Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

CD47 masks pro-phagocytic ligands in cis on tumor cells to suppress antitumor immunity

Nature Immunology volume 24, pages 2032–2041 (2023)Cite this article

Subjects

Abstract

Cancer cells often overexpress CD47, which triggers the inhibitory receptor SIRPα expressed on macrophages, to elude phagocytosis and antitumor immunity. Pharmacological blockade of CD47 or SIRPα is showing promise as anticancer therapy, although CD47 blockade has been associated with hematological toxicities that may reflect its broad expression pattern on normal cells. Here we found that, in addition to triggering SIRPα, CD47 suppressed phagocytosis by a SIRPα-independent mechanism. This mechanism prevented phagocytosis initiated by the pro-phagocytic ligand, SLAMF7, on tumor cells, due to a cis interaction between CD47 and SLAMF7. The CD47–SLAMF7 interaction was disrupted by CD47 blockade and by a first-in-class agonist SLAMF7 antibody, but not by SIRPα blockade, thereby promoting antitumor immunity. Hence, CD47 suppresses phagocytosis not only by engaging SIRPα, but also by masking cell-intrinsic pro-phagocytic ligands on tumor cells and knowledge of this mechanism may influence the decision between CD47 blockade or SIRPα blockade for therapeutic purposes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: CD47, but not SIRPα, inhibits phagocytosis by SLAMF7.
Fig. 2: Lack of CD47 or SIRPα phenocopies the effects of blockade.
Fig. 3: Both CD47 and SIRPα inhibit FcR-mediated phagocytosis.
Fig. 4: CD47 interacts in cis with SLAMF7 on tumor cells.
Fig. 5: Freeing SLAMF7 from CD47 promotes phagocytosis.
Fig. 6: First-in-class SLAMF7 mAb Z10 promotes phagocytosis.
Fig. 7: mAb Z10 frees SLAMF7 from CD47.

Similar content being viewed by others

Data availability

RNA-seq data supporting the findings of this study have been deposited into the Gene Expression Omnibus under accession no. GSE223059. All other relevant data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Minn, A. J. & Wherry, E. J. Combination cancer therapies with immune checkpoint blockade: convergence on interferon signaling. Cell 165, 272–275 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Sharma, P. & Allison, J. P. Dissecting the mechanisms of immune checkpoint therapy. Nat. Rev. Immunol. 20, 75–76 (2020).

    Article  CAS  PubMed  Google Scholar 

  3. Son, J. et al. Inhibition of the CD47-SIRPα axis for cancer therapy: a systematic review and meta-analysis of emerging clinical data. Front. Immunol. 13, 1027235 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Veillette, A. & Chen, J. SIRPα-CD47 immune checkpoint blockade in anticancer therapy. Trends Immunol. 39, 173–184 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Chen, J. et al. SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin. Nature 544, 493–497 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chao, M. P. et al. Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell 142, 699–713 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Advani, R. et al. CD47 blockade by Hu5F9-G4 and rituximab in non-Hodgkin’s lymphoma. N. Engl. J. Med. 379, 1711–1721 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Feng, M. et al. Phagocytosis checkpoints as new targets for cancer immunotherapy. Nat. Rev. Cancer 19, 568–586 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Matlung, H. L., Szilagyi, K., Barclay, N. A. & van den Berg, T. K. The CD47–SIRPα signaling axis as an innate immune checkpoint in cancer. Immunol. Rev. 276, 145–164 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. Zhao, X. W. et al. CD47-signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction. Proc. Natl Acad. Sci. USA 108, 18342–18347 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhao, X. W., Kuijpers, T. W. & van den Berg, T. K. Is targeting of CD47–SIRPα enough for treating hematopoietic malignancy? Blood 119, 4333–4334 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Petrova, P. S. et al. TTI-621 (SIRPαFc): a CD47-blocking innate immune checkpoint inhibitor with broad antitumor activity and minimal erythrocyte binding. Clin. Cancer Res. 23, 1068–1079 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. Weiskopf, K. et al. Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies. Science 341, 88–91 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Ring, N. G. et al. Anti-SIRPα antibody immunotherapy enhances neutrophil and macrophage antitumor activity. Proc. Natl Acad. Sci. USA 114, E10578–E10585 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tang, Z. et al. Inflammatory macrophages exploit unconventional pro-phagocytic integrins for phagocytosis and anti-tumor immunity. Cell Rep. 37, 110111 (2021).

