Sonicated Bordetella bronchiseptica Bacterin Can Protect Dendritic Cells from Differential Cytotoxicity Caused by Doxorubicin and Vincristine and Enhance Their Antigen-Presenting Capability
Round 1
Reviewer 1 Report
Improved enough.
Author Response
Thanks for your review comments.
Reviewer 2 Report
Please see the PDF.
Comments for author File: 
Comments.pdf
Author Response
Title: Sonicated Bordetella bronchiseptica Bacterin can Protect Dendritic Cells from
Doxorubicin- and Vincristine-induced Damage and Enhance Their Antigen-presenting Capability.
Manuscript ID: cimb-1787037
Authors: Sung JY & Joo HG
Journal: Current Issues in Molecular Biology.
Special Issue: Targeting Tumor Microenvironment for Cancer Therapy 2022.
In the present study, Sung & Joo evaluated the cytotoxic damage of two anti-cancer drugs, doxorubicin (DOX) and vincristine (VC), on DCs from mice, and investigated the cytoprotective effects of sonicated Bordetella bronchiseptica bacterin (sBb) on the DCs treated with the anti-cancer drugs. The authors concluded that sBb plays an important role in preventing the decrease of viability and functional impairment of DCs, but more relevant is its proposition that sBb has the potential as an immunostimulating and protective agent that can be used as an adjuvant with anti-cancer drugs. Authors should address the following questions to clarify their results and conclusions, and enrich the discussion:
In the present work, the measurements of mean fluorescence intensities of the immune response-related markers in control sBb-treated DCs (0.1 µg/mL) were completely different, with much lower values compared to results included in a previously published paper by authors (Lee, YJ.; Han, Y.; Joo, HG. Bordetella bronchiseptica is a potent and safe adjuvant that enhances the antigen-presenting capability of dendritic cells. Korean J Physiol Pharmacol. 2020, 24(1), 47-52. doi:10.4196/kjpp.2020.24.1.47):
MCH2: 54195 vs 9804
CD40: 19555 vs 4482
CD54: 48081 vs 13694
CD86: 19416 vs 16104
Q1. How do the authors explain this discrepancy in the adjuvant action of B. bronchiseptica using the same dose in both studies?
Response: Thanks for the comment. There were differences between the previous study (KJPP) and this study. In previous study, CytoFLEX (Beckman Coulter, Brea, CA, USA) was used for analysis and whereas LSR FortessaTM (BD Biosciences, Franklin Lakes, NJ, USA) was used in this study. In our experience, there was a big difference between two FACS instruments in their default settings. Plus, the voltage, the signals that is converted and displayed as ‘Fluorescence Intensities’ in histograms was also different between experiments. For this reason, we presented the results by converting the MFI into a percentage (The mean fluorescence intensity of control DCs was set at 100%.). The expression of the 4 DC markers was significantly increased in the sBb-treated DCs compared to the control DCs.
Q2. Does this mean that the availability of B. bronchiseptica as a vaccine adjuvant based on its activity in DCs is uncertain or highly variable?
Response: Sonicated Bordetella bronchiseptica itself would be effective vaccine adjuvant based on our previous study and this study. Although there are differences in the details, sBb-treated DCs consistently showed higher marker expression and cytokine production compared to those of control DCs. And thus, we think that the adjuvant effect of B. bronchiseptica (sBb) was evident.
Q3. Would it be a limitation of the use of sBb in preclinical models of cancer or in the clinical setting?
Response: sBb can be used in the pre-clinical phase as a candidate of vaccine adjuvant or a protective agent if the proper concentration is well established. Thus, we need to titrate the optimal concentration of sBb in vivo.
Q4. What would be the useful range of up-regulation of activation surface markers on DCs in the antitumor strategy?
Response: DCs treated with anti-cancer drugs must have impaired functional properties than control DCs. So, if the degree of surface marker expression is similar to that of control DCs or increases even a little by some substances or agents, they would play a role in preventing damaged DCs. Therefore, if DCs treated with anti-cancer drugs and sBb showed a statistically significant increase in marker expression than those of anti-cancer drug only treated DCs, in our opinion, we would say that sBb plays a role in preventing dendritic cells from being damaged by anticancer drugs.
Q5. The allostimulatory capacity of DC on spleen cells are similar to the presentation of a neoantigen or tumoral antigen in the scenario of tumor immunotherapy using DCs? This must be explained and contextualized. Lines 193-196 “..sBb significantly increased the antigen-presenting capability of control DCs by approximately 150%, and VC-treated DCs by 221%. Interestingly, VC-treated DCs showed marginally lower antigen-presenting capability than control DCs”. It is not clear to me and seems contradictory to say that the effect of VC is "marginally lower... than control DCs". Please explain or clarify this point.
