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Anti-CD47 –mediated phagocytosis of SEE COMMENTARY by 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

aInstitute 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)

Mobilization of the T-cell response against cancer has the potential efficacy of anti-CD47 blocking mAbs against xenograft human to achieve long-lasting cures. However, it is not known how to growing in immunodeficient mice, including cancers such as harness -presenting cells optimally to achieve an effective leukemia (5, 13), lymphoma (14), and multiple myeloma (15), solid antitumor T-cell response. In this study, we show that anti-CD47 tumors, including breast, colon, prostate, and bladder cancers, and antibody–mediated phagocytosis of cancer by macrophages can ini- sarcomas (6, 16). Whether the adaptive also can tiate an antitumor T-cell immune response. Using the ovalbumin be recruited against the cancer after anti-CD47 mAb treatment has fi model antigen system, anti-CD47 antibody–mediated phagocytosis not been tested, because the immunode cient mice used to establish of cancer cells by macrophages resulted in increased of OT-I the xenograft models lack T, B, and NK cells. In this study, we tested + – T cells [cluster of differentiation 8-positive (CD8 )] but decreased the hypothesis that anti-CD47 antibody mediated phagocytosis + + priming of OT-II T cells (CD4 ). The CD4 T-cell response was charac- of cancer cells can facilitate an antitumor T-cell immune response. + terized by a reduction in forkhead box P3-positive (Foxp3 ) regulatory Results T cells. Macrophages following anti-CD47–mediated phagocytosis + primed CD8 T cells to exhibit cytotoxic function in vivo. This re- Macrophages Phagocytose Cancer Cells in the Presence of Anti-CD47 sponse protected animals from tumor challenge. We conclude that Blocking Antibody. To follow the immune response to a model tu- anti-CD47 antibody treatment not only enables mor antigen, the human colon line DLD1 was trans- fected with a lentiviral vector for expressing cytoplasmic ovalbumin phagocytosis of cancer but also can initiate an antitumor cytotoxic (cOVA) and GFP (DLD1-cOVA-GFP) (Fig. S1). DLD1-cOVA- T-cell immune response. GFP cancer cells express CD47 and can be recognized by both CD47 mAbs, clones B6H12 and 2D3 (Fig. S1). Anti-CD47 B6H12 ntigen presentation is the process by which innate immune (blocking) mAb blocks the interaction between CD47 and SIRP-α, Acells such as macrophages and dendritic cells (antigen-pre- whereas anti-CD47 2D3 (non-blocking) antibody binds CD47 senting cells, APC) acquire and present them to T cells to but does not block its interaction with SIRP-α. Macrophages initiate the adaptive immune response. How APCs shape the im- phagocytoseDLD1-cOVA-GFPcancercellsinthepresenceof mune response by both degrading antigens and preserving antigens anti-CD47 B6H12, but not anti-CD47 2D3 mAbs, demonstrat- for presentation to T cells has been a longstanding area of interest ing that phagocytosis is dependent on the blockade of CD47/ (1). Recently, the mechanism of antigen recognition by APCs has SIRPα interactions and not entirely due to antibody opsoniza- been shown to affect the preference of MHC I versus MHC II tion effects (Fig. 1 and Fig. S2). Anti-CD47 mediated phago- antigen-presentation pathways. For instance, mannose receptor- cytosis of DLD1-cOVA-GFP cancer cells by macrophages leads mediated endocytosis on dendritic cells has been associated with to cross-presentation of ovalbumin peptide onto MHC-I, as MHC I , whereas scavenger receptor-mediated

