The Impact of Exercise Serum on Selected Parameters of CD4+ T Cell Metabolism

Round 1

Reviewer 1 Report

Pawlowski et al. The impact of exercise serum on the immunometabolism of T helper cells

Authors have conducted the experiment to rule out the effect of exercise serum on the immunometabolism of T helper cells. Many articles have been published on effect of exercise and T cells. However, this study added some more information on exercise and the immunometabolism of T helper cells.  Abstract is written more precisely. Introduction and conclusion are written in well manner. Overall manuscript is well written. However following points need to be address

Major:

1. Justification for selection of exercise protocol is not given with relevant reference, may be one of the reasons for authors not getting the impact of exercise serum on the immunometabolism of T-2 helper cells (of course authors discussed it).
2. Number of participants in Cohort 1 is 5 while 7 in cohort 2, justify for same. Did you do the power analysis for selection of number of subjects.
3. Procedure for control arm is not properly mentioned means how the control subjects were handled.
4. The cohort 1 protocol differed slightly due to logistics: Provide details for same.

Minor:

5. Figure 1 legend is mismatch with figure 1.
6. In figure 2b & 2c; in t0 it looks like a one subject data as there is no error bar. For cohort 2, 72h data collection (t72) is not mentioned in methods.
7. Figure 3 & 6: N=7 per group is mentioned, but as mentioned in methods for control cohort n is 5.

Author Response

We first thank the editor for being able to perform a major revision for this paper. We will respond in detail to each aspect of the reviewers' comments and at the same time prepare a revision of the manuscript that takes into account all comments.

Comments reviewer 1:

Authors have conducted the experiment to rule out the effect of exercise serum on the immunometabolism of T helper cells. Many articles have been published on effect of exercise and T cells. However, this study added some more information on exercise and the immunometabolism of T helper cells.  Abstract is written more precisely. Introduction and conclusion are written in well manner. Overall manuscript is well written. However following points need to be addressed.

Our response: On behalf of all authors, I thank you for the positive comments. We will gladly try to optimize any weaknesses of the paper on the basis of the comments.

Justification for selection of exercise protocol is not given with relevant reference, may be one of the reasons for authors not getting the impact of exercise serum on the immunometabolism of T-2 helper cells (of course authors discussed it).

Our response: We have chosen an exercise protocol that is feasible for untrained subjects on the one hand, but also exhaustive on the other. Therefore, we implemented a 30-minute endurance exercise with a final increase in intensity. We have explained this again in the revised manuscript (lines 113-115).

Number of participants in Cohort 1 is 5 while 7 in cohort 2, justify for same. Did you do the power analysis for selection of number of subjects.

Our response: Due to the lack of preliminary data, we were unable to perform a reliable and serious power analysis. We therefore calculated an approximate number of subjects based on our preliminary tests.

Procedure for control arm is not properly mentioned means how the control subjects were handled.

Our response: Our control conditions in each case is the condition before the athletic load. An additional unloaded control was not included, since we do not expect any change in the essential parameters over the time. The in vitro study included an incubation in serum, which was taken in an unloaded state, as a control condition. We have added this statement to the revised manuscript (lines 118, 139-140).

The cohort 1 protocol differed slightly due to logistics: Provide details for same.

Our response: The implementation of the protocols varied a little because the methodological requirements were not identical due to a laboratory move. However, we standardized all framework conditions in such a way that this had no influence on the practical execution, neither in load duration nor in load intensity. We have explained this accordingly in the revised manuscript.  

Figure 1 legend is mismatch with figure 1.

Our response: Corrected.

In figure 2b & 2c; in t0 it looks like a one subject data as there is no error bar. For cohort 2, 72h data collection (t72) is not mentioned in methods.

Our response: Reviewer 1 is right. The error bars are missing in figure 2b and 2c, because we seeded 1 mio. cells per well from every donor to standardize our experiments. Thus, starting conditions for all experiments are the same. In the revised manuscript we explained better which data belong to which cohort. In the first cohort we performed 72h incubation in the in vitro study, in the second cohort 48h for logistical reasons.  

