Macrophages show higher levels of engulfment after disruption of cis interactions between CD47 and the checkpoint receptor SIRPα.

The macrophage checkpoint receptor SIRPα signals against phagocytosis by binding CD47 expressed on all cells - including macrophages. Here, we found that inhibiting cis interactions between SIRPα and CD47 on the same macrophage increased engulfment ('eating') by approximately the same level as inhibiting trans interactions. Antibody blockade of CD47, as pursued in clinical trials against cancer, was applied separately to human-derived macrophages and to red blood cell (RBC) targets for phagocytosis, and both scenarios produced surprisingly similar increases in RBC engulfment. Blockade of both macrophages and targets resulted in hyper-phagocytosis, and knockdown of macrophage-CD47 likewise increased engulfment of 'foreign' cells and particles, decreased the baseline inhibitory signaling of SIRPα, and linearly increased binding of soluble CD47 in trans, consistent with cis-trans competition. Many cell types express both SIRPα and CD47, including mouse melanoma B16 cells, and CRISPR-mediated deletions modulate B16 phagocytosis, consistent with cis-trans competition. Additionally, soluble SIRPα binding to human CD47 displayed on Chinese hamster ovary (CHO) cells was suppressed by SIRPα co-display, and atomistic computations confirm SIRPα bends and binds CD47 in cis Safety and efficacy profiles for CD47-SIRPα blockade might therefore reflect a disruption of both cis and trans interactions.

Self inhibition of phagocytosis: the affinity of 'marker of self' CD47 for SIRPalpha dictates potency of inhibition but only at low expression levels.

Phagocytes engulf foreign cells but not 'self' in part because self cells express CD47 as a ligand for signal regulatory protein SIRPalpha, which inhibits phagocytosis. Motivated by reports of upregulation of CD47 on both normal and cancerous stem cells [1: Jaiswal et al., 2009] and also by polymorphisms in SIRPalpha [2: Takenaka et al., 2007], we show here that inhibition of engulfment correlates with affinity of CD47 for SIRPalpha - but only at low levels of CD47. One common human polymorph of SIRPalpha is studied and binds more strongly to human-CD47 than to mouse-CD47 (K(d) approximately 0.12 microM and 6.9 microM, respectively) and does not bind sheep red blood cells (RBCs) - which are well-established targets of human macrophages; in comparison, a common mouse polymorph of SIRPalpha binds with similar affinity to human and mouse CD47 (K(d) approximately 0.22 microM). Using immunoglobulin (IgG)-opsonized particles with varying levels of either human- or mouse-CD47, the effective inhibition constants K(i) for blocking phagocytosis are then determined with both human- and mouse-derived macrophages. Only human phagocytes show significant differences in man versus mouse K(i)'s and only at CD47 levels below normal densities for RBCs. While phospho-signaling through human-SIRPalpha shows similar trends, consistent again with the affinity differences, saturating levels of CD47 (>K(i)) can signal and inhibit phagocytosis regardless of man versus mouse. Quantitative analyses here prompt more complete characterizations of both CD47 levels and SIRPalpha polymorphisms when attempting to study in vivo effects of these key proteins in innate immunity.

"Marker of Self" CD47 on lentiviral vectors decreases macrophage-mediated clearance and increases delivery to SIRPA-expressing lung carcinoma tumors.

Lentiviruses infect many cell types and are now widely used for gene delivery in vitro, but in vivo uptake of these foreign vectors by macrophages is a limitation. Lentivectors are produced here from packaging cells that overexpress "Marker of Self" CD47, which inhibits macrophage uptake of cells when prophagocytic factors are also displayed. Single particle analyses show "hCD47-Lenti" display properly oriented human-CD47 for interactions with the macrophage's inhibitory receptor SIRPA. Macrophages derived from human and NOD/SCID/Il2rg-/- (NSG) mice show a SIRPA-dependent decrease in transduction, i.e., transgene expression, by hCD47-Lenti compared to control Lenti. Consistent with known "Self" signaling pathways, macrophage transduction by control Lenti is decreased by drug inhibition of Myosin-II to the same levels as hCD47-Lenti. In contrast, human lung carcinoma cells express SIRPA and use it to enhance transduction by hCD47-Lenti- as illustrated by more efficient gene deletion using CRISPR/Cas9. Intravenous injection of hCD47-Lenti into NSG mice shows hCD47 prolongs circulation, unless a blocking anti-SIRPA is preinjected. In vivo transduction of spleen and liver macrophages also decreases for hCD47-Lenti while transduction of lung carcinoma xenografts increases. hCD47 could be useful when macrophage uptake is limiting on other viral vectors that are emerging in cancer treatments (e.g., Measles glycoprotein-pseudotyped lentivectors) and also in targeting various SIRPA-expressing tumors such as glioblastomas.

