title: Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer.
abstract: Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.
authors.affiliation: 1] Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Twyman-Saint Victor
authors.affiliation: Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Rech
authors.affiliation: 1] Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Amit
authors.initials: A
authors.lastname: Maity
authors.affiliation: 1] Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Rengan
authors.affiliation: 1] Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Kristen E
authors.initials: KE
authors.lastname: Pauken
authors.affiliation: 1] Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Erietta
authors.initials: E
authors.lastname: Stelekati
authors.affiliation: 1] Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Benci
authors.affiliation: 1] Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Bihui
authors.initials: B
authors.lastname: Xu
authors.affiliation: 1] Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Hannah
authors.initials: H
authors.lastname: Dada
authors.affiliation: 1] Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Pamela M
authors.initials: PM
authors.lastname: Odorizzi
authors.affiliation: 1] Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Ramin S
authors.initials: RS
authors.lastname: Herati
authors.affiliation: 1] Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Kathleen D
authors.initials: KD
authors.lastname: Mansfield
authors.affiliation: Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Dana
authors.initials: D
authors.lastname: Patsch
authors.affiliation: 1] Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Amaravadi
authors.affiliation: 1] Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Lynn M
authors.initials: LM
authors.lastname: Schuchter
authors.affiliation: Division of Biostatistics, Department of Public Health Sciences, University of Miami, Miami, Florida 33136, USA.
authors.forename: Hemant
authors.initials: H
authors.lastname: Ishwaran
authors.affiliation: 1] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Mick
authors.affiliation: 1] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Pryma
authors.affiliation: 1] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Xu
authors.affiliation: 1] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Feldman
authors.affiliation: 1] Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Tara C
authors.initials: TC
authors.lastname: Gangadhar
authors.affiliation: 1] Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Hahn
authors.affiliation: 1] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [3] Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: E John
authors.initials: EJ
authors.lastname: Wherry
authors.affiliation: 1] Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [3] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [4] Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Vonderheide
authors.affiliation: 1] Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [2] Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [3] Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA [4] Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Andy J
authors.initials: AJ
authors.lastname: Minn
mesh_terms: D000818:Animals; D060890:B7-H1 Antigen / Q000037:antagonists & inhibitors* / Q000378:metabolism; D060908:CTLA-4 Antigen / Q000037:antagonists & inhibitors*; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D005260:Female; D006801:Humans; D008545:Melanoma / Q000188:drug therapy* / Q000276:immunology* / Q000473:pathology / Q000532:radiotherapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D011948:Receptors, Antigen, T-Cell / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000276:immunology / Q000528:radiation effects*; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology / Q000528:radiation effects
chemical_list: D060890:B7-H1 Antigen; D060908:CTLA-4 Antigen; D011948:Receptors, Antigen, T-Cell