Is CD47 an innate immune checkpoint for tumor evasion?
Liu et al. Journal of Hematology & Oncology
Is CD47 an innate immune checkpoint for tumor evasion?
Hyunwoo Kwon 0
Zihai Li 0 1
0 Department of Microbiology and Immunology, Hollings Cancer Center, Medical University of South Carolina , Charleston, SC , USA
1 First Affiliated Hospital, Zhengzhou University School of Medicine , Zhengzhou , China
Cluster of differentiation 47 (CD47) (also known as integrin-associated protein) is a ubiquitously expressed glycoprotein of the immunoglobulin superfamily that plays a critical role in self-recognition. Various solid and hematologic cancers exploit CD47 expression in order to evade immunological eradication, and its overexpression is clinically correlated with poor prognoses. One essential mechanism behind CD47-mediated immune evasion is that it can interact with signal regulatory protein-alpha (SIRPα) expressed on myeloid cells, causing phosphorylation of the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition motifs and recruitment of Src homology 2 domain-containing tyrosine phosphatases to ultimately result in delivering an anti-phagocytic-“don't eat me”-signal. Given its essential role as a negative checkpoint for innate immunity and subsequent adaptive immunity, CD47-SIRPα axis has been explored as a new target for cancer immunotherapy and its disruption has demonstrated great therapeutic promise. Indeed, CD47 blocking antibodies have been found to decrease primary tumor size and/or metastasis in various pre-clinical models. In this review, we highlight the various functions of CD47, discuss anti-tumor responses generated by both the innate and adaptive immune systems as a consequence of administering anti-CD47 blocking antibody, and finally elaborate on the clinical potential of CD47 blockade. We argue that CD47 is a checkpoint molecule for both innate and adaptive immunity for tumor evasion and is thus a promising target for cancer immunotherapy.
CD47; SIRPα; “Don't eat me” signal; Cancer immunotherapy; Chemotherapy; Clinical trial; Dendritic cell; Macrophage
Cluster of differentiation 47 (CD47), also known as
integrin-associated protein (IAP), is a ~50 kDa heavily
glycosylated, ubiquitously expressed membrane protein of the
immunoglobulin superfamily with a single IgV-like domain
at its N-terminus, a highly hydrophobic stretch with five
membrane-spanning segments and an alternatively spliced
cytoplasmic C-terminus . Each of the four alternatively
spliced cytoplasmic tails exists in vivo at different
frequencies (i.e., form 2 is the most abundant), but all lack a
substantial signaling domain . While CD47 was first
identified as a membrane protein involved in β3
integrinmediated signaling on leukocytes , it is now known to
also interact with thrombospondin-1, signal regulatory
protein-alpha (SIRPα), and others to regulate various
cellular functions including cell migration, axon extension,
cytokine production, and T cell activation [4–8]. However,
recent studies have focused most on CD47-SIRPα axis for
its inhibitory role in phagocytosis . SIRPα, also known as
Src homology 2 domain-containing protein tyrosine
phosphatase substrate 1/brain Ig-like molecule with
tyrosinebased activation motif/cluster of differentiation antigen-like
family member A (SHPS-1/BIT/CD172a), is another
membrane protein of the immunoglobulin superfamily that is
particularly abundant in the myeloid-lineage hematopoietic
cells such as macrophages and dendritic cells [10, 11]. The
ligation of SIRPα on phagocytes by CD47 expressed on a
neighboring cell results in phosphorylation of SIRPα
cytoplasmic immunoreceptor tyrosine-based inhibition (ITIM)
motifs, leading to the recruitment of SHP-1 and SHP-2
phosphatases. One resulting downstream effect is the
prevention of myosin-IIA accumulation at the phagocytic
synapse and consequently inhibition of phagocytosis [12–14].
Thus, CD47-SIRPα interaction functions as a negative
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
immune checkpoint to send a “don’t eat me” signal to
ensure that healthy autologous cells are not inappropriately
phagocytosed. Consistent with this notion, CD47−/− cells
are cleared rapidly when they are adoptively transferred to
the congeneic wild-type mice . However, it was recently
shown that CD47-SIRPα axis, while crucial, represents just
one mechanism that controls phagocytic behavior .
