was repeated on days 22, 43, and 64. Patients were re-imaged between days 81-88 and evaluated for responses in the non-irradiated measurable metastatic sites. Study code: NCT02221739 Mice. All animal experiments were carried out in compliance with French and European laws and regulations. Mice were used between 7 and 14 weeks of age. WT specific pathogen-free (SPF) C57BL/6J and BALB/c mice were obtained from Harlan(France)and Janvier(France) respectively, and were kept in SPF conditions at Gustave Roussy. C57BL/6 GF mice were obtained from Institut Pasteur and maintained in sterile isolators. 1-10- C57BL/6 mice and WT C57BL/6 control animals were kindly provided by Anne O Garra(National Institute for Medical Research, UK). NOD2, Il10,and NOD2-1110 mice(BALB/c background) were obtained from Institut Pasteur Lille C57BL/6 TIr2 mice were provided by Ivo Gompers Boneca(Institut Pasteur, Paris, France), TIr4 mice were bred and maintained in the animal facility of Gustave Roussy, Villejuif, France. Cell culture and reagents oVA-expressing mouse fibrosarcoma MCA205 cells, murine colon carcinoma MC38 cells, RET melanoma model (a transgene-enforced expression of the Ret protooncogene under the control of the metallothionein-1 promoter driving spontaneou melanomagenesis, kindly provided by Viktor Umansky)(class I MHC H-2, syngeneic for C57BL/6 mice)and mouse colon carcinoma CT26 cells(class I MHC H-2d syngeneic for BALB/c mice)were cultured at 37 C under 5% CO2 in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum(FBS), 100 units/ml penicillin G sodium, 100 ug/ml streptomycin sulfate, 2mM L-glutamine, ImM sodium pyruvate and non-essential amino acid (from this point on referred to as complete RPMI-1640; all reagents from Gibco-Invitrogen Carlsbad, CA, USA). OVA-expressing MCA205 cells were selected in complete RPMI-1640 medium(as above) though supplemented with 50 ug/ml hygromycin B(Invitrogen, Life Technologies) Tumor challenge and treatment Mice were subcutaneously injected into the right flank with 1 x 10 MCA205-OVA or MC38 with 0.5 eT or with 0.2 x 10 CT26 tumor cell lines When tumors reached a size of 20 to 40 mm(day 0), mice were injected intraperitoneally(i p with 100ug of anti-CTLA-4 mAb(clone 9D9)or isotype control(clone MPCI1) Mice were4 was repeated on days 22, 43, and 64. Patients were re-imaged between days 81-88 and evaluated for responses in the non-irradiated measurable metastatic sites. Study code: NCT02221739. Mice. All animal experiments were carried out in compliance with French and European laws and regulations. Mice were used between 7 and 14 weeks of age. WT specific pathogen-free (SPF) C57BL/6J and BALB/c mice were obtained from Harlan (France) and Janvier (France), respectively, and were kept in SPF conditions at Gustave Roussy. C57BL/6 GF mice were obtained from Institut Pasteur and maintained in sterile isolators. Il-10-/- C57BL/6 mice and WT C57BL/6 control animals were kindly provided by Anne O’Garra (National Institute for Medical Research, UK). NOD2-/-, Il10-/-, and NOD2-/-Il10-/- mice (BALB/c background) were obtained from Institut Pasteur Lille. C57BL/6 Tlr2-/- mice were provided by Ivo Gompers Boneca (Institut Pasteur, Paris, France), Tlr4-/- mice were bred and maintained in the animal facility of Gustave Roussy, Villejuif, France. Cell culture and reagents. OVA-expressing mouse fibrosarcoma MCA205 cells, murine colon carcinoma MC38 cells, RET melanoma model (a transgene-enforced expression of the Ret protooncogene under the control of the metallothionein-1 promoter driving spontaneous melanomagenesis, kindly provided by Viktor Umansky) (class I MHC H-2b , syngeneic for C57BL/6 mice) and mouse colon carcinoma CT26 cells (class I MHC H-2d , syngeneic for BALB/c mice) were cultured at 37°C under 5% CO2 in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 2mM L-glutamine, 1mM sodium pyruvate and non-essential amino acids (from this point on referred to as complete RPMI-1640; all reagents from Gibco-Invitrogen, Carlsbad, CA, USA). OVA-expressing MCA205 cells were selected in complete RPMI-1640 medium (as above) though supplemented with 50 μg/ml hygromycin B (Invitrogen, Life TechnologiesTM). Tumor challenge and treatment. Mice were subcutaneously injected into the right flank with 1 × 106 MCA205-OVA or MC38, with 0.5 x 106 RET or with 0.2 × 106 CT26 tumor cell lines. When tumors reached a size of 20 to 40 mm2 (day 0), mice were injected intraperitoneally (i.p) with 100μg of anti-CTLA-4 mAb (clone 9D9) or isotype control (clone MPC11). Mice were