Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response

Abstract

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 antibody-mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated 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-CD47-mediated 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.

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 anti-CD47 2D3 mAbs. The experiment was performed three times with similar results.

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 μM). 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.

Fig. 3.

After phagocytosis of cancer cells by anti-CD47, 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 μM). Analysis was performed on day 4, and the percentage of proliferating cells was determined. Macrophages were pulsed with OVA peptide 323–339 as a positive control. (B) RFP⁺ macrophages were stimulated with IFN-γ to up-regulate MHC II levels. Phagocytosis and priming of OT-II CD4⁺ cells were determined in the presence of anti-CD47 mAbs.

Fig. 4.

A reduction in Foxp3⁺ regulatory T cells occurs after anti-CD47 B6H12–mediated 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 quantified.

Fig. 5.

After anti-CD47–mediated phagocytosis of cancer cells, macrophages prime CD8⁺ T cells in vivo. (A) Experimental setup. BMDM, bone marrow-derived 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.

Fig. 6.

After anti-CD47–mediated phagocytosis of cancer cells, macrophages prime an antitumor CD8⁺ T-cell response in vivo. (A) After anti-CD47–mediated phagocytosis of cancer cells, macrophages prime effector cytotoxic T cells. CD8⁺ T cells were isolated from OT-I transgenic mice and were transferred i.v. to recipient mice. Macrophages (Mac) were cocultured with DLD1-cOVA-GFP cancer cells in vitro in the presence of IgG or anti-CD47 B6H12 (blocking) mAb. Macrophages were isolated by magnetic separation and were transferred subcutaneously (subQ) on the next day. After 4 d, target cells (CD45.1 splenocytes) were labeled as CFSE-high (10 μM) or -low (1 μM). CFSE-high cells were pulsed with 1 μM OVA class I-restricted peptide (SIINFEKL) to make them targets for OT-I cytotoxic T-cell function. CFSE-high (peptide-pulsed) and -low (unpulsed) cells were mixed in a 1:1 ratio and transferred i.v. Draining lymph nodes were analyzed 16 h later to determine the percentage of CFSE-high versus CFSE-low cells. The percentage of cell killing was 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.