Purpose: Multiple liver cell-types induce immunological tolerance. Indeed liver allografts are often spontaneously accepted in humans and mice. The role of host liver in the Medawar model of neonatal tolerance has not been examined, thus we studied events in the livers of newborn mice injected with a mixture of semi-allogeneic (F1) bone marrow (BMC) and spleen cells (SC).

Methods: GFP+ or CFSE-labelled F1-BMC/SC inoculum was injected iv into C3H/He(H-2k) neonates within 24 hr of birth. Images were acquired with Olympus-OV100 small animal imaging system (GFP) or AxioObserverZ1 (CFSE). Specific cell types were isolated with StemCell Technologies EasySep® purification kits. CFSE-labelled primary liver cells were co-cultured with CellTracker™ Orange CMRA-labelled HSC/progenitors in cell fusion experiments.

Results: Injected GFP+ F1-cells were detected in host liver, in addition to lymphoid organs. Liver fluorescence increased from days 1-6 after injection, with greater intensity in mice injected with the F1-BMC/SC mixture than either fraction alone. The majority of F1-cells in liver did not label with antibodies against T cells, B cells, monocytes or macrophages. Injected CFSE-donor cells appeared to fuse with host liver cells, given CFSE intensity differed between associated cells rather than being equal as expected in cell division. Consistent with cell fusion, CFSE-cells immunostained with antibody against albumin, a marker of hepatocytes. To identify donor cell type(s) that fused with hepatocytes, neonates were injected with different purified syngeneic GFP-cells: hematopoietic stem/progenitor cells (HSC), mesenchymal stem/progenitor cells, monocytes, thymocytes (total) and B cells. Only mice injected with HSC showed large distinct fluorescent spots in liver on day 6. To verify HSC could fuse with neonatal hepatocytes, these two cell fractions were differently labeled and co-cultured for 3 days. Imaging in culture showed a portion of cells labeled with both fluorochromes suggesting cell fusion; additionally, some expressed albumin.

Conclusion: Donor HSC/progenitors fuse with host hepatocytes in livers of neonatal mice tolerized by the classic Medawar model. Similar fusion events are reported when adult mice with diseased livers are injected with BMC. Given hepatocytes delete naïve CD8a T cells and generate Tregs, it is possible formation of donor-host hybrid hepatocytes in neonatal liver may represent a novel mechanism contributing to tolerance induction.

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