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Membrane fluctuations and acidosis regulate cooperative binding of 'marker of self' protein CD47 with the macrophage checkpoint receptor SIRP?.

ABSTRACT: Cell-cell interactions that result from membrane proteins binding weakly in trans can cause accumulations in cis that suggest cooperativity and thereby an acute sensitivity to environmental factors. The ubiquitous 'marker of self' protein CD47 binds weakly to SIRP? on macrophages, which leads to accumulation of SIRP? (also known as SHPS-1, CD172A and SIRPA) at phagocytic synapses and ultimately to inhibition of engulfment of 'self' cells - including cancer cells. We reconstituted this macrophage checkpoint with GFP-tagged CD47 on giant vesicles generated from plasma membranes and then imaged vesicles adhering to SIRP? immobilized on a surface. CD47 diffusion is impeded near the surface, and the binding-unbinding events reveal cooperative interactions as a concentration-dependent two-dimensional affinity. Membrane fluctuations out-of-plane link cooperativity to membrane flexibility with suppressed fluctuations in the vicinity of bound complexes. Slight acidity (pH 6) stiffens membranes, diminishes cooperative interactions and also reduces 'self' signaling of cancer cells in phagocytosis. Sensitivity of cell-cell interactions to microenvironmental factors - such as the acidity of tumors and other diseased or inflamed sites - can thus arise from the collective cooperative properties of flexible membranes.This article has an associated First Person interview with the first author of the paper.

SUBMITTER: Steinkuhler J

PROVIDER: S-EPMC6398477 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Membrane fluctuations and acidosis regulate cooperative binding of 'marker of self' protein CD47 with the macrophage checkpoint receptor SIRPα.

Steinkühler Jan J Różycki Bartosz B Alvey Cory C Lipowsky Reinhard R Weikl Thomas R TR Dimova Rumiana R Discher Dennis E DE

Journal of cell science 20180716 4

Cell-cell interactions that result from membrane proteins binding weakly in <i>trans</i> can cause accumulations in <i>cis</i> that suggest cooperativity and thereby an acute sensitivity to environmental factors. The ubiquitous 'marker of self' protein CD47 binds weakly to SIRPα on macrophages, which leads to accumulation of SIRPα (also known as SHPS-1, CD172A and SIRPA) at phagocytic synapses and ultimately to inhibition of engulfment of 'self' cells - including cancer cells. We reconstituted t ...[more]

PMID: 29777034

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Project description:BACKGROUND:Accumulating preclinical data indicate that targeting the SIRP?/CD47 axis alone or in combination with existing targeted therapies or immune checkpoint inhibitors enhances tumor rejection. Although several CD47-targeting agents are currently in phase I clinical trials and demonstrate activity in combination therapy, high and frequent dosing was required and safety signals (acute anemia, thrombocytopenia) were recorded frequently as adverse events. Based on the restricted expression pattern of SIRP? we hypothesized that antibodies targeting SIRP? might avoid some of the concerns noted for CD47-targeting agents. METHODS:SIRP?-targeting antibodies were generated and characterized for binding to human SIRP? alleles and blockade of the interaction with CD47. Functional activity was established in vitro using human macrophages or neutrophils co-cultured with human Burkitt's lymphoma cell lines. The effect of SIRP? versus CD47 targeting on human T-cell activation was studied using an allogeneic mixed lymphocyte reaction and a Staphylococcus enterotoxin B-induced T-cell proliferation assay. Potential safety concerns of the selected SIRP?-targeting antibody were addressed in vitro using a hemagglutination assay and a whole blood cytokine release assay, and in vivo in a single-dose toxicity study in cynomolgus monkeys. RESULTS:The humanized monoclonal IgG2 antibody ADU-1805 binds to all known human SIRP? alleles, showing minimal binding to SIRP?1, while cross-reacting with SIRP?, and potently blocking the interaction of SIRP? with CD47. Reduced Fc?R binding proved critical to retaining its function towards phagocyte activation. In vitro characterization demonstrated that ADU-1805 promotes macrophage phagocytosis, with similar potency to anti-CD47 antibodies, and enhances neutrophil trogocytosis. Unlike CD47-targeting agents, ADU-1805 does not interfere with T-cell activation and is not expected to require frequent and extensive dosing due to the restricted expression of SIRP? to cells of the myeloid lineage. ADU-1805 is cross-reactive to cynomolgus monkey SIRP? and upon single-dose intravenous administration in these non-human primates (NHPs) did not show any signs of anemia, thrombocytopenia or other toxicities. CONCLUSIONS:Blocking the SIRP?-CD47 interaction via SIRP?, while similarly efficacious in vitro, differentiates ADU-1805 from CD47-targeting agents with respect to safety and absence of inhibition of T-cell activation. The data presented herein support further advancement of ADU-1805 towards clinical development.

Project description:Rapid clearance of adoptively transferred Cd47-null (Cd47(-/-)) cells in congeneic WT mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of self, and its interaction with macrophage signal regulatory protein ? (SIRP?) triggers inhibitory signaling through SIRP? cytoplasmic immunoreceptor tyrosine-based inhibition motifs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotypes, Cd47(-/-) mice manifest no, or only mild, macrophage phagocytosis toward self-cells except under the nonobese diabetic background. Studying our recently established Sirp?-KO (Sirp?(-/-)) mice, as well as Cd47(-/-) mice, we reveal additional activation and inhibitory mechanisms besides the CD47-SIRP? axis dominantly controlling macrophage behavior. Sirp?(-/-) mice and Cd47(-/-) mice, although being normally healthy, develop severe anemia and splenomegaly under chronic colitis, peritonitis, cytokine treatments, and CFA-/LPS-induced inflammation, owing to splenic macrophages phagocytizing self-red blood cells. Ex vivo phagocytosis assays confirmed general inactivity of macrophages from Sirp?(-/-) or Cd47(-/-) mice toward healthy self-cells, whereas they aggressively attack toward bacteria, zymosan, apoptotic, and immune complex-bound cells; however, treating these macrophages with IL-17, LPS, IL-6, IL-1?, and TNF?, but not IFN?, dramatically initiates potent phagocytosis toward self-cells, for which only the Cd47-Sirp? interaction restrains. Even for macrophages from WT mice, phagocytosis toward Cd47(-/-) cells does not occur without phagocytic activation. Mechanistic studies suggest a PKC-Syk-mediated signaling pathway, to which IL-10 conversely inhibits, is required for activating macrophage self-targeting, followed by phagocytosis independent of calreticulin Moreover, we identified spleen red pulp to be one specific tissue that provides stimuli constantly activating macrophage phagocytosis albeit lacking in Cd47(-/-) or Sirp?(-/-) mice.

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Membrane fluctuations and acidosis regulate cooperative binding of 'marker of self' protein CD47 with the macrophage checkpoint receptor SIRP?.

Publications

Membrane fluctuations and acidosis regulate cooperative binding of 'marker of self' protein CD47 with the macrophage checkpoint receptor SIRPα.

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