Growth Factor Independence 1 (Gfi1) Regulates Cell-Fate Decision of the Bipotential Granulocytic-Monocytic Precursors Defined by Expression of CD48 As a New Marker,
Bibliographic record
Abstract
Abstract 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+GR1int 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+GR1lo bone marrow fraction that contains monocytes separates into two populations expressing low or high Gfi1. We FACS-sorted these two subsets (CD11b+GR1loGfi1hi and CD11b+GR1loGfi1lo) and observed that, 99% of CD11b+GR1loGfi1lo cells resembled monocytes, while over 90% of the CD11b+GR1loGfi1hi cells were myelocytes or metamyelocytes. After three days in culture in the presence of GM-CSF, almost all CD11b+GR1loGfi1lo cells developed into adhering macrophages, while 73% of the CD11b+GR1loGfi1hi 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+GR1int population found in Gfi1−/− mice (termed: CD11b+GR1loGfi1−/−). Unsupervised clustering analysis showed that CD11b+GR1loGfi1−/− cells are similar to CD11b+GR1loGfi1lo monocytes, while CD11b+GR1loGfi1hi cells are more closely related to mature granulocytes (CD11b+GR1hi). Gene-Set enrichment analysis (GSEA) revealed that genes associated with cell cycle progression were up regulated in CD11b+GR1loGfi1hi, while genes controlling the humoral immune response and chemokine receptor signaling activity were downregulated. Promoter analysis of differentially expressed genes showed for CD11b+GR1loGfi1hi an over-representation of binding sites for E2F, a regulator of cell cycle genes, and for CD11b+GR1loGfi1lo cells an over-representation of binding sites for STAT5, a transcription factor required for monocyte development. The comparison of CD11b+GR1loGfi1hi 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+GR1loGfi1hi cells show features of faster cycling, less differentiated precursor cells compared to monocytes (CD11b+GR1loGfi1lo) and granulocytes (CD11b+GR1hi). It is thus likely that CD11b+GR1loGfi1hi 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+GR1loGfi1hi and CD11b+GR1loGfi1lo 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. GR1loCD48hi cells gave rise to adherent macrophages in the presence of M-CSF but generated monocytes and granulocytes in the presence of GM-CSF, while GR1loCD48lo 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+Gr1int 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. Disclosures: Duehrsen: Alexion: Honoraria.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".