9:45 AM - SB09.04.04
Biomimetic Molecular-Scale Devices for the Spatial Control and Study of Activating-Inhibitory Balance in Natural Killer Cells
Mark Schvartzman1,Esti Toledo1,Guillaume Le Saux1,Avichai Edri1,Uzi Hadad1,Angel Porgador1
Ben-Gurion University of the Negev1
The cytotoxic activity of lymphocytes is regulated by a gentle balance between activating and inhibitory signals. Understanding the molecular mechanism of this balance is of fundamental importance, and is essential for the rational design of the future based immunotherapies. For this purpose, the exact function of each receptor must be investigated individually and in combination with each other. In particular, to understand the mechanism of the spatial integration of activating and inhibitory receptors, individual receptors should be manipulated and controlled. Spatial control of receptor with molecular resolution has been possible by exposing cells to arrays of patterned nanodots functionalized with the ligands for the studied receptors1–3. Such state-of-the-art arrays, however, could only control receptors of one type, and therefore could not be used to study how different receptors with complementary function integrate their signals. An experimental platform that allows simultaneous spatial control of two or more different receptors within the cell membrane has not been demonstrate up to date. Here, we developed a novel nanochip approach that allows simultaneous spatial control of individual transmembrane receptors of two types. We applied this nanochip approach to reveal how the nanoscale segregation between NKG2D and KIRDL1, which are the activating and inhibitory receptors in Natural Killer (NK) cells, respectively, regulate the cytotoxicity of NK cells. Our nanochips are based on tunable arrays of paired 10 nm nanodots of different metals, which are selectively functionalized with activating and inhibitory ligands for NK cells. The functionalized arrays, in turn, are used as a stimulation platform for NK cells, which encodes the arrangement of the two receptors within the cell membrane, and allows to monitor the cytotoxic response of NK cells the variations in this arrangement. To realize the nanochips, we first fabricated heterogeneous arrays of Ti and Au nanodots by nanoimprint lithography, followed double angle evaporation of two metals, and liftoff. Here, the spacing between the nanodots, which was ranged from zero to a few tens of nm, is precisely controlled by the metal evaporation angle. We then selectively functionalized Ti and Au nanodots with (i) MHC class I polypeptide-related sequence A (MICA) – a ligand for NKG2D, and (ii) monoclonal antibody for KIR2DL1, using biotin-avidin and Nitryltriacetic acid (NTA)-Histidine conjugations, respectively4. We stimulated NK cell on the chip surfaces, and assessed their degree of activation through the expression of CD107a – a commonly used degranulation marker. We found that KIR2DL1-regulated inhibition of NKG2D signaling indeed depends on the spacing between the two receptors, and is mostly effective when the two receptors are separated by 30 nm. Our results shed the light on the way by which the innate immune function is spatially regulated by the activating and inhibitory signaling crosstalk. Furthermore, our novel nanochip technology opens a general pathway to complex, multifunctional nanomaterials which can be used as experimental platforms for the nanoscale study of the function and structure of immunological synapse, as well as other interfaces between cells and their environment.
(1) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blümmel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. ChemPhysChem 2004, 5 (3) 383–388.
(2) Schvartzman, M.; Palma, M.; Sable, J.; Abramson, J.; Hu, X.; Sheetz, M. P.; Wind, S. J. Nano Lett. 2011, 11 (3).
(3) Cai, H.; Muller, J.; Depoil, D.; Mayya, V.; Sheetz, M. P.; Dustin, M. L.; Wind, S. J. Nat. Nanotechnol. 2018.
(4) Le Saux, G.; Edri, A.; Keydar, Y.; Hadad, U.; Porgador, A.; Schvartzman, M. ACS Appl. Mater. Interfaces 2018, 10 (14),