Page 312 - Haematologica April 2020

M. Hetzel et al.
CD45+ cells in BALF (Online Supplementary Figure S4A and B), which was accompanied by decreased turbidity, total protein, cholesterol, GM-CSF, M-CSF and MCP-1 in BALF of these mice nine months after HSC-GT (Online Supplementary Figure S4C-I).
Thus, Lv.Csf2rb HSC-GT can reverse the herPAP dis- ease phenotype and provide a long-term cure.
Rapid restoration of functional alveolar macrophages following hematopoietic stem cell transplantation
Given the efficient seeding of the lung with transplanted cells and the marked improvements in disease phenotype present already three months after HSC-GT, we next aimed to thoroughly characterize and better understand the early phases of pulmonary cell engraftment and its association with disease parameters. For these studies, lin– HSPC derived from healthy CD45.1+ were utilized and
transplanted intravenously into lethally irradiated Csf2rb-/- mice. The fate of CD45.1+ donor cells was analyzed in PB, BM, lungs and BALF one, two, four and eight weeks after HSCT (Figure 6A). Substantial contribution of CD45.1+ donor cells to the total hematopoietic pool of CD45+ cells was observed for the BM already one week after HSCT and increased to >90% at the 4-week time point. However, donor cells were low in the PB (4.3±0.8%) and virtually absent in the BALF and lungs at one week, and only increased moderately at the two week time point. The contribution of donor cells to overall blood cells rap- idly increased to 53±14% in the PB, 39±5% in BALF, and 41±15% in lungs at four weeks and reached 64±10% in blood, 73±5% in BALF and 67±5% in lungs eight weeks after HSCT. A more detailed characterization of these donor-derived cells in the BALF using AM typical CD11c/Siglec-F marker combination revealed that the few
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H
Figure 5. Improvement of disease-related parameters in bronchoalveolar lavage fluid (BALF) of secondary recipi- ents. (A and B) Representative picture of BALF (A) and BALF turbidity (B) measured as optical density at 600 nm (OD600). Lv.Csf2rb n=4, CD45.1 and Lv.GFP n=3, in two independent experiments. Circles belong to the first exper- iment, triangles to the second experiment; equal symbols in (B-H) represent the same mice. (C) Total protein concen- tration (μg/mL) in BALF. Lv.Csf2rb n=4, CD45.1 and Lv.GFP n=3 in two independent experiments. Cholesterol (μg/mL) (D), granulocyte/macrophage-colony-stimulating factor (GM-CSF) (pg/mL) (E), macrophage colony-stimulat- ing factor (M-CSF) (pg/mL) (F), and monocyte chemoattractant protein-1 (MCP-1) (pg/mL) (G) levels in BALF. Lv.Csf2rb n=4, CD45.1 and Lv.GFP n=3 in two independent experiments. (H) Vector copy number (VCN) in total bone marrow (BM) 12 weeks after Lv.Csf2rb hematopoietic stem cell-based gene therapy (HSC-GT); n=4 in two independ- ent experiments.
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