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  1. 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.
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  2. 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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  3. 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.

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  4. 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.

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  5. 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).

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