    Article  CAS  PubMed  Google Scholar 

  16. Guo, H., Cruz-Munoz, M. E., Wu, N., Robbins, M. & Veillette, A. Immune cell inhibition by SLAMF7 is mediated by a mechanism requiring src kinases, CD45, and SHIP-1 that is defective in multiple myeloma cells. Mol. Cell. Biol. 35, 41–51 (2015).

    Article  PubMed  Google Scholar 

  17. Lu, Y. et al. Immunological conversion of solid tumours using a bispecific nanobioconjugate for cancer immunotherapy. Nat. Nanotechnol. https://doi.org/10.1038/s41565-022-01245-7 (2022).

  18. Johnson, L. D. S. et al. Targeting CD47 in Sezary syndrome with SIRPαFc. Blood Adv. 3, 1145–1153 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Querfeld, C. et al. Intralesional TTI-621, a novel biologic targeting the innate immune checkpoint CD47, in patients with relapsed or refractory mycosis fungoides or Sezary syndrome: a multicentre, phase 1 study. Lancet Haematol. 8, e808–e817 (2021).

    Article  CAS  PubMed  Google Scholar 

  20. Strati, P. et al. Interim results from the first clinical study of CC-95251, an anti-signal regulatory protein-α (SIRPα) antibody, in combination with rituximab in patients with relapsed and/or refractory non-Hodgkin lymphoma (R/R NHL). Blood 138, 2493 (2021).

    Article  Google Scholar 

  21. Delidakis, G., Kim, J. E., George, K. & Georgiou, G. Improving antibody therapeutics by manipulating the Fc domain: immunological and structural considerations. Annu. Rev. Biomed. Eng. 24, 249–274 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kang, T. H. & Jung, S. T. Boosting therapeutic potency of antibodies by taming Fc domain functions. Exp. Mol. Med. 51, 1–9 (2019).

    PubMed  Google Scholar 

  23. Lo, M. et al. Effector-attenuating substitutions that maintain antibody stability and reduce toxicity in mice. J. Biol. Chem. 292, 3900–3908 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sekar, R. B. & Periasamy, A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J. Cell Biol. 160, 629 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cao, E. et al. NTB-A receptor crystal structure: insights into homophilic interactions in the signaling lymphocytic activation molecule receptor family. Immunity 25, 559–570 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Hatherley, D. et al. Paired receptor specificity explained by structures of signal regulatory proteins alone and complexed with CD47. Mol. Cell 31, 266–277 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Goyette, M.-A. et al. The receptor tyrosine kinase AXL is required at multiple steps of the metastatic cascade during HER2-positive breast cancer progression. Cell Rep. 23, 1476–1490 (2018).

    Article  CAS  PubMed  Google Scholar 

  28. Blanchard, E. L. et al. Proximity ligation assays for in situ detection of innate immune activation: focus on in vitro-transcribed mRNA. Mol. Ther. Nucleic Acids 14, 52–66 (2019).

    Article  CAS  PubMed  Google Scholar 

  29. Zhao, Y. et al. Antigen-presenting cell-intrinsic PD-1 neutralizes PD-L1 in cis to attenuate PD-1 signaling in T cells. Cell Rep. 24, 379–390 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhao, Y. et al. PD-L1:CD80 cis-heterodimer triggers the co-stimulatory receptor CD28 while repressing the inhibitory PD-1 and CTLA-4 pathways. Immunity 51, 1059–1073 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhao, Y. et al. cis-B7: CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity. Immunity https://doi.org/10.1016/j.immuni.2023.04.005 (2023).

  32. Ritchie, D. & Colonna, M. Mechanisms of action and clinical development of elotuzumab. Clin. Transl. Sci. 11, 261–266 (2018).

