Activin A Promotes Regulatory T-cell-Mediated Immunosuppression in Irradiated Breast Cancer.

Increased regulatory T cells (Treg) after radiotherapy have been reported, but the mechanisms of their induction remain incompletely understood. TGFß is known to foster Treg differentiation within tumors and is activated following radiotherapy. Thus, we hypothesized that TGFß blockade would result in decreased Tregs within the irradiated tumor microenvironment. We found increased Tregs in the tumors of mice treated with focal radiotherapy and TGFß blockade. This increase was mediated by upregulation of another TGFß family member, activin A. In vitro, activin A secretion was increased following irradiation of mouse and human breast cancer cells, and its expression was further enhanced upon TGFß blockade. In vivo, dual blockade of activin A and TGFß was required to decrease intratumoral Tregs in the context of radiotherapy. This resulted in an increase in CD8+ T-cell priming and was associated with a reduced tumor recurrence rate. Combination of immune checkpoint inhibitors with the dual blockade of activin A and TGFß led to the development of tumor-specific memory responses in irradiated breast cancer. Supporting the translational value of activin A targeting to reduce Treg-mediated immunosuppression, retrospective analysis of a public dataset of patients with breast cancer revealed a positive correlation between activin A gene expression and Treg abundance. Overall, these results shed light on an immune escape mechanism driven by activin A and suggest that dual targeting of activin A and TGFß may be required to optimally unleash radiation-induced antitumor immunity against breast cancer.

Exosomes Shuttle TREX1-Sensitive IFN-Stimulatory dsDNA from Irradiated Cancer Cells to DCs.

Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell-derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. In vivo, RT-TEX elicited tumor-specific CD8+ T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. Cancer Immunol Res; 6(8); 910-20. ©2018 AACR.

DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity.

Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12-18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-ß by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-ß production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8+ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy.

TGFß Is a Master Regulator of Radiation Therapy-Induced Antitumor Immunity.

T cells directed to endogenous tumor antigens are powerful mediators of tumor regression. Recent immunotherapy advances have identified effective interventions to unleash tumor-specific T-cell activity in patients who naturally develop them. Eliciting T-cell responses to a patient's individual tumor remains a major challenge. Radiation therapy can induce immune responses to model antigens expressed by tumors, but it remains unclear whether it can effectively prime T cells specific for endogenous antigens expressed by poorly immunogenic tumors. We hypothesized that TGFß activity is a major obstacle hindering the ability of radiation to generate an in situ tumor vaccine. Here, we show that antibody-mediated TGFß neutralization during radiation therapy effectively generates CD8(+) T-cell responses to multiple endogenous tumor antigens in poorly immunogenic mouse carcinomas. Generated T cells were effective at causing regression of irradiated tumors and nonirradiated lung metastases or synchronous tumors (abscopal effect). Gene signatures associated with IFNγ and immune-mediated rejection were detected in tumors treated with radiation therapy and TGFß blockade in combination but not as single agents. Upregulation of programmed death (PD) ligand-1 and -2 in neoplastic and myeloid cells and PD-1 on intratumoral T cells limited tumor rejection, resulting in rapid recurrence. Addition of anti-PD-1 antibodies extended survival achieved with radiation and TGFß blockade. Thus, TGFß is a fundamental regulator of radiation therapy's ability to generate an in situ tumor vaccine. The combination of local radiation therapy with TGFß neutralization offers a novel individualized strategy for vaccinating patients against their tumors.