assessed by staining for the SIINFEKL-H2kb complex on the MEDICAL SCIENCES endocytosis has been associated with MHC II presentation (2). cell surface (Fig. S3). Costimulatory molecule CD86 is up- Moreover, the functional outcomes of antigen presentation have regulated, but not coinhibitory molecule B7-H1 (Fig. S4). Anti- been shown to be context dependent. For instance, targeting anti- CD47 B6H12–mediated phagocytosis of cancer cells leads to gens to DEC-205 using monoclonal induced tolerance macrophage release of proinflammatory . For example, under noninflammatory conditions but mediated immunogenicity IL-12p40, TNF-α, regulated upon activation normal ex- under activating conditions by cluster of differentiation 40 pressed and secreted (RANTES), and monocyte chemotactic (CD40L) (3). Harnessing APCs to enhance the antitumor T-cell -3 (MCP-3) levels increase after anti-CD47 response offers an exciting strategy for cancer . The B6H12-mediated phagocytosis (Fig. S5). Next, the ability of ability of the T-cell immune response to be mobilized successfully the APCs, macrophages and dendritic cells, were tested for against cancer has been demonstrated through preclinical and phagocytic activity in response to anti-CD47 mAbs. Com- clinical studies of anti-CTLA4 antibody for T-cell activation (4). pared to dendritic cells, macrophages effectively phagocytose Phagocytosis by macrophages relies on the cell’s recognition of DLD1-cOVA-GFP cancer cells in the presence of anti-CD47 prophagocytic (“eat me”) and antiphagocytic (“don’teatme”) sig- nals on target cells. Anti-CD47 blocking monoclonal antibodies (mAbs) induce macrophage phagocytosis of cancer cells by Author contributions: D.T., J.-P.V., S.B.W., and I.L.W. designed research; D.T., J.-P.V., S.B.W., inhibiting an important antiphagocytic signal, allowing propha- H.C.-T., J.W.F., and N.B.F. performed research; D.T., J.-P.V., and K.W. contributed new gocytic signals to dominate (5, 6). CD47 is highly expressed on 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. cancer cells as compared with normal cells (5, 6) and interacts analyzed data; and D.T., J.-P.V., and I.L.W. wrote the paper. with the ligand signal regulatory protein α (SIRP-α) on macro- Conflict of interest statement: I.L.W. owns Amgen Inc. stock and is a Director of Stem phages (7). This interaction results in phosphorylation of immu- Cells, Inc. noreceptor tyrosine-based inhibition (ITIM) motifs on SIRP-α’s Freely available online through the PNAS open access option. cytoplasmic tail and the recruitment of Src homology phosphatase- See Commentary on page 10886. 1 (SHP-1) and SHP-2 phosphatases, which is thought to block 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. phagocytosis by preventing myosin-IIA accumulation at the phago- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cytic synapse (8–12). We have demonstrated the therapeutic 1073/pnas.1305569110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1305569110 PNAS | July 2, 2013 | vol. 110 | no. 27 | 11103–11108 Downloaded by guest on September 28, 2021 a carboxyfluorescein succinimidyl ester (CFSE)-dilution assay A -6 -6 IgG B6H12 2D3 p<5*10 p<5*10 was used to measure the proliferative response of OVA-specific 5 5 5 25 + + 10 10 10 fl 4 4 4 CD8 T cells (OT-I). Red uorescent protein-positive (RFP ) 10 10 10 20 103 103 103 15 macrophages were cocultured with DLD1-cOVA-GFP cancer 102 1.6 102 21.7 102 1.9 10 0 0 0 cells in the presence of IgG, anti-CD47 B6H12 (blocking), or 5 0103 104 105 0103 104 105 0103 104 105 anti-CD47 2D3 (non-blocking) mAbs. Lymph nodes were har- GFP (cancer) 0 + Phagocytosis (%) IgG B6H12 2D3 vested from OT-I (CD8 ) transgenic mice and were labeled with + B CFSE (0.5 μM) and CD8 cells enriched by magnetic separation. IgG B6H12 2D3 20 5 5 5 -5 -5 10 10 10 p<5*10 p<5*10 On day 3, the percentage of proliferating OT-I T cells was 104 104 104 quantified based on the percentage of cells that had diluted the 103 103 103 15

102 2.2 102 16.4 102 2.0 CFSE dye (CFSE-low). The percentage of proliferating OT-I

0 0 0 0103 104 105 0103 104 105 0103 104 105 10 T cells increased following anti-CD47 B6H12-mediated phagocy-

105 105 105 tosis of cancer by macrophages (Fig. 2A). To verify that the

104 104 104 5 p<0.05 p<0.05 proliferative response of OT-I T cells was an antigen-specific re- 103 103 103 Phagocytosis (%)

102 1.3 102 2.3 102 1.3 sponse, macrophages were allowed to phagocytose DLD1-cOVA-

