Class-Switching of B Lymphocytes by DNA and Cell Immunization for Stereospecific Monoclonal Antibodies against Native GPCR
Round 1
Reviewer 1 Report
Generating highly specific high-affinity class-switched monoclonal antibodies is essential for a variety of diagnostic as well as therapeutic applications. In this manuscript, the authors describe an optimized method of generating such monoclonal antibodies using a combination of DNA immunizations and cell-based immunization, which allows the antibodies to be specific for antigens in their native conformation. This is an important study and I would recommend the publication of this manuscript, given the following concerns are addressed:
- The authors describe in the methods section that antigen-specific B cells were selected from the splenic B cell suspension using huCRHR1-expressing myeloma cells. There are several points missing in the description:
- Single B cell-myeloma cell interaction: In order to obtain monoclonal antibodies, it is essential to fuse a single B cell with a myeloma cell in order to generate a hybridoma that secretes antibodies with single clonality. In this experiment, it is not clear if the authors used single-cell suspension in separate wells during fusion with myeloma cells. If not, the possibility of multiple B cells recognizing distinct antigens on the CRHR1 might get attached to a single myeloma cell, resulting in polyclonal antibodies. This is an important point that should be made clear before accepting the manuscript.
- Time: The BCRs on the B cells interact with the membrane-bound CRHR1 on the myeloma cells, resulting in the formation of the immune synapse. This is stable only for few hours. The authors need to mention the time for incubation of B cells with myeloma cells.
- After immunization, the initial immune response is dominated by IgM antibodies due to the generation of IgM secreting short-lived plasma cells and IgM+ memory B cells. As the immune reaction proceeds, it is dominated by IgG antibodies. After various immunizations protocols mentioned in this study, the mice would be generating both IgM+ and IgG+ memory B cells specific for the antigens on CRHR1. The observed difference in the proportion of IgG+ and IgM+ hybridomas could be due to the difference in frequency of IgG+ and IgM+ B cells, or the difference in binding of IgG+ and IgM+ B cells to myeloma cells. Hence, it would be useful to show the frequency of IgM+ and IgG+ antigen-specific B cells present in the spleen after the immunizations in order to correlate it with the frequency of IgG+ and IgM+ hybridomas.
Author Response
<Answer to Review 1>
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
( ) |
( ) |
(x) |
( ) |
|
Are the methods adequately described? |
( ) |
(x) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
( ) |
(x) |
( ) |
( ) |
Comments and Suggestions for Authors
Generating highly specific high-affinity class-switched monoclonal antibodies is essential for a variety of diagnostic as well as therapeutic applications. In this manuscript, the authors describe an optimized method of generating such monoclonal antibodies using a combination of DNA immunizations and cell-based immunization, which allows the antibodies to be specific for antigens in their native conformation. This is an important study and I would recommend the publication of this manuscript, given the following concerns are addressed:
Our reply: Thank you very much for your favorable comments.
- The authors describe in the methods section that antigen-specific B cells were selected from the splenic B cell suspension using huCRHR1-expressing myeloma cells. There are several points missing in the description:
- Single B cell-myeloma cell interaction: In order to obtain monoclonal antibodies, it is essential to fuse a single B cell with a myeloma cell in order to generate a hybridoma that secretes antibodies with single clonality. In this experiment, it is not clear if the authors used single-cell suspension in separate wells during fusion with myeloma cells. If not, the possibility of multiple B cells recognizing distinct antigens on the CRHR1 might get attached to a single myeloma cell, resulting in polyclonal antibodies. This is an important point that should be made clear before accepting the manuscript.
Our reply : Thank you for your very interesting point. To tell the truth, we did not use single-cell suspension in separate wells during fusion with myeloma cells, because we aimed at verifying the ratio of IgG to IgM production in various hybridoma cells obtained by five protocols for immunization in Fusion I to Fusion V. For this purpose, we would believe that the mixtures of hybridoma cells after each fusion would be informative.
The following sentence was added on page 3, lines 132 to 133.
“Single-cell suspension in separate wells during fusion with myeloma cells was not used”
- Time: The BCRs on the B cells interact with the membrane-bound CRHR1 on the myeloma cells, resulting in the formation of the immune synapse. This is stable only for few hours. The authors need to mention the time for incubation of B cells with myeloma cells.