Indeed, CD47−/− mice do not manifest significant
selfdestruction phenotype unless they are in inflammatory
conditions. Inflammatory cytokines stimulate protein kinase
Cspleen tyrosine kinase (PKC-Syk) signaling pathway (which
IL-10 negatively regulates), which then activates
macrophage to target self cells . Combined, these findings
suggest a potential mechanism for anemia of chronic
disease and that rhesus (Rh)-null individuals, who have <25%
of normal CD47 levels, may be particularly vulnerable to
anemia under inflammatory conditions and infections .
Research has demonstrated overexpression of CD47 in
nearly all types of tumors, some of which include acute
myeloid leukemia, non-Hodgkin’s lymphoma, bladder
cancer, and breast cancer [18–25]. While CD47 is
implicated in the regulation of cancer cell invasion and
metastasis [18, 26], its most well-studied and important
function related to tumor development is prevention of
phagocytosis via ligating with SIRPα on the surrounding
phagocytes [18, 27, 28]. Also, CD47 expression on
cancer stem cells (CSCs) implies its role in cancer
recurrence. Particularly, a study has shown that CSCs have
increased CD47 expression to protect themselves from
immune-mediated elimination during conventional
antitumor therapies . This increases the chance of CSC
survival, which in turn could repopulate a new tumor
mass and cause a tumor relapse.
CD47 blockade for direct cancer cell killing
Given the important inhibitory function of CD47 in
phagocytosis of tumor cells, it has been extensively
investigated as a potential target for tumor therapy. In various
xenograft tumor models using NOD-scid-IL2Rgammanull
(NSG) mice, use of human CD47-blocking monoclonal
antibodies has demonstrated superb efficacy against
human acute lymphocytic leukemia, acute myeloid leukemia,
leiomyosarcoma, and solid tumors [18, 20, 27, 28, 30, 31].
Most work initially concluded that the therapeutic effects
of anti-human CD47 were dependent on the direct killing
of the tumor by phagocytes. However, it is important to
note that xenograft models might have some unique
features that favor innate immune-mediated tumor killings.
First, human CD47 binds well to SIRPα of NSG mice, but
not of other strains [32, 33]. This unique feature could put
human tumor cells under CD47-SIRPα control more so in
NSG mice than in other strains of mouse, making them
more susceptible to signaling blockade. Thus, use of
human SIRPα-transgenic recombination-activating gene
(Rag)2−/− IL2Rgamma−/− mice may be necessary to
accurately test such antibody’s therapeutic benefit . Second,
in xenograft models, only human tumor cells express
human CD47. Hence, human CD47-blocking monoclonal
antibodies can efficiently target human tumors without
being “absorbed” by other normal cells (such as red blood
cells) expressing mouse CD47. Third, xenograft tissue
could come under strong innate immune attack. For
example, lacking the mouse MHC class I “self” marker,
xenograft human tumor cells might be attacked by natural
killer (NK) cells if human leukocyte antigen (HLA) fails to
mediate inhibitory signaling. Consistent with this notion,
in syngeneic immunodeficient mouse models such as
athymic nude mice or Rag-deficient mice, mouse
antiCD47 blockade resulted in less impressive efficacy after
treatment . Fourth, lymphocyte-deficient mice
typically demonstrate stronger innate immune responses .
All reasons listed above suggest that contribution of direct
killing by phagocytes to the therapeutic impact of CD47
blockade may be significantly different in an
Role of CD8+ T cells upon CD47 blockade
Indeed, adaptive immune response, particularly that
mediated by T cells, plays an important role in mouse
anti-CD47 blockade-induced tumor control. In syngeneic
immunocompetent mouse models, mouse anti-CD47
blockade shows impressive anti-tumor effect especially
upon intratumoral delivery [35, 37]. Depletion of CD8+ T
cells—but not CD4+ T cells—diminishes the therapeutic
effect of anti-mouse CD47 antibody. Furthermore, after
anti-mouse CD47 treatment, significantly more interferon
(IFN)-γ spot-forming antigen-specific CD8+ T cells are
present in the tumor, and T cell-mediated memory
response is formed to protect mice from tumor
rechallenge. All of these experimental results demonstrate
that T cells are essential for anti-mouse CD47-mediated
tumor regression. Thus, CD47 is a checkpoint molecule
for both innate and adaptive immunity for tumor evasion.
