SEE COMMENTARY

Anti-CD47 antibodymediated phagocytosis of cancer

by macrophages primes an effective antitumor
T-cell response
Diane Tsenga,b,1, Jens-Peter Volkmera,b, Stephen B. Willinghama,b, Humberto Contreras-Trujilloa,b,
John W. Fathmana,b, Nathaniel B. Fernhoffa,b, Jun Seitaa,b, Matthew A. Inlaya,b, Kipp Weiskopfa,b,
Masanori Miyanishia,b, and Irving L. Weissmana,b,1
a

Institute for Stem Cell Biology and Regenerative Medicine and bthe Ludwig Cancer Center, Stanford University Medical Center, Stanford, CA 94305

Contributed by Irving L. Weissman, April 4, 2013 (sent for review February 12, 2013)

ntigen presentation is the process by which innate immune

cells such as macrophages and dendritic cells (antigen-presenting cells, APC) acquire antigens and present them to T cells to
initiate the adaptive immune response. How APCs shape the immune response by both degrading antigens and preserving antigens
for presentation to T cells has been a longstanding area of interest
(1). Recently, the mechanism of antigen recognition by APCs has
been shown to affect the preference of MHC I versus MHC II
antigen-presentation pathways. For instance, mannose receptormediated endocytosis on dendritic cells has been associated with
MHC I antigen presentation, whereas scavenger receptor-mediated
endocytosis has been associated with MHC II presentation (2).
Moreover, the functional outcomes of antigen presentation have
been shown to be context dependent. For instance, targeting antigens to DEC-205 using monoclonal antibodies induced tolerance
under noninammatory conditions but mediated immunogenicity
under activating conditions by cluster of differentiation 40 ligand
(CD40L) (3). Harnessing APCs to enhance the antitumor T-cell
response offers an exciting strategy for cancer immunotherapy. The
ability of the T-cell immune response to be mobilized successfully
against cancer has been demonstrated through preclinical and
clinical studies of anti-CTLA4 antibody for T-cell activation (4).
Phagocytosis by macrophages relies on the cells recognition of
prophagocytic (eat me) and antiphagocytic (dont eat me) signals on target cells. Anti-CD47 blocking monoclonal antibodies
(mAbs) induce macrophage phagocytosis of cancer cells by
inhibiting an important antiphagocytic signal, allowing prophagocytic signals to dominate (5, 6). CD47 is highly expressed on
cancer cells as compared with normal cells (5, 6) and interacts
with the ligand signal regulatory protein (SIRP-) on macrophages (7). This interaction results in phosphorylation of immunoreceptor tyrosine-based inhibition (ITIM) motifs on SIRP-s
cytoplasmic tail and the recruitment of Src homology phosphatase1 (SHP-1) and SHP-2 phosphatases, which is thought to block
phagocytosis by preventing myosin-IIA accumulation at the phagocytic synapse (812). We have demonstrated the therapeutic
www.pnas.org/cgi/doi/10.1073/pnas.1305569110

efcacy of anti-CD47 blocking mAbs against xenograft human

cancers growing in immunodecient mice, including cancers such as
leukemia (5, 13), lymphoma (14), and multiple myeloma (15), solid
tumors, including breast, colon, prostate, and bladder cancers, and
sarcomas (6, 16). Whether the adaptive immune response also can
be recruited against the cancer after anti-CD47 mAb treatment has
not been tested, because the immunodecient mice used to establish
the xenograft models lack T, B, and NK cells. In this study, we tested
the hypothesis that anti-CD47 antibodymediated phagocytosis
of cancer cells can facilitate an antitumor T-cell immune response.
Results
Macrophages Phagocytose Cancer Cells in the Presence of Anti-CD47
Blocking Antibody. To follow the immune response to a model tu-

mor antigen, the human colon cancer cell line DLD1 was transfected with a lentiviral vector for expressing cytoplasmic ovalbumin
(cOVA) and GFP (DLD1-cOVA-GFP) (Fig. S1). DLD1-cOVAGFP cancer cells express CD47 and can be recognized by both
CD47 mAbs, clones B6H12 and 2D3 (Fig. S1). Anti-CD47 B6H12
(blocking) mAb blocks the interaction between CD47 and SIRP-,
whereas anti-CD47 2D3 (non-blocking) antibody binds CD47
but does not block its interaction with SIRP-. Macrophages
phagocytose DLD1-cOVA-GFP cancer cells in the presence of
anti-CD47 B6H12, but not anti-CD47 2D3 mAbs, demonstrating that phagocytosis is dependent on the blockade of CD47/
SIRP interactions and not entirely due to antibody opsonization effects (Fig. 1 and Fig. S2). Anti-CD47 mediated phagocytosis of DLD1-cOVA-GFP cancer cells by macrophages leads
to cross-presentation of ovalbumin peptide onto MHC-I, as
assessed by staining for the SIINFEKL-H2kb complex on the
cell surface (Fig. S3). Costimulatory molecule CD86 is upregulated, but not coinhibitory molecule B7-H1 (Fig. S4). AntiCD47 B6H12mediated phagocytosis of cancer cells leads to
macrophage release of proinammatory cytokines. For example,
IL-12p40, TNF-, regulated upon activation normal T cell expressed and secreted (RANTES), and monocyte chemotactic
protein-3 (MCP-3) cytokine levels increase after anti-CD47
B6H12-mediated phagocytosis (Fig. S5). Next, the ability of
the APCs, macrophages and dendritic cells, were tested for
phagocytic activity in response to anti-CD47 mAbs. Compared to dendritic cells, macrophages effectively phagocytose
DLD1-cOVA-GFP cancer cells in the presence of anti-CD47

