Antitumor Immunity: Role of NK Cells and Extracellular Vesicles in Cancer Immunotherapy
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis is a well written review on NK cells and their EVs for the treatment of cancer. It appears to be thorough and comprehensive. I only have a few comments.
1. Do EVs contain enough cytokines to have biological activity? For example, if they contain enough IFNg, this could increase expression of some cell surface receptors. and enhance the anti-tumor response.
2. The authors should mention any trials where cytokines were expressed in genetically modified NK cells
3. Are there specific treatments that increase or decrease the EV production my NK cells (e.g.IL-12 + IL-18)?
4. Any evidence that CAR-NK cells have triggered autoimmunity?
5. The authors might want to explain why NK92 cells are being used.
Author Response
Comment: 1. Do EVs contain enough cytokines to have biological activity? For example, if they contain enough IFNg, this could increase expression of some cell surface receptors and enhance the anti-tumor response.
Reply: Thank you for your effort and attention to the manuscript. You are right; this is a key issue for the effectiveness of EVs. It has been shown that EVs contain a wide range of cytokines (doi: 10.1038/s41598-018-27190-x) and exhibit the biological activity of parent cells (doi: 10.1007/s00262-022-03161-0). Indeed, EVs contain enough IFNg, which can stimulate other immune cells to enhance the anti-tumor response (doi: 10.3389/fimmu.2020.00262). We understand that we did not pay enough attention to this issue in the article, so we have made the necessary changes (lines 352-355, text highlighted in blue).
Comment: 2. The authors should mention any trials where cytokines were expressed in genetically modified NK cells
Reply: Thank you for the feedback. We have expanded the description of the clinical study in which NK cells were transduced with a retroviral vector carrying genes encoding CAR-CD19, interleukin-15, and inducible caspase-9-based suicide gene (iC9) (doi: 10.1056/NEJMoa1910607). In preclinical studies, the authors demonstrated that NK cells efficiently kill CD19-expressing cell lines and primary leukemia cells in vitro, with a marked prolongation of survival in a xenograft Raji lymphoma murine model. Additionally, the production of interleukin-15 (IL-15) by the transduced NK cells significantly improved their function (doi: 10.1038/leu.2017.226). Furthermore, results from the clinical trial showed that the use of iC9/CAR.19/IL-15 NK cells targeting CD19-positive lymphoid malignancies is safe and effective in patients with CD19+ CLL and B-cell lymphoma. We have also made edits to the document (lines 313-320).
Comment: 3. Are there specific treatments that increase or decrease the EV production my NK cells (e.g.IL-12 + IL-18)?
Reply: Indeed, in the study by Dosil et al., it was found that culturing NK cells in the presence of IL-12 and IL-18, followed by the addition of IL-2, leads to an increased vesicle yield (doi: 10.7554/eLife.76319) (lines 407-409). Another method is discussed in the article by Zhu et al., where it was shown that NK cells, pre-activated with IL-15, produced a greater quantity of EVs compared to non-activated IL-15 NK cells (lines 409-411).
Comment: 4. Any evidence that CAR-NK cells have triggered autoimmunity?
Reply: In the study by Liu et al., it was shown that the use of genetically modified CAR-NK cells was not associated with the development of cytokine release syndrome, graft-versus-host reaction, and an increase in inflammatory cytokine levels (doi: 10.1056/NEJMoa1910607). The reason is that NK cells, unlike T cells, do not respond to foreign MHC, thus they do not elicit an immune response during transplantation, which is an advantage (lines 307-311).
Comment: 5. The authors might want to explain why NK92 cells are being used.
Reply: We have added a discussion in the manuscript regarding why NK92 cells are being used: On one hand, the use of the NK92 cell line enables a less costly production of NK cell-based therapeutics; on the other hand, NK92 cells are not without drawbacks (lines 263-272).
Reviewer 2 Report
Comments and Suggestions for AuthorsCurrent review article outlined previous findings and present perspectives of the roles played by natural killer (NK) cells and extracellular vesicles (EVs) in involving in antitumor immunity. This manuscript is overall well written with current knowledge on the research area presumably relevant to immune-therapeutics against cancer and also conveys authors’ scientific messages.
In general, functions of NK cells have been somewhat considerably documented in the field of anticancer-related studies although drug development remains incomplete and is in a state of still ongoing. But, as shown just in later part (section 5; line 311~), current understanding and knowledge of antitumor (or even pro-tumor) effects of NK-EVs looks to be relatively quite less described compared to those of their source NK cells. To my understanding, more discussions or even speculations on the roles and functions of NK-EVs need to be thus included into this review article, because sufficient discussions, authors’ hypothetical descriptions, or speculations of studies on NK-EVs look to be short as shown in current form of the manuscript. Some parts in section 5 of the manuscript look to less discuss about potential roles of NK-EVs shown in antitumor studies.
