Abstract Immuno-radiotherapy is a promising strategy for generation of immunologic cancer recognition, rejection and induction of anti-cancer memory. Radiotherapy (RT) causes single or double strand DNA breaks in a dose dependent manner leading to immunogenic cell death of cancer cells and induction of type 1 interferons. Furthermore, RT increases MHCI expression on cancer cells and infiltration of cytotoxic T cells. Combined, these effects make RT a potent primer of anti-cancer immune responses. The most promising approaches in anti-cancer immunotherapy is to augment the adaptive immune response through danger associated molecular patterns signaling, including toll-like receptor (TLR) agonists. Optimally, these interventions should be given when tumor-associated antigen (TAA) uptake and presentation peaks. Previous studies on TAA uptake have mostly focused on in vitro or ex vivo assays to mimic the in vivo mechanisms. In the present study, we investigate the in vivokinetics of TAA uptake and trafficking using stably transfected mCherry-transfected B16F10 melanoma. B16F10 transfected with mCherry, a pH-stable fluorescent protein, allows tracking by e.g. flow cytometry in both cancer cells and phagocytosing populations. In vivo trafficking of mCherry fluorescence was investigated in mice treated with RT alone or in combination with TLR7 agonists given locally or systemically. Following treatment, TAA-uptake, -trafficking, and activation of relevant immune populations was evaluated in tumors or tumor draining lymph nodes (tdLNs). Increased TAA uptake was observed in MHCII high populations compared to MHCII low across all phagocytosing populations in tumors. The highest uptake (MFI of mCherry) was found in patrolling monocytes (CD11b+CD11c-Ly6c-CX3CR1high) and tumor-associated macrophages (CD11b+CD11c+CD64+). Interestingly, we exclusively found increased mCherry signal in resident cDC1s in tdLNs after treatment. RT led to a diminished population of migratory CD103+ cDC1s in tumors and we did not observe any increase in mCherry+ CD103+ cDC1s in tdLNs. We did not observe any mCherry signal in spleens - indicating that no cancer-specific immune response are mounted from the spleen. The results also indicate that TAA antigen is trafficked to tdLNs primarily by monocytes or by lymphatic drainage. Citation Format: Esben Christensen, Lars Ringgaard, Anja Brus, Andreas Kjaer, Anders E. Hansen, Thomas L. Andresen. Tumor associated antigen uptake tracking following immuno-radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1505.