Author contributions: D.T., J.-P.V., S.B.W., and I.L.W. designed research; D.T., J.-P.V., S.B.W.,
H.C.-T., J.W.F., and N.B.F. performed research; D.T., J.-P.V., and K.W. contributed new
reagents/analytic tools; D.T., J.-P.V., S.B.W., J.W.F., N.B.F., J.S., M.A.I., M.M., and I.L.W.
analyzed data; and D.T., J.-P.V., and I.L.W. wrote the paper.
Conict of interest statement: I.L.W. owns Amgen Inc. stock and is a Director of Stem
Cells, Inc.
Freely available online through the PNAS open access option.
See Commentary on page 10886.
1

To whom correspondence may be addressed. E-mail: dianet@gmail.com or irv@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1305569110/-/DCSupplemental.

PNAS | July 2, 2013 | vol. 110 | no. 27 | 1110311108

MEDICAL SCIENCES

Mobilization of the T-cell response against cancer has the potential

to achieve long-lasting cures. However, it is not known how to
harness antigen-presenting cells optimally to achieve an effective
antitumor T-cell response. In this study, we show that anti-CD47
antibodymediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin
model antigen system, anti-CD47 antibodymediated phagocytosis
of cancer cells by macrophages resulted in increased priming of OT-I
T cells [cluster of differentiation 8-positive (CD8+)] but decreased
priming of OT-II T cells (CD4+). The CD4+ T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3+) regulatory
T cells. Macrophages following anti-CD47mediated phagocytosis
primed CD8+ T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that
anti-CD47 antibody treatment not only enables macrophage
phagocytosis of cancer but also can initiate an antitumor cytotoxic
T-cell immune response.

2D3

105

105

105

104

104

104

103

103

1.6

102

103

21.7

102

103

104

1.9

102

105

103

104

105

103

104

105

GFP (cancer)

RFP (Mac)

IgG

2D3

105

105

104

104

104

103

103

103

2.2

102

16.4

102

103

104

103

104

105

105

104

104

2.3

102

0
103

104

105

1.3

102

105

103

104

IgG

B6H12

2D3

p<5*10-5 p<5*10-5

105

103

104

10

0
0

104

103

103

1.3

102

103

104
103

2.0

105

105

a carboxyuorescein succinimidyl ester (CFSE)-dilution assay

was used to measure the proliferative response of OVA-specic
CD8+ T cells (OT-I). Red uorescent protein-positive (RFP+)
macrophages were cocultured with DLD1-cOVA-GFP cancer
cells in the presence of IgG, anti-CD47 B6H12 (blocking), or
anti-CD47 2D3 (non-blocking) mAbs. Lymph nodes were harvested from OT-I (CD8+) transgenic mice and were labeled with
CFSE (0.5 M) and CD8+ cells enriched by magnetic separation.
On day 3, the percentage of proliferating OT-I T cells was
quantied based on the percentage of cells that had diluted the
CFSE dye (CFSE-low). The percentage of proliferating OT-I
T cells increased following anti-CD47 B6H12-mediated phagocytosis of cancer by macrophages (Fig. 2A). To verify that the
proliferative response of OT-I T cells was an antigen-specic response, macrophages were allowed to phagocytose DLD1-cOVAGFP versus DLD1-GFP cancer cells (the latter not expressing
OVA) in the presence of anti-CD47 B6H12 mAb before the addition of CFSE-labeled OT-I T cells. Increased OT-I T-cell proliferation was observed only after anti-CD47mediated phagocytosis
of DLD1-cOVA-GFP cancer cells but not DLD1-GFP cancer cells,
indicating an antigen-specic effect (Fig. 2B).

p<5*10-6

20

105

p<5*10-6
25
20
15
10

15

102

0
0

RFP (DC)

B6H12

105

Phagocytosis (%)

B6H12

IgG

Phagocytosis (%)

RFP (Mac)

105

GFP (cancer)

p<0.05 p<0.05

IgG B6H12 2D3

IgG B6H12 2D3

Macrophages

Dendritic cells

Fig. 1. Macrophages effectively phagocytose cancer cells in the presence of

anti-CD47 B6H12 antibody. (A) RFP+ macrophages (Mac) were cocultured with
DLD1-cOVA-GFP cancer cells in the presence of IgG or anti-CD47 B6H12
(blocking) or 2D3 (nonblocking) mAbs. Percentage of phagocytosis was
determined by the percentage of GFP+ cells within RFP+ macrophage cell
gate. (B) RFP+ macrophages versus dendritic cells (DCs) were cocultured with
DLD1-cOVA-GFP cancer cells in the presence of IgG, anti-CD47 B6H12, or antiCD47 2D3 mAbs. The experiment was performed three times with similar results.

