Article

In vivo discovery of immunotherapy targets in the tumour microenvironment

  • Nature volume 506, pages 5257 (06 February 2014)
  • doi:10.1038/nature12988
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Abstract

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments.

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Acknowledgements

This work was supported by the National Institutes of Health (Transformative Research Award 1R01CA173750 to K.W.W.), the Melanoma Research Alliance (to K.W.W.), the DF/HCC–MIT Bridge Project and the Lustgarten Foundation (to K.W.W., J.C.L. and H.L.P.), Novartis Institutes of Biomedical Research (to K.W.W.), the Koch Institute Support Grant P30-CA14051 from the National Cancer Institute, the American Cancer Society John W. Thatcher, Jr Postdoctoral Fellowship in Melanoma Research (to D.R.S.), the Terri Brodeur Breast Cancer Foundation Postdoctoral Fellowship (to P.Z.) and a NIH T32 grant (AI07386 to D.A.A.A.).

Author information

Author notes

    • Penghui Zhou
    •  & Donald R. Shaffer

    These authors contributed equally to this work.

    • Donald R. Shaffer
    •  & Kutlu Elpek

    Present address: Jounce Therapeutics, Cambridge, Massachusetts 02138, USA.

Affiliations

  1. Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA

    • Penghui Zhou
    • , Donald R. Shaffer
    • , Diana A. Alvarez Arias
    • , Yukoh Nakazaki
    • , Wouter Pos
    • , Viviana Cremasco
    • , Kutlu Elpek
    • , Shannon J. Turley
    • , Glenn Dranoff
    • , Harvey Cantor
    •  & Kai W. Wucherpfennig
  2. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA

    • Alexis J. Torres
    •  & J. Christopher Love
  3. Whitehead Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA

    • Stephanie K. Dougan
    •  & Hidde L. Ploegh
  4. Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • Glenn S. Cowley
    • , David E. Root
    •  & Nir Hacohen
  5. Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA

    • Jennifer Brogdon
  6. Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, USA

    • John Lamb

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Contributions

K.W.W., P.Z., S.J.T., G.D. and H.C. contributed to the overall study design; K.W.W., P.Z. and D.R.S. designed experiments; P.Z., D.A.A.A. and H.C. developed procedure for lentiviral infection of T cells and optimized approaches for adoptive T-cell therapy; P.Z., D.R.S. and D.A.A.A. performed shRNA screen; G.S.C., D.E.R. and N.H. provided pooled shRNA library and advice on shRNA screen; Y.N. and G.D. provided B16-Ova cell line and advice on tumour model; A.J.T. and J.C.L. performed nano-well analysis of cytokine production, V.C. and S.J.T. performed histological studies, W.P. performed protein quantification by mass spectrometry, S.K.D. and H.L.P. provided mouse models; J.B., K.E. and J.L. performed microarray analysis; K.W.W., P.Z. and D.R.S. wrote the paper.

Competing interests

K.W.W. and G.D. served as consultants to Novartis.

Corresponding author

Correspondence to Kai W. Wucherpfennig.

The access number for microarray data is GSE53388 in the Genomic Spatial Event (GSE) database.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    This file contains the results from primary and secondary screens. It lists gene name, gene ID, shRNA clone ID, targeting sequence and enrichment of shRNAs in tumors relative to spleen. The first tab shows results from primary screens, the second tab data from secondary screens.

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