Figure 4. Spatial arrangement of ligands within nanoclusters regulates engulfment.

(A) Schematics (top) depict 4-ligand origami pegboards presenting ligands at the positions indicated in red. Beads were functionalized with 0-ligand ‘blank’ (gray) origami pegboards, 4T (orange) origami pegboards, 4M (green) origami pegboards, or 4S (cyan) origami pegboards at equal amounts and fed to DNA-CARγ-expressing macrophages. Representative confocal images (middle) depict bead (bilayer in magenta) engulfment by macrophages (green). Internalized beads are denoted with a white sphere. Quantification of the engulfment assay is shown in the graph below depicting the number of beads engulfed per macrophage normalized to the maximum observed eating in that replicate. (B) Schematics of the receptor DNA (blue) paired with the medium-affinity 13 base pair DNA-ligand (red) used in all previous experiments including (A) and the high-affinity 16 base pair ligand-DNA (yellow) used for experiment shown in the graph below. Beads were functionalized with 0-ligand ‘blank’ (gray), high-affinity 4T (orange), high-affinity 4M (green), or high-affinity 4S (cyan) origami pegboards and fed to DNA-CARγ-expressing macrophages. Graph shows the number of beads engulfed per macrophage normalized to the maximum observed eating in that replicate. Each data point represents the mean of an independent experiment, shapes denote data from the same replicate, and bars show the mean ± SEM (A, B). * denotes p<0.05, *** denotes p<0.0005, **** denotes p<0.0001, and n.s. denotes p>0.05 as determined by an ordinary one-way ANOVA with Holm–Sidak’s multiple comparison test (A, B).

Figure 4—figure supplement 1. Ligand clustering enhances engulfment in RAW macrophages expressing DNA-CARs with endogenous FcγR transmembrane domains and in THP1s.

(A) Graph shows the average Atto647N fluorescence intensity from the beads used in Figure 4A measured using confocal microscopy. (B) Beads were functionalized with the indicated ligand-presenting origami pegboards in amounts calculated to equalize the total number of origami pegboards and ligands across conditions. Schematics (left) depict the origami utilized, where the positions presenting a ligand (red dots) and the positions not occupied by a ligand (light blue) are indicated. Graph (right) depicts the average number of the indicated type of beads internalized per DNA-CARγ-expressing THP1, normalized to the maximum bead eating in that replicate. (C) Graph shows the average Atto647N647 fluorescence intensity from the beads used in Figure 4B measured using confocal microscopy. (D) Schematics below the graph depict the DNA-CAR constructs designed with varying transmembrane domains. Beads were functionalized with 4T origami pegboards (orange), 4S origami pegboards (cyan), or 0-ligand ‘blank’ origami pegboards (gray) and fed to macrophages expressing the DNA-CAR receptor depicted below each section of the graph. Graph depicts the number of beads engulfed per macrophage normalized to the maximum observed eating in that replicate. (E) Graph shows the average Atto647N fluorescence intensity from the beads used in (D) measured using confocal microscopy. (F) DNA-CAR receptors used in (D) are expressed and trafficked to the membrane at similar levels. Fluorescent intensity at the cell cortex of the DNA-CAR-infected macrophage was quantified using the mean intensity of a two-pixel width linescan at the cell membrane, with the mean intensity of a linescan immediately adjacent to the cell subtracted for local background. The fluorescence intensity was normalized to the average intensity of the DNA-CAR_adhesion in each experiment. Each dot represents an individual cell, and data is pooled from three independent experiments, with red lines denoting mean ± SEM. n.s. denotes p>0.05, * denotes p<0.05, ** denotes p<0.005, *** denotes p<0.0005, and **** indicates p<0.0001 as determined by an ordinary one-way ANOVA with Holm–Sidak’s multiple comparison test (A–F).