The transcription factor Bach2 negatively regulates murine natural killer cell maturation and function

  1. Shasha Li
  2. Michael D Bern
  3. Benpeng Miao
  4. Changxu Fan
  5. Xiaoyun Xing
  6. Takeshi Inoue
  7. Sytse J Piersma
  8. Ting Wang
  9. Marco Colonna
  10. Tomohiro Kurosaki
  11. Wayne M Yokoyama

  • Curated by eLife

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    Evaluation Summary:

    The strength of this paper is identifying a novel factor Bach2 that might be involved in the generation and/or maintenance of NK cells. To date, the full molecular network guiding the development, maturation and maintenance of NK cells has not been fully defined and Bach2 has not yet been investigated.

    (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. The reviewers remained anonymous to the authors.)

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Abstract

BTB domain And CNC Homolog 2 (Bach2) is a transcription repressor that actively participates in T and B lymphocyte development, but it is unknown if Bach2 is also involved in the development of innate immune cells, such as natural killer (NK) cells. Here, we followed the expression of Bach2 during murine NK cell development, finding that it peaked in immature CD27 + CD11b + cells and decreased upon further maturation. Bach2 showed an organ and tissue-specific expression pattern in NK cells. Bach2 expression positively correlated with the expression of transcription factor TCF1 and negatively correlated with genes encoding NK effector molecules and those involved in the cell cycle. Lack of Bach2 expression caused changes in chromatin accessibility of corresponding genes. In the end, Bach2 deficiency resulted in increased proportions of terminally differentiated NK cells with increased production of granzymes and cytokines. NK cell-mediated control of tumor metastasis was also augmented in the absence of Bach2. Therefore, Bach2 is a key checkpoint protein regulating NK terminal maturation.

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  1. Version published to 10.7554/elife.77294 on eLife
  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6914092.

    This full PREreview is the result of a live-streamed preprint journal club organized and hosted by PREreview and eLife as the final module of the Open Reviewers workshop. We thank all eLife Ambassadors who contributed to the discussion and made it possible for us to provide feedback to this preprint. 

    Summary paragraph

    In this manuscript, the authors studied the role of Bach2, a transcription repressor known to be involved in B and T cell development, and found it is also involved in natural killer (NK) cell development. In this study, Bach2 was proposed as a checkpoint protein regulating NK cell maturation. Intriguingly, the authors found that (1) Bach2 expression is correlated with the expression of the transcription factor TCF1 and is indirectly correlated with tumor metastasis, (2) Bach2 does not play a role in the maturation and function of NK cells, which earlier was studied only in B and T cells, (3) NK cells in the knockout mice had more cytotoxic activity and those cells were able to limit the metastasis of tumor cells more than the NK cells in the control mice and (4) The authors showed that Bach2 expression is inversely correlated with NK maturation and associated with potential impacts in restricting tumor growth.

    The study forwards an intriguing hypothesis of Bach2 being a potential target for cancer immunotherapy. The findings in this preprint will be helpful for future researchers in the cancer therapeutics field.  

    Below we list major and minor issues and suggestions on how to address them, hoping that the authors will find our feedback helpful and constructive. 

    List of major issues and feedback

    The reviewers believe that addressing the following "major" issues would significantly strengthen the conclusion of the study: 

    1. The limited number of animals used in the study may reduce the power of the statistical analysis. This calls for additional validation of the findings using an animal cohort with sufficient statistical power. The authors could discuss this as a proof-of-concept study and what hypotheses were drawn or would be useful to validate in a larger animal cohort. Additionally, some findings were based on 3 animals and some on 4. Please address this number inconsistency.

    2. We found the abstract challenging for a broad readership. The content was highly field-specific and the significance of their findings unclear to the wider scientific community. 

    3. The introduction was too broad and made it difficult for the reader to identify the key concepts the paper would be based on. For example – only those proteins and their relationships related to the study should represent the focus of the introduction. Authors could improve the introduction to make it clear and concise. For example, the authors could add a schematic representation of the key proteins involved in the pathway.

    4. The changes in mRNA level found might not show a significant reflection in protein expression. Therefore, Western Blot results from knock-out mice would have strengthened their evidence of the claim made in this paper. 

    5. It is good that most of the author's conclusions are supported by data and results based on correlations. However, further experiments are crucial to validate the effects of Bach2 as a target for immunotherapy. The direct interaction between the Bach2 and other maturation factors could be investigated. This comment also applies to the NK cell reduction – for example, it is key to understanding how complete the KO is (complete loss at protein level, or any leakiness of mouse genetics). Substantial IP experiments could be included to justify the conclusions. Please rewrite the conclusion to state that these are promising experiments requiring further validation as well as citing relevant literature.