RFP (DC)RFP (Mac) RFP (Mac) RFP 0 0 0 0 IgG B6H12 2D3 IgG B6H12 2D3 0103 104 105 0103 104 105 0103 104 105 GFP versus DLD1-GFP cancer cells (the latter not expressing GFP (cancer) Macrophages Dendritic cells OVA) in the presence of anti-CD47 B6H12 mAb before the ad- dition of CFSE-labeled OT-I T cells. Increased OT-I T-cell pro- Fig. 1. Macrophages effectively phagocytose cancer cells in the presence of + liferation was observed only after anti-CD47–mediated phagocytosis anti-CD47 B6H12 antibody. (A)RFP macrophages (Mac) were cocultured with of DLD1-cOVA-GFP cancer cells but not DLD1-GFP cancer cells, DLD1-cOVA-GFP cancer cells in the presence of IgG or anti-CD47 B6H12 fi B (blocking) or 2D3 (nonblocking) mAbs. Percentage of phagocytosis was indicating an antigen-speci c effect (Fig. 2 ). + + determined by the percentage of GFP cells within RFP macrophage cell + + gate. (B)RFP macrophages versus dendritic cells (DCs) were cocultured with Macrophages Do Not Prime OT-II (CD4 ) T cells After Phagocytosis of Cancer Cells by Anti-CD47 B6H12 Blocking Antibody. To assess priming DLD1-cOVA-GFP cancer cells in the presence of IgG, anti-CD47 B6H12, or anti- + CD47 2D3 mAbs. The experiment was performed three times with similar results. of CD4 T cells after anti-CD47–mediated phagocytosis by mac- rophages, a CFSE-dilution assay was used to measure the prolifera- + tive response of OVA-specificCD4 T cells (OT-II). Macrophages B6H12 mAb (Fig. 1). Consistent with this result, SIRP-α, the li- werecoculturedwithDLD1-cOVA-GFP cancer cells in the pres- gand for CD47, is expressed at high levels on macrophages but at ence of IgG, anti-CD47 B6H12 (blocking), or anti-CD47 2D3 (non- + lower levels on dendritic cells (Fig. S6). blocking) mAbs, and CFSE-labeled OT-II (CD4 ) T cells were added to cultures. Interestingly, the percentage of proliferating + Macrophages Prime OT-I (CD8 ) T Cells After Phagocytosis of Cancer OT-II T cells was reduced following anti-CD47-mediated phago- + Cells by Anti-CD47 Blocking Antibody. To assess priming of CD8 T cytosis compared with baseline levels (Fig. 3A). Because of the possi- + cells after anti-CD47–mediated phagocytosis by macrophages, bility that cell-surface MHC II (I-Ab ) on macrophages might be

A p<0.005 100 70 80 p<0.01 p<0.05 60 60

40

IgG 20.2 50 20

0 0102 103 104 105 40

120 30 p<0.005 90 20 p<0.005 60 49.5 30 10 B6H12

0 0102 103 104 105 Proliferating cells (%) 0 200 Mac ++++++- -- 150 DLD1-cOVA +++- - -+-- 100 19.1 2D3 50 Antibody IgG B6H12 2D3 - --B6H12 B6H12 B6H12

0 T cell 2 3 4 5 +++++++++ 010 10 10 10 + CFSE Peptide ----++--- Fig. 2. Macrophages prime CD8 T cells to pro- liferate after phagocytosis of cancer cells by anti-CD47 + B6H12 mAb. (A)RFP macrophages were cocultured p<0.01 B with DLD1-cOVA-GFP colon cancer cells in the pres- -5 -5 p<0.01 p<0.01 20 p<5*10 p<5*10 60 ence of IgG, anti-CD47 B6H12 (blocking), or anti-CD47 p<5*10-4 + -6 -6 2D3 (nonblocking) mAbs. The next day, CD8 T cells p<5*10 p<5*10 50 from OT-I transgenic mice were magnetically en- 15 riched and labeled with CFSE (0.5 μM). Analysis was 40 p<5*10-4 performed on day 3, and the percentage of pro- 10 30 p<5*10-6 liferating cells was determined. Macrophages were pulsed with OT-I peptide (OVA257-264, SIINFEKL) as 20 a positive control. The experiment was performed 5 + 10 three times with similar results. (B)RFP macrophages Phagocytosis (%) were cocultured with DLD1-cOVA-GFP cancer cells or 0 Proliferating cells (%) 0 IgG B6H12 2D3 DLD1-GFP cancer cells not expressing cOVA. (Left) Mac +++- Phagocytosis was determined by the percentage of + + GFP cells within the RFP macrophage cell gate. DLD1-cOVA ++++ + DLD1-cOVA-GFP Antibody IgG B6H12 2D3 B6H12 (Right) CFSE-labeled CD8 T cells from OT-I mice were added to cultures, and the percentage of proliferating DLD1-GFP T cell ++++cells was determined.

11104 | www.pnas.org/cgi/doi/10.1073/pnas.1305569110 Tseng et al. Downloaded by guest on September 28, 2021 A SEE COMMENTARY 20 -4 45 p<5*10 15 40 10 35 IgG 5 22.1 30 -5 -6 0 p<5*10 p<5*10 25 80 20 60 15 40 10

20 B6H12 5.6 5 Proliferating cells (%) 0 0102 103 104 105 0 30 Mac ++++++- -- 20 DLD1-cOVA +++- - -+-- 2D3 10 Antibody IgG B6H12 2D3 B6H12 B6H12 B6H12 13 - -- T cell +++++++++ 0 0102 103 104 105 Peptide CFSE ----++--- B p<5*10-5 p<5*10-5 -5 15 p<5*10-6 p<5*10