Thus, more discussions about roles of functional molecules of NK-EVs, encompassing proteins (e.g., adhesion molecules), miRNAs, or any other cargoes possibly acting in regulating metastasis or oncogenesis, would be better to be included in this review, if available. Also, it can be considered to also include the roles of other immune cells’ (e.g., CD8 T cells’) EVs in associating with anticancer, as well as the NK-EVs which is main player, which can be advantageous in addressing any functional distinctiveness of NK-EVs.
Eventually, this review needs to be further revised along with citing pertinent references, based on the comments above, and newly add the Figure(s) illustrating proposed models of potential mechanisms or strategies with NK-EVs in developing antitumor treatment.
Comments on the Quality of English Language
I do not feel that English language of overall text in this manuscript requires extensive editing. But, minor checking of the manuscript including typos can be recommended.
Author Response
Comment: To my understanding, more discussions or even speculations on the roles and functions of NK-EVs need to be thus included into this review article, because sufficient discussions, authors’ hypothetical descriptions, or speculations of studies on NK-EVs look to be short as shown in current form of the manuscript.
Reply: Thank you for your detailed comments and time devoted to this. Indeed, the antitumor effects of NK-EVs are still not thoroughly understood. To enhance the description of the roles and functions of NK-EVs, we have added the following description and discussion: "In the study by Wu et al., it was demonstrated that NK-EVs have high levels of perforin and granzyme A, moderate levels of granulysin and granzyme B, and a low amount of FasL. The combination of cytotoxic proteins contributes to the cytotoxicity of EVs. Moreover, it has been shown that NK-EVs can cleave SET and HMG2, promoting the death of target cells via caspase-dependent apoptotic pathways, and induce the release of cytochrome C from mitochondria in target cells (doi: 10.1080/20013078.2019.1588538). However, information on the interaction between NK cell-derived vesicles and tumor cells is limited. Based on numerous studies on the interaction of vesicles from human cells with target cells (doi: 10.3402/jev.v3.24641), it is suggested that interaction occurs through plasma membrane fusion (doi: 10.1084/jem.183.3.1161), clathrin-mediated endocytosis, or receptor-mediated internalization (doi: 10.3389/fimmu.2021.658698)." We have incorporated this discussion on the roles and functions of NK-EVs into the manuscript (lines 384-393).
Comment: Some parts in section 5 of the manuscript look to less discuss about potential roles of NK-EVs shown in antitumor studies. Thus, more discussions about roles of functional molecules of NK-EVs, encompassing proteins (e.g., adhesion molecules), miRNAs, or any other cargoes possibly acting in regulating metastasis or oncogenesis, would be better to be included in this review, if available.
Reply: In the study by Neviani et al., it was demonstrated that exosomal miR-186 directly suppresses MYCN and AURKA in neuroblastoma cells, potentially negatively impacting their survival. Transfection of NK cells with the tumor suppressor miR-186 led to the downregulation of TGFBR1 and TGFBR2, thereby preventing TGFβ1-dependent inhibition of NK cell cytotoxicity (doi: 10.1158/0008-5472.CAN-18-0779) (lines 346-352).
Comment: Also, it can be considered to also include the roles of other immune cells’ (e.g., CD8 T cells’) EVs in associating with anticancer, as well as the NK-EVs which is main player, which can be advantageous in addressing any functional distinctiveness of NK-EVs.
Reply: In the study by Dosil et al., it was demonstrated that extracellular vesicles (EVs) from NK cells contain microRNAs that impact the function of T cells, promoting Th1-like responses characterized by increased levels of IFN-γ and IL-2 release. Additionally, NK-EVs influence monocytes, stimulating their activation and enhanced presentation, providing an additional anti-tumor effect (doi: 10.7554/eLife.76319) (lines 355-359).
Comment: Eventually, this review needs to be further revised along with citing pertinent references, based on the comments above, and newly add the Figure(s) illustrating proposed models of potential mechanisms or strategies with NK-EVs in developing antitumor treatment.
Reply: We tried to address all the comments, supplemented the missing information and discussion, significantly revised the manuscript. In addition, we created a graphical abstract illustrating the proposed model or strategy with NK-EVs in developing antitumor treatment.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have addressed my comments.
Comments on the Quality of English LanguageI do not feel that English language grammar of overall text in this manuscript requires extensive editing. But, minor checking of the manuscript is recommended.
