DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity Radiotherapy is being investigated for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. This study shows 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-β 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-β 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. Treatment with antibodies that target regulatory receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed death-1 (PD-1) in T cells to improve their activation, and effector function induces durable responses in a variable percentage of patients with metastatic disease across different malignancies. However, the majority of patients does not respond to the blockade of these immune checkpoints, often because their tumours are less immunogenic and do not elicit a sufficient immune reaction. Thus, to enhance responses it is necessary to identify treatments that synergize with immune checkpoints inhibitors (ICI) by stimulating anti-tumour T cell responses to poorly immunogenic tumours. Radiotherapy is under investigation in the clinic for its ability to induce anti-tumour T cells, and enhance responses to immune checkpoint inhibitors and other immunotherapies. A variety of radiation doses, fractionation and delivery schedules have been used to induce anti-tumour T cells in preclinical studies. However, in the absence of a mechanistic understanding of the relationship between the dose and fractionation of radiation and its immunogenicity, most clinical trials testing the ability of radiation to enhance responses to immunotherapy are guided by standard-of-care or empirical choices that may not be optimal. Here we report the results of our studies that identify the DNA exonuclease Trex1 as an upstream regulator of radiation-induced anti-tumour immunity, and show that Trex1 expression is dependent on the radiation dose. When radiation is delivered at high dose in a single fraction, with a threshold that varies between 12 and 18 Gy in different cancer cells, Trex1 is induced at levels sufficient to degrade the DNA that accumulates in the cytosol of irradiated cancer cells,DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity Radiotherapy is being investigated for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. This study shows 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-β 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-β 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. Treatment with antibodies that target regulatory receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed death-1 (PD-1) in T cells to improve their activation, and effector function induces durable responses in a variable percentage of patients with metastatic disease across different malignancies. However, the majority of patients does not respond to the blockade of these immune checkpoints, often because their tumours are less immunogenic and do not elicit a sufficient immune reaction. Thus, to enhance responses it is necessary to identify treatments that synergize with immune checkpoints inhibitors (ICI) by stimulating anti-tumour T cell responses to poorly immunogenic tumours. Radiotherapy is under investigation in the clinic for its ability to induce anti-tumour T cells, and enhance responses to immune checkpoint inhibitors and other immunotherapies. A variety of radiation doses, fractionation and delivery schedules have been used to induce anti-tumour T cells in preclinical studies. However, in the absence of a mechanistic understanding of the relationship between the dose and fractionation of radiation and its immunogenicity, most clinical trials testing the ability of radiation to enhance responses to immunotherapy are guided by standard-of-care or empirical choices that may not be optimal. Here we report the results of our studies that identify the DNA exonuclease Trex1 as an upstream regulator of radiation-induced anti-tumour immunity, and show that Trex1 expression is dependent on the radiation dose. When radiation is delivered at high dose in a single fraction, with a threshold that varies between 12 and 18 Gy in different cancer cells, Trex1 is induced at levels sufficient to degrade the DNA that accumulates in the cytosol of irradiated cancer cells,