- IFNg Unstained 10 + IFNg I-Ab stained

5 Phagocytosis (%)

b 0 I-A (MHC II) IgG B6H12 2D3

p<5*10-4 60 p<0.05 p<0.005 p<0.05 - IFNg 50 Fig. 3. After phagocytosis of cancer cells by anti- + IFNg + CD47, macrophages do not prime CD4 T cells to 40 + p<5*10-6 p<0.005 proliferate. (A)RFP macrophages were cocultured 30 with DLD1-cOVA-GFP cancer cells in the presence of -4 p<5*10 p<0.01 IgG, anti-CD47 B6H12 (blocking), or anti-CD47 2D3 20 + (nonblocking) mAbs. The next day, CD4 T cells 10 were isolated from OT-II transgenic mice and were μ

Proliferating cells (%) labeled with CFSE (0.5 M). Analysis was performed 0 on day 4, and the percentage of proliferating cells Mac ++++++ - -- was determined. Macrophages were pulsed with DLD1-cOVA +++ - - - + -- OVA peptide 323–339 as a positive control. (B)RFP+ Antibody IgG B6H12 2D3 + --B6H12 B6H12 B6H12 macrophages were stimulated with IFN-γ to up- MEDICAL SCIENCES regulate MHC II levels. Phagocytosis and priming of T cell +++++++++ + OT-II CD4 cells were determined in the presence of Peptide ----++--- anti-CD47 mAbs.

+ limiting OT-II (CD4 ) T cell proliferation after anti-CD47-mediated double-transgenic mice (Fig. S8). These mice express 25% + + + phagocytosis, we measured the percentage of macrophages ex- Foxp3-GFP cells within the CD4 CD25 population and ex- pressing MHC II on the cell surface. Interestingly, the percentage hibit T cell receptor V alpha 2 (Vα2) restriction (Fig. S8A). + + + of I-Ab macrophages increased after anti-CD47 B6H12–me- In addition, CD4 Foxp3-GFP T cells are responsive to OVA + diated phagocytosis of cancer cells, despite the decrease in CD4 peptide 323–339 and can be induced to differentiate in the + T-cell priming (Fig. S7). To determine whether IFN-γ stimula- context of TGF-β and all-trans-retinoic acid (Fig. S8B). RFP + tion of macrophages could overcome the decrease in CD4 Tcell macrophages were cocultured with DLD1-cOVA-GFP cancer proliferation, IFN-γ was used to up-regulate surface MHC II cells in the presence of IgG, anti-CD47 B6H12 (blocking), or + levels on macrophages for evaluation in phagocytosis and T cell anti-CD47 2D3 (non-blocking) mAbs. The next day, CD4 T cells + proliferation assays. IFN-γ–stimulated macrophages efficiently from OT-II/Foxp3-GFP double-transgenic mice were magneti- phagocytosed cancer in the presence of anti-CD47 B6H12 mAb, cally enriched and were added to the cultures. After 4 d, the per- + but the OT-II CD4 T-cell response was also diminished compared centage of regulatory T-cells was quantified by the percentage of + + + with baseline (Fig. 3B). CD4 Foxp3-GFP cells (Fig. 4). A reduction in Foxp3 regula- tory T cells was observed after anti-CD47-mediated phagocytosis Reduction in Forkhead Box P3-Positive Regulatory T Cells After Anti- of cancer cells. CD47–Mediated Phagocytosis of Cancer by Macrophages. To assess the functional effects of anti-CD47 B6H12–mediated phagocytosis After Anti-CD47–Mediated Phagocytosis of Cancer Cells, Macrophages + on CD4 regulatory T cells, we crossed OT-II transgenic mice Prime OT-I (CD8+) T Cells to Proliferate in Vivo. To evaluate the + with forkhead box 3P (Foxp3)-GFP reporter mice to generate effects of anti-CD47 B6H12–mediated phagocytosis on CD8