Our reply : We completely agree to your suggestion. The time for incubation of sensitized B cells with antigen-expressing myeloma cells was 1 hour. A cell suspension containing sensitized B lymphocytes and antigen-expressing myeloma cells was allowed to stand for 30 min and then rotated for 30 min to form a B cell-myeloma cell complex.
We added the following sentence on page 3, lines 128 to 129.
“ by incubating for 30 min at 4 °C after slowly spinning down the cell suspension, followed by gentle rotation for another 30 min at 4 °C.”
- After immunization, the initial immune response is dominated by IgM antibodies due to the generation of IgM secreting short-lived plasma cells and IgM+ memory B cells. As the immune reaction proceeds, it is dominated by IgG antibodies. After various immunizations protocols mentioned in this study, the mice would be generating both IgM+ and IgG+ memory B cells specific for the antigens on CRHR1. The observed difference in the proportion of IgG+ and IgM+ hybridomas could be due to the difference in frequency of IgG+ and IgM+ B cells, or the difference in binding of IgG+ and IgM+ B cells to myeloma cells. Hence, it would be useful to show the frequency of IgM+ and IgG+ antigen-specific B cells present in the spleen after the immunizations in order to correlate it with the frequency of IgG+ and IgM+ hybridomas.
Our reply : We respect your opinion. We would think that the difference in the proportion of IgG+ and IgM+ hybridomas could correlate with the difference in binding of IgG+ and IgM+ B cells to antigen-expressing myeloma cell, because the rate of production for IgG-type monoclonal antibodies was much higher than IgM-type monoclonal antibodies in Figs. 3D and 3E. These data suggest that B lymphocytes producing higher affinity of IgG-type antibodies could be preferentially selected by antigen-expressing myeloma cells through BCRs to generate hybridoma cells in the optimized SST technique.
As to this concern, as you kindly suggested, we should have conducted the experiment on how frequently IgM+ and IgG+ antigen-specific B cells could be obtained from the spleen after the immunization in the present work. We would consider it as an important question which will be closely investigated in the future.
Submission Date
30 September 2021
Date of this review
16 Oct 2021 23:49:09
Reviewer 2 Report
Dear Author,
the present manuscript describes the yield of specific IgG directed towards GPCR (huCRHR1) by means of SST vaccination.
The approach is interesting and the article is well written. My concerns are:
1) which is the antigen presenting cell after DNA vaccination (DNA introduced during vaccination cycles before the cell-based vaccination of mice)?
2) please, make some hypotesis on the possibility that the antigen is processed before presentation in the spleen. Also, consider that the G-coupled protein at the surface of CHO-K1 should be exposed in its native conformation. Am I correct?
2) there are no error bars in figure n.2 and n.3. Error bars and statistic tests are necessary.
3) the number of mice per group should be specified, also in the figure legends.
Author Response
<Answers to Review 2 >
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
(x) Moderate English changes required
( ) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
( ) |
(x) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
( ) |
(x) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
(x) |
( ) |
( ) |
( ) |
Comments and Suggestions for Authors
Dear Author,
the present manuscript describes the yield of specific IgG directed towards GPCR (huCRHR1) by means of SST vaccination.
The approach is interesting and the article is well written.
Our reply: Thank you very much for your favorable comments.
My concerns are:
1) which is the antigen presenting cell after DNA vaccination (DNA introduced during vaccination cycles before the cell-based vaccination of mice)?
Our reply : Thank you for your very nice point. Most genes are introduced into muscle cells and keratinocytes after DNA vaccination, where huCRHR1 is expressed on the surface of the transfected cells to initiate an immune response. Please see “Introduction” on page 1, lines 26 to 29 in the original manuscript.
2) please, make some hypotesis on the possibility that the antigen is processed before presentation in the spleen.
Our reply :Thank you for your very good advice. We are pleased to tell that we have already described some hypothesis on the possibility in which the antigen is processed before presentation in the spleen. Please see page 7, lines 254 to 263 and page 8, lines 298 to 309 in the original manuscript.
Also, consider that the G-coupled protein at the surface of CHO-K1 should be exposed in its native conformation. Am I correct?