Response:
- Anti-cancer drugs treated DCs are less likely to interact with lymphocytes due to their impaired function. Through this study, we compared the antigen presenting capability of the anti-cancer drug treated DCs and that of anti-cancer + sBb treated DCs, to see if sBb can restore or even increase that of DCs.
- We first expected that VC-treated DCs would show markedly reduced antigen presenting capability compared to that of control DCs. However, the antigen presenting capability of VC-treated DCs did not differ significantly from that of control DCs, whereas DOX-treated DCs showed significantly lower antigen presenting capability than that of control DCs. The description was written based on these results.
Q5. VC (sBb?) be considered an adjuvant according to the results, mainly the expression of
surface markers on DCs, and the promotion of the allostimulatory capacity of DCs?
Response: In our previous study, DCs treated with sBb were compared with those treated with LPS and cytokines production, markers expression, and antigen presenting capability were measured, suggesting that sBb could be a safe and potential vaccine adjuvant.
In this study, VC did not impair the viability and function of DCs, and furthermore VC + sBb treated DCs showed higher antigen presenting capability than control DCs (even control + sBb treated DCs). The combination of VC + sBb is considered to have an adjuvant effect from the aspect of antigen presenting capability.
Q6. What is the mechanism of VC on DCs as an anti-cancer drug?
Response: VC acts by binding tubulin in tumor cells, inhibiting the formation of microtubule and causing the cell to be unable to separate its chromosomes during metaphase. However, the study on how VC specifically works in DCs is lacking yet. Through this study, we speculate that anticancer drugs related to microtubules do not damage DCs, but rather have an effect and mechanism that stimulate them to a certain level.
Q6. What is the mechanism of sBb on DCs and to reverse the effect of DOX on DCs?
Response: This will be confirmed through further (ongoing) research. So far, it is estimated that sBb may be involved in enhanced cytokine production, the DC activation and protection by acting as agonists for Toll-like receptors or as ligands for nod-like receptors or NLRP3 inflammasome, but further studies will need to verify this.
Q7. Does vincristine really induce damage to DCs? The title would probably have to be modified!
Response: Thank you for the suggestion. We revised the title as follows.
Sonicated Bordetella bronchiseptica Bacterin can Protect Dendritic Cells from Differential Cytotoxicity caused by Doxorubicin and Vincristine and Enhance Their Antigen-presenting Capability.
Additional questions and suggestions:
- Explain why female mice were used?
Response: We wanted to control the variables in the experiment as much as possible. Thus, we usually select one type of gender in certain age of mice.
- Preparation of DCs. Why C57BL/6 mice? Balb/c isn't it? Between the two strains of used mice, there are critical immunogenetic differences. Are the results found using dendritic cells from C57BL/6 mice extrapolated to Balb/c mice, for example in the context of preclinical models of cancer?
Response: In allogeneic mixed lymphocyte response (MLR) to measure antigen presenting capability of DCs, two type of mice with distinct genetic background should be used. In this study, C57BL/6 DCs and Balb/c spleen cells were selected for MLR. It is a commonly used combinations.
- All figures. Authors should describe and explain the results of the figures and not
just repeat what is written in materials and methods.
Response: According to the reviewer’s comment, figure legends were revised.
Author Response File: 
Author Response.docx
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This is an interesting study. However, key data is not strong enough to support the major conclusion as stated in the title. The authors used one-way MLR to determine the antigen-presenting capability of DCs. It is not the correct assay that is used to determine the antigen presenting capability, because MLR measures T-cell response from one donor that is stimulated by antigen presenting cells from a second donor due to an HLA mismatch. One correct way to measure the antigen presenting capability is to measure the percentage of antigen-specific T cells generated. However, the results presented in Fig. 5 were corresponding to the results in Fig. 3b. In Fig. 3b, MHC II expression was statistically significant between VC + sBb and VC, but not DOX + sBb and DOX. That could explain why the MLR results were seen statistically significant in VC but not DOX group in presence of sBb.
Minor issues:
- In section 3.1, the authors mentioned that the concentrations of 0.25 and 0.5µg/mL for both DOX and VC. But in the Fig. 2 and 4, it was 0.25µg/mL for both drugs. Why?
- What is the implication of MMP? DOX and VC did not affect MMP by itself. The authors should give more discussion on MMP in terms of DC function.
- BCG may act similarly as sBb does. BCG has been approved for non-muscle-invasive bladder cancer (NMIBC) by FDA in 1990s. Based on the current data of sBb, the potential for translating current finding of sBb to clinical study is very low. Thus, the novelty of current finding was not sufficient for publication.
