Natural Killer Cell Death Mediated By NKG2D-Trogocytosis
Authors: Nakamura Kyohei , Nakayama Masafumi , Kawano Mitsuko , Ishii Tomonori , Harigae Hideo , Ogasawara Kouetsu 
Published: October 2019

Abstract

Introduction
The activating receptor, NKG2D, plays crucial role in natural killer (NK) cell-mediated effector function. NKG2D is involved not only in host defense against tumor and viral infection, but also in autoimmune diseases by recognizing stress-induced self-ligands (NKG2DLs). However, the negative feedback regulation of NKG2D has not been fully understood. It has been reported that NK cells undergo rapid apoptosis upon interaction with NK-sensitive tumor cells, suggesting that activation-induced NK cell death can be triggered in certain situations. In this study, we aimed to elucidate underlying mechanism of NK cell death, especially focused on NKG2D-NKG2DLs interaction.

Methods
NK cells were purified from splenocytes of C57BL/6, perforin-/-, and DAP10-/-/12-/- mice, and cultured with rhIL-2 (1000 U/ml) for 5 days. We used these IL-2-activated NK cells as effector cells and three target cell lines: mouse T cell lymphoma RMA cells (RMA), RMA lacking MHCI expression (RMA-S), and RMA stably expressing an NKG2DL, Rae-1δ (RMA/Rae-1δ). CFSE-labeled NK cells were co-cultured with target cells for 2 hours, and stained with anti-NK1.1 mAb propidium iodide (PI). The percentage of PI-positive cells within CFSE+ NK1.1+ population was measured by flowcytometry. The cell surface expression of Rae-1 on NK cells after co-culture with target cells was evaluated by flowcytometry and confocal microscopy.

Results
NK cells from WT mice rapidly underwent cell death when co-cultured with Rae-1δ, but not with RMA or RMA-S, suggesting that NKG2D-Rae-1 interaction is involved in NK cell death. NK cells from perforin-/-, and DAP10-/-/12-/- mice did not undergo cell death, highlighting the importance of the NKG2D pathway for NK cell death. However, cross-linking of NKG2D receptor failed to induce NK cell death. To understand underlying the mechanism of NK cell death, we evaluated the cell surface expression of NKG2DLs on NK cells after co-culture with tumor cells. We found that cell surface expression of Rae-1 on NK cells was remarkably induced after co-culture with RMA/Rae-1δ, but no with RMA or RMA-S, implying that these Rae-1-positive NK cells may be lysed by NK cells through NKG2D-induced perforin pathway. The cell surface induction of Rae-1 on NK cells was very rapid (within 5min), and it occurred cell-cell contact dependent manner. Interestingly, NK cells from C57/BL6 mice rapidly became BALB/c allele Rae-1γ-positive after co-culture with RMA/Rae-1γ, indicating that NK cells acquire tumor-derived Rae-1. Consistently, acquisition of Rae-1 by NK cells was confirmed by confocal microscopy. Therefore, NK cells rapidly dress tumor-derived Rae-1 after interaction with tumor cells through intercellular membrane transfer, namely trogocytosis. Trogocytosis of Rae-1 was significantly inhibited in NK cells from DAP10-/-/12-/- mice and by chemical inhibitors of PI3K and Syk, indicating that it requires NKG2D-signaling. To confirm whether Rae-1-dressed NK cells can be recognized and lysed by other NK cells, we used sort-purified Rae-1-dressed NK cells as target cells. Rae-1-dressed NK cells were lysed by WT NK cell in an E/T-ratio dependent manner through NKG2D-induced perforin pathway. Furthermore, adoptively transferred Rae-1-dressed NK cells were significantly eliminated in Rag-1-deficient mice, indicating that Rae-1-dressed NK cells are also recognized and lysed in vivo.

Conclusion
Upon interaction with NKG2DLs-expressing tumor cells NK cells rapidly dress tumor-derived NKG2DLs, and subsequently undergo fratricide. Our results provide novel insights into activation-induced NK cell death via dynamic intercellular communications.

Disclosures:
No relevant conflicts of interest to declare.