Janus Nanoparticles for T Cell Activation: Clustering Ligands to Enhance Stimulation

Abstract

The in vitro activation of T cells by synthetic particles is a promising technique for adoptive cancer immunotherapy. While it is known that cell-surface receptors form clusters during T cell activation, the use of clustered ligands on synthetic particles to modulate T cell response is a largely unexplored concept. Building upon our previous finding that T cells respond differently to various micro-sized patterns of ligands, we here investigate the effect of nano-sized ligand clusters on T cell activation. Two-faced Janus nanoparticles were fabricated to display ligands of different functions in spatially segregated clusters on single nanoparticles. Going beyond our earlier qualitative study, here we precisely quantified and controlled the surface density and the total amount of ligands on single nanoparticles. We show that nanoparticles with clustered ligands activate T cells to a greater level than ones uniformly coated with the same number of ligands. The enhanced effect is due to increased local surface density of ligands. The results demonstrate that the spatial arrangement of ligands on particles influences activation response of T cells and may be used as a new strategy to increase T cell stimulation in the presence of insufficient amount of stimuli. This fundamental study also represents an initial step in using nanoscale Janus particles for manipulating immune cell responses.

Figure 1

Fabrication and characterization of Janus nanoparticles. (a) Silica particles (500 nm in diameter) were first functionalized with azide groups. A piece of PDMS elastomer “inked” with biotin-BSA (shown in blue) was pressed against a monolayer of azide-functionalized silica particles to transfer a patch of biotin-BSA proteins onto the particle surface. After lifting off the PDMS elastomer, particles remained embedded and their exposed surface was functionalized with alkyne-tagged anti-CD28 antibodies via click chemistry. After click chemistry, particles were sonicated off the PDMS and functionalized with biotinylated anti-CD3 antibody using biotin-streptavidin (SA) conjugation. (b) Super-resolution fluorescence images from structured illumination microscopy (SIM) show the spatial segregation of the anti-CD3 (shown in red) and anti-CD28 (shown in green) clusters. Scale bar: 500 nm. (c) Histogram showing the distribution of the size of anti-CD3 clusters (N = 28). The average surface coverage of anti-CD3 is 0.11 ± 0.02 μm² per particle.

Figure 2

(a) A schematic illustration of the calcium response of T cells upon stimulation by three different types of nanoparticles: Janus particle (JP), uniform particle with the same number of ligands (U#), and uniform particles with the same density of ligands (Ud). (b) Color-coded plots show the calcium response of T cells stimulated by different types of nanoparticles as indicated. Each horizontal line indicates the calcium response of a single T cell as a function of time. The calcium response, measured as the fluorescence intensity of the calcium indicator Fluo-4, was normalized and color-coded on the same scale. (c) Quantification of T cell activation. Response fraction, defined as the fraction of time when the normalized fluorescence intensity of a T cell is greater than the threshold, quantifies the persistence of a calcium response. Average fluorescence amplitude, defined as the time-average of normalized fluorescence intensities on the entire calcium plot for each T cell, measures the average intensity of a calcium response. Both parameters extracted from results shown in (b) are plotted in a scatter plot together with a box plot. Each box plot indicates the median and the interquartile range from 25% to 75% of the corresponding data set. Statistical significance is highlighted by p-values (student’s t test) as indicated.

Figure 3

Calcium response of T cells stimulated by three types of nanoparticles coated only with anti-CD3. (a) Color-coded plots show the calcium response of T cells stimulated by different types of nanoparticles as indicated. (b) Quantification of T cell activation. Two parameters, response fraction and average fluorescence amplitude, extracted from results shown in (a) are plotted in a scatter plot together with a box plot. Each box plot indicates the median and the interquartile range from 25% to 75% of the corresponding data set. Statistical significance is highlighted by P-values (student’s t test) as indicated.