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The CD47-SIRP Pathway Regulates Phagocytosis

The CD47-SIRP alpha Pathway is an Innate Immune Checkpoint

In contrast to the T cell-related immune checkpoint pathways that are being targeted for cancer immunotherapy, the CD47/SIRP pathway is an innate immune checkpoint that suppresses the phagocytic activity of myeloid cells.1CD47, also known as Integrin-associated protein and OA3, is an atypical member of the immunoglobulin superfamily possessing a single IgV-like domain at its N-terminus, five transmembrane-spanning segments, and an alternatively spliced cytoplasmic C-terminus that lacks a signaling domain.2 CD47 is ubiquitously expressed on normal cells and serves as a marker of “self” to prevent healthy autologous cells from undergoing phagocytosis.3 As a result, it has also been called a “don’t eat me” signal.

Inhibition of phagocytosis is mediated by CD47 binding to SIRP alpha, another immunoglobulin superfamily member, that is highly expressed on the surface of myeloid cells, including macrophages, dendritic cells (DCs), neutrophils, and monocytes.3, 4 SIRP alpha contains an extracellular domain with a single N-terminal IgV-like domain that interacts with the IgV-like domain of CD47, followed by two IgC-like domains, a transmembrane segment, and a cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs).5 Binding of CD47 to SIRP alpha promotes phosphorylation of the ITIM domains in the cytoplasmic tail of SIRP alpha, leading to the recruitment of the SHP1/2 phosphatases, which subsequently dephosphorylate downstream targets to negatively regulate phagocytosis.6-8

In addition to SIRP alpha, CD47 also binds with significantly lower affinity to SIRP gamma, which is expressed on human T cells, and is thought to play a role in transendothelial migration.9 Other CD47-interacting proteins include the extracellular matrix protein, thrombospondin-1, and various integrins, including integrin alpha V beta 3, integrin alpha 2b beta 3, integrin alpha 2 beta 1, and integrin alpha 4 beta 1, which CD47 interacts with in cis.10-12 Significantly, the interaction of thrombospondin-1 with CD47 has also been reported to have immunosuppressive effects, which include the inhibition of T cell activation, generation of regulatory T cells from naïve or memory T cells, and negative regulation of dendritic cell activation and cytokine production.13-16

CD47 overexpression is common in a variety of hematologic and solid tumor cancers and it typically correlates with a poor prognosis, suggesting that tumor cells can exploit the SIRP alpha/CD47 pathway to evade phagocytic destruction.13-21 Blocking antibodies against CD47 have been shown to promote macrophage-mediated tumor cell phagocytosis in vitro and inhibit tumor growth and metastasis in multiple xenograft or syngeneic mouse cancer models.22, 23 Additionally, CD47 blockade has been shown to enhance antigen uptake and presentation, and cytokine secretion by dendritic cells and macrophages, resulting in an increase in the activation of tumor-specific cytotoxic T cells.3, 23-25 Studies in mouse tumor models have suggested that blockade of SIRP alpha or CD47, used in combination with other immune checkpoint inhibitors that target PD-1 or CTLA-4, may have synergistic anti-tumor effects.23

Bio-Techne offers R&D SystemsTM bioactive recombinant proteins for all five SIRP family proteins, with a variety of tags, along with recombinant human and mouse CD47 proteins and a wide selection of anti-CD47 and anti-SIRP alpha antibodies that are validated for a range of applications, including blocking/neutralization, flow cytometry, immunocytochemistry, and Western blot.

Tumor Cells can Exploit the CD47-SIRP alpha Pathway to Evade Phagocytic Destruction

The CD47-SIRP alpha phagocytosis checkpoint can be exploited by tumor cells, allowing them to evade immune detection.

The CD47-SIRP alpha signaling pathway is a myeloid cell-specific phagocytosis checkpoint that can be exploited by tumor cells. CD47 is a ubiquitously expressed protein that serves as a marker of “self”. Binding of CD47 to SIRP alpha on myeloid cells inhibits myeloid cell-mediated phagocytosis. By overexpressing CD47, tumor cells frequently exploit this pathway, allowing them to evade detection and phagocytic destruction. In addition to its interaction with SIRP alpha, CD47 interacts with Thrombospondin-1, and this interaction also has immunosuppressive effects including the inhibition of T cell and dendritic cell activation and the generation of regulatory T cells.

