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  1. Author Response:

    Reviewer #1 (Public Review):

    The authors show an important role of an RNA-binding protein (RBP), YTHDF2 in the accumulation of plasma cells. In addition, by a CRISPR/Cas9 knockout screening of RBPs, the authors suggest that some RBPs are involved in plasma cell differentiation. The roles of RBPs in a lymphocyte differentiation system are very interesting. The methods to detect germinal centre B cells and plasma cells could be improved.

    We thank the reviewer for their appraisal of our manuscript and have revised the manuscript to include some new data for clarity around technical issues and the interpretation of results.

    Reviewer #2 (Public Review):

    Turner et al. investigate the role for RNA binding proteins (RBPs) in regulating B cell to plasma cell differentiation in mice. They find sets of RBPs that control distinct phases of B cell differentiation including proliferation, survival, and the terminal differentiation of CD138+ plasma cells. They find only a few RBPs promote proliferation and hundreds of RBPs that control terminal differentiation. Follow up studies confirm the effect for select RBPs and the authors focus on the YTHDF2 gene which recognizes N6-methyladenosine in RNA. Using genetic deletion and bone marrow chimera models, the authors demonstrate a role for YTHDF2 in regulating plasma cell formation in response to NP-KLH immunization in both the spleen and bone marrow. Competitive bone marrow chimeras show that germinal centers and early B cell activation are normal in the absence of YTHDF2, but a significant decrease in bone marrow plasma cells is observed. The authors then using m6A-eCLIP and performed RNA-seq on the same cell types to define m6A modified transcripts. Contrary to the hypothesis, no enrichment for m6A-modified transcripts was observed for genes that repressed plasma cell formation and were predicted to be YTHDF2 targets.

    In its current form, the conclusions of the paper are not fully supported by the data. The number of samples per experimental group and whether experiments were reproducible across independent groups is not clear and needs to be clarified.

    Strengths: The area of RBP biology is underexplored in immune system function and the authors establish a powerful CRISPR/Cas9 sgRNA pool that will be a resource in the B cell field. Additionally, the use of sophisticated tools such as the two bone marrow chimera models, the tracking of NP-specific immune responses following NP-KLH immunization, and mapping of m6A by eCLIP allows for clear conclusions to be made.

    Weaknesses: It is not clear if sufficient replicates or statistics were used to demonstrate reproducibility and support the conclusions. For example, experiments in Fig 1C and 1F are critical to independently validate the results of the CRISPR/Cas9 screen, yet only 2-3 data points are presented, and no indication is given if the experiments were independently replicated across more than one cohort. Also, the same concern of independent replicates is raised for the data in Fig 2 and 3. Additionally, no evidence is provided that the ratios of Cas9+/Cas9- cells are statistically different from the NT controls. The fold-changes are small compared to the NT sample, and without flow cytometry data showing the percentage of CD138+ cells it is difficult to interpret what the true effect size is. Without this information, the authors conclusion that the CCR4-CNOT complex plays any role in plasma cell differentiation is not well supported.

    We thank the reviewer for their appraisal of the strengths and weaknesses of our manuscript and have used their feedback to improve the presentation and conclusions drawn. We have updated Figures 1C and 1F to include the requested statistical information. We have plotted the percentage of CD138+ cells in the Cas9- and Cas9+ cell populations for each sgRNA against our target genes and updated supplementary figure 1I. Independent replicates of the in vitro B cell culture tend to be variable in the proportion of accumulated CD138+ cells; within WT or non-targeting control conditions between 15-30% of cells may be CD138+. Hence, to enable the comparison of independent replicates we chose to use a coculture of Cas9+/Cas9- cells and compare the ratio of CD138+Cas9+/CD138+Cas9- cells.

    The data do not support the authors conclusion that IRF4 only affects B cell differentiation. IRF4 falls on the diagonal in the scatter plot in Fig 1D, indicating it also affects proliferation/survival. In fact, IRF4 has been previously shown to regulate B cell proliferation (Sciammas et al. 2006 Immunity) and differentiation to plasma cells.

    We thank the reviewer for pointing this out and have edited the text to clarify that our data supports a role for IRF4 in proliferation/survival quoting Sciammas et al.

    The validation of YTHDF2 and its role in plasma cell differentiation but not prior differentiation stages is a valuable section of the study. However, there are concerns about using only flow cytometry to measure very rare populations of plasma cells. From the data presented, roughly 8-10 plasma cells were counted per million cells.

    We are confident in the quantitative measurements that we’ve performed using flow cytometry because we analysed five million cells per sample to ensure that conclusions could be appropriately made on small populations. We show representative plots of our unimmunised controls to demonstrate the specificity of staining which underpins our confidence in these measurements.

    Reviewer #3 (Public Review):

    The mammalian genome contains thousands of RNA binding proteins. However, the importance of these proteins in regulating plasma cell differentiation is largely unknown. The authors sought to identify RNA binding proteins regulating the differentiation of plasma cells. They achieved this aim by using a Crispr-Cas9 screen to identify 292 RNA binding proteins that regulate the differentiation of CD138+ cells in vitro. This study effectively demonstrated the utility of Crispr-Cas9 screens in identifying factors regulating B cell differentiation.

    One limitation of this study is that the RNA binding proteins identified as regulating the differentiation of CD138+ cells in vitro may not necessarily have the same role in vivo. While the authors validated that the RNA m6A binding protein YTHFD2 regulated plasma cell differentiation following protein immunization, additional work will be required to determine the relevance of other RNA binding proteins identified in their screen. An additional limitation of this study is that the authors did not determine the mechanisms by which YTHFD2 promotes plasma cell differentiation. This lack of mechanistic insight limits the utility of this study in providing a conceptual advance in the understanding of the processes governing plasma cell differentiation. However, the results of their screen will still likely be a useful resource for the future work seeking to more precisely understand how RNA binding proteins regulate B cell differentiation.