    6. The RNA sequencing studies are missing details. The authors have not shown how they normalized the heatmap. Furthermore, the codes and detail parameters that were used to prepare the figures are missing. To address code reproducibility, please provide these details either in the Supplementary or upload code onto Zenodo for indexing. Additionally, please mention if this data would be made publicly available.

    7. Western blot figure (1E) does not have loading controls. The authors must add loading controls and show the corresponding uncropped Ponceau-stained gel images. The full image of WB should be included, not only the bands. There is a missing WB image as the authors mentioned in the text (Supplemental Fig S1D). 

    8. A graphical summary figure of the study could be included to help portray the research findings and enhance readability. 

    9. Limitations and future work to follow up on current findings should be addressed in the Discussion section.

    10. The figures need to be well-organized according to the numbers and placed with proper alignment. The panel mentioned to be included in supplementary figure 1 is missing. Please update this

    11. The study consists of findings on a single cell line. It would strengthen the study by mentioning why this was the case and what future experiments are planned for multiple cell lines.

    List of minor issues and feedback

    1. Consider less-crowded figure panels (for example in Figure 3). Too many figure panels in Fig. 2 made the paper difficult to follow. 

    2. The labeling of figure images needs extra care to adjust the axes of some graphs, labels, and margins.

    3. Authors should include appropriate units and labels for each figure corresponding to the wavelength of excitation/emission spectra to specify the X and Y-axis. 

    4. Discuss the number of replicates and cell population for the bulk-sequencing data

    5. Please ensure to go over the entire text to correct for grammar and typographical errors, eg. line 202: extra '.' after Cd69.

    6. Recommendations: It is highly recommended that the authors include a video of their methods in the Supplementary which would be great to enable open science. If possible, authors could have utilized CRISPR-Cas9 for knock-out mice and also done gene overexpression studies. 

    We thank the authors for sharing their work as a preprint. We hope our feedback above will be helpful as they consider any revisions to the manuscript or future lines of work.

    Competing interests

    The author declares that they have no competing interests.

  3. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6819295.

    Review Comment 11/07/2022

    The decision is: ACCEPTED after major revision

    Reviewer #1: I read carefully the manuscript entitle "The Transcription Factor Bach2 Negatively Regulates Natural Killer Cell Maturation and Function" needs major revision before publishing. This research aims to create a space to generate dialogue important in shaping and responding to questions about the drive in assessing the role of Bach2, a transcription repressor, in the process of maturation and functional state of NK cells. The manuscript offers a very interesting and important theme, for global agenda. The abstracts of the papers however, vary in the level of detail they each provide regarding their focus, methods and aims.

    · The abstract is not a true representative of the content and contributions of the paper. The abstract does not seem to properly convey the rigor of research. However, Abstract should lay out five key points:

    Rationale (1-2 sentences) - why was the research needed?

    Objective (1 sentence)- what were you trying to provide to meet that need?

    Method(s) (up to 3 sentences) - briefly summarize what and which parameters were measured.

    Results (up to 4-5 sentences)- what did you find? Please add some data to demonstrate the findings.

    Conclusions/Recommendations (1 sentence) - so what should be done with or in response to your findings?

    o Keywords are absents.

    o It is better to explain more about the novelty of manuscript in introduction section. Please explain about its novelty. Thus, the introduction section need to be improved, it needs significant amount of reorganization. It could be strengthened by adding more recent references. The authors should mention the relevant and recent related studies to give information to readers about the current knowledge.

    o The language can be substantially improved for clarity, concision, better grammar and logic. Thus, the manuscript needs to be properly formatted and resubmitted?

    o The purpose or purported significance of the article is not explicitly stated. The authors should explicitly state the novel contribution of this work, the similarities and the differences of this work with their previous publications.

    o The research contributions of the paper should be articulated more clearly.

    o You have a lot of hanging statement in your work.

    o The justification of the study is missing. Justification for using a specific methodology or instrument will make it more understandable. Adding more details in this section can give more clarity to the readers.

    o Authors should use transitional words mindfully to highlight clear and thoughtful connections among ideas.

    o Author research study methods need to be improved. Kindly focus on three basic elements of the methods section.

    a. How the study was designed?

    b. How the study was carried out?

    c. How the data were analyzed?

    o Also, authors materials and methods should come under Results

    o The result section needs to be written clearly for better understanding. More explanation about details of result is needed.

    o Make sure that all the tables and figures are quoted in the text and in correct order with proper labelling, too.

    o Discussion section is in line.

    o Make sure that all the tables and figures are quoted in the text and in correct order, too.

    o Use your data analysis to expand reader understanding why each of these variables matter and its implication.

    o Conclusion is absent.

    o Add the specific value, problems and challenges of the findings in the conclusion.

    o Add 2-3 lines about future recommendation or implications of research in last portion.

    o Each citation and reference need careful checking for accuracy of comment in the text. This is a very important and basic point in writing scientific papers; if the paper does not say what the authors are saying then it MUST NOT be cited in the text.

    o There is enough new content in this paper to distinguish it from other works.

    o The submission provides enough new material for journal publication. This suggestion would strengthen the study further and when addressed will improve the manuscript.

