Supplementary methods WST-1 assay of cell viability/proliferation. Raw264.7 or πBCL1 cells cultured with liposomes encapsulating dichloromethylene-bisphosphonate (Cl 2 MBP; clodronate) or PBS as control for 48 hours before viability/proliferation was assessed using the WST-1 assay (Roche, U.K.). Measurement of tumor antigen-specific T cells. Peripheral tail bleeds were sampled 72 hours after therapy, red blood cells were lysed (BD Pharmlyse, U.K.) and stained using SIINFEKL-MHC-I pentamers (ProImmune, U.K.) and counterstained with CD8 and CD19. Experimental groups contained at least 7 mice pooled into 3 samples and are representative of 2 independent experiments. Immunohistochemistry Tissues were harvested from mice immediately post mortem and frozen in isopentane cooled in liquid nitrogen. Staining of CD169 (clone 3D6.112, AbD Serotec, U.K.) was visualized using the rat-on- mouse horse radish peroxidase (HRP)-polymer kit (Biocare Medical, UK). In vivo studies Mice were housed under specific pathogen free conditions in Tecniplast 1284 IVC cages holding a maximum of 7 animals with aspenchips-2 bedding, sizzlenest (envirodry) nesting material, and a cardboard tunnel. Mice were housed on a 12/12 light/dark cycle and were given filtered water and fed ad libitum on Teklad Global 19 % protein extruded rodent diet. Animal experiments were approved by a local ethical committee and performed under a United Kingdom Home Office license. Female C57Bl/6 or Balb/c mice were used at around 8 weeks of age at around 17-20g in weight. Assessment of weights and endpoints related to tumor burden was performed by technical staff to ensure a lack of bias. For therapy studies group sizes were 5-7 animals and experiments were repeated at least once.

A B Supplementary Figure 1. Clodronate encapsulated liposomes deplete macrophages in vitro and in vivo. A, WST-1 assay of RAW macrophages and πBCL1 tumor cells co-cultured overnight with either PBS (PBS-lip) or clodronate (Clod-lip) encapsulated liposomes. ** P<0.01 (2-tailed Student t test). B, Frequency of F4/80 + macrophages in the spleen of mice 24 hours after administration of liposomes. * P<0.05 (Mann-Whitney test). Experimental groups contained at least 4 mice and are representative of 2 independent experiments.

Supplementary Figure 2. Depletion of DC abrogates the priming of tumour antigen specific CD8 + T cells following treatment with RT and αCD40 mAb. A, Frequency of SIINFEKL-MHC-I restricted CD8 + T- cells (expressed as a percentage of tumor naïve control mice) 72 hours after therapy in peripheral blood of EG7 tumor-bearing mice. P=0.075 (Mann-Whitney test). Experimental groups contained at least 7 mice pooled into 3 samples and are representative of 2 independent experiments.

A PBS+ DT CD169 B Supplementary Figure 3. Depletion of CD169 + macrophages does not impact on the efficacy of combination therapy with RT and αCD40 mAb. A, CD169-DTR mice were inoculated with EL4 tumor cells and received 10 Gy RT in combination with αCD40 mAb. For depletion of CD169 + cells animals received 100 ng DT i.p. 3qw for up to 2 weeks i.p. n/s P>0.05 compared to combination therapy (log- rank; Mantel-Cox test). Experimental groups contained at least 5 mice and are representative of at least 2 independent experiments. B, immunohistochemistry of CD169 staining in CD169-DTR mice 24 hours after i.p. administration of 100ng DT / PBS.

CD8IFNγ A B Supplementary Figure 4. Combination therapy with RT and a systemically administered TLR-7 agonist leads to the generation of durable anti-tumor immune responses capable of preventing disease recurrence in mice bearing established EL4 lymphoma. A and B, representative dot blot of IFNγ production (A) and frequency of IFNγ+ CD8+ T-cells (B) isolated from either tumor naïve, or LTS mice originally treated with RT and R848 following co-culture with 50 Gy irradiated EL4 cells for 5 days, followed by priming with 50 Gy irradiated EL4 cells. ** P<0.01 (Mann-Whitney test). Experimental groups contained at least 5 mice and are representative of 2 independent experiments.