Prdm1 Positively Regulates Liver Type 1 ILCs Cancer Immune Surveillance and Preserves Functional Heterogeneity

Reviewer #1 (Public Review):

He et al. investigate the requirement and function of Blimp1 (encoded by Prdm1) in murine NK cells and ILC1. Employing a conditional knockout mouse model (Prdm1flox x Ncr1cre), the authors describe impaired abundance and maturation of Prdm1-deficient NK cells and ILC1 in different tissues. Blimp1-deficient NK cells have reduced expression of cytotoxic molecules (Gzmb, Prf1) and, in some instances, Ifng production, and Prdm1flox x Ncr1cre mice show impaired tumor control in experimental metastasis models. Using single-cell RNA sequencing analysis, the authors propose that Prdm1 regulates JunB expression and NK cell maturation. Based on in silico analyses, the authors suggest manifold intercellular communication between NK/ILC1 and macrophages. Without following up on any of these potentially interesting suggestions, the authors conclude their study reiterating that Prdm1 regulates IFNg-production of tumor-infiltrating NK cells and ILC1.

Many of the reported functions of Blimp1 in NK cells have previously been identified using a mixed-chimera strategy comparing Prdm1 WT and KO NK cells (Kallies et al., Blood 2011). Here, the authors expand on these findings using a conditional model to delete Prdm1 in NK/ILC1 and single-cell sequencing and provide a more refined analysis of the functions of Blimp1 in these cells. Cell-chat analysis suggests close interactions of Blimp-dependent NK/ILC1 subsets with hepatic macrophages, but these suggestions are not followed up by experiments. Potentially interesting differences in the macrophage compartment of Ncr1-Cre x Prdm1-fl/fl mice are suggested by the scc-RNA-Seq data but are not validated e.g. by FACS. The study falls short in providing new mechanistic insights. Nevertheless, it is an interesting confirmation of "old" suggestions in a more refined setting, and the provided single-cell mRNA-Seq data represents a potentially valuable resource for the community. There are some control analyses that are required to support the conclusions of the authors, and I have a few suggestions that would help to improve the manuscript.

Major comments:

- The authors do not control for the potential effects of Cre expression. Expression of Cre from within the Ncr1 locus (using the mouse model established by Narni-Mancinelli et al.) has significant effects on NK cells and especially ILC1s (reducing their frequency and absolute numbers and altering their functionality. The authors should characterize the Ncr1cre mice used here (developed by Shanghai Model Organism Center) in this regard and should use proper controls (Ncr1Cre+ Prdm1wt/wt as control for Ncr1Cre+ Prdm1fl/fl, instead of WT littermates) for all of their key data, e.g. those depicted in Fig 1FG, 2ADFH, 7D, S2,3,4.

- Several of the phenotypic findings on NK cells have been described before by Kallies et al. in 2011 (Ref 29), although using a different genetic Prdm1-ablation model (Prdm1-GFP/GFP knockin/knockout model). This study reported impaired NK cell maturation, reduced Gzmb expression, impaired in vivo cytotoxicity against subcutaneous RMA-S cells, impaired in vitro proliferation, comparable in vitro killing, increase in BM NK cell numbers. The authors should discuss/mention this more prominently in their manuscript, and highlight where they confirm or refine these previous findings, and where they actually provide new information.

- What is the reason to refer to the enriched cluster in Blimp1-deficient NK cells as "Junbhi"? There is no follow-up for a function of Junb, and there are many other genes upregulated in these cells. Most critically, these cells seem to represent exactly the c-Kithi cells that Kallies et al. already showed and discussed in their paper. The authors should stain for Kit, and also refer to this. Also, MacKay et al. performed Blimp1-Chip-Seq (in T cells), maybe it would be interesting to check to which of the identified DEGs Blimp1 can bind.

- cNK cells are considered circulating cells, that transiently pass through the liver. Previous studies have suggested almost identical gene expression patterns in hepatic and splenic NK cells. In functional tests, they often "perform" identically. I am therefore a bit surprised that the authors find a differential dependency of Blimp1 for the IFNg production of splenic (no role of Blimp1) versus hepatic (Blimp1 regulating IFNg production) NK cells (Fig S3). Do the authors have any suggestions on that? The analyses are performed by 12+4h stimulations with IL12/18, which could involve the effects of altered bystander cells (as suggested by Figure 6). Therefore, these analyses should be provided upon standard 4h stimulations with IL12/18 and also with PMA/I under BFA. Note: liver and splenic cNK cells look quite different in the chosen histograms in Figures 7 A, B, C, yet there is massive variability in these analyses - is there any systematic/technical problem?

- Figure 4 H/I - In contrast to NK cells in Fig 4E, F, the KO and WT ILC1s seem to co-cluster largely. Authors should validate differentially expressed genes. How strong is the effect of Blimp1 in ILC1s? Or is Blimp1 a critical TF driving effector differentiation in NK cells, while it has only subtle effects in ILC1 (these may be regulated by Hobit?)? This seems an interesting finding that should at least be discussed. For these types of small differences in ILC1, FACS confirmation analyses should be performed and findings be reevaluated using Cre-expressing controls (see above).

The authors describe and discuss some of Figure 1 and 2 data as if Blimp1 would be involved in alternative NK versus ILC1 fates, but there is no evidence for this.

- There are several recent studies suggesting a role for Hobit, homologue of Blimp1, in NK cells and in ILC1, and in the control of liver metastases. The authors should discuss similar and unique functions of Hobit and Blimp1, also in the regulation of gene expression patterns, and should refer to these studies.

- Figure 4: The authors should discuss (and cross-validate) their liver gene expression analyses in the context of published datasets of NK and ILC1, such as the ones by Lopez et al, Friedrich et al, Ducimetiere et al and Yomogida et al.

Reviewer #2 (Public Review):

Summary:
This study offers a significant advancement in understanding liver innate lymphoid cell (ILC) biology by elucidating the role of the transcription factor Prdm1. It shows that Prdm1 is crucial in maintaining the balance between conventional natural killer (cNK) cells and ILC1s in the liver, with knockout models revealing a vital role in cancer defense mechanisms. Despite not affecting direct cytotoxicity, Prdm1 deficiency leads to increased cancer metastasis and reduced secretion of key molecules like IFN-γ, pointing to its importance in immune regulation. The use of single-cell RNA sequencing further underscores Prdm1's role in cellular communication within the liver's immune milieu. This study is a robust contribution to the field, providing insights that could inform new immunotherapy approaches for liver cancer.

Strengths:
The study's strength lies in its comprehensive approach, combining the specificity of Prdm1 conditional deletion in Ncr1-cre mice with integrative omics analyses and cutting-edge cytometry to delineate Prdm1's role in liver Type 1 ILC biology and its functional implications in tumor immunity. This multifaceted strategy not only clarifies Prdm1's influence on ILC composition and maturation but also conveys potential therapeutic insights for liver cancer immunotherapy.

Weaknesses:
A notable weakness of the study is the limited scope of in vivo disease models, primarily relying on the B16F10 melanoma model, which may not fully capture the complex behavior of Type 1 ILCs across diverse cancer types. Furthermore, the absence of direct human data, such as the effects of PRDM1 deletion in human NK cells or stem cells during their differentiation into NK and ILC1, leaves a gap in translating these findings to clinical settings.