed differences in chromosomal radiosensitivity between newborn and adult T-lymphocytes could potentially be linked to their immunophenotypic profile.“

Please state:

This agrees with previous published results that….

Introduction:

Shorten and focus the introduction, especially line 94-109

Line 111-113: clearly state that this is a repetion of a previous study to demonstrate the general principle of radiosensitive differences also in a different population of South Africans.

Results:

Table 2: Replace mean percentage expression … in  mean expression [%]

Figure 5: please describe in the legend what is really shown - box plots….., quartils,….

Discussion

Line 307-308: Please indicate again that this study validates previous results

Line 388-396: The numbers investigated (from authors response for whites e.g. only 2 samples) do not allow conclusions at all. Numbers should be given here, not only the percentage.

Conclusions

This study validates previous findings……

Citations

Citation 8 and 45 is the same:

45. Gomolka, M.;Oestreicher, U.; Rößler, U.; Samaga, D.; Endesfelder, D.; Lang, P.; Neumaier, K.; Belka, C.; Niemeyer, M.; 625Kiechle, M.; et al. Age-dependent differences in DNA damage after in vitro CT exposure. Int. J. Radiat. Biol.2018, 94, 272–281.

Reviewer 2 Report

Thank you to the authors for taking the time to address my comments. However, I still have some concerns over the results and conclusions, I have a few specific comments, which should be considered by the authors before publishing this work (See attached PDF).

However. even if these issues were resolved, I still have concerns more generally regarding the novelty of this work. While the original intentions of the authors may have been to analyse racial differences in radiosensitivity; due to factors out of their control the final product does not reflect this. As the paper stands additional experiments need to be carried out in order for this work to contribute new knowledge to the field. The authors may consider re-writing the publication emphasising that this is the first study of its kind in South African population and validates previous findings but in a different population, since this is where the novelty of this study is.

Comments for author File: Comments.pdf