Observation of Immune Activation Through Simultaneous Cell Receptor Monitoring Using Lanthanide Doped Upconverting Nanoparticles

Immune synapse (IS) formation, maintenance, and communication are conducted through the bonding of cell receptors. Long-duration cell receptor tracking throughout the synaptic activation process is difficult with current organic and inorganic fluorophores and quantum dots due to issues of biocompatibility, quenching, and photobleaching. Lanthanide doped upconverting nanoparticles (UCNP) are photostable over hours, and have the ability to convert incident photons to a higher frequency allowing for biologically transparent Near-IR (NIR) excitation and visible emission. Here, we show how UCNPs can be used as in-situ optical emitters to simultaneously observe multiple cell receptors throughout the stages of immune activation during a 6-8 hour IS interaction. <br/><br/>We colloidally synthesize 15 nm diameter hexagonal phase NaYF₄ nanoparticles, each doped with a different Lanthanide emitter — 2% Er, 0.5% Tm, or 2% Ho as well as the sensitizer Yb at 20%. The nanoparticles are shelled with inert, 4 nm thick NaYF₄ to minimize quenching. To disperse these UCNP in aqueous solutions, we wrap them in poly(maleic anhydride-alt-1-octadecene) (PMAO) hydrolyzed with 4-Dimethylaminopyridine (DMAP). Upon illumination at 980 nm these particles upconvert to visible light in the red, blue, and green. To target immune cells, we use a carbodiimide crosslinker to functionalize each nanoparticle with one of three antibodies targeting the cell receptors — CD69 (early activation marker), ICOS (co-stimulatory), and PD1 (co-inhibitory) — through neutravidin-biotin linkages. We confirm the functionalization of these nanoparticles using TEM, FTIR, UV-Vis, and fluorescence characterization methods. Live-dead assays with cloned cytotoxic T lymphocyte (CTL) cells that recognize cytomegalovirus (CMV) confirm the nanoparticles lack of cytotoxicity. We then image these nanoparticles on immune cells using scanning confocal microscopy. We demonstrate the ability to simultaneously track cell receptor expression through the stages of immune cell activation over the course of 8 hours without signal instability. As a control we parallely track these activation factors using Fluorescent Activated Cell Sorting (FACS). Additionally, we demonstrate the ability of UCNP to dynamically observe receptor binding kinetics in response to temperatures ranging between 20 and 40°C, as well as binding kinetics of the cytokine immunotherapy, Aldesleukin or IL2. Our work lays the foundation for future in-situ studies of simultaneous cell receptor tracking using a larger assortment of UCNPs with a functionalization platform that can be generalized to a variety of proteins and cell types.