Figure 3 & 6: N=7 per group is mentioned, but as mentioned in methods for control cohort n is 5.

Our response: We thank reviewer 1 for this comment, both figures are based on the data of cohort 2. We have assigned the data to the corresponding cohorts in Figure 3 and 6 and also added the missing information in the revised manuscript.

Reviewer 2 Report

The manuscript aims to identify changes in CD4⁺ T-cell immunometabolism upon exercise. The study is thought out but present some critical caveats.

My concerns are as followed:

1. The study does not provide enough evidence to be considered novel given the previous publications in the field (Alack et. al. Sci Rep 2020). It is understandable that the data can be different due to different exercise protocol, but more results are needed to support that and especially analysis of proteins presented in the referenced study.
2. Authors shows the difference in HK1 expression and glucose uptake. This data needs to be supported by measuring glucose uptake ability of CD4⁺ T-cell using 2-NBDG. Changes in intracellular glucose is closely linked to T-cell activation and effector function, shown using intracellular cytokine assays. Authors also needs to discuss the reason of decrease in HK1 expression.
3. Fig 5 shows oxygen in cell culture but as authors points by themselves, this needs to be properly evaluated to be acknowledged as changes in cell respiration. Cellular respiration is a direct measure of T-cell activation upon TCR engagement. To evaluate this, experiments should be performed using seahorse assays to measure changes in OCR and ECAR.
4. Fig 6 shows significant reduction in IFNgamma-R1 in control group, which can be due to reduced activation. Although more supportive experiments are needed to describe this results as no change in the expression of surface activation markers were observed between two groups. 

Minor comments : 

1. Line 136: CD3/28 beads from STEMCELL Tech. Stemcell does not make CD3/28 beads for activation. They provide CD3/28 tetramer cocktail. Please correct this.
2. Fig 5B: different groups are not clear in the graph. Please use either combination of dotted and solid lines or colored lines to differentiate between the groups.
3. Fig 7: Statistical bars overlays error bars.

 

Author Response

The study does not provide enough evidence to be considered novel given the previous publications in the field (Alack et. al. Sci Rep 2020). It is understandable that the data can be different due to different exercise protocol, but more results are needed to support that and especially analysis of proteins presented in the referenced study.

Our response: Our experimental approach is not found in this way in the literature, not even in the study from our working group cited here.  In previous studies, hardly any data have been shown that specifically concern the metabolism of T cells after acute exercise. Furthermore, most previous studies (as well as the cited paper) have looked at the total lymphocyte fraction (which is undoubtedly very heterogeneous), not - as here - the isolated CD4+ cells. Moreover, the PreSens technology for analyzing cellular respiration has not been used in the context of T cells and exercise.

Authors shows the difference in HK1 expression and glucose uptake. This data needs to be supported by measuring glucose uptake ability of CD4+ T-cell using 2-NBDG. Changes in intracellular glucose is closely linked to T-cell activation and effector function, shown using intracellular cytokine assays. Authors also needs to discuss the reason of decrease in HK1 expression.

Our response: We thank reviewer 2 for this helpful recommendation. Unfortunately, we can not longer make up the measurement of glucose with the mentioned method, but we are considering this for future study. We have added this as a limitation. We have also discussed the altered HK1 expression in more detail (lines 363-368).

Fig 5 shows oxygen in cell culture but as authors points by themselves, this needs to be properly evaluated to be acknowledged as changes in cell respiration. Cellular respiration is a direct measure of T-cell activation upon TCR engagement. To evaluate this, experiments should be performed using seahorse assays to measure changes in OCR and ECAR.