Tseng et al. PNAS | July 2, 2013 | vol. 110 | no. 27 | 11105 Downloaded by guest on September 28, 2021 IgG B6H12 2D3 p<0.05 p<0.05 an “invisibility cloak” for both innate and adaptive . 5 105 6.1 105 0.8 105 3.7 4 Treatment with anti-CD47 blocking mAbs led to adaptive T-cell 104 104 104 3 3 3 3 immune responses, thereby providing an additional mechanism of 10 10 10 + 2 fi 102 102 102 action for anti-CD47 antibodies. OVA-speci c OT-I (CD8 ) and 0 0 0 1 + Foxp3-GFP 0102 103 104 105 0102 103 104 105 0102 103 104 105 0 OT-II (CD4 ) T-cell clones were used to follow the outcomes of CD4 (%) Foxp3-GFP IgG B6H12 2D3 antigen presentation by macrophages after anti-CD47–mediated + Fig. 4. A reduction in Foxp3 regulatory T cells occurs after anti-CD47 phagocytosis of cancer cells engineered to express cytoplasmic + OVA. Using in vitro and in vivo assays, we show that antigens are B6H12–mediated phagocytosis of cancer cells by macrophages. RFP mac- + presented to CD8 T cells effectively, but the proliferative re- rophages were cocultured with DLD1-cOVA-GFP cancer cells in the presence + of IgG or anti-CD47 B6H12 (blocking) or 2D3 (nonblocking) mAbs. The next sponse of OT-II CD4 T cells to loaded macrophages was di- + + day, CD4 T cells from OT-II/Foxp3-GFP transgenic mice were magnetically minished compared with baseline levels. The baseline level of + + + enriched and were added to cultures. On day 4, the percentage of CD4 OT-I CD8 proliferation and OT-II CD4 proliferation was 20% + Foxp3-GFP cells was quantified. (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 T-cell priming in vivo, OT-I (CD8 ) T cells (CD45.2) were CFSE- pathways. Together, our results suggest that anti-CD47–mediated labeled and adoptively transferred to CD45.1 recipient mice (Fig. phagocytosis of cancer cells results in the presentation of negative + + + 5A). The next day, RFP macrophages were cocultured with signals to CD4 T cells and positive signals to CD8 T cells. In + DLD1-cOVA-GFP cancer cells in the presence of IgG or anti- addition, the CD4 T-cell response was characterized by reduced CD47 B6H12 mAb. Macrophages were isolated by magnetic en- regulatory T cells. This reduction might be attributed either to richment, and phagocytosis was verified by FACS analysis before decreased proliferation of regulatory T cells in response to pep- subcutaneous (subQ) transfer into the footpad. After 4 d, the tide or to less efficient regulatory T-cell differentiation. The in popliteal lymph node was analyzed for the percentage of pro- + vivo priming of an antitumor T-cell response by macrophages liferating cells (CFSE-low) within the CD45.2 gate. There was after anti-CD47–mediated phagocytosis of cancer protects mice an increase in proliferating OT-I T cells in mice receiving mac- from tumor challenge. Anti-CD47 mAbs may represent a thera- rophages that phagocytose cancer cells via anti-CD47 B6H12 B peutic strategy for overcoming the regulatory T-cell contribution mAb (Fig. 5 ). to immune evasion by cancer and initiating an effective antitumor + cytotoxic T-cell response. Macrophages Prime an Antitumor CD8 T-Cell Response in Vivo After In this system macrophages are the primary APCs that phago- Anti-CD47–Mediated Phagocytosis of Cancer Cells. We next evalu- + cytose cancer in response to anti-CD47 antibody and present an- + ated the functional effects of OT-I (CD8 ) T-cell priming after tigen to CD8 T cells. Perhaps this observation is due to higher anti-CD47–mediated phagocytosis of cancer cells by macrophages. + levels of SIRP-α expressed on macrophages than on dendritic To assess the efficacy of CD8 T-cell killing of OVA peptide- + cells or because macrophages phagocytose whole cells more ef- displaying targets, CD8 T cells were isolated from OT-I transgenic A + fectively. Because subsets of dendritic cells have been reported to mice and were i.v. transferred to recipient mice (Fig. 6 ). RFP vary in their levels of SIRP-α expression (19), it is possible that macrophages were cocultured with DLD1-cOVA-GFP cancer cells other subsets may more effectively phagocytose can- in vitro in the presence of IgG or anti-CD47 B6H12 mAb. After 2-h cer in response to anti-CD47 antibody in vivo. While dendritic cells incubation, macrophages were isolated and injected into the foot- are well known for their function in antigen presentation, in this pad. After 4 d, mice were challenged with target cells (CD45.1 study we show that macrophages also have the capacity to stimulate splenocytes) to assess cytotoxic activity. CFSE-high splenocytes + aCD8 T-cell immune response in the context of blocking the were pulsed with 1 μM OVA class I-restricted peptide (OVA257- CD47-SIRPalpha axis. Prior to this work, the ability to present 264, SIINFEKL) to make them targets for OT-I cytotoxic T cells + – exogenous antigens to CD8 T cells has been largely attributed to and then were mixed in a 1:1 ratio with non peptide-pulsed + fi CFSE-low cells before i.v. transfer. Analysis of draining lymph the CD8 subtype of DCs. Understanding the roles of tissue-speci c nodes 16 h later showed increased cell killing of peptide-pulsed macrophages and dendritic cells in response to anti-CD47 mAbs CFSE-high 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 + A immune response was evaluated. CD8 TcellsfromOT-Imicewere OT I C57BL/6 Analyze transferred into recipient animals (Fig. 6B). Macrophages were (CD45.2) (CD45.1) Popiteal LN cocultured with DLD1-cOVA-GFP cancer cells in vitro in the Purify CD8 T cells Day 1 CFSE label presence of IgG or anti-CD47 B6H12 mAb; then the macro- Day 0 Footpad Day 5 phages were transferred into the footpad on days 1 and 10. Ani- Injection mals were challenged with EG.7 (OVA-expressing EL4) cancer BMDM Co-culture with cancer Mac-1 enrichment + (IgG vs anti-CD47) for macrophages cells on day 14, and tumor growth was monitored over time. CD8 B T cells primed by macrophages following anti-CD47-mediated 30 B 105 p<0.05 phagocytosis protected mice from tumor challenge (Fig. 6 ). 4 10 0.5 20 100