Author Response
Reviewer 1
This is an interesting study. However, key data is not strong enough to support the major conclusion as stated in the title. The authors used one-way MLR to determine the antigen-presenting capability of DCs. It is not the correct assay that is used to determine the antigen presenting capability, because MLR measures T-cell response from one donor that is stimulated by antigen presenting cells from a second donor due to an HLA mismatch. One correct way to measure the antigen presenting capability is to measure the percentage of antigen-specific T cells generated. However, the results presented in Fig. 5 were corresponding to the results in Fig. 3b. In Fig. 3b, MHC II expression was statistically significant between VC + sBb and VC, but not DOX + sBb and DOX. That could explain why the MLR results were seen statistically significant in VC but not DOX group in presence of sBb.
Response: We agree the reviewer’s comment regarding MLR. Ag-specific T-cell response is more accurate assay to measure the antigen presenting capability of DCs. In this study, we used allogeneic MLR, measuring overall allogeneic antigen presenting capability of DCs rather than specific antigens. Thanks for your comments regarding VC + sBb. We agree that MHC II expression should be closely correlated to MLR results.
Minor issues:
- In section 3.1, the authors mentioned that the concentrations of 0.25 and 0.5µg/mL for both DOX and VC. But in the Fig. 2 and 4, it was 0.25 µg/mL for both drugs. Why?
Response: In ELISA, we wanted to determine the effect of drugs on DCs at two concentrations (weak, strong). In the case of viability and marker expression, it was thought to be reasonable to use 0.25 µg/mL, which is relatively less toxic to DCs.
- What is the implication of MMP? DOX and VC did not affect MMP by itself. The authors should give more discussion on MMP in terms of DC function.
Response: Mitochondria membrane potential (MMP) is closely related to the viability of cells.
According to figure 3a and 3b, DOX did affect MMP by itself, but VC did not. We have written additional information about this. In case of VC, we described out interpretation in discussion. (VC seems to be involved in activating/maturing DCs, even though it is an anti-cancer drug with an anti-microtubule property. Therefore, further research on this seems to be needed.)
- BCG may act similarly as sBb does. BCG has been approved for non-muscle-invasive bladder cancer (NMIBC) by FDA in 1990s. Based on the current data of sBb, the potential for translating current finding of sBb to clinical study is very low. Thus, the novelty of current finding was not sufficient for publication.
Response: We appreciate your insightful suggestions. As you stated above, we are further planning to investigate the effect of sBb with clinical aspects in detail. Though, in my opinion, based on our previous study [reference 16] and this study, we confirmed the specific immunostimulating and protective effects of sBb on anticancer drug-treated DCs.
Author Response File: Author Response.pdf
Reviewer 2 Report
- "However, studies on the toxicity of DOX and VC on DCs are lacking." - I found several papers studying the effect of doxorubicin or vincristine on DC in vitro (for example, https://0-doi-org.brum.beds.ac.uk/10.1186/1479-5876-7-58 , https://0-doi-org.brum.beds.ac.uk/10.1038/sj.bjc.6694366)
- The materials and methods section suffers from a lack of information, making it difficult to reproduce the results: no detailed information about manufacturing and composition (solvent) of Bordetella bronchiseptica bacterin (sBb) – main chemical of this article; insufficient information about the antibodies used (for example, fluorophores conjugated to them) etc.
- Histograms on the Figures 3 and 4 are difficult to analyze - there are no gates, the meaning of the numbers indicated on them (right top) is not clear.
- The conclusion ”sBb increases the antigen-presenting capability of DCs” was made on the basis of measuring the total number of cells in co-cultures (Cell Counting Kit-8), which can be explained by many factors.
- The obvious question - how Bordetella bronchiseptica bacterin affects the proliferation of cancer cells (for example, lymphoma) has not been studied in any way and is not discussed in the article. At the same time, judging by Figure 1, this reagent can stimulate the proliferation of dendritic cells.
Comments for author File: Comments.pdf
Author Response
Reviewer 2
- "However, studies on the toxicity of DOX and VC on DCs are lacking." - I found several papers studying the effect of doxorubicin or vincristine on DC in vitro (for example, https://0-doi-org.brum.beds.ac.uk/10.1186/1479-5876-7-58 , https://0-doi-org.brum.beds.ac.uk/10.1038/sj.bjc.6694366)
Response: Thank you for your point out. We corrected as follow; however, studies on the immunostimulatory agents to protect DCs from DOX- and VC-induced toxicity are lacking.
- The materials and methods section suffers from a lack of information, making it difficult to reproduce the results: no detailed information about manufacturing and composition (solvent) of Bordetella bronchiseptica bacterin (sBb) – main chemical of this article; insufficient information about the antibodies used (for example, fluorophores conjugated to them) etc.
Response: We corrected as follow;
- Bordetella bronchiseptica in PBS was sonicated under condition of amplitude 60%, 3 cycles of 30 sec with a 30 sec interval using a Q125 sonicator (Qsonica, Newtown, CT, USA). then the amount of protein was measured by the Bradford assay and stored in -20℃ freezer.