Role of dendritic cells upon CD47 blockade
Since macrophages have been shown to play an
important role in tumor cell phagocytosis in the xenograft
model, they were assumed to be the major
antigenpresenting cells for cytotoxic T lymphocyte (CTL)
induction. Supporting this, enhancement of
crosspriming by macrophages was observed in response to
anti-human CD47 treatment . However, using the
syngeneic mouse model, we have recently shown that
dendritic cells—not macrophages—appeared to play a more
important role for CTL cross-priming and anti-tumor
therapy based on the following observations . First, in
the presence of anti-mouse CD47 antibody, bone
marrowderived dendritic cells (BMDCs) were able to cross-prime
CD8+ T cells to a greater extent than bone
marrowderived macrophages (BMDMs) in general. Second, ex
vivo isolated dendritic cells (DCs) were more potent for
cross-priming of CTL than macrophages after anti-mouse
CD47 treatment. Third, the therapeutic effect of
antimouse CD47 antibody was severely impaired following
DC depletion but not macrophage depletion. Apparent
contradiction between the two studies likely resulted from
differences in experimental approaches. Indeed, when
BMDCs were cultured without serum (similar to in vitro
phagocytosis/priming assays in ), they demonstrated
increased apoptosis (as measured by increased annexin V
stain) which would likely impact their functional capacity.
In contrast, macrophages demonstrated very minimal
change in annexin V stain in the presence/absence of the
Also, it seems that although macrophages can
phagocytose more tumor cells, DCs are more potent than
macrophages in antigen presentation . Macrophages
are good at scavenging and destroying phagocytosed
tumor cells, but at the same time, tumor antigens and
danger signals are overly degraded . In contrast, DCs
have developed means to preserve useful information
from the ingested tumor cells that serve to initiate
adaptive immune responses .
How anti-CD47 blockade boosts DC-mediated antigen
cross-presentation and CTL induction is an intriguing
question that we have started to answer. We found that
after anti-mouse CD47 treatment, DCs—but not
macrophages—express more Ifna mRNA . Blocking type I
IFN signaling by intratumoral injection of interferon
alpha/beta receptor (IFNAR)-blocking antibody impaired
the therapeutic effect of anti-mouse CD47, suggesting an
important role of type I IFN signaling on DC activation.
Supporting this, conditional deletion of Ifnar 1 in CD11c+
cells markedly reduced the therapeutic effect of CD47
blockade on tumor growth. These data also confirm the
essential role of DCs as antigen-presenting cells (APCs) in
vivo for CTL induction. Interestingly, our data further
demonstrated that cytosolic DNA sensor stimulator of
interferon genes (STING)—but not classical Toll-like
receptor (TLR)-myeloid differentiation primary
response gene 88 (MyD88) pathway—is required for type
I IFN production and the therapeutic effect of
antiCD47. This raises a fascinating scenario that upon
antiCD47 treatment, DNA is released from tumor cells and
taken up by DCs, resulting in the activation of STING
and the production of type I IFN, which activates DCs
for antigen cross-presentation (Fig. 1). The detailed
mechanisms remain to be investigated in the future.
Fig. 1 Working model of CD47 blockade for enhancing antigen cross-presentation by dendritic cells and increased T cell priming. Upon
CD47-SIRPa blockade, tumor cells are phagocytosed and their DNA can gain access to the cytosol of intratumoral dendritic cells. Recognition
of cytosolic DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) and generation of cGAMP lead to the activation of STING, resulting in the production
of type I IFN. DCs are activated by type I IFN to cross-present tumor antigens to CD8+ T cells, which then proliferate and kill tumor cells
Targeting CD47-SIRPα signaling axis for therapy
As of November 13, 2016, there are eight phase I clinical
trials that are investigating the effect of blocking
CD47SIRPα signaling axis in various cancer patients
(summarized in Table 1). Among the six, NCT02216409, led by
Forty Seven, Inc., is the first in-human trial and the only
one yet whose data have been presented . Briefly, in
this study, humanized monoclonal anti-CD47 antibody
(“Hu5F9-G4”)  was administered to patients with
diverse solid tumors who are no longer candidates for
conventional therapies. As a phase I clinical trial, it
sought to determine the appropriate dosage of
Hu5F9G4 and to perform the initial pharmacodynamic and
-kinetic studies. Patients tolerated priming (starting)
dose of 0.1, 0.3, and 1 mg/kg well, while those receiving
3 mg/kg experienced a dose-limiting toxicity (abdominal
pain, RBC hemagglutination, and headache). Hence,
1 mg/kg was decided as the priming dose, and currently,
work is being done to determine the optimal
maintenance dose. Hu5F9-G4-related adverse events, majority of
which were reversible, included anemia,
hyperbilirubinemia, headache, hemagglutination, nausea, and retinal
toxicity. It would be interesting to see in the future how
other two therapeutic agents compare to Hu5F9-G4 in
terms of their safety profiles.
