Tissue-Specific Homing of Natural Killer Cells in Allogeneic Bone Marrow Transplantation.
Authors: Olson Janelle A. , Zeiser Robert , Beilhack Andreas , Negrin Robert S. 
Published: April 2020

Abstract
Cell trafficking to distinct anatomical sites is critical for in vivo function. Natural Killer (NK) cells have been demonstrated to suppress graft-versus-host disease (GVHD) while inducing a graft-versus-tumor response (GVT). However, little is known about the homing of NK cells following bone marrow transplantation (BMT), their proliferative capacity and how long they persist in vivo. To investigate this, we transplanted highly purified DX5+ CD3− NK cells from FVB L2G85 luciferase+ mice into lethally irradiated syngeneic and BALB/c allogeneic recipients. Bioluminescence imaging (BLI) of transplant recipients revealed distinct NK cell migration to and proliferation in secondary lymphoid organs, which was confirmed by CFSE proliferation analysis. The allogeneic NK cells persisted for 20–30 days after which the signal gradually declined. Proliferation in lymphoid organs was undetectable in syngeneic recipients, but was enhanced by daily intraperitoneal injection of IL-2. Moderate proliferation in lymphoid organs and dramatic expansion in the thymus and peritoneal cavity were observed, which persisted for the duration of IL-2 administration. Exogenous IL-2 also increased NK cell proliferation in the allogeneic recipient thymus and peritoneal cavity, and even more robustly enhanced proliferation in the secondary lymphoid organs. Corresponding to this observed thymic homing, splenic NK cells reisolated from allogeneic recipients 5 days after transplant upregulated CCR9, a receptor important in thymocyte migration and homing of T cells to the gut. In this allogeneic setting, ex vivo imaging of the spleen and gut region 3 and 5 days after transplant confirmed localization of NK cells to the mesenteric lymph nodes, and also revealed NK cell infiltration of the small intestine, a major site of GVHD pathology in addition to the skin and liver. Additionally, donor NK cells were visible in the skin of transplanted animals by immunohistochemistry staining. Similar to T cells, a subset of freshly isolated NK cells express α4β7 and P-selectin ligand, expression of which are required for homing to the gut and skin, respectively. FACS analysis revealed that both these markers were upregulated on splenic NK cells reisolated from allogeneic recipients 5 days post-transplant. However, in contrast to T cells, CD62L was not downregulated on these NK cells. These results raised the question of whether the NK cell homing pattern observed in vivo and reflected in the upregulation of tissue-specific homing receptors is a consequence of the conditioning and transplant regime and associated inflammatory conditions, or whether NK cell alloreactivity can induce a specific trafficking pattern. To address this question, purified FVB luc+ NK cells were transplanted into unirradiated allogeneic BALB/c RAG2−/− γc −/− recipients, which lack T, B and NK cells. Moderate proliferation was seen in the spleen and lymph nodes, and infiltration of the gut tissue was observed. This implies that the inflammatory environment caused by tissue damage due to irradiation of recipients is not required for NK cell infiltration into GVHD target organs such as the gut. These studies indicate that NK cells are capable of proliferation in vivo either due to alloresponses or cytokine stimulation. Further, NK cells infiltrate GVHD target organs yet do not cause significant GVHD pathology possibly due to reduced tissue damage. NK cells may further reduce T cell proliferation in GVHD target sites through production of anti-inflammatory cytokines or by modulating antigen presentation.