DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity

Abstract

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-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.

Figure 1. Radiation-induced activation of type-I interferon pathway correlates with radiation's ability to induce abscopal responses in combination with anti-CTLA4.

(a,b) In mice with bilateral TSA tumours one tumour was irradiated (RT) and mice received anti-CTLA4 antibody as indicated (a). Growth of irradiated and abscopal tumours in mice treated with 0 Gy, 0 Gy+anti-CTLA4, 8GyX1, 8GyX1+anti-CTLA4, 30GyX1, 30GyX1+anti-CTLA4, 8GyX3 and 8GyX3+anti-CTLA4. Ratios indicate the number of mice free from the irradiated tumour. (Duplicate; asterisks indicate P values for the comparison of irradiated tumours in each group versus 0 Gy controls, *P<0.05; **P<0.005; hashs indicate P values for the comparison of tumours treated or not with anti-CTLA4 within each radiation level, ^##P<0.005; two-way ANOVA; n=7). (b). (c) Heat map of gene expression in TSA tumours 24 h after radiation in vivo (n=3). (d) qRT-PCR (n=4) and IFNβ secretion (n=3) 24 h after in vitro irradiation of TSA cells (Triplicate; *P<0.05; **P<0.005; ***P<0.0005: t-test). (e) Number of CD11c⁺CD8α⁺ DCs infiltrating TSA tumours 5 days after irradiation and CD70 mean fluorescence intensity (MFI) gated on CD11c⁺CD8α⁺ cells (n=5). (Duplicate; *P<0.05; **P<0.005; ***P<0.0005: t-test). All data are mean±s.e.m.

Figure 2. IFNAR1 expression by TSA cancer cells is required for optimal therapeutic response of mice treated with 8GyX3 and anti-CTLA4.

(a) Upregulation of Ifnar1 expression measured by qRT-PCR 24 h after radiation in vitro in tetracycline-treated TSA^shNS cells (black bars) is abrogated in TSA^shIfnar1 cells (white bars) (Duplicate; *P<0.05; **P<0.005; ***P<0.0005: t-test; n=3). (b) pTRIPZ lentiviral vector with tetracycline-inducible shRNA and TurboRFP fluorescent protein (tRFP) expression, and microscopic image of tetracycline-treated TSA^shNS cells (magnification= × 20). (c–f) Mice bearing irradiated TSA^shIfnar1 or TSA^shNS tumours and TSA^shNS tumours in the abscopal site were treated with tetracycline and 0Gy (TSA^shNS=black; TSA^shIfnar1=dashed line), 8GyX3 (TSA^shNS=green; TSA^shIfnar1=yellow), 8GyX3+anti-CTLA4 (TSA^shNS=blue; TSA^shIfnar1=red). (c) Growth of irradiated and abscopal tumours. Duplicate; *P<0.05; **P<0.005: comparison of irradiated tumour outgrowth; two-way ANOVA; n=7. (d) Survival. Numbers indicates fraction of tumour-free mice. Log-rank (Mantel–Cox) test for the survival experiment; n=7 (e) IFNγ production by tumour-draining lymph node (TDLN) cells in response to the CD8 T cell epitopes AH1A5 (full circles) or control peptide MCMV (open circles). Each symbol represents one animal. Horizontal lines indicate the mean of antigen-specific (solid lines) or control (dashed lines) IFNγ concentration. Duplicate; *P<0.05; **P<0.005; ***P<0.0005: t-test; n=3 (f) Individual tumour growth from (d) showing recurrence of all abscopal tumours in mice bearing irradiated TSA^shIfnar1 tumours (red) while 3/7 mice with irradiated TSA^shNS tumours (blue) remained tumour-free. All data are mean±s.e.m.

Figure 3. Trex1 is induced by high dose radiation and degrades IFN-inducing cytoplasmic DNA.

