Figure 1. A DNA-based system for controlling engulfment.

(A) Schematic shows the endogenous (left box) and DNA-based (middle and right boxes) engulfment systems. Engulfment via endogenous FcγRs (left box) is induced through anti-biotin IgG bound to 1-oleoyl-2-(12-biotinyl(aminododecanoyl))-sn-glycero-3-phosphoethanolamine (biotin-PE) lipids incorporated into the bilayer surrounding the silica bead targets. Engulfment induced via the DNA-based system uses chimeric antigen receptors (CAR) expressed in the macrophage and biotinylated ligand DNA that is bound to the lipid bilayer surrounding the silica bead. The DNA-CARγ (middle box) consists of a single-stranded DNA (ssDNA) (receptor DNA) covalently attached to an extracellular SNAP-tag fused to a CD86 transmembrane domain, the intracellular domain of the FcRγ chain, and a fluorescent tag. The DNA-CAR_adhesion (right box) is identical but lacks the signaling FcRγ chain. (B) Example images depicting the engulfment assay. Silica beads were coated with a supported lipid bilayer (magenta) and functionalized with neutravidin and the indicated density of ligand DNA (Figure 1—figure supplement 1A). The functionalized beads were added to RAW264.7 macrophages expressing either the DNA-CARγ or the DNA-CAR_adhesion (green) and fixed after 45 min. The average number of beads engulfed per macrophage was assessed by confocal microscopy. Scale bar denotes 5 µm here and in all subsequent figures. Internalized beads are denoted with a white sphere in the merged images. (C) The number of beads engulfed per cell for DNA-CARγ (blue) or DNA-CAR_adhesion (gray) macrophages was normalized to the maximum bead eating observed in each replicate. Dots and error bars denote the mean ± SEM of three independent replicates (n ≥ 100 cells analyzed per experiment). (D) DNA-CARγ-expressing macrophages were incubated with bilayer-coated beads (gray) functionalized with anti-biotin IgG (magenta), neutravidin (black), or neutravidin and saturating amounts of ssDNA (blue). The average number of beads engulfed per cell was assessed. Full data representing the fraction of macrophages engulfing specific numbers of IgG or ssDNA beads is shown in Figure 1—figure supplement 1. Each data point represents the mean of an independent experiment, denoted by symbol shape, and bars denote the mean ± SEM. n.s. denotes p>0.05, * indicates p<0.05, ** indicates p<0.005, and **** indicates p<0.0001 by a multiple t-test comparison corrected for multiple comparisons using the Holm–Sidak's method (C) or Student’s t-test (D).

Figure 1—figure supplement 1. DNA-based engulfment system reflects endogenous engulfment.

(A) Graph depicts the calibration used to determine the surface density of single-stranded DNA (ssDNA) on beads used in Figure 1B, C. The intensity of Alexa Fluor 647 fluorescent bead standards (black dots) was measured, and a simple linear regression (red line) was fit to the data. The fluorescence intensity of Alexa Fluor 647-ssDNA coated beads (blue dots) was measured, and the surface density was interpolated using the regression determined from the fluorescent bead standards. The concentration of ssDNA used for each bead coupling condition is indicated next to the blue points on the graph. (B) Macrophages expressing the DNA-CARγ (blue) or the DNA-CAR_adhesion (gray) engulfed similar distributions of IgG-functionalized beads. Data is pooled from two independent replicates. (C) Graph depicts the fraction of macrophages engulfing the indicated number of IgG (magenta) or ssDNA (blue) beads from data pooled from the three independent replicates presented in Figure 1D. (D) Graph shows the average number of neutravidin (black), ligand-DNA (blue), or IgG (magenta) functionalized beads engulfed by the monocyte-like cell line THP1. Lines denote the mean engulfment from each independent replicate, and bars denote ± SEM. p values were calculated using the Mann–Whitney test (B, C) and n.s. denotes p>0.05 as determined by the Student’s t-test (D).