Our response: Reviewer 2 is right. We changed the wording cellular respiration to basal oxygen consumption (line 84), because cellular respiration was misleading. There are other methods that allow real-time measurement of cellular respiration as we stated in the discussion. Unfortunately, we do not have access to them in our laboratory. The PreSens technology should measure in a similar way but is –as seahorse assays are too- limited in sensitivity due to new oxygen resolving at the surface of the medium. As proof, we have added a citation that shows the methodological possibilities of PreSens.

Renner K, Bruss C, Schnell A, Koehl G, Becker HM, Fante M, Menevse AN, Kauer N, Blazquez R, Hacker L, Decking SM, Bohn T, Faerber S, Evert K, Aigle L, Amslinger S, Landa M, Krijgsman O, Rozeman EA, Brummer C, Siska PJ, Singer K, Pektor S, Miederer M, Peter K, Gottfried E, Herr W, Marchiq I, Pouyssegur J, Roush WR, Ong S, Warren S, Pukrop T, Beckhove P, Lang SA, Bopp T, Blank CU, Cleveland JL, Oefner PJ, Dettmer K, Selby M, Kreutz M. Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy. Cell Rep. 2019 Oct 1;29(1):135-150.e9. doi: 10.1016/j.celrep.2019.08.068. PMID: 31577944.

Fig 6 shows significant reduction in IFNgamma-R1 in control group, which can be due to reduced activation. Although more supportive experiments are needed to describe this results as no change in the expression of surface activation markers were observed between two groups.

Our response: Reviewer 2 is right. It could be related to the activation state of the cells. Unfortunately, it is no longer possible for us to carry out further experiments on this. There is no more material for PCR and no more isolated cells left, so we have addressed this again in detail in the discussion of the revised manuscript (lines 385-388).

Line 136: CD3/28 beads from STEMCELL Tech. Stemcell does not make CD3/28 beads for activation. They provide CD3/28 tetramer cocktail. Please correct this.

Our response: We thank reviewer 2 for this note. We corrected this in the revised manuscript (line 139).

Fig 5B: different groups are not clear in the graph. Please use either combination of dotted and solid lines or colored lines to differentiate between the groups.

Our response: We used a combination of dotted and solid lines in the revised manuscript to better differentiate between groups in the revised manuscript.

Fig 7: Statistical bars overlays error bars.

Our response: Optimized in the revised manuscript.

Reviewer 3 Report

In this manuscript, Palmowski et al show that the impact of exercise serum on the immunometabolism of T helper cells. Although results are encouraging and have future potential in the role of exercise in human T cell immunometabolism, the lack of novelty and the mechanistic basis of the observations, and the very poor presentation of the results with lots of discrepancies between the results described and the title of the article.

Author Response

Although results are encouraging and have future potential in the role of exercise in human T cell immunometabolism, the lack of novelty and the mechanistic basis of the observations, and the very poor presentation of the results with lots of discrepancies between the results described and the title of the article.

Our response: We thank reviewer 3 for the feedback. Based on Reviewer 3's comment, we have changed the title to better match the results to ‘The impact of exercise serum on selected parameters of CD4+ T cell metabolism‘. The novelty of the study is due to the fact that that we primarily addressed direct and indirect parameters of the metabolism of isolated CD4+ cells. This type of study has not been found in the field of exercise immunology before. Most previous studies analyzed lymphocyte subsets within the whole lymphocyte population and are biased by the effects of shifts in T cell subpopulation after exercise. We methodically avoided this problem by taking cells in the resting state and incubated them in serum taken before (control) or after acute exercise. Furthermore, this work is the first study in which, in the context of acute exercise and T cells, the basal oxygen consumption of CD4+ T cells was analyzed.

Round 2

Reviewer 2 Report

The authors have answered my concerns, although sue to lack of samples it is a pity that requested data cannot be obtained. I suggest careful design of experiments in order to utilize the samples as much as possible to obtain a wholesome story. 

Reviewer 3 Report

The authors have adequately addressed reviewers major concerns, thus
accept this manuscript in its current form.