Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
American Type Culture Collection
- CD47 m:
CD47-translational blocking morpholino
Cytotoxic T-lymphocyte-associated protein 4
Myeloid-derived suppressor cells
National Cancer Institute
Cancer/testis antigen 1B
The Cancer Genome Atlas
Salomon B, Bluestone JA (2001) Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu Rev Immunol 19:225–252. https://doi.org/10.1146/annurev.immunol.19.1.225
Walunas TL, Bakker CY, Bluestone JA (1996) CTLA-4 ligation blocks CD28-dependent T cell activation. J Exp Med 183:2541–2550
Eggermont AM, Chiarion-Sileni V, Grob JJ, Dummer R, Wolchok JD, Schmidt H et al (2015) Adjuvant ipilimumab versus placebo after complete resection of high-risk stage III melanoma (EORTC 18071): a randomised, double-blind, phase 3 trial. Lancet Oncol. 16:522–530. https://doi.org/10.1016/S1470-2045%5b15%5d70122-1
Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L et al (2015) Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med 372:320–330. https://doi.org/10.1056/NEJMoa1412082
Ribas A, Puzanov I, Dummer R, Schadendorf D, Hamid O, Robert C et al (2015) Pembrolizumab versus investigator-choice chemotherapy for ipilimumab-refractory melanoma (KEYNOTE-002): a randomised, controlled, phase 2 trial. Lancet Oncol. 16:908–918. https://doi.org/10.1016/S1470-2045%5b15%5d00083-2
Barker CA, Postow MA (2014) Combinations of radiation therapy and immunotherapy for melanoma: a review of clinical outcomes. Int J Radiat Oncol Biol Phys 88:986–997. https://doi.org/10.1016/j.ijrobp.2013.08.035
Schoenhals JE, Skrepnik T, Selek U, Cortez MA, Li A, Welsh JW (2017) Optimizing radiotherapy with immunotherapeutic approaches. Adv Exp Med Biol 995:53–71. https://doi.org/10.1007/978-3-319-53156-4_3
Young KH, Baird JR, Savage T, Cottam B, Friedman D, Bambina S et al (2016) Optimizing timing of immunotherapy improves control of tumors by hypofractionated radiation therapy. PLoS ONE 11:e0157164. https://doi.org/10.1371/journal.pone.0157164
Vonderheide RH (2015) CD47 blockade as another immune checkpoint therapy for cancer. Nat Med 21:1122–1123. https://doi.org/10.1038/nm.3965
Chao MP, Weissman IL, Majeti R (2012) The CD47-SIRPalpha pathway in cancer immune evasion and potential therapeutic implications. Curr Opin Immunol 24:225–232. https://doi.org/10.1016/j.coi.2012.01.010
Soto-Pantoja DR, Terabe M, Ghosh A, Ridnour LA, DeGraff WG, Wink DA et al (2014) CD47 in the tumor microenvironment limits cooperation between antitumor T-cell immunity and radiotherapy. Cancer Res 74:6771–6783. https://doi.org/10.1158/0008-5472.CAN-14-0037-T
Liu X, Pu Y, Cron K, Deng L, Kline J, Frazier WA et al (2015) CD47 blockade triggers T cell-mediated destruction of immunogenic tumors. Nat Med 21:1209–1215. https://doi.org/10.1038/nm.3931
Maxhimer JB, Soto-Pantoja DR, Ridnour LA, Shih HB, Degraff WG, Tsokos M et al (2009) Radioprotection in normal tissue and delayed tumor growth by blockade of CD47 signaling. Sci Transl Med 1:3ra7. https://doi.org/10.1126/scitranslmed.3000139
Roberts DD, Miller TW, Rogers NM, Yao M, Isenberg JS (2012) The matricellular protein thrombospondin-1 globally regulates cardiovascular function and responses to stress via CD47. Matrix Biol 31:162–169. https://doi.org/10.1016/j.matbio.2012.01.005
Soto-Pantoja DR, Kaur S, Roberts DD (2015) CD47 signaling pathways controlling cellular differentiation and responses to stress. Crit Rev Biochem Mol Biol 50:212–230. https://doi.org/10.3109/10409238.2015.1014024
Li Z, Calzada MJ, Sipes JM, Cashel JA, Krutzsch HC, Annis DS et al (2002) Interactions of thrombospondins with alpha4beta1 integrin and CD47 differentially modulate T cell behavior. J Cell Biol 157:509–519. https://doi.org/10.1083/jcb.200109098
Grimbert P, Bouguermouh S, Baba N, Nakajima T, Allakhverdi Z, Braun D et al (2006) Thrombospondin/CD47 interaction: a pathway to generate regulatory T cells from human CD4 + CD25- T cells in response to inflammation. J Immunol 177:3534–3541. https://doi.org/10.4049/jimmunol.177.6.3534