Our reply: Thank you for your very important point. Yes, you are correct. As we used a full length of huCRHR1 gene, huCRHR1 must be expressed on the cell surface of CHO-K1 cells retaining the native conformation. We have already verified the intact expression of huCRHR1 on CHO-K1 cells in the previous study [Ref. 10, Int. Immunopharmacol. 2021, 98, 107872] based on immunofluorescence analysis.
2) there are no error bars in figure n.2 and n.3. Error bars and statistic tests are necessary.
Our reply : Thank you for your very important suggestion. We added error bars in Figure 2. Since each well in Figure 3 was presented by a single measurement, which are typical data, we did not add any error bars. We are afraid that no statistic tests were performed. Standard deviation was added in the revised Figure 2. The following sentence was added on page 5, lines 202 to 203.
“Results are shown as the mean ± standard deviation (n = 2). Some error bars are hidden behind each symbol due to the small standard deviation values.”
3) the number of mice per group should be specified, also in the figure legends.
Our reply : We completely agree to your suggestion. We added the following sentences on page 3, lines 118 to 120 and on page 6, lines 226 to 227.
“Three mice per group were sensitized by DNA and cell immunization at one time. One of the sensitized mice was used for each experiment”
Submission Date
30 September 2021
Date of this review
19 Oct 2021 13:45:14
Round 2
Reviewer 1 Report
The authors have included information about the conditions for generating hybridomas. Since they have clearly mentioned that they have not used single-cell suspension, the possibility still holds that multiple B cells recognizing distinct epitopes on the CRHR1 would get attached to the myeloma cells expressing CRHR1, resulting in polyclonal antibodies generated by the resulting hybridoma. This should be made clear in the conclusion as well as the discussion section of the paper before it is accepted for publication.
Author Response
<Answer to Reviewer 1>
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
( ) |
(x) |
( ) |
( ) |
Comments and Suggestions for Authors
The authors have included information about the conditions for generating hybridomas. Since they have clearly mentioned that they have not used single-cell suspension, the possibility still holds that multiple B cells recognizing distinct epitopes on the CRHR1 would get attached to the myeloma cells expressing CRHR1, resulting in polyclonal antibodies generated by the resulting hybridoma. This should be made clear in the conclusion as well as the discussion section of the paper before it is accepted for publication.
Our replay : Thank you so much for your very valuable suggestion. We completely agree to your opinion. In our experimental procedures, as you expected, multiple cells are treated with the electrical pulse in a single chamber, and thus, some portion of the cell suspension might contain aggregates consisting of several B-cells and myeloma cells. Interestingly, when microscopically observing the diluted suspension that we prepared after mixing both cells, we used to observe a pair of a small B-cell tightly associated with a single myeloma cell, which is shown with an attached file (Ref. 9, Yamasaki et al. J. Immunol. Methods, 2020, 484-485, 112813, Fig. 1b). This would imply that the pair may happen to be selected among aggregated cells upon cell-fusion in our protocol. We have never been able to find a single hybridoma secreting multiple types of antibodies, and so, further limiting dilution could probably result in production of a monoclonal antibody in this case. However, of course, the present data could not completely rule out the possibility that hybridoma secreting multiple types of antibodies should be generated in the wells because IgM- and IgG-secreting cells were detected therein. As mentioned above, we believe that monoclonal antibody-expressing hybridoma cells can be obtained after conducting further limiting dilution.
To make it clearer about this point, we would like to add the following sentence to “Results and Discussion” on page 6, lines 215 to 220.
“For this purpose, we used mixtures of hybridoma cells obtained by fusion of each of sensitized B lymphocytes with a single myeloma cell using the optimized SST technique, resulting in producing polyclonal antibodies consisting of various kinds of monoclonal antibodies. It is noted that the previous microscopic observation suggested that a pair of a B-cell and a myeloma cell can often be selected to fuse with each other when applying the electrical pulse with our method [9].”
Submission Date
30 September 2021
Date of this review
01 Nov 2021 03:59:38
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Thanks for the explanations.
Author Response
<Answer to Reviewer 2>
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
( ) |
(x) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
( ) |
(x) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
( ) |
(x) |
( ) |
( ) |
Comments and Suggestions for Authors
Thanks for the explanations.
Our replay : Thank you so much for your favorable comments.
Submission Date
30 September 2021
Date of this review
04 Nov 2021 10:54:36