It is still unclear, however, if administration of
Hu5F9-G4 alone will result in therapeutic benefits that
are expected based on the promising results of many
pre-clinical studies. Indeed, effective clinical responses
are generally rare and statistically inconclusive in phase
I trials, mainly due to small numbers of patients and
inability to optimally administer the therapeutic agent
(i.e., the dosage). Phase II and III trials will be critical
for evaluating the ability to either delay disease
progression or perhaps even cause its remission.
Given that blockade of CD47-SIRPα signaling axis has
(and continues to) demonstrate success in more
preclinical tumor models, more entries into clinical trials
involving the CD47-SIRPα axis are anticipated. Below, we
offer some suggestions and important considerations to
potentially improve the specificity and efficacy of therapy.
Chemotherapy influences anti-mouse CD47 effects
Many patients might have previously received or continue
to receive chemotherapy during anti-CD47 treatment.
Since chemotherapy can suppress the immune system by
killing recently activated immune cells [42, 43], it is possible
that chemotherapy may blunt the therapeutic effects of
CD47 blockade. However, on the other hand,
chemotherapy may increase the release of tumor antigen and DNA
from dying tumor cells, which may synergize with CD47
blockade. These possibilities have been experimentally
evaluated . It was found that chemotherapy administered
after anti-CD47 therapy has a detrimental effect on the
development of beneficial anti-tumor memory immune
responses. In contrast, chemotherapy administered before
anti-CD47 therapy not only synergized with anti-CD47 for
tumor control but also preserved the host memory
response against relapsing tumors. Several possibilities exist
for the synergistic effect of chemotherapy and anti-CD47
treatment. First, chemotherapy may induce the release of
tumor DNA from dying tumor cells, which could augment
STING-mediated cytosolic DNA sensing. Second,
chemotherapy may sensitize tumor cells by the upregulation of
“eat me” signals, such as surface calreticulin, which could
synergistically amplify the CTL induction in combination
with “don’t eat me” blockade. Third, it is also possible that
the chemotherapy preconditions the tumor
microenvironment with more infiltrating inflammatory cells, allowing
Clinical trial identifiers Study start date
Disease in recruited patients
and NCT02953509 use cetuximab
and rituximab, respectively,
Humanized anti-human CD47 1. NCT02216409
monoclonal antibody 2. NCT02678338
Generated by grafting 3. NCT02953782
complementarity 4. NCT02953509
determining region (CDR)
onto a human IgG4
A soluble recombinant
SIRPα-Fc fusion protein
Generated by combining
the sequences encoding the
N-terminal portion of human
SIRPα with the Fc region of
1. October 2016
2. January 2016
1. March 2015
2. March 2016
1. August 2014
2. November 2015
3. November 2016
4. November 2016
1. Solid malignancy
2. Acute myeloid leukemia
3. Solid malignancy and advanced colorectal cancer
4. Relapsed/refractory B cell
1. Solid malignancy and mycosis fungoides
2. Hematologic malignancy
1. Solid and hematologic malignancies
2. Acute myeloid leukemia and myelodysplastic syndrome
anti-CD47 blockade to work. Therefore, proper
combination therapy of chemotherapeutic drugs and anti-CD47
antibody may depend on the type, timing, dose of these
agents, and tumor types. Further studies are needed to
uncover the underlying synergistic mechanisms for a rational
Intratumoral CD47-SIRPα blockade
Given the ubiquitous expression of CD47 on normal cells,
tumor-specific delivery of CD47 blockade would generate
better anti-tumor effect with fewer side effects than
systemic administration. Indeed, the possibility of attack
against healthy self cells warrants a concern. For example,
patients, especially those under chronic inflammatory
conditions or infection, may become severely anemic
upon CD47 blockade . Thus, how to block
CD47SIRPα inside the tumor tissues specifically becomes the
challenge. Tumor-targeting antibodies may be conjugated
with anti-CD47 or SIRPα-Ig to increase specificity . In
the selection of a conjugation partner, two kinds of
partners can be exploited. One is pro-phagocytic Fc receptor
