In this report, we provide the first description of NK cell trafficking in allogeneic and syngeneic BMT using an in vivo BLI strategy. This study provides critical new information regarding the behavior of NK cells in BMT, which has important clinical application as NK cells hold promise as a cellular therapy in reducing GVHD in allogeneic BMT, especially from a haploidential donor. Additionally, donor NK cells have been shown to promote bone marrow engraftment in allogeneic recipients, while not causing GVHD themselves (26
). GVHD has a characteristic tissue-specific induction pattern; thus, the description of the tissue-specific homing of NK cells in BMT provides valuable insights into their therapeutic potential in reducing GVHD. Our study describes the homing and proliferative capabilities of transplanted NK cells and examines some of the factors responsible for these biological characteristics.
The duration of the luc+
NK cell BLI signal in our studies is consistent with NK cell survival time in vivo as reported in the literature. Jamieson et al. (18
) have reported a half-life of NK cells of 17 days based on BrdU-labeling studies. We are able to visualize the BLI signal up to 4 wk after transplantation. This duration of the BLI signal was likely due to the limited life span of NK cells, but could also be due to the reconstitution of donor NK cells from bone marrow at this time point, limiting further expansion of the donor population of NK cells. There is also a small population of mature NK cells in the TCD-BM that could be competing for space with the adoptively transferred NK cells. Previous studies have demonstrated a correlation between the BLI signal intensity from a luc+
tumor line in vivo and the number of luc+
tumor cells reisolated from this tumor-bearing animal (25
). Therefore, taken together, the increase in the BLI signal shown in as well as the CFSE studies demonstrate that NK cells have a greater proliferative capacity in allogeneic recipients.
Interestingly, the striking differences in NK cell proliferation between irradiated allogeneic and syngeneic recipients does not appear to be related to Ly49/MHC class I-mediated interactions by the donor NK cells in the allogeneic environment. In preliminary studies, the Ly49 repertoire of transplanted NK cells pre- and post-transplant did not reflect an expansion of the alloreactive subset of NK cells (Ly49C/I+) in allogeneic recipients (our unpublished observations). This finding indicates that factors other than MHC interactions may be driving NK cell proliferation in allogeneic recipients.
The in vivo trafficking patterns of NK cells in allogeneic transplant recipients are remarkably similar to allogeneic T cell trafficking. In addition to homing to the same organs and tissues, spleen, lymph nodes, and gastrointestinal tract, the cells also acquire a similar phenotype regarding homing receptor up-regulation. Like allogeneic T cells, NK cells lose expression of L-selectin in vivo upon activation and proliferation; however, this is not observed among syngeneic NK cells, which up-regulate homing markers without down-regulating CD62L. Both cell populations up-regulate the gut-homing receptor α4β7 and the skin-homing molecule P-selectin ligand. However, they differ in the duration of time they persist in the tissues as measured by BLI. These findings are especially interesting when taken in combination with the knowledge that T cells cause GVHD in an allogeneic environment, whereas NK cells do not. Based upon these observations, the reason for this difference in GVHD induction potential is not because NK cells are not reaching GVHD priming sites and target tissues; however, they do not cause the tissue damage at those sites characteristic of the GVHD response.
It is interesting to note that there is an increase in bioluminescence from lymph node sites, while the NK cells in the allogeneic setting down-regulate CD62L, a lymph node-homing molecule. Based upon the fact that syngeneic NK cells also have increased bioluminescence in lymph nodes without down-regulating CD62L and our CFSE analysis of NK cells reisolated from lymph nodes (data not shown), we can conclude that this is due to cell proliferation in these sites rather solely to an accumulation of cells from the peripheral blood.
Previous work from our laboratory assessed the impact of anti-CD62L Ab on donor T cell trafficking in GVHD induction with a similar blocking regimen, and parallel observations were made with regard to the impact of this Ab on NK cell trafficking. The anti-CD62L Ab, when administered together with anti-MAdCAM-1, prevented T cell entry into cLN and other lymph nodes as well as Peyer’s patches (36
). The treatment with anti-CD62L Ab in our studies reduced NK cell trafficking into lymph nodes, although some luc+
NK cells were visible at some nodal sites during Ab administration, albeit with a lower BLI signal. This is due to either insufficient blocking at the indicated Ab dose or more likely redundant molecules responsible for NK cell trafficking into these lymphoid tissues.
It has been well demonstrated that the pretransplant conditioning regimen causes tissue damage that is associated with GVHD severity (34
). This tissue injury activates host cells which secrete proinflammatory cytokines, such as IL-1 and TNF-α
), which can activate host dendritic cells and contribute to GVHD pathophysiology (38
). Because NK cells express receptors for many proinflammatory chemokines and migrate in response to them, we reasoned that our observations of NK cell trafficking in allogeneic BMT could be a result of inflammation induced by the irradiation. However, using RAG2−/− γ
recipients, which do not require conditioning since they lack endogenous T and NK cells and are incapable of rejecting the donor-derived allogeneic NK cells, demonstrates that the conditioning regimen necessary in BMT is not solely responsible for the observed NK cell trafficking patterns. NK cell trafficking to the lymph nodes is slightly delayed and lessened in the RAG2−/− γ
animals, possibly due to the decreased lymphoid compartment and organ size in these immunodeficient animals. We observed NK cell trafficking to the bone marrow compartment in these recipient animals, indicating that their trafficking and expansion may be driven more by homeostatic mechanisms in this transplant setting. Therefore, the irradiation does not seem to be solely responsible for NK cell trafficking, since the cells are still able to enter lymph nodes and gut tissue in the absence of irradiation.
NK cells require IL-2 for activation and survival and a number of clinical trials have been performed with IL-2 in addition to NK cells. Indeed, exogenous IL-2 augmented the proliferation of transplanted NK cells, especially in recipients of syngeneic NK cells, but did not alter or enhance the expression of homing molecules toward lymphoid or GVHD target tissues. These findings indicate that the homing receptor expression of the NK cells is not enhanced with the addition of an activating cytokine. The large increase in the BLI signal in the peritoneal cavity represented a localized accumulation of NK cells at the site of IL-2 administration. This study has functional consequences, as we observed increased tumor clearance in the presence of NK cells which was augmented with exogenous IL-2. This increased tumor clearance is presumably due to either increased proliferation of NK cells and thus greater cell numbers or enhanced functional capacity of IL-2-activated NK cells.
In conclusion, these studies provide the first visualization of NK cells following transplantation. The results demonstrate that NK cells proliferate in response to allogeneic signals and cytokines, survive for ~30 days following adoptive transfer, and infiltrate a variety of organs and tissues without GVHD induction. These results provide important insights into the biology of adoptively transferred NK cells and form the basis of future studies aimed at modulating NK cell survival and function.