    We thank the reviewer for their analysis and feedback of our presentation which we have used to produce an improved manuscript. We agree that the cellular and molecular mechanisms by which YTHDF2 regulates plasma cell accumulation is not explained by this paper. We submitted this manuscript for consideration as a short paper. The work on YTHDF2 was intended to further validate one of our screen hits as being required for plasma cell accumulaiton. An in depth resolution of the mechanism of YTHDF2 action will require several additional years of study. The findings on YTHDF2 that we have made are consistent with those of Grenov et al whose paper has since been published.

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  2. Evaluation Summary:

    This paper utilizes an elegant Crispr-Cas9 screen to identify RNA binding proteins that may regulate B cell differentiation. With some additional work to verify that the identified proteins are important in vivo, the paper will be of interest to a broad audience of immunologists studying the signals regulating B cell differentiation during an immune response.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The authors show an important role of an RNA-binding protein (RBP), YTHDF2 in the accumulation of plasma cells. In addition, by a CRISPR/Cas9 knockout screening of RBPs, the authors suggest that some RBPs are involved in plasma cell differentiation. The roles of RBPs in a lymphocyte differentiation system are very interesting. The methods to detect germinal centre B cells and plasma cells could be improved.

    Read the original source
    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    Turner et al. investigate the role for RNA binding proteins (RBPs) in regulating B cell to plasma cell differentiation in mice. They find sets of RBPs that control distinct phases of B cell differentiation including proliferation, survival, and the terminal differentiation of CD138+ plasma cells. They find only a few RBPs promote proliferation and hundreds of RBPs that control terminal differentiation. Follow up studies confirm the effect for select RBPs and the authors focus on the YTHDF2 gene which recognizes N6-methyladenosine in RNA. Using genetic deletion and bone marrow chimera models, the authors demonstrate a role for YTHDF2 in regulating plasma cell formation in response to NP-KLH immunization in both the spleen and bone marrow. Competitive bone marrow chimeras show that germinal centers and early B cell activation are normal in the absence of YTHDF2, but a significant decrease in bone marrow plasma cells is observed. The authors then using m6A-eCLIP and performed RNA-seq on the same cell types to define m6A modified transcripts. Contrary to the hypothesis, no enrichment for m6A-modified transcripts was observed for genes that repressed plasma cell formation and were predicted to be YTHDF2 targets.

    In its current form, the conclusions of the paper are not fully supported by the data. The number of samples per experimental group and whether experiments were reproducible across independent groups is not clear and needs to be clarified.

    Strengths:
    The area of RBP biology is underexplored in immune system function and the authors establish a powerful CRISPR/Cas9 sgRNA pool that will be a resource in the B cell field. Additionally, the use of sophisticated tools such as the two bone marrow chimera models, the tracking of NP-specific immune responses following NP-KLH immunization, and mapping of m6A by eCLIP allows for clear conclusions to be made.

    Weaknesses:
    It is not clear if sufficient replicates or statistics were used to demonstrate reproducibility and support the conclusions. For example, experiments in Fig 1C and 1F are critical to independently validate the results of the CRISPR/Cas9 screen, yet only 2-3 data points are presented, and no indication is given if the experiments were independently replicated across more than one cohort. Also, the same concern of independent replicates is raised for the data in Fig 2 and 3. Additionally, no evidence is provided that the ratios of Cas9+/Cas9- cells are statistically different from the NT controls. The fold-changes are small compared to the NT sample, and without flow cytometry data showing the percentage of CD138+ cells it is difficult to interpret what the true effect size is. Without this information, the authors conclusion that the CCR4-CNOT complex plays any role in plasma cell differentiation is not well supported.

    The data do not support the authors conclusion that IRF4 only affects B cell differentiation. IRF4 falls on the diagonal in the scatter plot in Fig 1D, indicating it also affects proliferation/survival. In fact, IRF4 has been previously shown to regulate B cell proliferation (Sciammas et al. 2006 Immunity) and differentiation to plasma cells.

    The validation of YTHDF2 and its role in plasma cell differentiation but not prior differentiation stages is a valuable section of the study. However, there are concerns about using only flow cytometry to measure very rare populations of plasma cells. From the data presented, roughly 8-10 plasma cells were counted per million cells.

    Read the original source
    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    The mammalian genome contains thousands of RNA binding proteins. However, the importance of these proteins in regulating plasma cell differentiation is largely unknown. The authors sought to identify RNA binding proteins regulating the differentiation of plasma cells. They achieved this aim by using a Crispr-Cas9 screen to identify 292 RNA binding proteins that regulate the differentiation of CD138+ cells in vitro. This study effectively demonstrated the utility of Crispr-Cas9 screens in identifying factors regulating B cell differentiation.

    One limitation of this study is that the RNA binding proteins identified as regulating the differentiation of CD138+ cells in vitro may not necessarily have the same role in vivo. While the authors validated that the RNA m6A binding protein YTHFD2 regulated plasma cell differentiation following protein immunization, additional work will be required to determine the relevance of other RNA binding proteins identified in their screen. An additional limitation of this study is that the authors did not determine the mechanisms by which YTHFD2 promotes plasma cell differentiation. This lack of mechanistic insight limits the utility of this study in providing a conceptual advance in the understanding of the processes governing plasma cell differentiation. However, the results of their screen will still likely be a useful resource for the future work seeking to more precisely understand how RNA binding proteins regulate B cell differentiation.

    Read the original source
    Was this evaluation helpful?