Kaur S, Kuznetsova SA, Pendrak ML, Sipes JM, Romeo MJ, Li Z et al (2011) Heparan sulfate modification of the transmembrane receptor CD47 is necessary for inhibition of T cell receptor signaling by thrombospondin-1. J Biol Chem 286:14991–15002. https://doi.org/10.1074/jbc.M110.179663
Miller TW, Kaur S, Ivins-O’Keefe K, Roberts DD (2013) Thrombospondin-1 is a CD47-dependent endogenous inhibitor of hydrogen sulfide signaling in T cell activation. Matrix Biol 32:316–324. https://doi.org/10.1016/j.matbio.2013.02.009
Kaur S, Chang T, Singh SP, Lim L, Mannan P, Garfield SH et al (2014) CD47 signaling regulates the immunosuppressive activity of VEGF in T cells. J Immunol 193:3914–3924. https://doi.org/10.4049/jimmunol.1303116
Lamy L, Foussat A, Brown EJ, Bornstein P, Ticchioni M, Bernard A (2007) Interactions between CD47 and thrombospondin reduce inflammation. J Immunol 178:5930–5939. https://doi.org/10.4049/jimmunol.178.9.5930
Oldenborg PA, Zheleznyak A, Fang YF, Lagenaur CF, Gresham HD, Lindberg FP (2000) Role of CD47 as a marker of self on red blood cells. Science 288:2051–2054. https://doi.org/10.1126/science.288.5473.2051
Tseng D, Volkmer JP, Willingham SB, Contreras-Trujillo H, Fathman JW, Fernhoff NB et al (2013) Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response. Proc Natl Acad Sci U S A 110:11103–11108. https://doi.org/10.1073/pnas.1305569110
Nath PR, Gangaplara A, Pal-Nath D, Mandal A, Maric D, Sipes JM et al (2018) CD47 expression in natural killer cells regulates homeostasis and modulates immune response to lymphocytic choriomeningitis virus. Front Immunol 9:2985. https://doi.org/10.3389/fimmu.2018.02985
Nath P, Pal-Nath, D, Mandal, A, Cam, MC, Schwartz AL, Roberts DD (2019) CD47 in the tumor microenvironment and CD47 antibody blockade regulate natural killer cell recruitment and activation. Cancer Immunol Res 7:1547–1561. https://doi.org/10.1158/2326-6066.CIR-18-0367
Matlung HL, Szilagyi K, Barclay NA, van den Berg TK (2017) The CD47-SIRPalpha signaling axis as an innate immune checkpoint in cancer. Immunol Rev 276:145–164. https://doi.org/10.1111/imr.12527
Sikic BI, Lakhani N, Patnaik A, Shah SA, Chandana SR, Rasco D et al (2019) First-in-human, first-in-class phase I trial of the anti-CD47 antibody Hu5F9-G4 in patients with advanced cancers. J Clin Oncol 37:946–953. https://doi.org/10.1200/JCO.18.02018
Kim MJ, Lee JC, Lee JJ, Kim S, Lee SG, Park SW et al (2008) Association of CD47 with natural killer cell-mediated cytotoxicity of head-and-neck squamous cell carcinoma lines. Tumour Biol 29:28–34. https://doi.org/10.1159/000132568
Vanpouille-Box C, Alard A, Aryankalayil MJ, Sarfraz Y, Diamond JM, Schneider RJ et al (2017) DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity. Nat Commun. 8:15618. https://doi.org/10.1038/ncomms15618
Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO et al (2013) Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 6:l1. https://doi.org/10.1126/scisignal.2004088
Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA et al (2012) The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2:401–404. https://doi.org/10.1158/2159-8290.CD-12-0095
Soto-Pantoja DR, Miller TW, Pendrak ML, DeGraff WG, Sullivan C, Ridnour LA et al (2012) CD47 deficiency confers cell and tissue radioprotection by activation of autophagy. Autophagy 8:1628–1642. https://doi.org/10.4161/auto.21562
Topalian SL, Solomon D, Rosenberg SA (1989) Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. J Immunol 142:3714–3725
Britten CM, Janetzki S, Butterfield LH, Ferrari G, Gouttefangeas C, Huber C et al (2012) T cell assays and MIATA: the essential minimum for maximum impact. Immunity 37:1–2. https://doi.org/10.1016/j.immuni.2012.07.010
Willingham SB, Volkmer JP, Gentles AJ, Sahoo D, Dalerba P, Mitra SS et al (2012) The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci U S A 109:6662–6667. https://doi.org/10.1073/pnas.1121623109
Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD Jr et al (2009) CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell 138:286–299. https://doi.org/10.1016/j.cell.2009.05.045
Sensi M, Nicolini G, Petti C, Bersani I, Lozupone F, Molla A et al (2006) Mutually exclusive NRASQ61R and BRAFV600E mutations at the single-cell level in the same human melanoma. Oncogene 25:3357–3364. https://doi.org/10.1038/sj.onc.1209379
