Horizontal Gene Transfer through Apoptotic Bodies Confers a Possible Mechanism of Epithelial Chimerism after Allogeneic Hematopoietic Cell Transplantation.
Authors: Waterhouse Miguel , Themeli Maria , Finke Jurgen , Spyridonidis Alexandros 
Published: April 2020

Abstract
Animal and human studies have shown that after allogeneic hematopoietic cell transplantation (HCT) low percentage of epithelial cells containing donor-derived genome emerge. The mechanisms underlying this phenomenon are unclear. We investigated whether fusion or horizontal gene transfer between donor lymphocytes and recipient epithelial cells may explain epithelial chimerism after allo-HCT. In an in vitro model we analyzed whether and how genomic material could be transferred between cells. Briefly, keratinocyte HaCaT cells (Y-chromosome neg) were cocultivated with non-apoptotic Jurkat cells (Y+) or Jurkat cells in which apoptosis was induced with Camptothecin (4μM, 16h). Jurkat cells were labeled with CMFDA or BrdU in order to track the fate of genomic material. HaCaT cells co-cultivated with non-apoptotic Jurkat cells for 24–72h did not show any CMFDA, BrdU or FISH-Y chromosome signal, excluding fusion between cells in this experimental model. In contrast, co-cultivation of HaCaT cells with Jurkat apoptotic bodies for 48h resulted in CMFDA signal in 28% of the HaCaT cells, BrdU signal in about 5% and FISH Y-chromosome signal in about 3% of the cells. Importantly, the BrdU and the Y-chromosome signals were observed not only in the cytoplasm but also within the nucleus of the HaCaT cells as evaluated by confocal microscopy (Leica Confocal Microscope) and 3D analysis. Moreover, we obtained metaphases from the BrdU+ HaCaT cells and confirmed that the BrdU signal was located within the isolated chromosomes suggesting the creation of hybrid chromosomes. The incorporation of the transferred genomic material in the HaCaT host genome was inhibited by cytochalasin (inhibitor of phagocytosis) and increased 2-fold by bafylomicin (inhibitor of lysosomes) and aphidicolin (blocks the cell cycle in G1 phase). No transfer of genomic material was observed when the apoptotic bodies were separated from the HaCat cells by a semipermeable membrane in a transwell system. We isolated by a MoFlo FACS sorter the BrdU+ HaCaT cells bearing the Jurkat derived genomic material and found that 40% of them had an increased proliferating capacity (detected with CFSE labeling) as compared to untreated HaCaT cells. In order to evaluate expression of the horizontally transferred genomic material, we co-cultivated HacaT cells with intact or apoptotic GFP-transfected JvM cells. Cocultivation with non-apoptotic GFP+ cells resulted in no GFP expression n Hacat cells. In contrast, cocultivation of Hacat cells with GFP+ apoptotic bodies resulted in a 6-fold increase in the mean GFP fluorescence in HaCaT cells. When GFP+ apoptotic bodies were pretreated with DNase I no GFP expression in Hacat cells was observed indicating that the GFP expression of HaCaT cells after cocultivation with the apoptotic bodies was through horizontal transfer of DNA and not of mRNA material. Taken together, our in vitro model suggests horizontal gene transfer through apoptotic bodies as a possible mechanism explaining epithelial chimerism after allogeneic HCT. A possible scenario is that after allo-HCT apoptotic hematopoietic cells charge constantly the host environment with donor DNA which is phagocytated by epithelial cells. Part of this DNA could escape lysosomal degradation and integrate within the recipient DNA resulting in epithelial cells containing donor-derived genome.