(a–c) Cytoplasmic dsDNA accumulation (a), Trex1 gene expression (b), IFNβ secretion and Mx1 and Ifnar1 gene expression (c) in TSA cells treated with various radiation doses (n=3). (d–g) TSA^{KI Trex1} cells were cultured without (grey bars) or with doxycycline (white bars) to induce Trex1 expression (Supplementary Fig. 3b) before measuring cytoplasmic dsDNA accumulation (d), Mx1 (e), Ifnar1 (f) and Ifnb1 (g) gene expression induced by radiation (n=3). (Duplicate; *P<0.05; **P<0.005; ***P<0.0005; ****P<0.0001: t-test). All data are mean±s.e.m.

Figure 4. Trex1 knockdown restores cytosolic dsDNA accumulation and induction of ISGs in TSA cells treated with 20 Gy.

(a–c) TSA^shNS and TSA^shTrex1 cells were treated with doxycycline to induce Trex1 knockdown and tRFP expression, then exposed to a radiation dose of 8 Gy or 20 Gy. (a) After 24 h cells were fixed, permeabilized and stained with an antibody specific for dsDNA, followed by detection with Alexa fluor 488-conjugated secondary antibody. Representative micrographs show DAPI-stained nuclei (blue), cytoplasmic dsDNA (green), cytoplasmic tRFP (red), and the three channels combined. Magnification: × 400; duplicate; n=3 per group. White bars, 10 μm. (b,c) After 24 h cells were harvested for measurement of cytoplasmic dsDNA (b) or Trex1, Ifnb1 and Mx1 expression by qRT-PCR (c). Duplicate; *P<0.05; **P<0.005; ***P<0.0005: t-test; n=3. All data are mean±s.e.m.

Figure 5. Cancer cell-intrinsic type-I IFN activation is mediated by cGAS-STING pathway and is required for radiation-induced abscopal responses.

(a) Doxycycline-inducible shRNA-mediated knockdown of cGAS (TSA^shcGAS) or STING (TSA^shSTING) in TSA cells completely abrogated IFNβ release and Ifnb1 and Mx1 gene expression induced by viral infection and 8GyX3 radiation in vitro. (b–f) Mice with TSA^shcGAS, or non-silencing shRNA (TSA^shNS) in the irradiated tumour and TSA^shNS in the abscopal tumour were treated with doxycycline, 8GyX3 and anti-CTLA4. IFNγ production by TDLN cells (b) and percentage of IFNγ⁺ CD8⁺ T cells in spleen (c) in response to CD8 epitope AH1A5 (full circles) or control peptide MCMV (open circles). Each symbol represents one animal. Horizontal lines indicate the mean of antigen-specific (solid lines) or control (dashed lines). (d) Representative fields (× 200) showing CD8⁺ cells (green), DAPI+ nuclei (blue), and RFP+ TSA cells (red), and mean number±s.d. of CD8⁺ cells per field in abscopal tumours harvested at day 22 from 8GyX3+anti-CTLA4-treated mice with irradiated TSA^shcGAS (red squares) or TSA^shNS (blue squares) tumours. White bars, 25 um. (e) Growth of irradiated and abscopal tumour in mice with TSA^shNS cells treated with 0Gy (black), 8GyX3 (green), 8GyX3+anti-CTLA4 (blue), and mice with TSA^shcGAS cells treated with 0Gy (dashed line), 8GyX3 (yellow), 8GyX3+anti-CTLA4 (red). (f) Survival of mice from 8GyX3+anti-CTLA4 that rejected the irradiated and abscopal tumour (n=4) and were rechallenged at day 100 with a tumorigenic inoculum of TSA cells, together with a group of naïve mice (n=5). (a-d) Duplicate; *P<0.05; **P<0.005; ***P<0.0005: t-test; n=3. (e) Duplicate; *P<0.05; **P<0.005: comparison of irradiated tumour outgrowth; two-way ANOVA; n=7; ^##P<0.005: comparison of abscopal tumour outgrowth; two-way ANOVA; n=7. All data are mean±s.e.m.