- The treated cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-mouse MHC class â…¡ (I-Ab), phycoerythrin (PE)-labeled anti-mouse CD40, PerCP Cy5.5-labeled anti-mouse CD54, and allophycocyanin (APC)-labeled anti-mouse CD86.
- Histograms on the Figures 3 and 4 are difficult to analyze - there are no gates, the meaning of the numbers indicated on them (right top) is not clear.
Response: We counted all cells except particles since MMP was not used to analyze cell viability. We also edit the number at the right top of Figure 3 to make it clear.
- The conclusion ”sBb increases the antigen-presenting capability of DCs” was made on the basis of measuring the total number of cells in co-cultures (Cell Counting Kit-8), which can be explained by many factors.
Response: Thank you for the suggestion. There are many factors that affect the optical density. In this study, we focused on the protective effect of sBb on DCs affected by two anti-cancer drugs, so we measured the expression of markers through flow cytometry and antigen-presenting capability by MLR to observe changes by sBb. In MLR using Cell Counting Kit-8 assay, the increase in O.D. is affected by the increase of DC-activated lymphocyte rather than by DC itself. Also, lymphocyte activation by DCs is attributed to the marker expression on DCs.
- The obvious question - how Bordetella bronchiseptica bacterin affects the proliferation of cancer cells (for example, lymphoma) has not been studied in any way and is not discussed in the article. At the same time, judging by Figure 1, this reagent can stimulate the proliferation of dendritic cells.
Response: Previous studies in our lab suggested that sBb can be used as a potential vaccine adjuvant because it activates DCs and is less toxic than conventional O26 LPS [reference 16], and this study focuses on the protective effect of sBb on DCs treated with two anti-cancer drugs frequently used in lymphoma.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The manuscript has been improved.
Just a note: The type of this manuscript is "Article" (Currently listed as "Article, Review, Communication, etc).
Reviewer 2 Report
My comments on the answers to the questions of the first round
1. Q1 "However, studies on the toxicity of DOX and VC on DCs are lacking." - I found several papers studying the effect of doxorubicin or vincristine on DC in vitro (for example, https://0-doi-org.brum.beds.ac.uk/10.1186/1479-5876-7-58 , https://0-doi-org.brum.beds.ac.uk/10.1038/sj.bjc.6694366)
Response: Thank you for your point out. We corrected as follow; however, studies on the immunostimulatory agents to protect DCs from DOX- and VC induced toxicity are lacking.
Comments: The authors did not make a clarification, but simply removed this phrase from the introduction. Thus, the reader is left in the dark about the toxicity of these drugs to dendritic cells. They then added the phrase "However, studies on the immunostimulatory agents to protect DCs from DOX- and VC-induced toxicity are lacking.", which again mentions such toxicity without any supporting references. I think that my remark was not taken into account.
2. Q2 was taken into account, but the section still remained very concise.
3. Q3 The authors did not improve the presentation of Figure 3 in any way, but despite the poor description, I still realized that gates are really not needed in this experiment. However, what the number in the upper part of the histograms means must be written in the legend. Much more unsatisfactory condition with figure 4 - histograms are the result of immunostaining of cells, gates are absolutely necessary in this case. Unfortunately, it is clear from the descriptions of the experiment that these results poorly reflect the expression of surface markers on DCs. Dendritic cells express Fc Receptors that can strongly bind fluorescently labeled antibodies. The use of isotype control during staining and gating for such cells would clarify the question of the reliability of staining. Blocking Fc Receptors on staining would also reduce such non-specific staining. However, neither of these was done. Thus, the results described in section 3.4 were obtained incorrectly and do not allow us to draw the above conclusions.
4. Q4 was taken into account. Unfortunately, it is not known from the description of the experiment at what point the agent sBb was added. In co-culture, sBb could directly stimulate lymphocyte proliferation, in the case of pre-treatment, dendritic cells presenting bacterial antigens could themselves cause more lymphocyte activation, not due to greater antigen-presenting capacity, but due to a more foreign set of presented antigens. In this regard, the conclusion drawn from the results of this experiment does not seem correct.
5. Q5 The obvious question - how Bordetella bronchiseptica bacterin affects the proliferation of cancer cells (for example, lymphoma) has not been studied in any way and is not discussed in the article. At the same time, judging by Figure 1, this reagent can stimulate the proliferation of dendritic cells.
Response: Previous studies in our lab suggested that sBb can be used as a potential vaccine adjuvant because it activates DCs and is less toxic than conventional O26 LPS [reference 16], and this study focuses on the protective effect of sBb on DCs treated with two anti-cancer drugs frequently used in lymphoma.
Comments: I think that my remark was not taken into account.
Comments for author File: Comments.docx
