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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.