Evaluating the IgMi mouse as a novel tool to study B‐cell biology

The IgMi mouse fails to secrete antibodies or class switch its BCR from IgM. Our study reveals that other cellular compartments, including B‐cell subsets, DC subsets, GC B cells and TFH cells are perturbed in the IgMi mouse, thus presenting important additional considerations when using the mouse to explore the role of secreted antibody.

In more details, we think that you should modify the manuscript so that: A revised version of your manuscript that takes into account the comments of the referees will be reconsidered for publication. Should you disagree with any of the referees™ concerns, you should address this in your point-by-point response and provide solid scientific reasons for why you will not make the requested changes.
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Once again, thank you for submitting your manuscript to European Journal of Immunology and we look forward to receiving your revision. Therefore, this study is of potential interest to clarify the regulatory role of soluble antibodies and the regulatory capacity of B cells.

Comments:
The abstract does not really provide all essential information about the IgMi mouse model. Not everyone is familiar with this model. Please explain the relevance of this mouse model in more detail.
In their manuscript the authors use a mouse model named IgMi and refer to the following reference: Waisman et al., 2007. In Waisman et al., 2007 Based on the finding that IgMi mice harbor more GL7+ GC cells: What about plasmablasts and plasma cells. Are they generated in this mouse? Is there an enrichment of IgM+ plasmablasts?

Statistics:
Please clearly state the total number of mice used for every experiment. Sometimes it is not clear; Figure   2: "Data are pooled from two experiments, n=4". Is that n=8 in total? Or was it two times 2 animals resulting in n=4. Please clarify.
For n=4 a t-test is not appropriate because of its low statistical power for small n. Please use other appropriate statistical tests.

Reviewer: 2
Comments to the Author In this study, Sahputra et al. report the characterization of cell subsets in IgMi mice that cannot secrete antibodies compared to controls. They find alteration in B cell subsets, DC subsets, and TFH cells.
Although this is possibly of interest for people who are considering working with these mice, these descriptive results do not reach the level of significance necessary for publication in the European Journal of Immunology. Part of these findings are already known eg B cell subsets abnormalities in the absence of secreted Abs (muS mice or Aid-/-muS mice). Figure 3 is missing in the figure files (cited in line 148).

Reviewer: 3
Comments to the Author Overall this is a very useful study. Personally, I have wondered why more information on the IgMi mouse was not available, so this is a welcome addition to the literature.
There is always more to do, therefore I will not ask for additional experimentation. I have only minor comments.
It would be helpful for the authors to emphasize the degree and timing with which WT and IgMi/i mice were housed together. This is particulary important for the microbiota data in Figure 6, but may also be relevant to several other figures. Moreover, the authors should consider emphasizing the caution with which the microbiota data are discussed. The microbiota literature is becoming clogged with conflicting reports, and at least some of this reflects different approaches to housing and co-housing genotype-disparate mice. Really drilling down on these issues can be quite involved (see PMID 28614717, for example).

05-Sep-2018
Responses to Reviewers Throughout this response letter, the comments of reviewers are in normal text and underlined and our responses are italicised, and indicated by referring to text lines where the amends appear in our revised manuscript Executive Committee: Please convert your manuscript to a Letter to the Editor, focus on the major findings and use referees' comments as a guideline to improve the study. We have now converted our manuscript to a Letter to the editor style   We have now analysed transitional B cells on the spleen of IgMi and WT littermate controls. We find that this population of B cells is increased. We have now included this data in Figure 1G and commented in the text line 24.