Macrophages Do Not Prime OT-II (CD4+) T cells After Phagocytosis of

Cancer Cells by Anti-CD47 B6H12 Blocking Antibody. To assess priming

of CD4+ T cells after anti-CD47mediated phagocytosis by macrophages, a CFSE-dilution assay was used to measure the proliferative response of OVA-specic CD4+ T cells (OT-II). Macrophages
were cocultured with DLD1-cOVA-GFP cancer cells in the presence of IgG, anti-CD47 B6H12 (blocking), or anti-CD47 2D3 (nonblocking) mAbs, and CFSE-labeled OT-II (CD4+) T cells were
added to cultures. Interestingly, the percentage of proliferating
OT-II T cells was reduced following anti-CD47-mediated phagocytosis compared with baseline levels (Fig. 3A). Because of the possibility that cell-surface MHC II (I-Ab+) on macrophages might be

B6H12 mAb (Fig. 1). Consistent with this result, SIRP-, the ligand for CD47, is expressed at high levels on macrophages but at
lower levels on dendritic cells (Fig. S6).
Macrophages Prime OT-I (CD8+) T Cells After Phagocytosis of Cancer
Cells by Anti-CD47 Blocking Antibody. To assess priming of CD8+ T

cells after anti-CD47mediated phagocytosis by macrophages,

A
Proliferating cells (%)

100

IgG

80
60

20.2

40
20
0

0 102

103

104

105

B6H12

120
90
60

49.5

30
0

0 102

103

104

105

200

2D3

19.1

50

0
0 102

103

104

p<0.01 p<0.05

60
50
40
30

p<0.005

20

p<0.005

10
0

Mac
+
+
+
DLD1-cOVA +
+
+
IgG B6H12 2D3
Antibody
T cell
+
+
+
Peptide
-

150

100

p<0.005

70

105

CFSE

+
+
-

+
+
+

+
-

B6H12 B6H12

+
+

+
-

+
-

B6H12

+
-

p<0.01
p<5*10

-5

p<5*10

p<5*10-6

-5

p<5*10-6

15
10
5
0

IgG

B6H12

DLD1-cOVA-GFP
DLD1-GFP

2D3

Proliferating cells (%)

20

Phagocytosis (%)

60

p<0.01

p<0.01
p<5*10-4

50
40
30

p<5*10-4

p<5*10-6

20
10
0

Mac
+
DLD1-cOVA +
IgG
Antibody
T cell
+

11104 | www.pnas.org/cgi/doi/10.1073/pnas.1305569110

+
+

+
+

B6H12

2D3

B6H12

Fig. 2. Macrophages prime CD8+ T cells to proliferate after phagocytosis of cancer cells by anti-CD47
B6H12 mAb. (A) RFP+ macrophages were cocultured
with DLD1-cOVA-GFP colon cancer cells in the presence of IgG, anti-CD47 B6H12 (blocking), or anti-CD47
2D3 (nonblocking) mAbs. The next day, CD8+ T cells
from OT-I transgenic mice were magnetically enriched and labeled with CFSE (0.5 M). Analysis was
performed on day 3, and the percentage of proliferating cells was determined. Macrophages were
pulsed with OT-I peptide (OVA257-264, SIINFEKL) as
a positive control. The experiment was performed
three times with similar results. (B) RFP+ macrophages
were cocultured with DLD1-cOVA-GFP cancer cells or
DLD1-GFP cancer cells not expressing cOVA. (Left)
Phagocytosis was determined by the percentage of
GFP+ cells within the RFP+ macrophage cell gate.
(Right) CFSE-labeled CD8+ T cells from OT-I mice were
added to cultures, and the percentage of proliferating
cells was determined.

Tseng et al.

IgG

15

10

22.1

B6H12

80

60

40

20

5.6

0
0 102

103

104

105

2D3

20

10

13

0
0 102

103

104

p<5*10-4

45
40
35
30

p<5*10-5 p<5*10-6

25
20
15
10
5
0

Mac
+
+
+
DLD1-cOVA +
+
+
IgG B6H12 2D3
Antibody
T cell
+
+
+
Peptide
-

30

105

CFSE

SEE COMMENTARY

20

Proliferating cells (%)

+
+
-

+
+
+

Phagocytosis (%)

+
+

+
-

+
-

B6H12

+
-

p<5*10-5

- IFNg
+ IFNg

10

I-Ab (MHC II)