(FcR)-activating antibodies, such as the anti-CD20
antibody, since CD47-SIRPα interruption can synergize with
antibody-dependent cellular phagocytosis [20, 44]. The
other partner can be adaptive check point blockade
antibodies including anti-programmed death ligand 1 (PDL1)
for unleashing both an innate and adaptive anti-tumor
response . While cytotoxic T lymphocyte-associated
protein 4 (CTLA4) or programmed cell death protein 1
(PD1) blockade monotherapy has gained enormous
attention for its potential to result in a durable clinical response
and prolonged overall survival with tolerable toxicity
compared to standard chemotherapy, not all patients respond
. Discovery that nivolumab and ipilimumab dual
therapy is more efficacious than ipilimumab monotherapy in
patients with untreated metastatic melanoma highlights
the importance of combination therapy and search for
other molecular targets . It is possible that
combination therapy of anti-CD47 antibody, which increases the
tumor cell phagocytosis and priming of anti-tumor CD8+
T cell responses, and anti-CTLA4/PD1, which
reinvigorates exhausted T cells, may give greater synergism by
improving different steps to generating effective
antitumor immunity. Such idea that tumor-targeted delivery
of the CD47 checkpoint antagonist can work as a potential
booster to synergize with other tumor-targeting antibodies
for better cancer immunotherapy is being actively
investigated, as reflected by phase I clinical trials testing its
combination therapy with cetuximab or rituximab (Table 1).
Many solid and hematologic malignancies express CD47
on their cell surface to display an anti-phagocytic signal
to SIRPα-expressing myeloid cells and evade destruction
by innate and adaptive immune system. Administration
of anti-CD47 blocking antibodies has been enormously
successful in various pre-clinical models, mechanism of
which likely involves both phagocyte-mediated direct
killing and their cross-priming of cytotoxic T cells. Our
recent work has illustrated a critical role for dendritic
cells and the STING pathway, as well as CD8+ T cells, to
achieving therapeutic effect of CD47 blockade.
Currently, there are eight clinical trials in progress related to
CD47-SIRPα blockade and more entries are anticipated.
In the future, a combinational design including
antiCD47 antibody with appropriate chemotherapy and
immune-modulating agents such as anti-tumor
antibodies, type I IFN, STING agonists, immune checkpoint
modulators, and others should be intensely investigated
for achieving synergistic and tumor-specific effect for
APCs: Antigen-presenting cells; BMDC: Bone marrow-derived dendritic cells;
CD47/IAP: Cluster of differentiation 47/integrin-associated protein;
cGAMP: Cyclic GMP-AMP; cGAS: cGAMP synthase; CSC: Cancer stem cell;
CTL: Cytotoxic T lymphocyte; CTLA4: Cytotoxic T lymphocyte-associated
protein 4; DCs: Dendritic cells; DNA: Deoxyribonucleic acid; FcR: Fc receptor;
GMP-AMP: Guanosine-adenosine monophosphate; HLA: Human leukocyte
antigen; IFN: Interferon; IFNAR: Interferon alpha/beta receptor;
Ig: Immunoglobulin; IL10: Interleukin 10; ITIM motifs: Immunoreceptor
tyrosine-based inhibition motifs; MHC: Major histocompatibility complex;
mRNA: Messenger ribonucleic acid; MyD88: Myeloid differentiation primary
response gene 88; NK: Natural killer; NSG: NOD-scid-IL2Rgammanull;
PD1: Programmed cell death protein 1; PDL1: Programmed death ligand 1;
PKC: Protein kinase C; RAG: Recombination-activating gene; Rh: Rhesus;
SIRPα/SHPS1/BIT/CD172a: Signal regulatory protein-alpha/Src homology 2
domain-containing protein tyrosine phosphatase substrate 1/brain Ig-like
molecule with tyrosine-based activation motif/cluster of differentiation
antigen-like family member A; STING: Stimulator of interferon genes;
Syk: Spleen tyrosine kinase; TLR: Toll-like receptor
This work was supported by the Ministry of Science and Technology of
China grant (No. 2011DFA31250) to Y-XF and the US National Institutes of
Health grants CA141975 to Y-XF. HK was supported by a scholarship from
the Abney Foundation. ZL is supported by the US National Institutes of
Health grants DK105033, CA186866, CA188419, and AI070603.
Consent for publication
This is not applicable for this review.
Ethics approval and consent to participate
This is not applicable for this review.
