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Identification and characterization of an alternative cancer-derived PD-L1 splice variant

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Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

Therapeutic blockade of the PD-1/PD-L1 axis is recognized as an effective treatment for numerous cancer types. However, only a subset of patients respond to this treatment, warranting a greater understanding of the biological mechanisms driving immune evasion via PD-1/PD-L1 signaling and other T-cell suppressive pathways. We previously identified a head and neck squamous cell carcinoma with human papillomavirus integration in the PD-L1 locus upstream of the transmembrane domain-encoding region, suggesting expression of a truncated form of PD-L1 (Parfenov et al., Proc Natl Acad Sci USA 111(43):15544–15549, 2014). In this study, we extended this observation by performing a computational analysis of 33 other cancer types as well as human cancer cell lines, and identified additional PD-L1 isoforms with an exon 4 enrichment expressed in 20 cancers and human cancer cell lines. We demonstrate that cancer cell lines with high expression levels of exon 4-enriched PD-L1 generate a secreted form of PD-L1. Further biochemical studies of exon 4-enriched PD-L1 demonstrated that this form is secreted and maintains the capacity to bind PD-1 as well as to serve as a negative regulator on T cell function, as measured by inhibition of IL-2 and IFNg secretion. Overall, we have demonstrated that truncated forms of PD-L1 exist in numerous cancer types, and have validated that truncated PD-L1 can be secreted and negatively regulate T cell function.

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Abbreviations

ATCC:

American type culture collection

CCLE:

Cancer cell line encyclopedia

CST:

Cell signaling technology

EBV:

Epstein–Barr virus

ELISA:

Enzyme linked immunosorbent assay

FPKM:

Fragments per kilobase of transcript per million mapped reads

GTeX:

Genotype-Tissue Expression

HA:

Hemagglutinin

HNSCC:

Head and neck squamous cell carcinoma

HPV:

Human papillomavirus

HRP:

Horseradish Peroxidase

NCI:

National Cancer Institute

PD-L1:

Programmed death-ligand-1

PHA:

phytohemagglutinin

PSG:

Penicillin/streptomycin/glutamine

RIPA:

Radioimmunoprecipitation assay

RT:

Room temperature

TCA:

Trichloroacetic acid

TCGA:

The Cancer Genome Atlas

TMB:

3,3′,5,5′-Tetramethylbenzidine

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Acknowledgements

The authors would like to acknowledge the Dana-Farber Cancer Institute Flow Cytometry Core (Suzan Lazo-Kallanian, John Daley), and UT Southwestern Children’s Research Institute Flow Cytometry Core for help with flow cytometry. The authors would also like to thank Gordon Freeman for PD-L1 antibodies.

Funding

This work was supported by the NCI R01 CA205150, CA196932, and K08 CA163677 grants, as well as the Starr Consortium for Cancer Research and Stand up to Cancer awards to Peter Hammerman. This work was also supported by the Young Investigator Award from the International Association for the Study of Lung Cancer, Career Enhancement Award (5P50CA070907) through the National Institutes of Health, and Cancer Prevention and Research Institute of Texas Scholar Award (RR160080) to Esra Akbay. Venkat Malladi was supported by the Cancer Prevention and Research Institute of Texas award (RP150596).

Author information

Author notes

  1. Nadia B. Hassounah, Glenn Dranoff & Peter S. Hammerman

    Present address: Novartis Institutes for Biomedical Research, Cambridge, MA, USA

  2. Nadia B. Hassounah and Venkat S. Malladi contributed equally to this study and are joint first authors of this paper.

Authors and Affiliations

  1. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Nadia B. Hassounah, Ellen M. Beauchamp, Shohei Koyama, Nicholas Souders, Sunil Martin, Glenn Dranoff, Kwok-Kin Wong, Chandra S. Pedamallu & Peter S. Hammerman

  2. Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Venkat S. Malladi

  3. Bioinformatics Core Facility, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Venkat S. Malladi

  4. Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390, USA

    Yi Huang & Esra A. Akbay

  5. Simmons Comprehensive Cancer Center, Dallas, TX, USA

    Yi Huang & Esra A. Akbay

  6. Cancer Program, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA

    Samuel S. Freeman, Chandra S. Pedamallu & Peter S. Hammerman

  7. Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

    Glenn Dranoff, Kwok-Kin Wong & Peter S. Hammerman

  8. Department of Medical Oncology and Cancer Vaccine Center, Dana Farber Cancer Institute, Boston, MA, USA

    Glenn Dranoff

  9. Ludwig Institute for Cancer, Boston, MA, USA

    Kwok-Kin Wong

  10. Belfer Institute for Applied Cancer Science, Boston, MA, USA

    Kwok-Kin Wong

Authors
  1. Nadia B. Hassounah
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  2. Venkat S. Malladi
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  3. Yi Huang
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  4. Samuel S. Freeman
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  6. Shohei Koyama
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  8. Sunil Martin
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  9. Glenn Dranoff
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  10. Kwok-Kin Wong
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  11. Chandra S. Pedamallu
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  12. Peter S. Hammerman
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  13. Esra A. Akbay
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Contributions

NBH and VSM contributed equally to experimental design, data collection and analysis, and manuscript writing. YH and EMB helped with data collection and analysis. SSF and CSP helped with bioinformatics. SK, SM, NS, K-KW and GD aided with experimental design and provided reagents. PSH and EAA helped with experimental design, data collection, providing reagents, and manuscript writing and editing.

Corresponding author

Correspondence to Esra A. Akbay.

Ethics declarations

Conflict of interest

The authors have no relevant conflicts to disclose.

Informed consent

Informed consent was obtained from blood donors from the Brigham and Women’s Blood Bank under the approved IRB protocol 02-180.

Cell line authentication

HEK293T cells were purchased from ATCC, and RKO, CAL62, and RERF-LC-Ad1 cells were obtained from the CCLE [34] through the Broad Institute of Massachusetts Institute of Technology and Harvard. All cells were used within 6 months of initial passage and not further authenticated.

Additional information

Poster/abstract publication in Cancer Immunology Research, 2017; 5 (3 Suppl); Abstract nr A56; Proceedings of the American Association for Cancer Research Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 21; Boston, MA, USA.

This paper is published together with the following paper https://doi.org/10.1007/s00262-018-2282-1.

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Hassounah, N.B., Malladi, V.S., Huang, Y. et al. Identification and characterization of an alternative cancer-derived PD-L1 splice variant. Cancer Immunol Immunother 68, 407–420 (2019). https://doi.org/10.1007/s00262-018-2284-z

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  • DOI: https://doi.org/10.1007/s00262-018-2284-z

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