Growth Factor Independence 1 (Gfi1) Regulates Cell-Fate Decision of the Bipotential Granulocytic-Monocytic Precursors Defined by Expression of CD48 As a New Marker,

Abstract 3217

Gfi1 regulates the expression of genes important for survival, proliferation and differentiation of hematopoietic cells and is required for normal multi-lineage blood cell development. Mutations affecting the function of Gfi1 are implicated in leukemia, lymphoma, auto-immune disease and neutropenia. Gfi1 deficient mice are severely neutropenic and accumulate an aberrant CD11b⁺GR1^int myeloid population, which was assumed to contain precursors either arrested in granulocytic differentiation or redirected into the monocytic lineage. To further analyze the function of Gfi1 during granulopoiesis, we used bone marrow cells from Gfi1-GFP knock-in mice, which enable to follow Gfi1 expression by simply measuring green fluorescence. By this, we found that the CD11b⁺GR1^lo bone marrow fraction that contains monocytes separates into two populations expressing low or high Gfi1. We FACS-sorted these two subsets (CD11b⁺GR1^loGfi1^hi and CD11b⁺GR1^loGfi1^lo) and observed that, 99% of CD11b⁺GR1^loGfi1^lo cells resembled monocytes, while over 90% of the CD11b⁺GR1^loGfi1^hi cells were myelocytes or metamyelocytes. After three days in culture in the presence of GM-CSF, almost all CD11b⁺GR1^loGfi1^lo cells developed into adhering macrophages, while 73% of the CD11b⁺GR1^loGfi1^hi cells developed into mature neutrophils, suggesting that Gfi1 expression drives myeloid precursors into the granulocytic lineage.

We determined the genome-wide expression patterns of these two populations and as a control also of CD11b⁺GR1⁺ granulocytes and the aberrant CD11b⁺GR1^int population found in Gfi1^−/− mice (termed: CD11b⁺GR1^loGfi1^−/−). Unsupervised clustering analysis showed that CD11b⁺GR1^loGfi1^−/− cells are similar to CD11b⁺GR1^loGfi1^lo monocytes, while CD11b⁺GR1^loGfi1^hi cells are more closely related to mature granulocytes (CD11b⁺GR1^hi). Gene-Set enrichment analysis (GSEA) revealed that genes associated with cell cycle progression were up regulated in CD11b⁺GR1^loGfi1^hi, while genes controlling the humoral immune response and chemokine receptor signaling activity were downregulated. Promoter analysis of differentially expressed genes showed for CD11b⁺GR1^loGfi1^hi an over-representation of binding sites for E2F, a regulator of cell cycle genes, and for CD11b⁺GR1^loGfi1^lo cells an over-representation of binding sites for STAT5, a transcription factor required for monocyte development. The comparison of CD11b⁺GR1^loGfi1^hi cells with granulocytes also showed a higher expression of cell cycle associated genes and a lower expression of humoral immune response genes. This indicated that CD11b⁺GR1^loGfi1^hi cells show features of faster cycling, less differentiated precursor cells compared to monocytes (CD11b⁺GR1^loGfi1^lo) and granulocytes (CD11b⁺GR1^hi). It is thus likely that CD11b⁺GR1^loGfi1^hi monocyte bone marrow subset contains the precursors committed to granulocytic differentiation.

The unsupervised clustering analysis also revealed that CD48, a 40–45 kD cell surface glycoprotein, is inversely correlated to the Gfi1 expression and might enable to differentiate between CD11b⁺GR1^loGfi1^hi and CD11b⁺GR1^loGfi1^lo cells and to find a definition for precursors committed for granulopoiesis. We found that CD48 can replace Gfi1/GFP as well as CD11b in a flow cytometric analysis and using GR1, CD48 staining only, we were able to clearly separate granulocytic precursors from other monocytic cells and mature granulocytes as shown by Wright-Giemsa staining and in vitro culture of sorted cells in the presence of M-CSF or GM-CSF. GR1^loCD48^hi cells gave rise to adherent macrophages in the presence of M-CSF but generated monocytes and granulocytes in the presence of GM-CSF, while GR1^loCD48^lo cells can only develop into granulocytes in the presence of GM-CSF. Thus CD48 allowed the separation of bipotential precursors from other precursors that are fully committed to granulopoiesis. Analysis of Gfi1^−/− bone marrow cells using the GR1, CD48 markers clearly showed that the aberrant CD11b⁺Gr1^int population represents regular monocytes that accumulated at the expense of granulocytes. This suggests that Gfi1 regulates cell-fate decision in bipotential granulocytic-monocytic precursors, which can be precisely defined by expression of GR1 and CD48. This allows for the first time a more precise definition of monocytic-, granulocytic and bipotential precursors in mice on a phenotypic basis.