Modifications in Immune Response Patterns Induced by Kynurenine and One-Residue-Substituted T Cell Epitopes in SARS-CoV-2-Specific Human T Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The report by Tokano et al. describes how Kynurenine modifies the immune response patterns after administration of a Peptide (p176-190) designed on SARS-CoV-2 spike protein and how the modification within peptide sequence (a leucine for neutral hydrophilic serine) completely abolishes the cytokine responses of a cloned T cells, specific for the peptide, as reported in the title.

My observations:

·       Fig 1 A the concentration tested gaps from 10 to 100, why intermediate concentrations have not been tested? Why also not more than 100? A toxicity curve of Kynurenine has to be performed and reported within the manuscript

·       Also the concentration curve of peptide is necessary

·       Legend to figure 2: (C) TM45.2 cells were cultured were cultured Please correct

 

·       Discussion:

a)      When TM45 cells were incubated with kynurenine for 7 days, they produced IL-8 in a dose-dependent manner; however, there was no increase in the production of GM-CSF from TM45 cells. As requested before, a toxicity curve of Kynurenine has to be performed

b)      This suggests that the oversecretion induced by kynurenine is specific to the IL-8 response. Specific is a not correct term. Have you ever tested other markers?

Please also check english typos

 

Comments on the Quality of English Language

english has to be improved

Author Response

Detailed Response to Reviewers:

 

Thank you very much for reviewing our manuscript and offering valuable advice.

We have addressed your comments with point-by-point responses and revised the manuscript accordingly.

 

Reviewer #1:

Fig 1 A the concentration tested gaps from 10 to 100, why intermediate concentrations have not been tested? Why also not more than 100? A toxicity curve of Kynurenine has to be performed and reported within the manuscript

Because the T-cell line was not immortalized, the number of cells obtained was limited, so we were unable to examine many concentrations. However, the concentration of kynurenine we examined this time sufficiently covers the physiological concentration of kynurenine in viral infection (see line 207-211). The viability of TM45 cells co-cultured with 100μM　kynurenine were >90% (trypan blue stain). Based on this result, we concluded that 100μM kynurenine has no toxicity. I added words in lines 142-143.

 

Also the concentration curve of peptide is necessary

The concentration of the peptide was determined according to a previous study (reference 19). The T cells used in this study showed a similar peptide dose response patterns. Therefore, we did not examine wide range of concentration.

 

Legend to figure 2: (C) TM45.2 cells were cultured were cultured Please correct

I have revised as you suggested.

 

Discussion:

1. a) When TM45 cells were incubated with kynurenine for 7 days, they produced IL-8 in a dose-dependent manner; however, there was no increase in the production of GM-CSF from TM45 cells. As requested before, a toxicity curve of Kynurenine has to be performed

Dose-dependent increase of IL-8 response well testifies the lack of toxicity. The viability of TM45 cells co-cultured with 100μM　kynurenine were >90% (Trypan Blue Stain). I added words in lines 142-143.

 

1. b) This suggests that the oversecretion induced by kynurenine is specific to the IL-8 response. Specific is a not correct term. Have you ever tested other markers?

GM-CSF, IFNγ, and IL-5 did not show a dose-dependent increase with kynurenine co-culture. I added words in lines 144-145.

 

Again, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We hope that these revisions persuade you to accept our submission.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Please, comment in the introduction aspects about the function of the kynurenine and why it was used in the research.

In line 112 what do you refer to cytokine cross-reactivity? Please, comment

What methods were used to observe that peptides rotated? Please, comment

I could not see the figures 3 and 4. Are there supplementary material?

The COVID-19 vaccine contains the “harmful” epitope p176-190? Please, comment about a potential danger or not of its use.

The discussion repeats the results. Please, avoid that. 

Author Response

Detailed Response to Reviewers:

 

Thank you very much for reviewing our manuscript and offering valuable advice.

We have addressed your comments with point-by-point responses and revised the manuscript accordingly.

 

Reviewer #2:

Please, comment in the introduction aspects about the function of the kynurenine and why it was used in the research.

Certain viral infections such as coronaviruses and Zika virus are reported to induce kynurenine production, which activates AHR [13-17]. The blood concentration of kynurenine in COVID-19 patients has been reported to be around 10-15 μM [27]. Based on these observations, we used kynurenine in our study. Please refer to lines 41-42, 207-208.

Kynurenine is produced from tryptophan by indoleamine 2,3-dioxygenase (IDO). For information on the functions of kynurenine, please refer to lines 202-207.

 

In line 112 what do you refer to cytokine cross-reactivity? Please, comment

We checked whether the ELISA kit measures cytokines other than the intended target cytokine.

 

What methods were used to observe that peptides rotated? Please, comment

This is an established observation that has already been proven in previous studies (references 22, 23).

 

I could not see the figures 3 and 4. Are there supplementary material?

Figure 3 was a mistake for Figures 2A and 2B, and Figure 4 was a mistake for Figure 2C. I have corrected the relevant section.

 

The COVID-19 vaccine contains the “harmful” epitope p176-190? Please, comment about a potential danger or not of its use.

It is difficult to obtain the complete sequences of the epitopes targeted by each COVID-19 vaccine currently in circulation. The Receptor Binding Domain (RBD) of SARS-CoV-2 is highly mutable, and it is possible that the sequences of the epitopes targeted by the vaccines have also changed. We do not believe that the epitope p176-190 is harmful on its own. We would like to argue that this technique (a single-residue substitution) should be able to eliminate potentially harmful T cell epitopes, while preserving beneficial B-cell epitopes. See line 227-228.

 

The discussion repeats the results. Please, avoid that.

I have removed a part of the relevant section as you pointed out.

 

Again, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We hope that these revisions persuade you to accept our submission.