1. Brown EJ , Frazier WA. Integrin-associated protein (CD47) and its ligands . Trends Cell Biol . 2001 ; 11 ( 3 ): 130 - 5 .
2. Reinhold MI , Lindberg FP , Plas D , Reynolds S , Peters MG , Brown EJ . In vivo expression of alternatively spliced forms of integrin-associated protein (CD47) . J Cell Sci . 1995 ; 108 (Pt 11): 3419 - 25 .
3. Brown E , Hooper L , Ho T , Gresham H. Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins . J Cell Biol . 1990 ; 111 ( 6 Pt 1 ): 2785 - 94 .
4. Gao AG , Lindberg FP , Finn MB , Blystone SD , Brown EJ , Frazier WA. Integrinassociated protein is a receptor for the C-terminal domain of thrombospondin . J Biol Chem . 1996 ; 271 ( 1 ): 21 - 4 .
5. Liu Y , Merlin D , Burst SL , Pochet M , Madara JL , Parkos CA . The role of CD47 in neutrophil transmigration . Increased rate of migration correlates with increased cell surface expression of CD47 . J Biol Chem . 2001 ; 276 ( 43 ): 40156 - 66 .
6. Lindberg FP , Bullard DC , Caver TE , Gresham HD , Beaudet AL , Brown EJ . Decreased resistance to bacterial infection and granulocyte defects in IAPdeficient mice . Science . 1996 ; 274 ( 5288 ): 795 - 8 .
7. Miyashita M , Ohnishi H , Okazawa H , Tomonaga H , Hayashi A , Fujimoto TT , Furuya N , Matozaki T. Promotion of neurite and filopodium formation by CD47: roles of integrins , Rac, and Cdc42 . Mol Biol Cell . 2004 ; 15 ( 8 ): 3950 - 63 .
8. Reinhold MI , Lindberg FP , Kersh GJ , Allen PM , Brown EJ . Costimulation of T cell activation by integrin-associated protein (CD47) is an adhesion-dependent, CD28-independent signaling pathway . J Exp Med . 1997 ; 185 ( 1 ): 1 - 11 .
9. Chao MP , Majeti R , Weissman IL . Programmed cell removal: a new obstacle in the road to developing cancer . Nat Rev Cancer . 2011 ; 12 ( 1 ): 58 - 67 .
10. Okazawa H , Motegi S , Ohyama N , Ohnishi H , Tomizawa T , Kaneko Y , Oldenborg PA , Ishikawa O , Matozaki T. Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system . J Immunol. 2005 ; 174 ( 4 ): 2004 - 11 .
11. van Beek EM , Cochrane F , Barclay AN , van den Berg TK . Signal regulatory proteins in the immune system . J Immunol . 2005 ; 175 ( 12 ): 7781 - 7 .
12. van den Berg TK , van der Schoot CE . Innate immune 'self' recognition: a role for CD47-SIRPalpha interactions in hematopoietic stem cell transplantation . Trends Immunol . 2008 ; 29 ( 5 ): 203 - 6 .
13. Barclay AN , Van den Berg TK . The interaction between signal regulatory protein alpha (SIRPalpha) and CD47: structure, function, and therapeutic target . Annu Rev Immunol . 2014 ; 32 : 25 - 50 .
14. Tsai RK , Discher DE . Inhibition of “self” engulfment through deactivation of myosin-II at the phagocytic synapse between human cells . J Cell Biol . 2008 ; 180 ( 5 ): 989 - 1003 .
15. Oldenborg PA , Zheleznyak A , Fang YF , Lagenaur CF , Gresham HD , Lindberg FP . Role of CD47 as a marker of self on red blood cells . Science . 2000 ; 288 ( 5473 ): 2051 - 4 .
16. Bian Z , Shi L , Guo YL , Lv Z , Tang C , Niu S , Tremblay A , Venkataramani M , Culpepper C , Li L , et al. Cd47-Sirpalpha interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells . Proc Natl Acad Sci U S A . 2016 ; 113 ( 37 ): E5434 - 5443 .
17. Avent ND , Reid ME . The Rh blood group system: a review . Blood . 2000 ; 95 ( 2 ): 375 - 87 .
18. Jaiswal S , Jamieson CH , Pang WW , Park CY , Chao MP , Majeti R , Traver D , van Rooijen N , Weissman IL . CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis . Cell . 2009 ; 138 ( 2 ): 271 - 85 .