    Article  PubMed  Google Scholar 

  33. Howden, A. J. M. et al. Quantitative analysis of T cell proteomes and environmental sensors during T cell differentiation. Nat. Immunol. 20, 1542–1554 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bian, Z. et al. Cd47-Sirpα interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells. Proc. Natl Acad. Sci. USA 113, E5434–E5443 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Guo, H. et al. Deletion of Slam locus in mice reveals inhibitory role of SLAM family in NK cell responses regulated by cytokines and LFA-1. J. Exp. Med. 213, 2187–2207 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Abraham, N., Miceli, M. C., Parnes, J. R. & Veillette, A. Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature 350, 62–66 (1991).

    Article  CAS  PubMed  Google Scholar 

  37. Bouchon, A., Cella, M., Grierson, H. L., Cohen, J. I. & Colonna, M. Activation of NK cell-mediated cytotoxicity by a SAP-independent receptor of the CD2 family. J. Immunol. 167, 5517–5521 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. von Boehmer, L. et al. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat. Protoc. 11, 1908–1923 (2016).

    Article  Google Scholar 

  39. Davidson, D. et al. The Csk-associated adaptor PAG inhibits effector T cell activation in cooperation with phosphatase PTPN22 and Dok adaptors. Cell Rep. 17, 2776–2788 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zhong, M. C. et al. SLAM family receptors control pro-survival effectors in germinal center B cells to promote humoral immunity. J. Exp. Med. 218, e20200756 (2021).

    Article  CAS  PubMed  Google Scholar 

  41. Roszik, J., Szollosi, J. & Vereb, G. AccPbFRET: an ImageJ plugin for semi-automatic, fully corrected analysis of acceptor photobleaching FRET images. BMC Bioinform. 9, 346 (2008).

    Article  Google Scholar 

Download references

Acknowledgements

We thank members of the Veillette laboratory for useful discussions. We also acknowledge T. van den Berg (Amsterdam) and T. Matozaki (Kobe) for providing hybridomas. This work was supported by grants from the Canadian Institutes of Health Research (MT-14429, MOP-82906, FDN-143338, PJT-178314 and PJT-183593), the Terry Fox Research Institute (1190-02) and the Ministère de l’économie et de l’innovation (Québec) to A.V.; and US National Institutes of Health grant R37 CA239072 to E.H. A.V. received a contract from Bristol Myers-Squibb to study the mechanism of action of SLAMF7 monoclonal antibody elotuzumab against multiple myeloma. Z.T. received a Fellowship from the Cole Foundation; C.C.G. received a Studentship from the Fonds de la recherche en santé – Québec; J.L. receives a Studentship from the Chinese Science Council; and A.V. holds the Canada Research Chair on Signaling in the Immune System.

Author information

Authors and Affiliations

  1. Laboratory of Molecular Oncology, Institut de recherches cliniques de Montréal (IRCM), Montréal, Québec, Canada

    Zhenghai Tang, Ming-Chao Zhong, Jin Qian, Cristian Camilo Galindo, Dominique Davidson, Jiaxin Li & André Veillette

  2. Department of Medicine, McGill University, Montréal, Québec, Canada

    Cristian Camilo Galindo, Jiaxin Li & André Veillette

  3. Department of Cell and Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA

    Yunlong Zhao & Enfu Hui

  4. Department of Medicine, University of Montréal, Montréal, Québec, Canada

    André Veillette

Authors
  1. Zhenghai Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ming-Chao Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jin Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cristian Camilo Galindo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dominique Davidson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jiaxin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yunlong Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Enfu Hui
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. André Veillette
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.T., M.C.-Z., J.Q., D.D., Y.Z., E.H. and A.V. designed experiments. Z.T., M.C.-Z., J.Q., C.C.G., J.L. and Y.Z. performed experiments. All authors interpreted the results. Z.T. and A.V. wrote the manuscript. All authors commented on the manuscript. A.V. and E.H. provided funding.

Corresponding author

Correspondence to André Veillette.

Ethics declarations

Competing interests

Members of the Veillette laboratory will file a patent on the use of monoclonal antibody Z10 for the treatment of SLAMF7-positive human tumors. The authors declare no other competing interests.