B6H12 B6H12

p<5*10-6

15

p<5*10-5

p<5*10-5

Unstained
I-Ab stained

+
-

IgG

B6H12

2D3

p<0.05

p<0.005

p<0.05

- IFNg
+ IFNg

50
40
30

p<5*10-6

p<0.005

p<5*10-4

p<0.01

20
10
0

Mac
+
DLD1-cOVA +
IgG
Antibody
T cell
+
Peptide
-

+
+

+
+

B6H12

2D3

+
-

+
-

+
+
+
-

+
+
+

+
-

B6H12

B6H12

+
+

+
-

limiting OT-II (CD4+) T cell proliferation after anti-CD47-mediated

phagocytosis, we measured the percentage of macrophages expressing MHC II on the cell surface. Interestingly, the percentage
of I-Ab+ macrophages increased after anti-CD47 B6H12mediated phagocytosis of cancer cells, despite the decrease in CD4+
T-cell priming (Fig. S7). To determine whether IFN- stimulation of macrophages could overcome the decrease in CD4+ T cell
proliferation, IFN- was used to up-regulate surface MHC II
levels on macrophages for evaluation in phagocytosis and T cell
proliferation assays. IFN-stimulated macrophages efciently
phagocytosed cancer in the presence of anti-CD47 B6H12 mAb,
but the OT-II CD4+ T-cell response was also diminished compared
with baseline (Fig. 3B).
Reduction in Forkhead Box P3-Positive Regulatory T Cells After AntiCD47Mediated Phagocytosis of Cancer by Macrophages. To assess

the functional effects of anti-CD47 B6H12mediated phagocytosis

on CD4+ regulatory T cells, we crossed OT-II transgenic mice
with forkhead box 3P (Foxp3)-GFP reporter mice to generate
Tseng et al.

+
-

B6H12

+
-

Fig. 3. After phagocytosis of cancer cells by antiCD47, macrophages do not prime CD4+ T cells to
proliferate. (A) RFP+ macrophages were cocultured
with DLD1-cOVA-GFP cancer cells in the presence of
IgG, anti-CD47 B6H12 (blocking), or anti-CD47 2D3
(nonblocking) mAbs. The next day, CD4+ T cells
were isolated from OT-II transgenic mice and were
labeled with CFSE (0.5 M). Analysis was performed
on day 4, and the percentage of proliferating cells
was determined. Macrophages were pulsed with
OVA peptide 323339 as a positive control. (B) RFP+
macrophages were stimulated with IFN- to upregulate MHC II levels. Phagocytosis and priming of
OT-II CD4+ cells were determined in the presence of
anti-CD47 mAbs.

double-transgenic mice (Fig. S8). These mice express 25%

Foxp3-GFP+ cells within the CD4+ CD25+ population and exhibit T cell receptor V alpha 2 (V2) restriction (Fig. S8A).
In addition, CD4+ Foxp3-GFP+ T cells are responsive to OVA
peptide 323339 and can be induced to differentiate in the
context of TGF- and all-trans-retinoic acid (Fig. S8B). RFP+
macrophages were cocultured with DLD1-cOVA-GFP cancer
cells in the presence of IgG, anti-CD47 B6H12 (blocking), or
anti-CD47 2D3 (non-blocking) mAbs. The next day, CD4+ T cells
from OT-II/Foxp3-GFP+ double-transgenic mice were magnetically enriched and were added to the cultures. After 4 d, the percentage of regulatory T-cells was quantied by the percentage of
CD4+ Foxp3-GFP+ cells (Fig. 4). A reduction in Foxp3+ regulatory T cells was observed after anti-CD47-mediated phagocytosis
of cancer cells.
After Anti-CD47Mediated Phagocytosis of Cancer Cells, Macrophages
Prime OT-I (CD8+) T Cells to Proliferate in Vivo. To evaluate the

effects of anti-CD47 B6H12mediated phagocytosis on CD8+

PNAS | July 2, 2013 | vol. 110 | no. 27 | 11105

MEDICAL SCIENCES

Proliferating cells (%)

p<5*10-4
60

104

103

103

103

102

102

102
0

0
0 102

103

104

105

2D3
3.7

105

0 102

103

104

105

0 102

CD4

103

104

105

p<0.05

p<0.05

5
4
3
2
1
0

IgG

B6H12

2D3

Fig. 4. A reduction in Foxp3 regulatory T cells occurs after anti-CD47

B6H12mediated phagocytosis of cancer cells by macrophages. RFP+ macrophages were cocultured with DLD1-cOVA-GFP cancer cells in the presence
of IgG or anti-CD47 B6H12 (blocking) or 2D3 (nonblocking) mAbs. The next
day, CD4+ T cells from OT-II/Foxp3-GFP+ transgenic mice were magnetically
enriched and were added to cultures. On day 4, the percentage of CD4+
Foxp3-GFP+ cells was quantied.

