Genomic Analysis Defines Increased Circulating, Leukemia-Induced Macrophages That Promote Immune Suppression in Mouse Models of FGFR1-Driven Leukemogenesis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I read a research article entitled “Genomic analysis defines increased circulating, monocyte-derived macrophages that promote immune suppression in mouse models of FGFR1-driven leukemogenesis.” written by Dr. Ting Zhang et al. This research defined the characteristics of circulating monocyte-derived macrophages in in vivo model mouse, which exerted immunosuppressive property.

 

 

Major comments

1. What is the property of predominant presence of macrophage in leukemic mouse? This is a result of leukemia or a cause of leukemogenesis? Or both?
2. IL-4 and IL-13 is crucial cytokine associated in suppressive macrophages. How was the serum concentration in murine model or in vitro study?
3. As you mentioned, FLT-3 is a key molecule in some population of leukemia. You indicated the flt3 (CD135) gene inactivation. How was the tyrosine kinase activity?
4. How did you think that the macrophage mediated-immune suppression is shown only in FGFR1-driven leukemia? How about other leukemia?
5. The survival benefit in mouse treated with GW2580 CSFIR inhibitor looked non-significant shown by Kaplan Meier survival curve. Because you used only five animals in each group setting. More modest description is preferable. Otherwise please perform additional experiment with more mice.

 

Minor comments

1. Please tell the details for statistical method you performed in this study.
2. What is the biological sense of a downregulation of cardiac macrophages pathway?

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a very interesting paper describing cutting-edge findings in a murine model of FGFR1-driven leukemia. The clinical potential of the data would be increased by a few addition to the manuscript.

What markers overlapping in mice and humans have potential to indicate the presence and kinetic of aberrant monocytres in this model, possibly by flow cytometry (so to be more easily translated in the clinic)?

Did authors observe an increase in Tregs during disease progression? What about NKs? What about exhaustion markers in T cell subsets? 

 

 

 

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript investigates the emergence of a large population of immunosuppressive, circulating, monocyte-derived “circulating” macrophages in murine models of FGFR1-driven leukemogenesis (SCLL). Using CyTOF, scRNA-seq, trajectory inference, and functional assays, the authors demonstrate that these cells derive from classical monocytes via a non-classical intermediate and exert T cell suppression. Pharmacologic depletion with GW2580 (CSF1R inhibitor) restores immune responsiveness and prolongs survival, suggesting therapeutic relevance. This work is a follow-up study from the same group, which demonstrated an increase in PMN-MDSC in the same model. Here they reanalyzed their previously published scRNA-seq focusing on non-classical monocytes and performed mass cytometry characterization of macrophagic populations. This works brings interesting new insights on this class of macrophages, although their differences between ncMono and MDSC is not so clear.

 

Specific comments

• The changes in the proportion of macrophages is clear as compared to other immune cells. It is however, expected that all hematopoiesis is disrupted in leukemic mice and all lineages affected. It could thus be that macrophages are less sensitive to leukemic progression. The total number of CD45+ cells should be provided in naïve and leukemic mice the number of macrophages should be estimated.
• The definition of “circulating macrophages” is not so clear. Is F4/80 specific of macrophages or can it be expressed in activated monocytes. Are they different from resident macrophages? It would have been interesting to compare these circulating macrophages with macrophages in tissue samples (bone marrow, liver, spleen).
• To characterize their immunosuppressive activity on T-cells, the authors compare PB macrophages in leukemic mice to bone marrow macrophages in naïve mice. Is there evidence that PB macrophages have the same behavior as BM or tissue/resident macrophages?
• For the CyTOF experiments, the number of cells that are analyzed are very low, 1191-1391 cells for naïve mice, 1400 for leukemic mice. This questions the statistical relevance of the findings
• Is the CSF1R inhibitor having a direct effect on the leukemic cells? The expression of CSF1R should be measured at the surface of the cancer cells and their sensitivity to the drug could be analyzed in vitro.
• The difference between ncMono and MDSC are not so clear. It would be helpful to provide a scheme that recapitulates the hierarchy between the different cell types including the specific markers for each of them
• The findings should be discussed in light of the specificities of FGFR1-driven SCLL model. Are the findings likely to apply to other hematological malignancies (AML, ALL)
• Circulating macrophages are described in 2 different ways: Circulating monocyte-derived macrophages and Leukemia-induced macrophage-like ncMonos. This introduces some confusion and should be homogenized.
• There should be a section for statistical analysis in the method part and the statistical tests explained for each figure.
• The font size is too small in many figures, making it impossible to read.
• The method part is not exhaustive enough. For example, there is no description of the T-Cell proliferation assays
• The immune populations should be better described on Fig 1
• The same coloring for the naïve and leukemic conditions should be kept throughout the figures

Minor

• “nave” instead of “naive” line 253
• Discussion: line 523 “pesudotime” instead of “pseudotime”

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have successfully addressed my previous comments