Peer review

Peer review information

Nature Immunology thanks David Raulet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ioana Visan, in collaboration with the Nature Immunology team. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Generation of Fc-silent mAbs.

a, The three mutations (‘LALAPG’) introduced in the Fc portion of mAbs to render them Fc-silent are depicted. LALAPG is L234A (‘LA’), L235A (‘LA’), and P329G (‘PG’). b, Binding assay of Fc-intact and Fc-silent variants of mIgG2a (mAb MOPC21) or hIgG1 (mAb Z10) to FcRs on BMDMs assessed by flow cytometry. c, Binding of Fc-silent mSIRPα mAbs #23, #27 and MY-1 to EL-4 cells and EL-4 cells expressing full-length mSIRPα or a variant of mSIRPα containing only the first Ig-like variable (V) domain assessed by flow cytometry. d, Binding of a soluble mCD47-Fc fusion protein to EL-4 cells, expressing or not expressing mSIRPα, studied by flow cytometry. MFI, mean fluorescence intensity. e, Binding of Fc-silent mSIRPα mAbs #23, #27 and MY-1 to mSIRPα-Fc, mSIRPβ1a-Fc, mSIRPβ1b-Fc and mSIRPβ1c-Fc, determined by ELISA. f, Binding of Fc-silent Ctrl IgG MOPC21, hCD47 mAbs B6H12 and AO-176 or hSIRPα-Fc fusion proteins TTI-621 and ALX148 to WT and CD47−/− Daudi cells evaluated by flow cytometry. g, Binding of Fc-silent hSIRPα mAbs to Sirpa-/- BMDMs, transduced with retroviruses encoding or not encoding the V1 or V2 version of hSIRPα, studied by flow cytometry. All data are means ± s.e.m. ****p < 0.0001. Flow cytometry profiles are representative of 3 (c,f, g) or 2 (b) independent experiments. Results are pooled from 5 (d) and 3 (e) independent experiments.

Extended Data Fig. 2 CD47, but not SIRPα, inhibits SLAMF7-mediated phagocytosis.

a,b, Representative flow cytometry profiles (a) and compiled data from 3 independent experiments (b) of phagocytosis assays of L1210 cells by WT BMDMs, assessed by flow cytometry using the pHrodo dye as in Fig. 1b. c, Phagocytosis assays of L1210 cells by WT BMDMs as in Fig. 1b. BMDMs and L1210 cells were pre-treated with indicated mAbs, followed or not by extensive washing, prior to the phagocytosis assay. d, Tumors from the experiment depicted in Fig. 1e were dissected, weighed, measured, and analyzed by flow cytometry. e, Phagocytosis of Raji cells by Sirpa-/- BMDMs, expressing or not expressing hSIRPα version V1 or V2 as in Fig. 1f. f,g, Representative flow cytometry profiles (f) and compiled data from 4 independent experiments (g) of phagocytosis assays of WT and Cd47-/- L1210 cells by WT or Sirpa-/- BMDMs, assessed by flow cytometry using the pHrodo dye as in Fig. 2a. h, Phagocytosis assays of WT and Cd47-/- L1210 cells by WT or Sirpa-/- BMDMs, with or without pre-treatment of lipopolysaccharide (LPS), as in Fig. 2a. i, Tumors from the experiment depicted in Fig. 2e were dissected, weighed, measured and analyzed by flow cytometry. One mouse from ‘Rag1-/- mice + Cd47-/- L1210’ group showed no clinically detectable tumor on day 17. All data are means ± s.e.m. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Flow cytometry profiles are representative of 3 (a) or 4 (f) independent experiments. Results are pooled from 3 (b,c,e) or 4 (g,h) mice studied in 3 (b,c,e) or 4 (g,h) independent experiments, 14 (d) mice from 3 independent experiments, and 11 (WT L1210) or 10 (Cd47-/- L1210) mice from 2 independent experiments (i). Each symbol represents one mouse.

Extended Data Fig. 3 Loss of SIRPa does not alter macrophage differentiation.

a, RNA sequence of Sirpa-/- vs WT BMDMs was analyzed. b, expression of various cell surface markers (red lines) in WT and Sirpa-/- BMDMs assayed by flow cytometry. Filled curves, Ctrl IgG. c, expression of various cell surface markers (red lines) in WT and Sirpa-/- BMDMs from a second Sirpa-/- C57BL/6 mouse strain (#54) assayed by flow cytometry. Filled curves, Ctrl IgG. d,e, Phagocytosis of L1210 cells (d) and Raji cells (e), expressing or not expressing CD47, by WT or Sirpa-/- BMDMs as in Fig. 2a. All data are means ± s.e.m. ns, not significant; **p < 0.01, ***p < 0.001, and ****p < 0.0001. 3 pairs of mice were studied in one experiment (a). Flow cytometry profiles are representative of 3 (b,c) independent experiments. Results are pooled from a total of 3 (d,e) mice studied in 3 independent experiments. Each symbol represents one mouse.