T-cell priming in vivo, OT-I (CD8+) T cells (CD45.2) were CFSElabeled and adoptively transferred to CD45.1 recipient mice (Fig.
5A). The next day, RFP+ macrophages were cocultured with
DLD1-cOVA-GFP cancer cells in the presence of IgG or antiCD47 B6H12 mAb. Macrophages were isolated by magnetic enrichment, and phagocytosis was veried by FACS analysis before
subcutaneous (subQ) transfer into the footpad. After 4 d, the
popliteal lymph node was analyzed for the percentage of proliferating cells (CFSE-low) within the CD45.2+ gate. There was
an increase in proliferating OT-I T cells in mice receiving macrophages that phagocytose cancer cells via anti-CD47 B6H12
mAb (Fig. 5B).
Macrophages Prime an Antitumor CD8+ T-Cell Response in Vivo After
Anti-CD47Mediated Phagocytosis of Cancer Cells. We next evalu-

ated the functional effects of OT-I (CD8+) T-cell priming after

anti-CD47mediated phagocytosis of cancer cells by macrophages.
To assess the efcacy of CD8+ T-cell killing of OVA peptidedisplaying targets, CD8+ T cells were isolated from OT-I transgenic
mice and were i.v. transferred to recipient mice (Fig. 6A). RFP+
macrophages were cocultured with DLD1-cOVA-GFP cancer cells
in vitro in the presence of IgG or anti-CD47 B6H12 mAb. After 2-h
incubation, macrophages were isolated and injected into the footpad. After 4 d, mice were challenged with target cells (CD45.1
splenocytes) to assess cytotoxic activity. CFSE-high splenocytes
were pulsed with 1 M OVA class I-restricted peptide (OVA257264, SIINFEKL) to make them targets for OT-I cytotoxic T cells
and then were mixed in a 1:1 ratio with nonpeptide-pulsed
CFSE-low cells before i.v. transfer. Analysis of draining lymph
nodes 16 h later showed increased cell killing of peptide-pulsed
CFSE-high lymphocytes in mice receiving macrophages that had
phagocytosed cancer cells by anti-CD47 B6H12 mAb (Fig. 6A).
Next, the ability of CD8+ effector T cells to prime an antitumor
immune response was evaluated. CD8+ T cells from OT-I mice were
transferred into recipient animals (Fig. 6B). Macrophages were
cocultured with DLD1-cOVA-GFP cancer cells in vitro in the
presence of IgG or anti-CD47 B6H12 mAb; then the macrophages were transferred into the footpad on days 1 and 10. Animals were challenged with EG.7 (OVA-expressing EL4) cancer
cells on day 14, and tumor growth was monitored over time. CD8+
T cells primed by macrophages following anti-CD47-mediated
phagocytosis protected mice from tumor challenge (Fig. 6B).
Discussion
Recent studies from the I.L.W. laboratory have shown that malignant cancers universally up-regulate the dont eat me signal
CD47, presumably in their progression to allow escape from
endogenous eat me signals that were induced as part of
programmed cell death and programmed cell removal (5, 6, 13,
14, 1618). These results indicate a role for human CD47-blocking antibodies in cancer therapy via induced phagocytosis. Our
previous experiments involved xenotransplantation of primary
human cancers into immunodecient mice. We now have examined the possible role of anti-CD47enabled phagocytosis in antigen presentation of tumor peptides to T cells of the adaptive
immune system. Here we have demonstrated that CD47 serves as
11106 | www.pnas.org/cgi/doi/10.1073/pnas.1305569110

an invisibility cloak for both innate and adaptive immunity.

Treatment with anti-CD47 blocking mAbs led to adaptive T-cell
immune responses, thereby providing an additional mechanism of
action for anti-CD47 antibodies. OVA-specic OT-I (CD8+) and
OT-II (CD4+) T-cell clones were used to follow the outcomes of
antigen presentation by macrophages after anti-CD47mediated
phagocytosis of cancer cells engineered to express cytoplasmic
OVA. Using in vitro and in vivo assays, we show that antigens are
presented to CD8+ T cells effectively, but the proliferative response of OT-II CD4+ T cells to loaded macrophages was diminished compared with baseline levels. The baseline level of
OT-I CD8+ proliferation and OT-II CD4+ proliferation was 20%
(Figs. 2 and 3), likely because of OVA released from cancer cells
that become endocytosed or pinocytosed by macrophages and
then processed for presentation to both MHC I and MHC II
pathways. Together, our results suggest that anti-CD47mediated
phagocytosis of cancer cells results in the presentation of negative
signals to CD4+ T cells and positive signals to CD8+ T cells. In
addition, the CD4+ T-cell response was characterized by reduced
regulatory T cells. This reduction might be attributed either to
decreased proliferation of regulatory T cells in response to peptide or to less efcient regulatory T-cell differentiation. The in
vivo priming of an antitumor T-cell response by macrophages
after anti-CD47mediated phagocytosis of cancer protects mice
from tumor challenge. Anti-CD47 mAbs may represent a therapeutic strategy for overcoming the regulatory T-cell contribution
to immune evasion by cancer and initiating an effective antitumor
cytotoxic T-cell response.
In this system macrophages are the primary APCs that phagocytose cancer in response to anti-CD47 antibody and present antigen to CD8+ T cells. Perhaps this observation is due to higher
levels of SIRP- expressed on macrophages than on dendritic
cells or because macrophages phagocytose whole cells more effectively. Because subsets of dendritic cells have been reported to
vary in their levels of SIRP- expression (19), it is possible that
other dendritic cell subsets may more effectively phagocytose cancer in response to anti-CD47 antibody in vivo. While dendritic cells
are well known for their function in antigen presentation, in this
study we show that macrophages also have the capacity to stimulate
a CD8+ T-cell immune response in the context of blocking the
CD47-SIRPalpha axis. Prior to this work, the ability to present
exogenous antigens to CD8+ T cells has been largely attributed to
the CD8+ subtype of DCs. Understanding the roles of tissue-specic
macrophages and dendritic cells in response to anti-CD47 mAbs