103 10 17.3 Discussion 102 75 0 0 0102 103 104 105 0102 103 104 105 Recent studies from the I.L.W. laboratory have shown that ma- 100 105 50 “ ’ ” 80

lignant cancers universally up-regulate the don t eat me signal CD45.2 4 10 1.7 60 CD47, presumably in their progression to allow escape from 103 40 80.2 25 2 10 20 endogenous “eat me” signals that were induced as part of B6H12 IgG 0 0 Proliferating cells (%) programmed cell death and programmed cell removal (5, 6, 13, 0102 103 104 105 0102 103 104 105 0 14, 16–18). These results indicate a role for human CD47-block- SSC-A CFSE IgG B6H12 ing antibodies in cancer therapy via induced phagocytosis. Our Fig. 5. After anti-CD47–mediated phagocytosis of cancer cells, macrophages + previous experiments involved xenotransplantation of primary prime CD8 T cells in vivo. (A) Experimental setup. BMDM, - + human cancers into immunodeficient mice. We now have exam- derived macrophages. (B) Adoptively transferred CFSE OT-I T cells were ana- + ined the possible role of anti-CD47–enabled phagocytosis in an- lyzed in the draining lymph node by gating on CD45.2 cells. The percentage of tigen presentation of tumor peptides to T cells of the adaptive proliferating cells was determined by gating on the CFSE-low population. . Here we have demonstrated that CD47 serves as n = 5 mice per group.

11106 | www.pnas.org/cgi/doi/10.1073/pnas.1305569110 Tseng et al. Downloaded by guest on September 28, 2021 Fig. 6. After anti-CD47–mediated phagocytosis of p<0.005 + IgG B6H12

A SEE COMMENTARY cancer cells, macrophages prime an antitumor CD8 50 60 55.6 44.2 87.3 12.6 T-cell response in vivo. (A) After anti-CD47–mediated 60 40

40 phagocytosis of cancer cells, macrophages prime 40 30 effector cytotoxic T cells. CD8+ T cells were isolated 20 20 20 from OT-I transgenic mice and were transferred i.v. 10 0 0 to recipient mice. Macrophages (Mac) were cocul- 0102 103 104 105

Cell killing (%) 0 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 B 3 350 IgG (subQ) on the next day. After 4 d, target cells 300 anti-CD47 * (CD45.1 splenocytes) were labeled as CFSE-high OT I Mac Mac EG.7 tumor 250 (10 μM) or -low (1 μM). CFSE-high cells were pulsed T cells iv subQ subQ challenge 200 * with 1 μM OVA class I-restricted peptide (SIINFEKL) * 150 to make them targets for OT-I cytotoxic T-cell func- Time (days) * tion. CFSE-high (peptide-pulsed) and -low (unpulsed) 100 * cells were mixed in a 1:1 ratio and transferred i.v. 0 1 10 14 50 * ** Draining lymph nodes were analyzed 16 h later 0 to determine the percentage of CFSE-high versus volume (mm Tumor 678 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-CD47–mediated 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.

