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Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types1,2,3,4,5. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8+ T cells (termed adaptive immune resistance)6,7. Here we show that pre-existing CD8+ T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8+ T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8+ T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance.

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  • 27 November 2014

    Extended Data Fig. 3 was revised to include missing labels on panel e.


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This study was funded in part by National Institutes of Health grants K08 AI091663, Kure It Research Grant, UL1TR000124 (to P.C.T.), P01 CA168585, U54 CA119347, R01 CA170689, the Ressler Family Fund, the Dr Robert Vigen Memorial Fund, the Wesley Coyle Memorial Fund, and the Garcia-Corsini Family Fund (to A.R.), P30 CA16042 to D.A.E. A.R. was supported by a Stand Up To Cancer—Cancer Research Institute Cancer Immunology Dream Team Translational Research Grant (SU2C-AACR-DT1012). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. M.S. was supported as a Howard Hughes Medical Institute Medical Research Fellow. Some of the studies were funded by Merck Sharp and Dome and by Adaptive Biotechnologies. L.R. was supported by the V Foundation-Gil Nickel Family Endowed Fellowship in Melanoma Research and a grant from the Spanish Society of Medical Oncology for Translational Research in Reference Centers. We acknowledge the Translational Pathology Core Laboratory for tissue sectioning and slide scanning, S. Roy, N. Kamsu-Kom, R. Guo, J. Pang, W. Li, A. Villanueva and K. Crawford for biopsy processing and clinical data, S. Hashaghan for assisting with quantitative imaging approaches, E. Penaflor for assisting in IHC assay optimization, execution and digital image generation, and B. Dogdas and S. Mehta who assisted with the proximity assay. We would like to thank S. Ebbinghaus, E. Rubin, S. P. Kang, R. L. Modlin, C. R. Taylor and C. Denny for critically reviewing the manuscript.

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  1. University of California Los Angeles (UCLA), Los Angeles, California 90095, USA

    • Paul C. Tumeh
    • , Christina L. Harview
    • , I. Peter Shintaku
    • , Emma J. M. Taylor
    • , Lidia Robert
    • , Bartosz Chmielowski
    • , Marko Spasic
    • , Gina Henry
    • , Voicu Ciobanu
    • , Alisha N. West
    • , Manuel Carmona
    • , Christine Kivork
    • , Elizabeth Seja
    • , Grace Cherry
    • , Antonio J. Gutierrez
    • , Tristan R. Grogan
    • , John A. Glaspy
    • , David A. Elashoff
    •  & Antoni Ribas
  2. Jonsson Comprehensive Cancer Center, Los Angeles, California 90095, USA

    • Paul C. Tumeh
    • , Bartosz Chmielowski
    • , John A. Glaspy
    • , David A. Elashoff
    •  & Antoni Ribas
  3. Merck & Co, Palo Alto, California 94304, USA

    • Jennifer H. Yearley
    •  & Robert H. Pierce
  4. Gustave Roussy and INSERM U981, Villejuif, Paris Sud, France

    • Christine Mateus
    • , Gorana Tomasic
    •  & Caroline Robert
  5. Adaptive Biotechnologies, Seattle, Washington 98102, USA

    • Ryan O. Emerson
    •  & Harlan Robins
  6. Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA

    • Harlan Robins


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P.C.T. and A.R. supervised the project and developed the concepts. P.C.T., C.L.H., C.R. and A.R. designed the experiments. P.C.T., C.L.H., M.S., C.R. and A.R. interpreted the data. A.R., P.C.T., C.L.H., C.R., J.H.Y., M.S., I.P.S., E.J.M.T., R.O.E., H.R. and R.H.P. gave conceptual advice and edited the manuscript. P.C.T., J.H.Y., I.P.S., A.N.W. and E.J.M.T. established IHC staining and/or imaging protocols. J.H.Y., I.P.S., E.J.M.T. and R.H.P. provided confirmatory pathology analyses. G.T. worked on IHC samples from the patients from Gustave Roussy. C.L.H., L.R., M.S., G.H., V.C., M.C., C.K. and E.S. provided technical support. P.C.T., T.R.G. and D.A.E. designed and implemented the predictive model and provided statistical support. P.C.T., B.C., A.J.G., C.M., J.A.G., G.C., C.R. and A.R. clinically evaluated patients in the trial.

Competing interests

R.O.E. has full-time employment and equity ownership at Adaptive Biotechnologies Corporation. H.S.R. has consultancy, patents and royalties, and equity ownership at Adaptive Biotechnologies Corporation. P.C.T., D.A.E. and A.R. have filed a patent related to this work.

Corresponding authors

Correspondence to Paul C. Tumeh or Antoni Ribas.

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