A OT I

C57BL/6
(CD45.1)

(CD45.2)
Purify CD8 T cells
CFSE label

Day 0
Co-culture with cancer
(IgG vs anti-CD47)

BMDM

Mac-1 enrichment
for macrophages

30

0.5

20

103
10

17.3

102
0
0
0 102

103

104

105

0 102

103

104

105

100
105

104

1.7

80

60
103
40
102

80.2

20

0
0
0 102

103

104

SSC-A

105

Footpad
Injection

Day 5

p<0.05

105

104

Analyze
Popiteal LN

Day 1

0 102

103

104

CFSE

105

Proliferating cells (%)

B6H12
0.8

104

B6H12
IgG
CD45.2

105

104

Foxp3-GFP (%)

Foxp3-GFP

IgG
6.1

105

100
75
50
25
0

IgG

B6H12

Fig. 5. After anti-CD47mediated phagocytosis of cancer cells, macrophages

prime CD8+ T cells in vivo. (A) Experimental setup. BMDM, bone marrowderived macrophages. (B) Adoptively transferred CFSE+ OT-I T cells were analyzed in the draining lymph node by gating on CD45.2+ cells. The percentage of
proliferating cells was determined by gating on the CFSE-low population.
n = 5 mice per group.

Tseng et al.

Cell killing (%)

60

60

40

40

20

103

104

105

warrants further investigation both in mouse and human systems

(2022).
Antibody-mediated uptake of antigens via Fc receptor-mediated endocytosis by dendritic cells can prime CD4+ and CD8+
T-cell responses in some circumstances (23, 24). In contrast, antiCD47mediated phagocytosis by macrophages predominantly
primes CD8+ T cells. This predominant CD8+ T cell response is
not explained entirely by opsonization of cancer cells, because
both the anti-CD47 clones B6H12 and 2D3 bind DLD1-cOVAGFP cancer cells, but only B6H12 is capable of inducing phagocytosis through its ability to block the interaction between CD47
on cancer cells and SIRP- on macrophages. The preferential
activation of CD8+ T cells may rely on routing phagocytosed
cancer antigens toward the MHC I pathway or might be explained
by SIRP-s role as a negative regulator of macrophage function.
Engagement of SIRP- by CD47 leads to phosphorylation of
SIRP-s cytoplasmic ITIM motifs by SHP-1 and SHP-2. This
pathway is well described to inhibit macrophage phagocytosis, but
more recently, SIRP- also has been shown to attenuate macrophage activation by LPS by sequestering SHP-2, which is needed for
activation of the MAPK and NF-B pathways (25). In addition,
inhibition of SHP-1 in dendritic cells has been reported to enhance
CD8+ and CD4+ T-cell responses and reduce regulatory T cells,
a response that protected mice against tumor challenge (26). SIRP also may negatively regulate cross-presentation by a mechanism
not yet understood, perhaps involving recruitment of cross-presentation machinery to phagosomes. Alternatively, prophagocytic
signals such as calreticulin have been described to elicit antitumor
immunity (2729). It may be that the unmasking of such prophagocytic signals may route antigens preferentially toward the crosspresentation pathway.
Other groups examining the effect of SIRP- on dendritic cells
interacting with CD47 on T cells have reported T-cell activation
or inhibition, depending on the context (3033). In contrast, our
experiments were designed to study the impact of antigen presentation after anti-CD47 mAbmediated phagocytosis of cancer cells by macrophages. In our system, anti-CD47 B6H12 mAb
did not have a direct effect on T-cell activation or inhibition
(Figs. 2 and 3).
The involvement of both the innate and adaptive immune systems in the mechanism of action of anti-CD47 antibody has several clinical implications. First, our ndings suggest a novel role of
anti-CD47 blocking mAbs as a vaccination strategy to enhance
CD8+ effector T cells recognizing antigens on phagocytosed
Tseng et al.

