CD47 in the tumor microenvironment limits cooperation between antitumor T-cell immunity and radiotherapy

Abstract

Although significant advances in radiotherapy have increased its effectiveness in many cancer settings, general strategies to widen the therapeutic window between normal tissue toxicity and malignant tumor destruction would still offer great value. CD47 blockade has been found to confer radioprotection to normal tissues while enhancing tumor radiosensitivity. Here, we report that CD47 blockade directly enhances tumor immunosurveillance by CD8(+) T cells. Combining CD47 blockade with irradiation did not affect fibrosarcoma growth in T cell-deficient mice, whereas adoptive transfer of tumor-specific CD8(+) T cells restored combinatorial efficacy. Furthermore, ablation of CD8(+) T cells abolished radiotherapeutic response in immunocompetent syngeneic hosts. CD47 blockade in either target cells or effector cells was sufficient to enhance antigen-dependent CD8(+) CTL-mediated tumor cell killing in vitro. In CD47-deficient syngeneic hosts, engrafted B16 melanomas were 50% more sensitive to irradiation, establishing that CD47 expression in the microenvironment was sufficient to limit tumor radiosensitivity. Mechanistic investigations revealed increased tumor infiltration by cytotoxic CD8(+) T cells in a CD47-deficient microenvironment, with an associated increase in T cell-dependent intratumoral expression of granzyme B. Correspondingly, an inverse correlation between CD8(+) T-cell infiltration and CD47 expression was observed in human melanomas. Our findings establish that blocking CD47 in the context of radiotherapy enhances antitumor immunity by directly stimulating CD8(+) cytotoxic T cells, with the potential to increase curative responses.

Fig. 1. CD47 blockade combined with irradiation increases the efficacy of adoptive T cell transfer

Athymic BALB/c nu/nu mice were injected in one hind limb with 15-12 RM fibrosarcoma cells expressing HIV gp160. Mice were injected IP with saline or 10 µM CD47 morpholino in saline or received these treatments in combination with IR at 10 Gy. (A), Tumor-bearing mice received adoptive transfer (I.V.) of RT-1 CD8⁺ T cells in the presence or absence of CD47 morpholino (CD47M) treatment (A and B) or received these treatments in combination with IR treatment (A and C) *P<0.05. At the end of the study (day 36) mice were sacrificed, and tumor volume (D) and wet weights (E) were measured. N = 6–8, * statistically significant versus vehicle control, P < 0.05, ** statistically significant CD47+CD8⁺+IR versus CD8⁺+IR.

Fig. 2. CD8⁺ T cells are necessary for blockade of CD47 to enhance radiation growth delay in an immunocompetent mouse syngeneic fibrosarcoma model

15-12RM cells were injected in immunocompetent BALB/c mice. Groups of mice were treated with saline or 10 µM CD47 morpholino (CD47M), and a subset from each treatment was exposed to IR. (A). At the end of the study (day 30) tumors were excised and wet weights were measured (B, N=6 *p<0.05). In a different set of experiments mice were treated in the same manner in combination with anti-CD8 to deplete CD8+ T cells (C). A concurrent control study was carried out (D, N=6). At the end of the study mice were sacrificed, and wet weight was measured (E). At the end of the study trunk blood was collected from each animal, and circulating CD8⁺ T cells were measured by flow cytometry to determine the effectiveness of depletion.

Fig. 3. Blockade of CD47 enhances CD8⁺ T cell recruitment into irradiated tumors

Sections from fibrosarcoma tumors grown in BALB/c athymic mice (A) or BALB/c immunocompetent mice (C) that received the indicated treatments were stained with an antibody to CD8⁺ cells. Sections were photographed under light microscopy (20×) and quantified using imageJ software (B and D). N=3,4 (4 sections/animal)*p<0.05 from saline #p<0.05 from IR.

Fig. 4. CD47 blockade enhances antigen-dependent T cell killing of 15-12RM fibrosarcoma when combined with irradiation

(A) 15-12RM target cells were seeded into 16-well plates. RT-1-derived effector T cells were co-cultured with target cells at a ratio of 1:5, and target cell growth and viability was dynamically monitored using the RT-CES system. Target cell viability monitored by surface impedance is presented as a normalized cell index (N=3 in triplicate, *p<0.05). Cells were plated into 96-well plates and effector and or targets cells were treated with 10 µM CD47 Morpholino (CD47 M; A, C and D). Mismatched morpholino (MM A and B) or mouse antibody to CD47 clone 301 (D), and LDH release was quantified after 24 h as a measure of cell cytotoxicity, N=3 *p<0.05.

Fig. 5. Lack of CD47 in the tumor microenvironment increases the IR-induced growth delay in a syngeneic mouse melanoma model

(A) Thirty-eight paired serial sections from 13 human melanoma tumors were stained with antibodies to human CD47 and CD8. Numbers of CD47+ and CD8+ cells were inversely correlated (p=0.021 by Pearson correlation). (B) Representative stained tumor sections (20×) from melanoma patients with low (upper panels) and high CD47 expression (lower panels). (C) B16F10 melanoma cells were injected into WT and CD47^−/− mice hind limbs and were irradiated at 10 Gy on day 5. Tumor dimensions were measured ever other day using calipers, and tumor volume was calculated as W² × L/2, where W = shortest diameter and L = longest diameter. (D) Tumors were excised, and weights were recorded at the end of the study. N=5, *p<0.05. (E) Immunohistochemical analysis to detect CD8⁺ infiltrating cells (Brown stain) in melanoma tumor sections counterstained Hematoxilin, sections were visualized under light microscopy (20×) and quantified using ImageJ software N=3 (4 sections/animal) *p<0.05.

Fig. 6. Deficiency of CD47 increases granzyme B and regulates Foxp3 gene expression in tumors

Expression of granzyme B and Foxp3 was determined by RT-PCR in 15-12RM tumors grown in athymic BALB/c mice (A,D), immunocompetent BALB/c mice (B,E), or B16 melanoma tumors grown in WT or CD47-null C57BL/6 mice (C,F) (*p<0.05.).

Fig. 7. Deficiency of CD47 increases granzyme B in CD8⁺ cells and in spleen tissue

Protein expression (A) and mRNA expression (B) was determined by Western Blot Hybridization and qRT-PCR respectively in CD8⁺ T cells used for cytotoxicity assays (*p<0.007). Tissue sections of spleens from WT and CD47-null mice bearing B16 melanoma tumors were stained with an antibody specific to granzyme B arrow denotes granular pattern of stain (C).