PREreview of The Transcription Factor Bach2 Negatively Regulates Natural Killer Cell Maturation and Function

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.