Figure 6. Trex1 regulates radiation-induced abscopal responses in combination with immune checkpoint inhibitors.

Half of the mice with doxycycline-inducible Trex1 in TSA cells (TSA^{KI Trex1}) in the irradiated tumour and parental TSA (TSA-WT) in the abscopal tumour were given doxycycline and all mice were then treated with 8GyX3 and anti-CTLA4 (a,b) or anti-PD-1 (c,d). Growth of irradiated (a) and abscopal (b) tumour in mice treated with 0 Gy (black), 0 Gy+doxycycline (dashed line), 3 × 8 Gy (green), 3 × 8 Gy+doxycycline (yellow); 3 × 8 Gy+anti-CTLA4 (blue) and 3 × 8 Gy+anti-CTLA4+doxycycline (red). Growth of irradiated (c) and abscopal (d) tumour in mice treated with 0 Gy (black), 0 Gy+doxycycline (dashed line), 3 × 8 Gy (green), 3 × 8 Gy+doxycycline (yellow); 3 × 8 Gy+anti-PD-1 (blue) and 3 × 8 Gy+anti-PD-1+doxycycline (red). Mice with TSA^shTrex1 (broken lines) or non-silencing shRNA (TSA^shNS) (solid lines) in the irradiated tumour and TSA^shNS in the abscopal tumour were treated with doxycycline to induce Trex1 knockdown (e,f). Growth of irradiated (e) and abscopal (f) tumour in mice treated with 0 Gy (black), 1 × 20 Gy (green), 1 × 20 Gy+anti-CTLA4 (blue), 2 × 20 Gy (yellow), 2 × 20 Gy+anti-CTLA4 (red). (Duplicate; *P<0.05; **P<0.005: comparison of irradiated tumour growth; two-way ANOVA; n=7; ^##P<0.005: comparison of abscopal tumour growth; two-way ANOVA; n=7). All data are mean±s.e.m.

Figure 7. Threshold for Trex1 induction by radiation in mouse cell lines.

Trex1 gene expressions and cytoplasmic dsDNA accumulations in TSA (a), MCA38 (b) and 4T1 (c) carcinoma cells measured 24 h after in vitro treatment. Duplicate; *P<0.05; **P<0.005; ***P<0.0005: t-test; n=3. All data are mean±s.e.m.

Figure 8. Threshold for Trex1 induction by radiation in human cell lines and induction of Trex1 and IFN-I pathway in a patient-derived lung adenocarcinoma xenograft.

(a,b) Trex1 expression and cytoplasmic dsDNA accumulations in MDA-MB-231 (a) and 4175TR (b) breast carcinoma cells 24 hrs after in vitro treatment. Duplicate; *P<0.05; **P<0.005; ***P<0.0005: t-test; n=3. All data are mean±s.e.m. (c) Trex1, Ifnb1 and Mx1 expression measured by qRT-PCR in a patient-derived tumour xenograft (PDTX) 24 h after mice treatment with tumour-directed radiation. Duplicate; *P<0.05; **P<0.005; ***P<0.0005: t-test; n=4. All data are mean±s.e.m.

Figure 9. Graphical summary.

Left, treatment with a radiation regimen that causes dsDNA accumulation in the cancer cells cytosol without inducing the DNAse Trex1 activates interferon type-I pathway via cGAS/STING. Downstream recruitment of DC and activation of CD8⁺ T cells is enabled and tumour rejection occurs in synergy with anti-CTLA4 or anti-PD-1 antibody. Right, in tumour treated with a dose of radiation above the threshold for Trex1 induction dsDNA is cleared from the cytosol precluding interferon-β release by the cancer cells. This leads to insufficient DC recruitment and activation and lack of CD8⁺ T cell activation resulting in absence of local and abscopal tumour regression in combination with immune checkpoint inhibitors.