Tight nanoscale clustering of Fcγ-receptors using DNA origami promotes phagocytosis

Background

Phagocytosis (from Ancient Greek (phagein) ‘to eat’, and (kytos) ‘cell’) is the process by which cells consume foreign particles from their environment and plays an essential role in humoral immunity (Fig. 1A). Macrophages recognize invading pathogens like bacteria and viruses after they have been identified and bound by antibodies such as IgG. This binding of pathogens by IgG is called opsonization. Once macrophages encounter an antibody-opsonized target, receptors on their cell surface bind to the Fc region of the IgG antibody. When macrophages have bound to a significant number of IgG molecules, they undergo extensive morphological changes to envelop and consume the opsonized target (Fig. 1A). A big question in the field is what constitutes a significant number of IgG molecules; what are the rules for phagocytosis? 

A key macrophage receptor that recognizes the Fc region is the Fc gamma receptor (FcɣR, Fig. 1B). Binding of FcɣR to Fc initiates an intracellular signaling cascade resulting in cytoskeletal and membrane protein rearrangements that facilitate phagocytosis. Previous studies have demonstrated that increased FcɣR engagement promotes phagocytosis, but the optimal spacing and density of engaged FcɣRs remains a major open question in the field. In this preprint, Kern et al. address this question with a synthetic biology approach. They make a chimeric FcɣR molecule that can bind to DNA oligonucleotide ligands and combine this approach with a new DNA origami-based method that allows them to precisely control the spacing and arrangement of these oligonucleotide ligands (Fig. 1B). This method allows them to exactly determine the optimal spacing and density of FcɣR ligands required to trigger phagocytosis. 

DNA origami uses single stranded DNA and “staple oligos” to anneal a DNA template into a three-dimensional structure of precise shape and orientation. In this study the authors are able to create a rectangular DNA origami  “pegboard” onto which the oligonucleotide ligands can be arranged with nanometer precision and anchored to silica beads (Fig. 1C, 1D). The authors use these ligand-opsonized beads to engage FcɣR receptors in human macrophages in various arrangements and densities, identifying optimal receptor positioning for phagocytosis of these synthetic particles.

• The authors engineered macrophages with synthetic Fcɣ receptors that could be attached to DNA oligonucleotides (DNA-CARɣ). Binding to DNA-ligands can induce clustering of these DNA-CARɣ molecules (Fig. 1A). 
• They combined these cells with silica beads coated with complimentary DNA oligonucleotide ligands that were precisely arranged using DNA-origami technology (Fig. 1C, 1D). Annealing of the oligo-ligands to the DNA-CARɣ molecules mimics the binding of an Fc region to the endogenous FcɣR and initiates phagocytosis.
• The DNA Origami platform is called a “pegboard” (analogous to a pegboard where tools can be hung in many different ways (Fig. 1C, 1D). Oligo-ligands arranged on the pegboard platform were used to activate DNA-CARɣ macrophages and allows manipulation of ligand spacing at the nanometer-resolution to quantitatively investigate receptor engagement and downstream signaling pathways (Fig. 2A, B).

Summary Figures (adapted from Kern et al)

Key Findings

• A cluster size of 8 ligands is the critical density threshold for FcɣR clusters needed to initiate FcɣR signalling and phagocytosis. 
• The strongest initiator of phagocytosis was the closest possible clustering of receptor-ligands (3.5 nm apart, Fig. 2A) on the particle. This experiment also suggested that avidity does not cause preferential engulfment.
• Tighter spacing between ligand-receptor molecules (4T origami pegboard, Fig. 2B) increases the probability of engulfment initiation as well as the overall frequency of successful completion of engulfment.
• 4T ligand spacing was still preferentially engulfed when 4T, 4M, and 4S ligands were replaced with higher-affinity DNA oligos (Fig. 2C), suggesting avidity does not play a role in engulfment efficiency.  
• Spatial organization of ligand-receptor molecules can affect downstream signaling events that occur in phagocytic cup formation, such as an increase in receptor phosphorylation observed in the more tightly clustered ligands. However, increasing the number of intracellular signalling modules (ITAMs) was not sufficient to surpass the threshold required to initiate phagocytic engulfment. 

 

Why we chose this prelight

The use of DNA origami to precisely space DNA-FcɣR ligands is a fascinating approach to answer the important question of optimal FcɣR spacing and density. This controlled and tunable system yielded convincing data supporting a specific orientation of DNA-FcɣR ligands that is optimal for phagocytosis. Previous work has not offered such specific and fine control over the spacing of ligands making this system an exciting advancement for the future investigation of the cell-target interactions that drive phagocytosis and other signalling systems as well as having therapeutic implications for optimizing antibody spacing or chimeric antigen receptors.

 

Questions for the authors

• Does the size of the particle itself impact phagocytosis? Would the same experiment on a smaller or larger bead yield similar results?
• How does the affinity for the DNA-FcɣR ligand compare to the endogenous Fc region and how might this impact phagocytosis? Have the authors consider probing the effects of using different combinations of affinity ligands to see how weak interactions may affect phagocytosis?
• Do 4T origami pegboard spatial dynamics of ligand-receptor interactions recapitulate biologically relevant conformations such as those found on opsonized viruses or bacteria?  
• According to Bakalar et al. (2018), antigen height can enhance/ impair phagocytic efficiency depending on the distance between the target cell and macrophage. We noticed that the height of the ligand remained unchanged across the experiments performed – do the authors believe that changing the height would affect which spatial arrangement (4T, 4M, 4S) or which cluster size leads to preferential engulfment?

 

References

Tight nanoscale clustering of Fcγ-receptors using DNA origami promotes phagocytosis
Nadja Kern, Rui Dong, Shawn M. Douglas, Ronald D. Vale, Meghan A. Morrissey
bioRxiv 2021.03.18.436011; doi: https://doi.org/10.1101/2021.03.18.436011

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