Extended Data Fig. 4 Both CD47 and SIRPα inhibit FcR-mediated tumor growth.

Tumors from the experiment depicted in Fig. 3d were dissected, weighed, measured and analysed by flow cytometry. One mouse from ‘Rag1-/- mice + Ctrl IgG (C1.18.4)’ group showed no clinically detectable tumor on day 18. All data are means ± s.e.m. ns, not significant; ****p < 0.0001. Results are pooled from a total of 15 mice [16 mice for ‘Rag1-/- mice + Tac mAb (7G7)’ group] from 3 independent experiments. Each symbol represents one mouse.

Extended Data Fig. 5 CD47 interacts in cis with SLAMF7.

a, Expression of Flag or CD47 (red lines) on L1210 cells and Cd47-/- L1210 cells, expressing or not expressing mCD47-Flag, assessed by flow cytometry. Permeabilization was used for the Flag staining. Filled curves, Ctrl IgG. b, Means of the normalized total ion current (TIC) for the potential interactors of CD47. c, Expression of hCD47 (red lines) on WT or CD47-/- 293 T cells was assessed by flow cytometry. Filled curves, Ctrl IgG. d, Fluorescence intensity of Dye 647 and Dye 547 in CD47-/- 293 T cells, transfected with tagged versions of mCD47 and mSLAMF7 and treated with or without Ctrl IgG, mCD47 mAb and mSLAMF7 mAb, assayed by flow cytometry. e, Binding of soluble mSIRPα-Fc fusion protein and expression of mCD47 on transfected CD47-/- 293 T cells assessed by flow cytometry. f, Binding of soluble mSLAMF7-Fc fusion protein and expression of mSLAMF7 on transfected BI-141 cells assessed by flow cytometry. g,h, Representative confocal microscopy images (g) and compiled data from 18 cells in 3 independent experiments (h) of FRET assays as in Fig. 4d,e. All data are means ± s.e.m. ns, not significant; ****p < 0.0001. Flow cytometry profiles are representative of 3 (a,c-f) independent experiments. Results are representative of 3 (g) independent experiments. Results are pooled from 3 (b,h) independent experiments.

Extended Data Fig. 6 Structure-function analyses of CD47 and SLAMF7.

a, Expression of mCD47 and mSLAMF7 and binding of mSIRPα-Fc (red lines) in L1210 derivatives assessed by flow cytometry. Filled curves, Ctrl IgG or Ctrl Fc fusion protein. b, Phagocytosis assays of L1210 derivatives by WT or Sirpa-/- BMDMs as in Fig. 2a. c, Expression of mSLAMF7 (red lines) in Slamf7-/- conA-activated CD4+ T cells, transduced with variants of mSLAMF7 constructs, assessed by flow cytometry. Filled curves, Ctrl IgG. d, Phagocytosis assays of conA-activated CD4+ T cells by WT or Sirpa-/- BMDMs as in Fig. 2a. e,f, Schematic representation (e) and time-course of normalized dye 505 fluorescence intensity (f) of LUV-based FRET assay of mSLAMF7 (donor) or hPD-L1 (donor) with mCD47 (acceptor) or hPD-1 (acceptor) monitored by a real time plate reader. g, Time-course of normalized dye 505 fluorescence intensity of LUV-based FRET assay of mSLAMF7 (donor) with mCD47 (acceptor), pre-treated with Ctrl IgG MOPC21, mSLAMF7 mAb 4G2 and mCD47 mAb Miap301, monitored by a real time plate reader. h, Binding of soluble hCD47-Fc fusion protein to WT or CD47-/- Raji cells assessed by flow cytometry. All data are means ± s.e.m. ns, not significant; **p < 0.01 and ***p < 0.001. Flow cytometry profiles are representative of 3 (a,c) or 2 (h) independent experiments. Results are representative of 3 (f) or 2 (g) independent experiments. Results are pooled from 4 (b) or 4 (d; for all variants, except 3 for SLAMF7R75A) independent experiments. Each symbol represents one mouse.