Casey SC, Tong L, Li Y, Do R, Walz S, Fitzgerald KN et al (2016) MYC regulates the antitumor immune response through CD47 and PD-L1. Science 352:227–231. https://doi.org/10.1126/science.aac9935
Wei SC, Levine JH, Cogdill AP, Zhao Y, Anang NAS, Andrews MC et al (2017) Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1 checkpoint blockade. Cell 170(1120–1133):e1117. https://doi.org/10.1016/j.cell.2017.07.024
Reinhold MI, Lindberg FP, Kersh GJ, Allen PM, Brown EJ (1997) Costimulation of T cell activation by integrin-associated protein (CD47) is an adhesion-dependent, CD28-independent signaling pathway. J Exp Med 185:1–11
Ticchioni M, Deckert M, Mary F, Bernard G, Brown EJ, Bernard A (1997) Integrin-associated protein (CD47) is a comitogenic molecule on CD3-activated human T cells. J Immunol 158:677–684
Kohlhapp FJ, Broucek JR, Hughes T, Huelsmann EJ, Lusciks J, Zayas JP et al (2015) NK cells and CD8 + T cells cooperate to improve therapeutic responses in melanoma treated with interleukin-2 (IL-2) and CTLA-4 blockade. J Immunother Cancer 3:18. https://doi.org/10.1186/s40425-015-0063-3
Ingram JR, Blomberg OS, Sockolosky JT, Ali L, Schmidt FI, Pishesha N et al (2017) Localized CD47 blockade enhances immunotherapy for murine melanoma. Proc Natl Acad Sci U S A 114:10184–10189. https://doi.org/10.1073/pnas.1710776114
Vanpouille-Box C, Formenti SC, Demaria S (2018) Toward precision radiotherapy for use with immune checkpoint blockers. Clin Cancer Res 24:259–265. https://doi.org/10.1158/1078-0432.CCR-16-0037
Kumar V, Patel S, Tcyganov E, Gabrilovich DI (2016) The nature of myeloid-derived suppressor cells in the tumor microenvironment. Trends Immunol 37:208–220. https://doi.org/10.1016/j.it.2016.01.004
Gabrilovich DI (2017) Myeloid-derived suppressor cells. Cancer Immunol Res 5:3–8. https://doi.org/10.1158/2326-6066.CIR-16-0297
Lazarevic V, Glimcher LH, Lord GM (2013) T-bet: a bridge between innate and adaptive immunity. Nat Rev Immunol 13:777–789. https://doi.org/10.1038/nri3536
Callahan MK, Postow MA, Wolchok JD (2014) CTLA-4 and PD-1 pathway blockade: combinations in the clinic. Front Oncol 4:385. https://doi.org/10.3389/fonc.2014.00385
Feliz-Mosquea YR, Christensen AA, Wilson AS, Westwood B, Varagic J, Melendez GC et al (2018) Combination of anthracyclines and anti-CD47 therapy inhibit invasive breast cancer growth while preventing cardiac toxicity by regulation of autophagy. Breast Cancer Res Treat 172:69–82. https://doi.org/10.1007/s10549-018-4884-x
Lo J, Lau EY, Ching RH, Cheng BY, Ma MK, Ng IO et al (2015) Nuclear factor kappa B-mediated CD47 up-regulation promotes sorafenib resistance and its blockade synergizes the effect of sorafenib in hepatocellular carcinoma in mice. Hepatology 62:534–545. https://doi.org/10.1002/hep.27859
Lo J, Lau EY, So FT, Lu P, Chan VS, Cheung VC et al (2016) Anti-CD47 antibody suppresses tumour growth and augments the effect of chemotherapy treatment in hepatocellular carcinoma. Liver Int 36:737–745. https://doi.org/10.1111/liv.12963
Cioffi M, Trabulo S, Hidalgo M, Costello E, Greenhalf W, Erkan M et al (2015) Inhibition of CD47 effectively targets pancreatic cancer stem cells via dual mechanisms. Clin Cancer Res 21:2325–2337. https://doi.org/10.1158/1078-0432.CCR-14-1399
Sockolosky JT, Dougan M, Ingram JR, Ho CC, Kauke MJ, Almo SC et al (2016) Durable antitumor responses to CD47 blockade require adaptive immune stimulation. Proc Natl Acad Sci U S A 113:E2646–2654. https://doi.org/10.1073/pnas.1604268113
Tao H, Qian P, Wang F, Yu H, Guo Y (2017) Targeting CD47 enhances the efficacy of anti-PD-1 and CTLA-4 in esophageal squamous cell cancer preclinical model. Oncol Res. https://doi.org/10.3727/096504017X14900505020895
Zhao XW, van Beek EM, Schornagel K, Van der Maaden H, Van Houdt M, Otten MA et al (2011) CD47-signal regulatory protein-alpha (SIRPalpha) interactions form a barrier for antibody-mediated tumor cell destruction. Proc Natl Acad Sci U S A 108:18342–18347. https://doi.org/10.1073/pnas.1106550108