    Best regards.

  4. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6604790.

    Review of: Li, et al. February 14, 2022. "The Transcription Factor Bach2 Negatively Regulates Natural Killer Cell Maturation and Function." BioRxiv. DOI: https://doi.org/10.1101/2022.02.14.480364

    This review was written by an undergraduate student at Mount Holyoke College (MA, USA) who selected this preprint for an assignment in a course on peer review taught by Dr. Rebeccah S. Lijek, Assistant Professor of Biological Sciences. 

    Disclosures: The review author declares no conflict of interest and has no personal or financial relationship with the study's authors. The reviewer acknowledges a limitation of this review, as it is written by an undergraduate student and not a practicing immunologist. We thank the authors for sharing their manuscript as a preprint.

    Summary

    The manuscript investigates the role of the transcription repressor, Bach2, in the development of natural killer cells. Splenic NK cells were used for RNA-Seq technology to study protein expression in Bach2 conditional knockout mice with qPCR and western blot validation. Flow cytometry was conducted to evaluate cellular maturation stages. The authors found that NK cells without Bach2 had increased terminal differentiation and augmentation in tumor metastasis. I believe that the findings presented in this manuscript have the potential to provoke future research in immunology and cancer biology.

    Strengths

    • This study's findings are novel, as the role of Bach2 in NK cell development has yet to be elucidated. We have a limited understanding of the numerous factors at play in natural killer cells and this research is crucial. Bach2 has been studied in B and T cells but looking at it from an innate perspective is interesting and could prove to be valuable for our understanding of cellular immune development and function. 
    • Descriptions of natural killer cell development and the unique transcription factors were well explained. The narrative of NK cell maturation was clear and well explained throughout. This improves the readability and understanding of the scientific concepts at work.
    • The results shown do a nice job in supporting the key conclusion that Bach2 negatively regulates NK maturation and function. These findings were well displayed and discussed to make the overarching interpretations understandable. 

     

    Major Critiques

    • The B16F10 metastasis assay had very interesting results. But the findings were suggested to be that the composition of the natural killer cell subset in the Bach2-deficient mice resulted in an increase in the control of tumor metastasis. This was determined by counting the number of black colonies formed in the lungs. It remains unclear to me how, or even if, the changes in NK subsets are causing these changes. Please either perform functional analyses to ascertain how the effector functions may be an influence on the metastatic control or include a statement regarding the limitations of these results.
    • I would appreciate further explanation surrounding the influence of Bach2 on the observed phenotypes. Specifically in the section 'Bach2-deficiency drives a transition from immature stem-like phenotype towards mature effector phenotype of NK cells.' This would benefit the reader's understanding of Bach2's mechanisms as well as having the potential to be of use to future manuscripts or studies that are looking at Bach2 or innate immune cell maturation. 
    • Some of the experimental groups used are small. I think it would be helpful to either acknowledge the limited sample size or perform a power analysis to show what a meaningful group would consist of. 

    Minor Critiques

    • In the section discussing RNA-seq analysis, cellular phenotypes were discussed as they related to the expressed genes. If effector phenotypes or terminal differentiation are going to be discussed without corresponding assays, it would be helpful to have additional information or citations to explain why we can presume that these phenotypic differences would be seen. 
    • Much of the data presented in the figures are a representation of the samples. I believe that including raw data and statistics, perhaps in a supplement, could allow readers to better evaluate the trends and consistency of the results. 
    • This study used splenic NK cells from mice throughout the study, this population is distinct and has a majority CD56dim CD16+ phenotype. Human NK cells and the CD56bright phenotype are brought up in the discussion section. It could be helpful to include how the results may differ - or not - based on the choice to use murine splenic NK cells.
  5. eLife

    Evaluation Summary:

    The strength of this paper is identifying a novel factor Bach2 that might be involved in the generation and/or maintenance of NK cells. To date, the full molecular network guiding the development, maturation and maintenance of NK cells has not been fully defined and Bach2 has not yet been investigated.

    (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. The reviewers remained anonymous to the authors.)

