T-Lymphocytes Activated by Dendritic Cells Loaded by Tumor-Derived Vesicles Decrease Viability of Melanoma Cells In Vitro

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors demonstrated the potential of Dendritic Cell based anti-tumor specific therapy using the tumor specific cytochalasin B-induced membrane vesicles to activate CD8+ T cells and initiate effective anti-tumor responses. The results reveal a proof of concept of tumor cell killing by activated immune cell populations.

 

1. How the authors determined the 78.3 ± 10.12) copies of GM-CSF transcripts per cell?

2. Provide the full blot images for the Fig 3B. Why the blot looks like a crop-edited image? Could you revise the blot with the original uncropped blot.

3. Provide the statistical correlations in the respective figure panels of Fig 3 and its figure legends.

Comments on the Quality of English Language

The authors demonstrated the potential of Dendritic Cell based anti-tumor specific therapy using the tumor specific cytochalasin B-induced membrane vesicles to activate CD8+ T cells and initiate effective anti-tumor responses. The results reveal a proof of concept of tumor cell killing by activated immune cell populations.

 

1. How the authors determined the 78.3 ± 10.12) copies of GM-CSF transcripts per cell?

2. Provide the full blot images for the Fig 3B. Why the blot looks like a crop-edited image? Could you revise the blot with the original uncropped blot.

3. Provide the statistical correlations in the respective figure panels of Fig 3 and its figure legends.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors provide interesting study about immune interaction in the neoantigen context for melanoma. However, the effect they are demonstrating does not show very strong result in the study. Could the authors provide better pictures for  the confocal. two cells per field seems rather low. Did the try ELISA for GM-CSF? Could they please properly annotate the western original data? How were the various fraction identified by flow cytometry? Why does mda-mb-231 cimv have similar killing for M14? should HLA-DR+/CD38+ cytotoxic T-lymphocytes should also be CD8+? How were the % calculated? The authors can include some of the flow cytometry data.

 

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, authors explored a T-cell activation approach by DCs loaded with cytochalasin-induced membrane vesicles (CIMVs) from cancer cells, and evaluated the anticancer activity of the activated T cells. Overall, this study has interests. The study design is logic, the experiments have been properly performed, and their conclusions can be supported by the results. In addition, the research background is well introduced. Several suggestions to further improve the present manuscript are listed below.

1. Table 2 is commonly used information and is recommended to be removed.

2. In fig.1B, authors should indicate which signal in the original WB corresponds to each signal in the cropped image.

3. In fig.7A, the results showed that co-culture of moDCs loaded with M14 CIMVs-GM-CSF with PBMCs led to the highest 29% increase in CTLs. Did the difference of activated CTLs between M14 CIMVs-GM-CSF and M14 CIMVs treatments show significant?

4. In fig.8A, as mentioned in the introduction, authors implicate that tumor-derived vesicles are as useful as TSAs in training DCs for the T cell activation. Therefore, it is of interest whether M14 CIMV-activated CTLs exhibit higher or comparable cytotoxicity to those activated by M14 crude extracts.

5. In fig.8B, the reason or rationale for assessing the cytotoxicity of activated PBMCs against MSCs should be further addressed.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript entitled “T-Lymphocytes Activated by Dendritic Cells Loaded by Tumor-Derived Vesicles Decrease Viability of Melanoma Cells In Vitro” written by Filin et al, has well demonstrated the loading of Dendritic cells in tumor models and its effect on cell viability, but the authors claimed that it alters the T-cell function and increases the activation and effectiveness of the T-cell response. The study lacks seriously with their reports,

1)      There are already a well demonstrated studies been reported on Dendritic cell-based immunotherapy <https://www.nature.com/articles/cr2016157>, and more on-going phase1 & 2 clinical trials, what is the significance of the study?

2)      The major setback of the was lack of functional assays, the authors extensively reported on the generation and characterization of the cell models.

3)      The real-time experiments are missing, it would be better if author did an in-vivo experiment by developing syngeneic mice models and studied, the toxicity and t-cell functions particularly, the % of CD3, CD4/CD8 ratio and Tregs FOXp3.

4)      It would be better if authors report the cytokine levels of IL6, IL-10, particularly, IL2. Cells stimulation with IFN-g

Minor comments:

1)      The author can remove table:2, it’s not necessary to show the composition of SDS-PAGE gels %, just include used 12% SDS-PAGE gel in method section.

2)      In section 3.1 author reported confirmation of GM-CSF was demonstrated by qPCR and WB, the manuscript shows only WB, remove qPCR.

3)      Fig 1.B WB should be replaced with better image, the lane 1 and 2 seems to cropped and placed

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors answered all the queries. I do not have any further questions. 

Reviewer 3 Report

Comments and Suggestions for Authors

Previous concerns were appropriately addressed and the manuscript was well revised. No further issues arise.