Analysis of the Binding Properties of R&D Systems CD47:SIRP alpha Proteins by SPR

Surface plasmon resonance data showing affinity measurements and binding kinetics between the CD47 and SIRP alpha proteins.

Affinity Measurements and Binding Kinetics of the CD47:SIRP alpha Interaction by Surface Plasmon Resonance. Sensorgram data of captured Avi-tag Biotinylated Recombinant Human CD47 Fc tag (R&D Systems, Catalog # AVI4670)binding to Recombinant Human SIRP alpha His tag (R&D Systems, Catalog # 9378-SA). The corresponding overlaid kinetic fits with the residual plot shown below. The concentration of Recombinant Human SIRP alpha His-tag ranged from 6.85 nM to 3.51 μM. The corresponding steady state affinity fit is shown at the bottom. The experiment was performed on a Biacore T200, GE Healthcare. 

Analysis of the Bioactivity of R&D Systems Recombinant Mouse SIRP alpha and CD47 Proteins

Analysis of the effect of Recombinant Mouse SIRP-alpha on red blood cell adhesion and neutralization by soluble CD47 protein.

Recombinant Mouse SIRP alpha Activity is Suppressed by a Soluble Version of the CD47 Receptor. Red blood cells from mouse whole blood were incubated with the indicated concentrations of immobilized Recombinant Mouse SIRP alpha Fc Chimera (R&D Systems, Catalog # 7154-SA) for 1 hour at 37˚C. The plate was washed to remove non-adhered cells and red blood adhesion was assessed (green line). When Recombinant Mouse SIRP alpha Fc Chimera was immobilized at 2 μg/mL, the effect could be antagonized by the addition of soluble Recombinant Mouse CD47 Fc Chimera (R&D Systems, Catalog # 1866-CD; blue line).

References

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3. Matlung, H.L. et al (2017) The CD47-SIRP-alpha signaling axis as an innate immune checkpoint in cancer. Immunol. Rev. 276 145 PMID: 28258703

4. Adams, S. et al (1998) Signal-regulatory protein is selectively expressed by myeloid and neuronal cells. J. Immunol. 161 1853 PMID: 9712053

5. van Beek, E.M. et al (2005) Signal regulatory proteins in the immune system. J. Immunol. 175 7781 PMID: 16339510

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7. Lienard, H. et al (1999) Signal regulatory proteins negatively regulate immunoreceptor-dependent cell activation. J. Biol. Chem. 274 32493 PMID: 10542295

10. Brown, E.J. & W.A. Frazier (2001) Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 11 130 PMID: 11306274

12. Isenberg, J.S. et al (2008) CD47: a new target in cardiovascular therapy. Arterioscler. Thromb. Vasc. Biol. 28 615 PMID: 18187671

13. Li, Z. et al (2001) Thrombospondin-1 inhibits TCR-mediated T lymphocyte early activation. J. Immunol. 166 2427 PMID: 11160302

16. Doyen, V. et al (2003) Thrombospondin 1 is an autocrine negative regulator of human dendritic cell activation. J. Exp. Med. 198 1277 PMID: 14568985

17. Rendtlew, J.M. et al (2007) Dysregulation of CD47 and the ligands thrombospondin 1 and 2 in multiple myeloma. Br. J. Haematol. 138 756 PMID: 17760807

19. Edris, B. et al (2012) Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma. Proc. Natl. Acad. Sci. USA 109 6656 PMID: 22451919

20. Willingham, S.B. et al (2012) The CD47-signal regulatory protein alpha (SIRP-alpha) interaction is a therapeutic target for human solid tumors. Proc. Natl. Acad. Sci. USA 109 6662 PMID: 22451913

21. Liu, X.J. et al (2017) Is CD47 an innate immune checkpoint for tumor evasion? J. Hematol. Oncol. 10 12 PMID: 28077173

22. Burugu, S. et al (2017) Emerging targets in cancer immunotherapy. Semin. Cancer Biol. 52 39 PMID: 28987965

24. Tseng, D. et al (2013) Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response. Proc. Natl. Acad. Sci. USA 110 11103 PMID: 23690610

25. Liu, X. et al (2015) CD47 blockade triggers T cell-mediated destruction of immunogenic tumors. Nat. Med. 21 1209 PMID: 26322579