Rebuttal Letter

Dear Dr. Cao,

RE: JoVE59531

We are pleased to learn that our manuscript was reviewed quickly and also thankful for the reviewers’ insightful comments. I have provided our point-by-point responses in this rebuttal document and certainly hope that you will find our revisions satisfactory.

Sincerely,

Dr. S.M. Mansour Haeryfar

Editorial comments:

Please include an ethics statement before your numbered protocol steps, indicating that the protocol follows the animal care guidelines of your institution.

We have now moved our Ethics Statement to the requested position within the manuscript.  

In the JoVE Protocol format, “Notes” should be concise and used sparingly. They should only be used to provide extraneous details, optional steps, or recommendations that are not critical to a step. Any text that provides details about how to perform a particular step should either be included in the step itself or added as a sub-step. Please consider moving some of the notes about the protocol to the discussion section.

Most of our Notes conform to their intended purpose as indicated above. We have now amalgamated several Notes into one (please see our revised manuscript). 

1.3: Is this done at room temperature?

Yes. We have now revised the text to make this clear. 

1.7: What volume of sterile cold PBS is used?

This is typically done in 10 mL of PBS. We have revised the text.

1.9: Are the cells counted before this step? Please specify.

Yes. We have modified the text to clarify this point. 

1.10, 3.1: Please specify the age and gender of the mouse.

Modified as requested!

9. 3.1, 8.1: Please specify the euthanasia method used.
Specified as requested!

4.2: Please specify the peptide used for each tube.

This may complicate the manuscript and make its flow less accessible to the readership. The readers should be able to readily refer to previous sections specifying the peptides to be used. Sorry!
 
4.3: What is the mesh size of the cell strainer?

Consistent with the rest of the protocol, cell strainers with 70-μm pores need to be used. We have specified this in the text now. 

5.8: Is step 5.7 also repeated?

We have added a short sentence to clarify this further.

7.1: Please specify from which step the source tubes are prepared.

We have now done this actually in an earlier step where we have referred to the ‘source tubes’. Accordingly, in step 6.5, we have referred to step 6.1. Please refer to our revised manuscript.   

Please highlight complete sentences (not parts of sentences). Please do not highlight any steps describing anesthetization and euthanasia.

We have removed the highlights from several section titles that do not form complete sentences. We did not have any highlighted texts referring to anesthetization and euthanasia to begin with.   

19. References: Please do not abbreviate journal titles.

The manuscript is now revised as instructed!

Please obtain explicit copyright permission to reuse any figures from a previous publication. Explicit permission can be expressed in the form of a letter from the editor or a link to the editorial policy that allows re-prints. Please upload this information as a .doc or .docx file to your Editorial Manager account. 

We have previously obtained such letters, which we will upload during the re-submission. 

Please upload each Table individually to your Editorial Manager account as an .xls or .xlsx file.

We have now prepared and uploaded separate tables. 

Table of Materials: Please revise to include the name, company, and catalog number of all relevant supplies, reagents, equipment and software, and sort the items in alphabetical order according to the name of material/equipment.
Revised as instructed!

Reviewer 1:

“The manuscript is well written and clear.” 

We thank the reviewer for her/his positive feedback.

“I have few minor points that needs to be clarified.”“1. In section 5, it is interesting to know if different dyes could be used instead of various CFSE titrations.”

This is definitely a possibility. However, many factors need to be considered (e.g., toxicity and stability/instability of other dyes) and various conditions also need to be optimized before such dyes can be used. CFSE has worked well in various in vivo killing assays using different setups.     

“2. In section 7.6, I suggest that the splenocyte mix is checked in FACS just before injection in the recipient mice to ensure the ratios between differently labeled cells is the same after the washing steps and the vortexing (sections 7.1-7.4).”

This is an important quality control measure. Steps 6.1-6.5 exactly address the reviewer’s point.   

“3. Figure 1.A and B are copied from a previous paper (Memarnejadian, A et al. J Immunol. 2017). I am not sure but some copyright issues might be concern regarding the figure. Additionally it is interesting to know why the authors did not performed cytofluorimetric analysis in order to recreate graphs represented in Figure 1.”

It is our understanding, based on JoVE’s instructions, that this is an acceptable practice. We did obtain formal permission from The Journal of Immunology (the flagship journal of the American Association of Immunologists) for the reproduction of figures 1 and 2, both of which are adopted from our own previously published articles. Two permission letters are now uploaded with this resubmission. We routinely perform in vivo killing assays and are confident about the reproducibility of our results. Our animal use protocols stipulate that we should sacrifice additional mice only if we absolutely have to.      

Reviewer #2:

“Minor Concerns: Can the authors discuss adaptability of this assay to draining lymph nodes (e.g. to derive target cells)?”

This is an important question. Lymph nodes are not typically used as a source of target cells simply because they do not provide a sufficient cellular yield even if they are pooled. 

Typically, CFSE-labeled target cells are not tracked in lymph nodes either since they are injected via tail vein. The intravenous route of target cell injections dictates that these cells end up in the spleen among several other organs, but not in lymph nodes, except potentially for peri-thymic nodes. It will be interesting to look for intravenously injected target cells in these nodes. We fully agree that it will also be curious to determine whether CFSE-labeled target cells that can be potentially injected subcutaneously (in other settings) show up and remain in sentinel lymph nodes draining the area of injection. 
Please note that our manuscript’s focus has been the application of in vivo cytotoxicity assays in evaluation of CD8+ T cell immunodominance. Therefore, we did not discuss the above possibilities in the interest of producing a coherent manuscript.