Quantum Dots as a Unique Nanoscaffold to Mimic Membrane Receptor Clustering Nadia Anikeeva 1) , Dimitry Gakamsky 2) and Yuri Sykulev 1) 1) Department of Microbiology and Immunology and Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia; 2) Edinburgh Instruments Ltd, Livingston ABSTRACT Clustering of immune receptors and their ligands play an important role in the regulation of various responses of immune cells. However, very few tools to evaluate the effect of receptor and ligand clustering on live immune cells are available. We propose here to model major histocompatibility complex (MHC) proteins clusters. Quantum dots (QD) were used as a scaffold to assemble peptide-MHC (pMHC) molecules, the natural ligands for T- cell antigen receptor (TCR), into highly ordered oligomers with coherent orientation of individual protein molecules. Using pMHC/QD conjugates as a unique tool, we have shown that self pMHC proteins productively interact with the surface of T cells in CD8-dependent manner and facilitates recognition of foreign (viral) pMHC ligands at very low densities. We have also shown that pMHC/QD serve as an elegant probe to visualize uptake of productively engaged TCR and could be used to probe the extent of TCR and CD8 co-receptor clustering on the surface of live T cells at various stages of T cell activation and differentiation. Keywords: quantum dots, membrane receptor clustering, major histocompatibility complex (MHC) proteins, T cells, T-cell antigen receptor and CD8 co-receptor 1 INTRODUCTION It is thought that cell surface receptors on immune cells are clustered and could form “protein islands” that regulate antigen recognition and downstream signaling. It has been reported that MHC clusters on the surface of antigen- presenting cells (APC) increase T cell sensitivity to low density of agonist peptide-MHC (pMHC) ligands [1; 2]. To model MHC patches we utilized fluorescent nanoparticles, quantum dots (QD), as a scaffold to assemble various immune receptors into highly ordered oligomers with coherent orientation of individual protein molecules [3]. High local concentration of ZnS that constitutes the shell of QD mediates strong binding of hexahystidyl (His 6 )-tagged proteins to the QD surface and the formation of stable protein/QD conjugates. QD of various sizes could accommodate from 10 to 40 protein molecules with molecular weight of about 50 kD. This allows assembly of various immune receptors or their ligands at designated ratios, to vary the distances between assembled molecules, and to evaluate how the extent of molecular clustering of these proteins influences immune recognition and kinetics of downstream signaling in live immune cells. Previously, we exploited fluorescent nanocrystals covered with dihydrolipoic acid (DHLA) to produce soluble, aggregate- free QD [3]. DHLA-capped QD are highly negatively charged and have proved to be very stable at alkaline pH, but they are prone to aggregation at neutral pH that is close to physiological conditions [4]. Here we present data comparing two types of QD carrying pMHC proteins that serve as ligands for antigen- specific T-cell receptor (TCR). These QD are differed from each other by the nature of QD cap, namely, DHLA-capped QD and QD encapsulated in lipid micelle. Inclusion of NiNTA (nickel-nitriloacetic acid) functionalized lipids into the micelle composition allows capturing His 6 -tagged proteins. Such QD, termed NiNTA-QD, appear to be more stable at neutral pH. Analysis of functional properties and the binding of both kinds of QD to live T cells demonstrate similar results. In addition, NiNTA-QD have a lower surface charge that is close to that of the cell membrane and accommodate a higher number of pMHC proteins per dot making these dots a more versatile tool to mimic cell surface pMHC patches. 2 RESULTS 2.1 The number of pMHC molecules attached to NiNTA-QD We compared the number of pMHC proteins conjugated with NiNTA-QD and DHLA-QD using experimental approach and theoretical calculations (Table 1). While His- tagged protein molecules interact with Zn 2+ ions of the DHLA-QD shell, NiNTA-QD bind His-tagged proteins through formation of coordinate bonds with Ni-NTA functional groups conjugated with the micelle outer surface. Core size of various QD has been reported [5]. The size of ZnS shell that usually contains 5 monolayers amounts to ~1.2 nm [5]. The size of NiNTA-QD mainly depends on the dimensions of micelle and has been found to be ~ 5-7.5 nm [6]. To measure the amount of the conjugated pMHC we used gel filtration chromatography. The QD were incubated with pMHC at different ratio and the mixture was loaded on the gel filtration column. The presence of an excess of the pMHC resulted in two peaks. The second peak defined the amount of unbound pMHC molecules. The obtained numbers of pMHC per dot were in a agreement with the numbers calculated from the geometrical size of QD and the NSTI-Nanotech 2011, www.nsti.org, ISBN 978-1-4398-7138-6 Vol. 3, 2011 443