19. Kim MJ , Lee JC , Lee JJ , Kim S , Lee SG , Park SW , Sung MW , Heo DS . Association of CD47 with natural killer cell-mediated cytotoxicity of headand-neck squamous cell carcinoma lines . Tumour Biol . 2008 ; 29 ( 1 ): 28 - 34 .
20. Chao MP , Alizadeh AA , Tang C , Myklebust JH , Varghese B , Gill S , Jan M , Cha AC , Chan CK , Tan BT , et al. Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma . Cell . 2010 ; 142 ( 5 ): 699 - 713 .
21. Chan KS , Espinosa I , Chao M , Wong D , Ailles L , Diehn M , Gill H , Presti Jr J , Chang HY , van de Rijn M , et al. Identification , molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumorinitiating cells . Proc Natl Acad Sci U S A . 2009 ; 106 ( 33 ): 14016 - 21 .
22. Manna PP , Frazier WA. CD47 mediates killing of breast tumor cells via Gi-dependent inhibition of protein kinase A. Cancer Res . 2004 ; 64 ( 3 ): 1026 - 36 .
23. Schulenburg A , Blatt K , Cerny-Reiterer S , Sadovnik I , Herrmann H , Marian B , Grunt TW , Zielinski CC , Valent P. Cancer stem cells in basic science and in translational oncology: can we translate into clinical application ? J Hematol Oncol . 2015 ; 8 : 16 .
24. Fan D , Li Z , Zhang X , Yang Y , Yuan X , Zhang X , Yang M , Zhang Y , Xiong D. AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells . J Hematol Oncol . 2015 ; 8 : 18 .
25. Fang X , Chen C , Xia F , Yu Z , Zhang Y , Zhang F , Gu H , Wan J , Zhang X , Weng W , et al. CD274 promotes cell cycle entry of leukemia-initiating cells through JNK/Cyclin D2 signaling . J Hematol Oncol . 2016 ; 9 ( 1 ): 124 .
26. Zhao H , Wang J , Kong X , Li E , Liu Y , Du X , Kang Z , Tang Y , Kuang Y , Yang Z , et al. CD47 promotes tumor invasion and metastasis in non-small cell lung cancer . Sci Rep . 2016 ; 6 : 29719 .
27. Majeti R , Chao MP , Alizadeh AA , Pang WW , Jaiswal S , Gibbs Jr KD , van Rooijen N , Weissman IL . CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells . Cell . 2009 ; 138 ( 2 ): 286 - 99 .
28. Willingham SB , Volkmer JP , Gentles AJ , Sahoo D , Dalerba P , Mitra SS , Wang J , Contreras-Trujillo H , Martin R , Cohen JD , et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors . Proc Natl Acad Sci U S A . 2012 ; 109 ( 17 ): 6662 - 7 .
29. Soltanian S , Matin MM . Cancer stem cells and cancer therapy . Tumour Biol . 2011 ; 32 ( 3 ): 425 - 40 .
30. Edris B , Weiskopf K , Volkmer AK , Volkmer JP , Willingham SB , ContrerasTrujillo H , Liu J , Majeti R , West RB , Fletcher JA , et al. Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma . Proc Natl Acad Sci U S A . 2012 ; 109 ( 17 ): 6656 - 61 .
31. Xiao Z , Chung H , Banan B , Manning PT , Ott KC , Lin S , Capoccia BJ , Subramanian V , Hiebsch RR , Upadhya GA , et al. Antibody mediated therapy targeting CD47 inhibits tumor progression of hepatocellular carcinoma . Cancer Lett . 2015 ; 360 ( 2 ): 302 - 9 .
32. Takenaka K , Prasolava TK , Wang JC , Mortin-Toth SM , Khalouei S , Gan OI , Dick JE , Danska JS . Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells . Nat Immunol . 2007 ; 8 ( 12 ): 1313 - 23 .
33. Yamauchi T , Takenaka K , Urata S , Shima T , Kikushige Y , Tokuyama T , Iwamoto C , Nishihara M , Iwasaki H , Miyamoto T , et al. Polymorphic Sirpa is the genetic determinant for NOD-based mouse lines to achieve efficient human cell engraftment . Blood . 2013 ; 121 ( 8 ): 1316 - 25 .