Tumor volume (mm3)

0 102

* *

**

* *

target cells. Second, in designing clinical trial protocols for testing

anti-CD47 therapy in patients, immune monitoring of T cells may
be important for understanding clinical response to treatment and
clinical outcomes. Third, anti-CD47 mAbs might be used clinically
in combination with adoptive T-cell therapy or T-cellactivating
antibodies to enhance the adaptive immune response against tumor antigens and minimize toxicity. We conclude that anti-CD47
mediated phagocytosis of cancer not only functions in directly
clearing cancer cells but also can initiate an antitumor T-cell response to eliminate cancers. Patients receiving anti-CD47 therapy
may benet from both the innate and adaptive immune responses
against cancer.
Materials and Methods
Mice. Mice, including C57BL/Ka (CD45.2), C57BL/Ka (CD45.1), and C57BL/Ka
Rosa26-mRFP1 mice, were bred and maintained at the Stanford University
Research Animal Facility in accordance with the Administrative Panel on
Laboratory Animal Care. All the animals were housed in sterile microinsulators and were given water and rodent chow ad libitum. OT-I TCR
transgenic mice, OT-II TCR transgenic mice, and Foxp3-GFP mice were purchased from the Jackson Laboratory. OT-I mice have transgenic TCRs specic
for OVA257-264 in the context of H2-kb. OT-II mice have TCRs specic for
OVA323-339 in the context of IAb. Both OT-I and OT-II mice have transgenic
V2 V5 TCRs.
Molecular Biology. cOVA was cloned from pCI-neo-cOVA (plasmid 25097;
AddGene) and was shuttled into the lentiviral pCDH-EF1-MCS-T2A-copGFP
vector (System Biosciences) using EcoRI and BamHI restriction sites. Lentiviral
production and concentration were accomplished using standard protocols.
Generation of Macrophages and Dendritic Cells. Whole bone marrow cells
were isolated from C57BL/Ka (CD45.2) or C57BL/Ka Rosa26-mRFP1 mice.
Macrophages were generated by incubating whole bone marrow in macrophage colony-stimulating factor (10 ng/mL) for 7 d and harvesting the
adherent fraction. Dendritic cells were generated in granulocyte-macrophage colony stimulating factor (GM-CSF) (1,000 U/mL); on days 2 and 4 cells
were washed and medium was replaced with fresh cytokine-containing
medium. Nonadherent cells were replated on day 6 and harvested on day 7.
In Vitro Phagocytosis Assay. For the in vitro phagocytosis assay, 2 104
macrophages or dendritic cells per well were plated in a 96-well ultra-lowadherent plate (Corning), along with 2 104 cancer cells (DLD1-cOVA-GFP)
in serum-free RPMI medium.
The indicated antibodies (10 g/mL) were added and incubated for 4 h at
37. Macrophages were washed twice and analyzed using a BD LSR Fortessa
Analyzer. The percentage of phagocytosis was calculated as the percentage

PNAS | July 2, 2013 | vol. 110 | no. 27 | 11107

MEDICAL SCIENCES

20

SEE COMMENTARY

p<0.005
Fig. 6. After anti-CD47mediated phagocytosis of
IgG
B6H12
50
cancer cells, macrophages prime an antitumor CD8+
55.6 44.2
87.3 12.6
40
T-cell response in vivo. (A) After anti-CD47mediated
phagocytosis of cancer cells, macrophages prime
30
effector cytotoxic T cells. CD8+ T cells were isolated
20
from OT-I transgenic mice and were transferred i.v.
10
to recipient mice. Macrophages (Mac) were cocul0
tured with DLD1-cOVA-GFP cancer cells in vitro in
CFSE
IgG
B6H12
the presence of IgG or anti-CD47 B6H12 (blocking)
mAb. Macrophages were isolated by magnetic
separation and were transferred subcutaneously
350
IgG
(subQ) on the next day. After 4 d, target cells
300
anti-CD47
OT I
Mac
Mac EG.7 tumor
(CD45.1 splenocytes) were labeled as CFSE-high
250
T cells iv subQ
subQ challenge
(10 M) or -low (1 M). CFSE-high cells were pulsed
200
with 1 M OVA class I-restricted peptide (SIINFEKL)
150
to make them targets for OT-I cytotoxic T-cell funcTime (days)
100
tion. CFSE-high (peptide-pulsed) and -low (unpulsed)
50
0
1
10
14
cells were mixed in a 1:1 ratio and transferred i.v.
Draining lymph nodes were analyzed 16 h later
0
to determine the percentage of CFSE-high versus
6 7 8 9 10 11 12 13 14 15
CFSE-low cells. The percentage of cell killing was
Days post tumor challenge
determined as described in Materials and Methods.
+
n = 10 mice. (B) After anti-CD47mediated phagocytosis of cancer cells, macrophages prime an antitumor CD8 T-cell response. OT-I CD8+ T cells were
transferred i.v. to recipient mice. Macrophages were cocultured with DLD1-cOVA-GFP cancer cells in vitro in the presence of IgG or anti-CD47 B6H12 mAb, and
then macrophages were transferred on days 1 and 10. Animals were challenged with EG.7 (EL4 mouse lymphoma cells expressing ovalbumin) cancer cells on
day 14, and tumor growth was monitored over time. n = 5 mice per group. *P < 0.05; **P < 0.01.