warrants further investigation both in mouse and human systems target cells. Second, in designing clinical trial protocols for testing (20–22). anti-CD47 therapy in patients, immune monitoring of T cells may Antibody-mediated uptake of antigens via Fcγ receptor-medi- be important for understanding clinical response to treatment and + + ated endocytosis by dendritic cells can prime CD4 and CD8 clinical outcomes. Third, anti-CD47 mAbs might be used clinically T-cell responses in some circumstances (23, 24). In contrast, anti- in combination with adoptive T-cell therapy or T-cell–activating CD47–mediated phagocytosis by macrophages predominantly antibodies to enhance the adaptive immune response against tu- + + primes CD8 T cells. This predominant CD8 T cell response is mor antigens and minimize toxicity. We conclude that anti-CD47– not explained entirely by opsonization of cancer cells, because mediated phagocytosis of cancer not only functions in directly both the anti-CD47 clones B6H12 and 2D3 bind DLD1-cOVA- clearing cancer cells but also can initiate an antitumor T-cell re- GFP cancer cells, but only B6H12 is capable of inducing phago- sponse to eliminate cancers. Patients receiving anti-CD47 therapy cytosis through its ability to block the interaction between CD47 may benefit from both the innate and adaptive immune responses on cancer cells and SIRP-α on macrophages. The preferential against cancer. + activation of CD8 T cells may rely on routing phagocytosed cancer antigens toward the MHC I pathway or might be explained Materials and Methods by SIRP-α’s role as a negative regulator of macrophage function. Mice. Mice, including C57BL/Ka (CD45.2), C57BL/Ka (CD45.1), and C57BL/Ka Engagement of SIRP-α by CD47 leads to phosphorylation of Rosa26-mRFP1 mice, were bred and maintained at the Stanford University SIRP-α’s cytoplasmic ITIM motifs by SHP-1 and SHP-2. This Research Animal Facility in accordance with the Administrative Panel on pathway is well described to inhibit macrophage phagocytosis, but Laboratory Animal Care. All the animals were housed in sterile micro- more recently, SIRP-α also has been shown to attenuate macro- insulators and were given water and rodent chow ad libitum. OT-I TCR transgenic mice, OT-II TCR transgenic mice, and Foxp3-GFP mice were pur- MEDICAL SCIENCES phage activation by LPS by sequestering SHP-2, which is needed for fi κ chased from the Jackson Laboratory. OT-I mice have transgenic TCRs speci c activation of the MAPK and NF- B pathways (25). In addition, for OVA257-264 in the context of H2-kb. OT-II mice have TCRs specificfor inhibition of SHP-1 in dendritic cells has been reported to enhance + + OVA323-339 in the context of IAb. Both OT-I and OT-II mice have transgenic CD8 and CD4 T-cell responses and reduce regulatory T cells, Vα2Vβ5 TCRs. a response that protected mice against tumor challenge (26). SIRP- α also may negatively regulate cross-presentation by a mechanism Molecular Biology. cOVA was cloned from pCI-neo-cOVA (plasmid 25097; not yet understood, perhaps involving recruitment of cross-pre- AddGene) and was shuttled into the lentiviral pCDH-EF1-MCS-T2A-copGFP sentation machinery to phagosomes. Alternatively, prophagocytic vector (System Biosciences) using EcoRI and BamHI restriction sites. Lentiviral signals such as calreticulin have been described to elicit antitumor production and concentration were accomplished using standard protocols. immunity (27–29). It may be that the unmasking of such propha- gocytic signals may route antigens preferentially toward the cross- Generation of Macrophages and Dendritic Cells. Whole bone marrow cells presentation pathway. were isolated from C57BL/Ka (CD45.2) or C57BL/Ka Rosa26-mRFP1 mice. Other groups examining the effect of SIRP-α on dendritic cells Macrophages were generated by incubating whole bone marrow in mac- interacting with CD47 on T cells have reported T-cell activation rophage colony-stimulating factor (10 ng/mL) for 7 d and harvesting the or inhibition, depending on the context (30–33). In contrast, our adherent fraction. Dendritic cells were generated in granulocyte-macro- experiments were designed to study the impact of antigen pre- phage 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 sentation after anti-CD47 mAb mediated phagocytosis of can- medium. Nonadherent cells were replatedonday6andharvestedonday7. cer cells by macrophages. In our system, anti-CD47 B6H12 mAb did not have a direct effect on T-cell activation or inhibition In Vitro Phagocytosis Assay. For the in vitro phagocytosis assay, 2 × 104 (Figs. 2 and 3). macrophages or dendritic cells per well were plated in a 96-well ultra-low- The involvement of both the innate and adaptive immune sys- adherent plate (Corning), along with 2 × 104 cancer cells (DLD1-cOVA-GFP) tems in the mechanism of action of anti-CD47 antibody has sev- in serum-free RPMI medium. eral clinical implications. First, our findings suggest a novel role of The indicated antibodies (10 μg/mL) were added and incubated for 4 h at anti-CD47 blocking mAbs as a vaccination strategy to enhance 37°. Macrophages were washed twice and analyzed using a BD LSR Fortessa + CD8 effector T cells recognizing antigens on phagocytosed Analyzer. The percentage of phagocytosis was calculated as the percentage

