Nanoscale imaging of CD47 informs how plasma membrane modifications shape apoptotic cell recognition

Abstract

CD47 recognized by its macrophage receptor SIRPα serves as a “don’t eat-me” signal protecting viable cells from phagocytosis. How this is abrogated by apoptosis-induced changes in the plasma membrane, concomitantly with exposure of phosphatidylserine and calreticulin “eat-me“ signals, is not well understood. Using STORM imaging and single-particle tracking, we interrogate how the distribution of these molecules on the cell surface correlates with plasma membrane alteration, SIRPα binding, and cell engulfment by macrophages. Apoptosis induces calreticulin clustering into blebs and CD47 mobility. Modulation of integrin affinity impacts CD47 mobility on the plasma membrane but not the SIRPα binding, whereas CD47/SIRPα interaction is suppressed by cholesterol destabilization. SIRPα no longer recognizes CD47 localized on apoptotic blebs. Overall, the data suggest that disorganization of the lipid bilayer at the plasma membrane, by inducing inaccessibility of CD47 possibly due to a conformational change, is central to the phagocytosis process.

Quantitatively Lighting up the Spatial Organization of CD47/SIRPα Immune Checkpoints on the Cellular Membrane with Single-Molecule Localization Microscopy

“…Besides, the density of SIRPα clusters on the stimulated M1 macrophages is quantified to be about 1.5 times compared with that of M1 macrophages without stimulation (Figure d). These results indicate that SIRPα was accumulated into large clusters upon binding with CD47, which is consistent with the previous report of the clustering of SIRPα on the cell membrane during interaction with CD47. , A similar clustering phenomenon of SIRPα on the membrane of M2 macrophages is also observed upon CD47 stimulation (Figure e). The magnified images and cluster area distribution analysis indicate that CD47 stimulation leads to a significant increase in the size of SIRPα clusters (Figures f and g).…”

“…CD47 is a crucial membrane receptor that mediates phagocytosis and immune homeostasis. The interaction of CD47 with SIRPα on macrophages transfers the “do not eat me” signal to immune systems. , Through the overexpression of CD47, cancer cells, such as bladder tumor cells and breast cancer, can evade immune surveillance and spread to distant organs. − Immune checkpoints such as CD47 are usually organized into oligomers, which offer a platform for receptor–ligand interaction investigation and protein trafficking. Immune checkpoint oligomers typically range in size from 5 to 300 nm, − and the interactions between immune checkpoints generally occur at the scale of roughly 10 nm .…”

Rituximab-IgG2 is a phagocytic enhancer in antibody-based immunotherapy of B-cell lymphoma by altering CD47 expression

“…A similar change in spatial distribution of CD47 has previously been reported in aged mouse erythrocytes and human Jurkat T-cells. 16,19 These studies, together with our observation, suggest that apoptosis trigger major structural alterations of the cell plasma membrane, which may either destabilize lateral molecular interactions needed for the proper CD47 “don’t-eat-me” signaling 16,19 or induce conformation changes of CD47, hindering its interaction with SIRP-α 44 .…”