Minimal "Self" peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles.

Foreign particles and cells are cleared from the body by phagocytes that must also recognize and avoid clearance of "self" cells. The membrane protein CD47 is reportedly a "marker of self" in mice that impedes phagocytosis of self by signaling through the phagocyte receptor CD172a. Minimal "Self" peptides were computationally designed from human CD47 and then synthesized and attached to virus-size particles for intravenous injection into mice that express a CD172a variant compatible with hCD47. Self peptides delay macrophage-mediated clearance of nanoparticles, which promotes persistent circulation that enhances dye and drug delivery to tumors. Self-peptide affinity for CD172a is near the optimum measured for human CD172a variants, and Self peptide also potently inhibits nanoparticle uptake mediated by the contractile cytoskeleton. The reductionist approach reveals the importance of human Self peptides and their utility in enhancing drug delivery and imaging.

The effect of CD47 modified polymer surfaces on inflammatory cell attachment and activation.

CD47 is a transmembrane protein that is a marker of "self". CD47 binding to its cognate receptor in leukocytes and macrophages, signal-regulatory protein alpha (SIRPα), causes inhibition of inflammatory cell attachment. We hypothesized that immobilization of recombinant CD47 on polymeric surfaces would reduce inflammation. Recombinant CD47 was appended to polyvinyl chloride (PVC) or polyurethane (PU) surfaces via photoactivation chemistry. Cell culture studies showed that CD47 immobilization significantly reduced human neutrophil (HL-60) and human monocyte derived macrophage (MDM) (THP-1) attachment to PVC and PU respectively. A neutralizing antibody, directed against SIRPα, inhibited THP-1 and HL-60 binding to PU and PVC surfaces respectively. This antibody also increased the level of SIRPα tyrosine phosphorylation, thereby indicating a direct role for SIRPα mediated signaling in preventing inflammatory cell attachment. Studies using human blood in an ex vivo flow-loop showed that CD47 modified PVC tubing significantly reduced cell binding and neutrophil activation compared to unmodified tubing or poly-2-methoxy-ethylacrylate (PMEA) coated tubing. In ten-week rat subdermal implants, CD47 functionalized PU films showed a significant reduction in markers of MDM mediated oxidative degradation compared to unmodified PU. In conclusion, CD47 functionalized surfaces can resist inflammatory cell interactions both in vitro and in vivo.

Inhibition of "self" engulfment through deactivation of myosin-II at the phagocytic synapse between human cells.

Phagocytosis of foreign cells or particles by macrophages is a rapid process that is inefficient when faced with "self" cells that display CD47-although signaling mechanisms in self-recognition have remained largely unknown. With human macrophages, we show the phagocytic synapse at cell contacts involves a basal level of actin-driven phagocytosis that, in the absence of species-specific CD47 signaling, is made more efficient by phospho-activated myosin. We use "foreign" sheep red blood cells (RBCs) together with CD47-blocked, antibody-opsonized human RBCs in order to visualize synaptic accumulation of phosphotyrosine, paxillin, F-actin, and the major motor isoform, nonmuscle myosin-IIA. When CD47 is functional, the macrophage counter-receptor and phosphatase-activator SIRPalpha localizes to the synapse, suppressing accumulation of phosphotyrosine and myosin without affecting F-actin. On both RBCs and microbeads, human CD47 potently inhibits phagocytosis as does direct inhibition of myosin. CD47-SIRPalpha interaction initiates a dephosphorylation cascade directed in part at phosphotyrosine in myosin. A point mutation turns off this motor's contribution to phagocytosis, suggesting that self-recognition inhibits contractile engulfment.