34. Strowig T , Rongvaux A , Rathinam C , Takizawa H , Borsotti C , Philbrick W , Eynon EE , Manz MG , Flavell RA . Transgenic expression of human signal regulatory protein alpha in Rag2-/-gamma(c)-/- mice improves engraftment of human hematopoietic cells in humanized mice . Proc Natl Acad Sci U S A . 2011 ; 108 ( 32 ): 13218 - 23 .
35. Liu X , Pu Y , Cron K , Deng L , Kline J , Frazier WA , Xu H , Peng H , Fu YX , Xu MM . CD47 blockade triggers T cell-mediated destruction of immunogenic tumors . Nat Med . 2015 ; 21 ( 10 ): 1209 - 15 .
36. Kim KD , Zhao J , Auh S , Yang X , Du P , Tang H , Fu YX. Adaptive immune cells temper initial innate responses . Nat Med . 2007 ; 13 ( 10 ): 1248 - 52 .
37. Soto-Pantoja DR , Terabe M , Ghosh A , Ridnour LA , DeGraff WG , Wink DA , Berzofsky JA , Roberts DD . CD47 in the tumor microenvironment limits cooperation between antitumor T-cell immunity and radiotherapy . Cancer Res . 2014 ; 74 ( 23 ): 6771 - 83 .
38. Tseng D , Volkmer JP , Willingham SB , Contreras-Trujillo H , Fathman JW , Fernhoff NB , Seita J , Inlay MA , Weiskopf K , Miyanishi M , et al. Anti-CD47 antibodymediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response . Proc Natl Acad Sci U S A . 2013 ; 110 ( 27 ): 11103 - 8 .
39. Savina A , Amigorena S. Phagocytosis and antigen presentation in dendritic cells . Immunol Rev . 2007 ; 219 : 143 - 56 .
40. Sikic BI , Narayanan S , Colevas AD , Padda SK , Fisher GA , Supan D , Wakelee HA , Aoki R , Pegram MD , Villalobos VM et al: A first-in-human, first-in-class phase I trial of the anti-CD47 antibody Hu5F9-G4 in patients with advanced cancers . J Clin Oncol . 2016 , 34 :(suppl; abstr 3019). http://meetinglibrary.asco. org/content/162472- 176 .
41. Liu J , Wang L , Zhao F , Tseng S , Narayanan C , Shura L , Willingham S , Howard M , Prohaska S , Volkmer J , et al. Pre-clinical development of a humanized anti-CD47 antibody with anti-cancer therapeutic potential . PLoS One . 2015 ; 10 ( 9 ): e0137345 .
42. Obeid M , Tesniere A , Ghiringhelli F , Fimia GM , Apetoh L , Perfettini JL , Castedo M , Mignot G , Panaretakis T , Casares N , et al. Calreticulin exposure dictates the immunogenicity of cancer cell death . Nat Med . 2007 ; 13 ( 1 ): 54 - 61 .
43. Obeid M , Tesniere A , Panaretakis T , Tufi R , Joza N , van Endert P , Ghiringhelli F , Apetoh L , Chaput N , Flament C , et al. Ecto-calreticulin in immunogenic chemotherapy . Immunol Rev . 2007 ; 220 : 22 - 34 .
44. Piccione EC , Juarez S , Tseng S , Liu J , Stafford M , Narayanan C , Wang L , Weiskopf K , Majeti R. SIRPalpha-antibody fusion proteins selectively bind and eliminate dual antigen-expressing tumor cells . Clin Cancer Res . 2016 ; 22 : 5109 - 19 .
45. Sockolosky JT , Dougan M , Ingram JR , Ho CC , Kauke MJ , Almo SC , Ploegh HL , Garcia KC . Durable antitumor responses to CD47 blockade require adaptive immune stimulation . Proc Natl Acad Sci U S A . 2016 ; 113 ( 19 ): E2646 - 2654 .
46. Ma W , Gilligan BM , Yuan J , Li T. Current status and perspectives in translational biomarker research for PD-1/PD-L1 immune checkpoint blockade therapy . J Hematol Oncol . 2016 ; 9 ( 1 ): 47 .
47. Postow MA , Chesney J , Pavlick AC , Robert C , Grossmann K , McDermott D , Linette GP , Meyer N , Giguere JK , Agarwala SS , et al. Nivolumab and ipilimumab versus ipilimumab in untreated melanoma . N Engl J Med . 2015 ; 372 ( 21 ): 2006 - 17 .