Introduction: We have previously demonstrated that liver NK cells play a pivotal role in islet graft loss during the early phase after intraportal islet transplantation (IT). However, the influence of NK cells on the engraftment of islets at the later phase remains to be elucidated. Liver DX5- NK cells have recently been reported to possess memory properties, distinguishing them from conventional DX5+ NK cells. Here, we demonstrated that DX5- NK cells, which express TNF-related apoptosis-inducing ligand (TRAIL), persistently expanded in livers after intraportal IT.

Methods: To investigate the relationship between instant blood-mediated inflammatory reaction (IBMIR) and NK cell activation, liver NK cells from C57BL6 mice were cultured for 24 h in the presence of IFNγ, TNFα, and IL-1β in vitro, which were actively produced under the IBMIR. To evaluate the phenotypical alteration of different subpopulation of NK cells during from the early to late phase after intraportal IT in vivo, liver NK cells were collected from mice at 24 h after syngeneic intraportal IT, and thereafter at monthly intervals for 3 months.

Results: Untreated liver NK cells contained approximately 30% of DX5- memory NK cells and 70% of DX5+ NK cells. The expressions of CD69, TRAIL, and chemokine receptor CXCR3 on the liver DX5- NK cells were significantly higher compared to those on the DX5+ NK cells on FCM analyses (84.1±7.5% vs. 12.8±4.2%, 70.8±9.3% vs. 4.9±2.2%, and 94.3±2.4% vs. 25.7±5.8%, respectively). When co-cultured with all three cytokines mimicking IBMIR, the population of DX5- NK cells expanded 2.4 times(p<0.01). Furthermore, the expression of both CD69 and CXCR3 on the liver NK cells was significantly increased during the culture. In the in vivo assay, the population of DX5- memory NK cells expanded robustly at 30 d after IT and continued to do so up to 90 d after IT (90 day/fresh ratio: 2.1, p < 0.01). In parallel, a significant increase in the expression of CD69, TRAIL, and CXCR3 was persistently shown.

Conclusion: We demonstrated persistent expansion of DX5- memory NK cells expressing CD69, TRAIL, and CXCR3 in the liver after intraportal IT. Taken together with the fact that islets express death receptor (DR) 5, a receptor for TRAIL, and secrete CXCL10, a ligand for CXCR3, it is likely that the expanded liver-resident DX5- memory NK cells target originally engrafted islets and even secondarily transplanted islets via TRAIL-DR5 and CXCR3-CXCL10 interactions.

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