Soto-Pantoja DR, Miller TW, Frazier WA, Roberts DD (2012) Inhibitory signaling through signal regulatory protein-alpha is not sufficient to explain the antitumor activities of CD47 antibodies. Proc Natl Acad Sci U S A 109:E2842. https://doi.org/10.1073/pnas.1205441109(author reply E2844–2845)
Soto-Pantoja DR, Ridnour LA, Wink DA, Roberts DD (2013) Blockade of CD47 increases survival of mice exposed to lethal total body irradiation. Sci Rep 3:1038. https://doi.org/10.1038/srep01038
Miller TW, Soto-Pantoja DR, Schwartz AL, Sipes JM, DeGraff WG, Ridnour LA et al (2015) CD47 receptor globally regulates metabolic pathways that control resistance to ionizing radiation. J Biol Chem 290:24858–24874. https://doi.org/10.1074/jbc.M115.665752
Li Z, He L, Wilson K, Roberts D (2001) Thrombospondin-1 inhibits TCR-mediated T lymphocyte early activation. J Immunol 166:2427–2436. https://doi.org/10.4049/jimmunol.166.4.2427
Arnon TI, Achdout H, Lieberman N, Gazit R, Gonen-Gross T, Katz G et al (2004) The mechanisms controlling the recognition of tumor- and virus-infected cells by NKp46. Blood 103:664–672. https://doi.org/10.1182/blood-2003-05-1716
Stojanovic A, Fiegler N, Brunner-Weinzierl M, Cerwenka A (2014) CTLA-4 is expressed by activated mouse NK cells and inhibits NK Cell IFN-gamma production in response to mature dendritic cells. J Immunol 192:4184–4191. https://doi.org/10.4049/jimmunol.1302091
Kojima Y, Volkmer JP, McKenna K, Civelek M, Lusis AJ, Miller CL et al (2016) CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis. Nature 536:86–90. https://doi.org/10.1038/nature18935
Logtenberg MEW, Jansen JHM, Raaben M, Toebes M, Franke K, Brandsma AM et al (2019) Glutaminyl cyclase is an enzymatic modifier of the CD47- SIRPalpha axis and a target for cancer immunotherapy. Nat Med 25:612–619. https://doi.org/10.1038/s41591-019-0356-z
Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12:323. https://doi.org/10.1186/1471-2105-12-323
We would like to thank the staff of NCI’s Pathology/Histotechnology Laboratory at Frederick for performing immunohistochemical staining for CD3.
This work was supported by the Intramural Research Program of the NIH/National Cancer Institute.
Conflict of interest
Anthony L. Schwartz is the Chief Executive Officer and shareholder of Morphiex Biotherapeutics which holds licensing rights to the CD47 Morpholino. All other authors of this paper declare no conflicts of interest.
Animal Studies: The B16 melanoma animal model was carried out under approved protocols (Protocol #LP-026, January 2016) following the guidelines of the National Cancer Institute’s Animal Care and Use Committee.
Human T-cell studies: TCGA data were obtained under informed consent as described (https://cancergenome.nih.gov/abouttcga/policies/informedconsent). No new human materials for T-cell experiments were obtained, thus did not require consent or IRB review.
C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME).
Cell line authentication
B16F10 (CRL-6475) mouse melanoma cell line was purchased from ATCC and were authenticated at the Frederick National Laboratory for Cancer Research (Frederick, MD). The origin of the T-cell lines was previously published, described and provided by the Surgery Branch, Center for Cancer Research, National Cancer Institute .
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The authors of this paper report on their T-cell assays transparently and comprehensively as per field-wide consensus, allowing the community a full understanding and interpretation of presented data as well as a comparison of data between groups. The electronic supplementary materials of this publication include a MIATA checklist. For more details, see http://miataproject.org/.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Schwartz, A.L., Nath, P.R., Allgauer, M. et al. Antisense targeting of CD47 enhances human cytotoxic T-cell activity and increases survival of mice bearing B16 melanoma when combined with anti-CTLA4 and tumor irradiation. Cancer Immunol Immunother 68, 1805–1817 (2019). https://doi.org/10.1007/s00262-019-02397-7
- Phosphorodiamidate morpholino oligomer