of GFP+ cells within RFP+ macrophages or F4/80+ macrophages. For in vivo

transfer assays, 5 105 macrophages and cancer cells were cocultured in the
presence of control IgG1 or anti-CD47 B6H12 mAb (10 g/mL) and were incubated for 2 h. Macrophages then were separated from the cultures during
antiMac-1 magnetic beads (Miltenyi Biotec).
T Cell Priming Assay. For in vitro T cell priming assays, 104 macrophages were
cocultured overnight with equal numbers of DLD1-cOVA-GFP cancer cells in
serum-free RPMI medium. The next day, equal volume of RPMI+ 20% (vol/
vol) FCS was added to the cultures. Peripheral lymph nodes were harvested
from OT-I or OT-II TCR transgenic mice and labeled with 0.5 mM CFSE
(Molecular Probes). T cells were isolated using biotinylated anti-CD8 or antiCD4 antibodies, followed by enrichment with anti-biotin magnetic beads
(Miltenyi Biotec). Then 5 104 T cells were added to the cultures and analyzed on day 3 (for OT-I T cells) or day 4 (for OT-II T cells). For in vivo T cell
priming assays, 2 106 CFSE-labeled OT-I T cells (CD45.2) were adoptively
transferred i.v. into recipient mice (CD45.1). Macrophages were isolated
from coculture with cancer cells as previously described and were injected
into the footpads of mice. Popliteal lymph nodes were analyzed on day 4 for
CFSE dilution within CD45.2+ cells.
Antibodies and Flow Cytometry Analysis. Mouse anti-human anti-CD47 mAb
B6H12 (IgG1) was obtained from Bio-XCell. Mouse anti-human anti-CD47
mAb 2D3 (IgG1) and mouse IgG1 mAb were obtained from eBioscience. For
verication of binding of anti-CD47 B6H12 and 2D3 to DLD1-cOVA-GFP
cancer cells, the cells were labeled with a saturating concentration of antiCD47 antibody, followed by phycoerythrin-conjugated donkey-anti-mouse
IgG (H&L) (eBioscience). Data were acquired using a BD LSR Fortessa Analyzer and analyzed using FlowJo software.

a 1:1 ratio. Draining lymph nodes were analyzed 16 h later. The percentage
of cytotoxicity was calculated as (1 percentage of CFSE-high/percentage of
CFSE-low) normalized to the ratio in control mice.
Tumor Challenge. CD8-enriched OT-I T cells (1 106) were adoptively transferred i.v. into recipient C57BL/Ka mice. Macrophages from syngeneic C57BL/
Ka mice were cocultured with DLD1-cOVA-GFP cancer cells as previously
described and then were isolated by magnetic enrichment and injected into
the footpads of mice. The tumor cell line E.G7 (EL.4 cells expressing the
chicken OVA cDNA) was used for tumor challenge of mice (Amercan Type
Culture Collection). E.G7 cells (1 105) were injected s.c. into the right
hindlimb of the mice in a 1:1 ratio with regular Matrigel. Tumor size was
measured every day using ne calipers, and tumor volume was calculated
based on length width height /6.
Cytokine Assay. Macrophages were cocultured overnight with equal numbers
of DLD1-cOVA-GFP cancer cells in serum-free RPMI medium. The next day,
supernatants were harvested and submitted to the Stanford Human Immune
Monitoring Core for cytokine analysis by mouse 26-plex Luminex assay
(Affymetrix).

In Vivo Cell-Killing Assay. In brief, splenocytes from C57BL/Ka (CD45.1) mice

were labeled with 10 M CFSE (CFSE-high) or 1 M CFSE (CFSE-low). CFSEhigh splenocytes then were pulsed in a six-well plate with 1 M SIINFEKL
peptide for 1 h. Cells then were mixed in a 1:1 ratio with nonpeptide-pulsed
CFSE-low cells before i.v. transfer. To account for variation in the CFSE-high/-low
ratio in the absence of peptide-specic lysis, control mice received CFSEhigh and -low splenocytes not pulsed with SIINFEKL peptide and mixed in

ACKNOWLEDGMENTS. We thank Theresa Storm and Libuse Jerabek for excellent laboratory management, Tejaswitha Naik for antibody conjugation,
Aaron McCarty for mouse breeding and management, Patty Lovelace and
Jennifer Ho for technical assistance with ow cytometry, the Stanford Human Immune Monitoring Core and Yael Rosenberg-Hasson for assistance
with the Luminex assay, and Suparna Dutt for intellectual discussions. Funding support for this work was provided by the Student Training and Research in Tumor Immunology (STaRT) Program of the Cancer Research
Institute (D.T.), the Virginia and D. K. Ludwig Fund for Cancer Research
(I.L.W., D.T., M.M.), the Joseph and Laurie Lacob Gynecologic/Ovarian Cancer Fund (J.V.P.), and by Grants R01 CA86017 (I.L.W.), P01 CA139490 (I.L.W.,
S.W., H.C.-T., K.W.), P30 CA124435 (I.L.W.), and F30 CA168059 (K.W.) from
the National Institutes of Health. D.T. is the recipient of a Howard Hughes
Medical Institute medical student fellowship. We also thank the Stanford
Program in Cancer Biology and Stanford Medical Scientist Training Program
for their support (D.T. and K.W.).

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Tseng et al.