Microenvironment and Immunology Abscopal Effects of Radiotherapy Are Enhanced by Combined Immunostimulatory mAbs and Are Dependent on CD8 T Cells and Crosspriming María E. Rodriguez-Ruiz 1,2 , Inmaculada Rodriguez 1 , Saray Garasa 1 , Benigno Barbes 1 , Jose Luis Solorzano 2 , Jose Luis Perez-Gracia 2 , Sara Labiano 1 , Miguel F. Sanmamed 1,3 , Arantza Azpilikueta 1 , Elixabet Bola ~ nos 1 , Alfonso R. Sanchez-Paulete 1 , M. Angela Aznar 1 , Ana Rouzaut 1 , Kurt A. Schalper 4,5 , Maria Jure-Kunkel 6 , and Ignacio Melero 1,2 Abstract Preclinical and clinical evidence indicate that the proim- mune effects of radiotherapy can be synergistically augmented with immunostimulatory mAbs to act both on irradiated tumor lesions and on distant, nonirradiated tumor sites. The combination of radiotherapy with immunostimulatory anti- PD1 and anti-CD137 mAbs was conducive to favorable effects on distant nonirradiated tumor lesions as observed in trans- planted MC38 (colorectal cancer), B16OVA (melanoma), and 4T1 (breast cancer) models. The therapeutic activity was cru- cially performed by CD8 T cells, as found in selective depletion experiments. Moreover, the integrities of BATF-3–dependent dendritic cells specialized in crosspresentation/crosspriming of antigens to CD8 þ T cells and of the type I IFN system were absolute requirements for the antitumor effects to occur. The irradiation regimen induced immune infiltrate changes in the irradiated and nonirradiated lesions featured by reductions in the total content of effector T cells, Tregs, and myeloid-derived suppressor cells, while effector T cells expressed more intracel- lular IFNg in both the irradiated and contralateral tumors. Importantly, 48 hours after irradiation, CD8 þ TILs showed brighter expression of CD137 and PD1, thereby displaying more target molecules for the corresponding mAbs. Likewise, PD1 and CD137 were induced on tumor-infiltrating lympho- cytes from surgically excised human carcinomas that were irradiated ex vivo. These mechanisms involving crosspriming and CD8 T cells advocate clinical development of immuno- therapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompanied by radiotherapy strat- egies, even if the disease is left outside the field of irradiation. Cancer Res; 76(20); 5994–6005. Ó2016 AACR. Introduction Radiotherapy is a solid pillar of cancer treatment used to treat localized stages of a broad variety of malignant diseases and to alleviate local complications in advanced or metastatic cases as a palliative treatment. The mechanism of action of ionizing radio- therapy against cancer is thought to mainly rely on catastrophic damage of genomic DNA, leading to apoptotic tumor cell death. Many cellular genetic and epigenetic factors affect the sensitivity of each tumor to radiotherapy approaches. Recently, the tumor stroma component has been found to play a key role in the outcome of irradiated tumors (1). When radiotherapy is pre- scribed to a patient, it is assumed that the normal nonmalignant tissue will also be irradiated giving rise to multifarious biological effects including inflammation and scarring (1). Radiotherapy can be performed by applying an external beam of irradiation or by the temporal surgical insertion of radiation sources guided by catheters into the cancer tissue using techniques collectively known as brachytherapy. Immunotherapy is emerging as another major pillar for the treatment of cancer treatment. mAbs acting on immune system receptors to derepress or agonistically augment antitumor immunity are being developed in the clinic (2). Antibodies against the inhibitory (checkpoint) receptor CTLA-4 were the first to be clinically developed with ipilimumab receiving FDA and European Medicines Agency (EMA) approval for metastatic melanoma (3). Among these checkpoint inhibitor monoclonal immunostimulatory antibodies, agents blocking the PD1/ PD-L1 receptor/ligand pair have already attained FDA and EMA approval for metastatic melanoma (4), non–small cell lung cancer (5–7), and renal cell carcinoma (8) and other indica- tions are under regulatory evaluation. This achievement was preceded by extensive and successful preclinical research in mouse models. Agonist antibodies crosslinking CD137 (4-1BB) were also shown to enhance antitumor immunity in mice to the point of 1 Division of Immunology and Immunotherapy, Center for Applied Medical Research (CIMA), University of Navarra and Instituto de Investigacion Sanitaria de Navarra (IdISNA), Pamplona, Spain. 2 Uni- versity Clinic, University of Navarra and Instituto de Investigacion Sanitaria de Navarra (IdISNA), Pamplona, Spain. 3 Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut. 4 Department of Pathology, Yale School of Medicine, New Haven, Connecticut. 5 Department of Medicine (Medical Oncology), Yale School of Medicine, New Haven, Connecticut 6 Bristol-Myers Squibb, Lawrenceville, New Jersey. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Authors: Ignacio Melero, University of Navarra and Instituto de Investigacion Sanitaria de Navarra (IdISNA), Av. Pio XII, 55, Pamplona, Navarra 31008, Spain. Phone: 349-4819-4700; Fax: 349-4819-4717; E-mail: imelero@unav.es; and Maria E. Rodríguez-Ruiz, mrruiz@unav.es doi: 10.1158/0008-5472.CAN-16-0549 Ó2016 American Association for Cancer Research. Cancer Research Cancer Res; 76(20) October 15, 2016 5994 on June 10, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst August 22, 2016; DOI: 10.1158/0008-5472.CAN-16-0549