  6. eLife

    Reviewer #1 (Public Review):

    The strength of this paper is identifying a novel factor that might be involved in the generation and/or maintenance of NK cells. To date, the full molecular network guiding the development, maturation and maintenance of NK cells has not been fully defined.

    While the approach aims to dissect out this network and identified a number of differentially expressed genes, relatively little refinement or validation of that network has been performed. Thus, the study is somewhat descriptive and awaits a more detailed analysis and confirmation of interacting molecular partners.

  7. eLife

    Reviewer #2 (Public Review):

    Transcription factors represent key regulators of NK cell development and function. This study compared two mouse strains with or without Bach2 expression in NK cells for NK cell development, maturation and function. The phenotype of wild-type and Bach2-deficient NK cells were investigated by flow cytometry and transcriptomic profiling. The effector function of NK cells was evaluated in a mouse model of NK cell-mediated protection of tumor metastasis in the lung. Authors conclude that Bach2 expression in NK cells negatively regulates NK cell maturation and effector function.

    The results support the key conclusion. However, some experimental information was missing and additional analysis of NK cell function in the tumor model can improve the knowledge gained from this study.

  8. eLife

    Reviewer #3 (Public Review):

    Motivated by previously described roles for BACH2 in T cells and B cells, Li et al examine the role of BACH2 in steady-state NK cell differentiation. They first examine the kinetics of BACH2 expression during NK cell development and differentiation using a reporter mouse line. After BACH2 initially decreases in expression once common lymphoid progenitors commit to the NK lineage, BACH2 expression is regained within mature NK cells across multiple anatomical sites. Interestingly, within mature NK cells, BACH2 expression is lost within terminally differentiated NK cells, which is analogous to the BACH2 expression patterns seen in exhausted CD8+ T cells. Subsequent phenotyping of mice with conditional BACH2 deletion within NK cells revealed an accumulation of more effector-like, terminally differentiated NK cells at the expense of less differentiated populations. Challenge of these conditional knock-out mice using a lung melanoma metastasis model known to be highly NK cell sensitive revealed superior tumour control. Collectively, this suggests that BACH2 restrains differentiation of terminally differentiated effector NK cell subsets and that targeting BACH2 in NK cells could represent a novel strategy for cancer immunotherapy.

    Strengths

    This is the first paper to comprehensively describe BACH2 expression patterns across NK cell development and differentiation, and the first paper to describe a function for BACH2 in NK cells. There are many interesting parallels between BACH2 expression patterns and function in NK cells, and those previously reported in CD8+ T cells, suggesting a conserved function for BACH2 in both contexts. Nevertheless, there are also some interesting differences. Of note, one of the key mechanisms by which BACH2 controls exhausted CD8+ T cell differentiation is through repression of Blimp-1 and BATF, however, neither factor appears to be up-regulated after BACH2 loss in NK cells suggesting key mechanistic differences. Finally, the authors provide interesting data suggesting that the boosted effector function of BACH2 knock-out NK cells leads to elevated tumour control, which represents a novel approach to boosting NK-dependent tumour control.

    Weaknesses

    There are a few key weaknesses that limit the impact of the study. In particular, the mechanistic explanation for how BACH2 mediates the observed phenotypes is very limited. Furthermore, published work on BACH2 biology rules out some of the authors' claims. For example, the authors speculate that BACH2 operates similarly in TCF1+ exhausted CD8+ T cells and NK cells, as in both cases knock-out cells lose TCF1 expression. However, as noted above, the effects of BACH2 on TCF1 in CD8s are indirect and due to both repression of Blimp-1 and BATF expression, and antagonism of RUNX3 and BATF binding site accessibility. Given that Blimp-1 and BATF expression is unchanged in BACH2 knock-out NK cells, this argues that there are significant mechanistic differences between how BACH2 operates in CD8+ T cells versus NK cells. Moreover, unlike CD8+ T cell phenotypes, many of the phenotypic changes in BACH2 knock-out NK cells are very subtle again hinting at underlying differences in the mechanism.

    The authors suggest that the changes in NK cell subset composition are responsible for the enhanced control of melanoma lung metastases in knock-out mice, but alternative explanations are not explored. For example, it is possible that enhanced NK cell homing to the lung could explain these phenotypes. Data examining knock-out NK cell effector functions (eg. ex vivo killing capacity and cytokine production) are also not included, so it remains unclear whether the subset changes in knock-out mice lead to a meaningful difference in effector capacity.

    Finally, many of the figures show representative data without pooled data points and statistics, making it difficult to evaluate how consistent the reported trends are. Additionally, some experimental groups are small and would benefit from more mice, particularly for key experiments (eg. the melanoma metastasis data).

  9. Version published to 10.1101/2022.02.14.480364v1 on bioRxiv