Is B cell development in the bone marrow completely normal? We have now conducted extra experiments analysing B cells in the bone marrow compartment of IgMi and WT C57BL/6 controls and
found that pre-B cells and immature B cells were increased whilst mature B cells were decreased in the IgMi mouse. We have added this data to the supporting data ( Supplementary Fig.1) and a description in the text at line 28-30.
In Figure 2 the authors present data that show an increase in B1 cells and MZB cells. Are the cell numbers normalized to the increased number of total splenocytes? Our data for B1 cells and MZB cells is presented as total cells and so is normalised to the increased number of total splenocytes. Total cell numbers are shown in Figure 2 What about CD4 and CD8 T cells in the Spleen (and the MLN)? We saw no significant differences in CD4 and CD8 T cells and have added a comment in the text, line 58-59 to mention this as "data not shown".
Is there more output from the bone marrow? We know that B cell development is altered in the bone marrow of IgMi mice as we have now conducted extra experiments as stated above. Thus we find that pre-B cells and immature B cells were increased whilst mature bone marrow B cells were decreased in the IgMi mouse. We have added this data to supporting data ( Supplementary Fig.2) and a description in the text at line 28-30.
Did the authors analyze the B1 compartment in the peritoneum as well? We did analyse B1 cells in the peritoneum. Please see Figure 1E,F In Figure 4 the authors nicely show an increase in GL7+ GC B cells.
Are IgMi B cells "hyperactive" in general? Is there in vitro data available (LPS Stimulation, anti CD40/IL4 etc..) regarding proliferation, GL7 upregulation, apoptosis? Please clarify this point. We have stimulated IgMi B cells with LPS and these cells certainly make significantly higher levels of IL-10 (Fig 2I,J). Further we also know that the number of GL7+ cells in IgMi are increased after an infection with an intestinal nematode parasite, compared to WT control levels (unpublished). We have now conducted extra experiments analysing proliferation/apoptosis of GC B cells in MLNs. We found that the proliferation of Ki67+ GC B cells was significantly increased in IgMi mice, whilst the apoptosis Casp-3+ GC B cells was significantly decreased. We have added this data in supplementary Figure 2 and have added a comment in the text, line 50-51.
Based on the finding that IgMi mice harbor more GL7+ GC cells: What about plasmablasts and plasma cells. Are they generated in this mouse? Is there an enrichment of IgM+ plasmablasts? Plasma cells and plasmablasts are generated in IgMi mice and littermate controls.
Levels are very low under the steady state conditions we have been analysing and do not differ between genotype. We have now added a comment to the text line 52-54 to mention this as "data not shown".

Statistics:
Please clearly state the total number of mice used for every experiment. Sometimes it is not clear; Figure   2: "Data are pooled from two experiments, n=4". Is that n=8 in total? Or was it two times 2 animals resulting in n=4. Please clarify. We apologise that this was not clear; we have amended our text to make it clear that n=8 in total For n=4 a t-test is not appropriate because of its low statistical power for small n. Please use other appropriate statistical tests. We have reanalysed our data using Mann-whitney test.

Reviewer: 2
Comments to the Author In this study, Sahputra et al. report the characterization of cell subsets in IgMi mice that cannot secrete antibodies compared to controls. They find alteration in B cell subsets, DC subsets, and TFH cells.
Although this is possibly of interest for people who are considering working with these mice, these descriptive results do not reach the level of significance necessary for publication in the European Journal of Immunology. Part of these findings are already known eg B cell subsets abnormalities in the absence of secreted Abs (muS mice or Aid-/-muS mice).
We are disappointed to hear that this reviewer didn't feel that our results were significant to warrant publication in EJI. We do note that both the other referees concurred that the data was important and useful to other workers in the field. We hope that by converting our manuscript to a Letter to the Editor the reviewer will now support publication Comments: Supplementary Figure 3 is missing in the figure files (cited in line 148). Our figures have now changed given the conversion to a Letter to the editor style.

Reviewer: 3
It would be helpful for the authors to emphasize the degree and timing with which WT and IgMi/i mice were housed together. This is particularly important for the microbiota data in Figure 6, but may also be relevant to several other figures.
Moreover, the authors should consider emphasizing the caution with which the microbiota data are discussed. The microbiota literature is becoming clogged with conflicting reports, and at least some of this reflects different approaches to housing and co-housing genotype-disparate mice. Really drilling down on these issues can be quite involved (see PMID 28614717, for example).
We agree that this is a very important point especially in the context of microbiota data. Throughout our studies we have bred our IgMi and WT mice from crossing heterozygote parents therefore mice are littermate controls and have been co-housed from birth. We have re-emphasised our breeding strategy and present caution when discussing our microbiota data (lines 75-78) Note: typo - Figure 1C is actually 1B. Apologies -figures have now changed with reformatting to Letter to editor style

21-Sep-2018
Dear Dr. Else, It is a pleasure to provisionally accept your manuscript entitled "Evaluating the IgMi mouse as a novel tool to study B cell biology" for publication in the European Journal of Immunology. For final acceptance, please follow the instructions below and return the requested items as soon as possible as we cannot process your manuscript further until all items listed below are dealt with.
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