Tseng et al. PNAS | July 2, 2013 | vol. 110 | no. 27 | 11107 Downloaded by guest on September 28, 2021 + + + of GFP cells within RFP macrophages or F4/80 macrophages. For in vivo a 1:1 ratio. Draining lymph nodes were analyzed 16 h later. The percentage transfer assays, 5 × 105 macrophages and cancer cells were cocultured in the of was calculated as (1 − percentage of CFSE-high/percentage of μ presence of control IgG1 or anti-CD47 B6H12 mAb (10 g/mL) and were in- CFSE-low) normalized to the ratio in control mice. cubated for 2 h. Macrophages then were separated from the cultures during – anti Mac-1 magnetic beads (Miltenyi Biotec). Tumor Challenge. CD8-enriched OT-I T cells (1 × 106) were adoptively trans-

ferred i.v. into recipient C57BL/Ka mice. Macrophages from syngeneic C57BL/ T Cell Priming Assay. For in vitro T cell priming assays, 104 macrophages were K mice were cocultured with DLD1-cOVA-GFP cancer cells as previously cocultured overnight with equal numbers of DLD1-cOVA-GFP cancer cells in a + described and then were isolated by magnetic enrichment and injected into 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 the footpads of mice. The tumor cell line E.G7 (EL.4 cells expressing the from OT-I or OT-II TCR transgenic mice and labeled with 0.5 mM CFSE chicken OVA cDNA) was used for tumor challenge of mice (Amercan Type (Molecular Probes). T cells were isolated using biotinylated anti-CD8 or anti- Culture Collection). E.G7 cells (1 × 105) were injected s.c. into the right CD4 antibodies, followed by enrichment with anti-biotin magnetic beads hindlimb of the mice in a 1:1 ratio with regular Matrigel. Tumor size was (Miltenyi Biotec). Then 5 × 104 T cells were added to the cultures and ana- measured every day using fine calipers, and tumor volume was calculated lyzed on day 3 (for OT-I T cells) or day 4 (for OT-II T cells). For in vivo T cell based on length × width × height × π/6. 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 Cytokine Assay. Macrophages were cocultured overnight with equal numbers from coculture with cancer cells as previously described and were injected of DLD1-cOVA-GFP cancer cells in serum-free RPMI medium. The next day, into the footpads of mice. Popliteal lymph nodes were analyzed on day 4 for + supernatants were harvested and submitted to the Stanford Human Immune CFSE dilution within CD45.2 cells. Monitoring Core for cytokine analysis by mouse 26-plex Luminex assay Antibodies and Flow Cytometry Analysis. Mouse anti-human anti-CD47 mAb (Affymetrix). B6H12 (IgG1) was obtained from Bio-XCell. Mouse anti-human anti-CD47 mAb 2D3 (IgG1) and mouse IgG1 mAb were obtained from eBioscience. For ACKNOWLEDGMENTS. We thank Theresa Storm and Libuse Jerabek for ex- verification of binding of anti-CD47 B6H12 and 2D3 to DLD1-cOVA-GFP cellent laboratory management, Tejaswitha Naik for antibody conjugation, Aaron McCarty for mouse breeding and management, Patty Lovelace and cancer cells, the cells were labeled with a saturating concentration of anti- Jennifer Ho for technical assistance with flow cytometry, the Stanford Hu- CD47 antibody, followed by phycoerythrin-conjugated donkey-anti-mouse man Immune Monitoring Core and Yael Rosenberg-Hasson for assistance IgG (H&L) (eBioscience). Data were acquired using a BD LSR Fortessa Ana- with the Luminex assay, and Suparna Dutt for intellectual discussions. Fund- lyzer and analyzed using FlowJo software. ing support for this work was provided by the Student Training and Re- search in Tumor Immunology (STaRT) Program of the Cancer Research Institute (D.T.), the Virginia and D. K. Ludwig Fund for Cancer Research In Vivo Cell-Killing Assay. In brief, splenocytes from C57BL/Ka (CD45.1) mice were labeled with 10 μM CFSE (CFSE-high) or 1 μM CFSE (CFSE-low). CFSE- (I.L.W., D.T., M.M.), the Joseph and Laurie Lacob Gynecologic/Ovarian Can- cer Fund (J.V.P.), and by Grants R01 CA86017 (I.L.W.), P01 CA139490 (I.L.W., high splenocytes then were pulsed in a six-well plate with 1 μM SIINFEKL – S.W., H.C.-T., K.W.), P30 CA124435 (I.L.W.), and F30 CA168059 (K.W.) from peptide for 1 h. Cells then were mixed in a 1:1 ratio with non peptide-pulsed the National Institutes of Health. D.T. is the recipient of a Howard Hughes CFSE-low cells before i.v. transfer. To account for variation in the CFSE-high/-low Medical Institute medical student fellowship. We also thank the Stanford ratio in the absence of peptide-specific lysis, control mice received CFSE- Program in Cancer Biology and Stanford Medical Scientist Training Program high and -low splenocytes not pulsed with SIINFEKL peptide and mixed in for their support (D.T. and K.W.).

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