Extended Data Fig. 7 mAb Z10 promotes phagocytosis with SIRPα blockade.

a,b, Phagocytosis assay of Daudi (a, left), MM.1 S (a, right), and Fc-silent CD3 + CD28 mAbs activated mouse CD8+ T cells expressing (b, right) or not expressing (b, left) hSLAMF7 by WT or Sirpa-/- BMDMs assayed by fluorescence microscopy. c, Schematic representations of the impact of mAb Z10 on macrophages or target cells. d, Tumors from experiment depicted in Fig. 6g were dissected, weighed, measured, and analysed by flow cytometry. One mouse showed no detectable tumor in the ‘SIRPα mAb (#27) + SLAMF7 mAb (Z10)’ group on day 24. All data are means ± s.e.m. ns, not significant; **p < 0.01, ***p < 0.001, and ****p < 0.0001. Results are pooled from 3 (a,b) mice studied in 3 (a,b) independent experiments; 8 [except 7 for ‘SLAMF7 mAb (Z10)’ and ‘SIRPα mAb (#27)’ groups] mice from 2 independent experiments (d). Each symbol represents one mouse.

Extended Data Fig. 8 Z10 disrupts CD47-SLAMF7 interaction and does not affect conjugate formation.

a, Co-immunoprecipitation assay of CD47 and SLAMF7 in CD47-/- 293 T cells expressing a Flag-tagged variant of hCD47 and a Myc-tagged variant of hSLAMF7, in the presence of Ctrl IgG MOPC21 or hSLAMF7 mAb Z10. b, Representative flow cytometry profiles (b, left) and compiled data from 3 independent experiments (b, right) of conjugate formation assays of Sirpa-/- BMDMs with Raji cells for the indicated time points, in the presence of Ctrl IgG or hSLAMF7 mAb Z10, detected by flow cytometry. c, Conjugate formation assays of Sirpa-/- BMDMs with Raji cells for 20 minutes, assessed by confocal microscopy. All data are means ± s.e.m. ns, not significant. Results are representative of 2 (a) independent experiments. Flow cytometry profiles are representative of 3 independent experiments (b). Results are pooled from a total of 3 (b,c) mice studied in 3 (b,c) independent experiments. Each symbol represents one mouse.

Source data

Extended Data Fig. 9 Z10 does not interfere with phagocytosis of CD47-deficient targets.

a,b, Phagocytosis of Raji (a) and Daudi (b), expressing or not expressing CD47 by WT or Sirpa-/- BMDMs in the presence of Ctrl IgG MOPC21 and hSLAMF7 mAb Z10 assayed by fluorescence microscopy. All data are means ± s.e.m. ns, not significant; **p < 0.01 and ****p < 0.0001. Results are pooled from 3 (a,b) mice studied in 3 independent experiments. Each symbol represents one mouse.

Extended Data Fig. 10 Z10 binds to the second Ig-like domain of SLAMF7.

a,b, Binding of hSLAMF7 mAbs Z10, Elo and Z8 to CD47-/- 293 T cells expressing various chimeras between hSLAMF7 and mSLAMF7 (a) or various mutants of hSLAMF7 (b), assayed by flow cytometry. For the chimeras, the residues in hSLAMF7 (numbered) were replaced by the equivalent residues in mSLAMF7. Flow cytometry profiles are representative of 3 (a,b, except 2 for hSLAMF7N177A, hSLAMF7P178A, hSLAMF7V179A, hSLAMF7S180A) independent experiments.

Supplementary information

Source data

Source Data Fig. 4

Unprocessed immunoblots.

Source Data Extended Data Fig. 8

Unprocessed immunoblots.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tang, Z., Zhong, MC., Qian, J. et al. CD47 masks pro-phagocytic ligands in cis on tumor cells to suppress antitumor immunity. Nat Immunol 24, 2032–2041 (2023). https://doi.org/10.1038/s41590-023-01671-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41590-023-01671-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer