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

  • Nadia B. Hassounah
  • Venkat S. Malladi
  • Yi Huang
  • Samuel S. Freeman
  • Ellen M. Beauchamp
  • Shohei Koyama
  • Nicholas Souders
  • Sunil Martin
  • Glenn Dranoff
  • Kwok-Kin Wong
  • Chandra S. Pedamallu
  • Peter S. Hammerman
  • Esra A. AkbayEmail author
Original Article
  • 324 Downloads

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.

Keywords

HPV PD-L1 Secreted PD-L1 Cancer immunology 

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

Notes

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.

Author 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.

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).

Compliance with ethical standards

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.

Supplementary material

262_2018_2284_MOESM1_ESM.pdf (14.3 mb)
Supplementary material 1 (PDF 14634 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Nadia B. Hassounah
    • 1
    • 11
  • Venkat S. Malladi
    • 2
    • 3
  • Yi Huang
    • 4
    • 5
  • Samuel S. Freeman
    • 6
  • Ellen M. Beauchamp
    • 1
  • Shohei Koyama
    • 1
  • Nicholas Souders
    • 1
  • Sunil Martin
    • 1
  • Glenn Dranoff
    • 1
    • 7
    • 8
    • 11
  • Kwok-Kin Wong
    • 1
    • 7
    • 9
    • 10
  • Chandra S. Pedamallu
    • 1
    • 6
  • Peter S. Hammerman
    • 1
    • 6
    • 7
    • 11
  • Esra A. Akbay
    • 4
    • 5
    Email author
  1. 1.Department of Medical OncologyDana-Farber Cancer InstituteBostonUSA
  2. 2.Department of BioinformaticsUniversity of Texas Southwestern Medical CenterDallasUSA
  3. 3.Bioinformatics Core FacilityUniversity of Texas Southwestern Medical CenterDallasUSA
  4. 4.Department of PathologyUniversity of Texas Southwestern Medical CenterDallasUSA
  5. 5.Simmons Comprehensive Cancer CenterDallasUSA
  6. 6.Cancer ProgramBroad Institute of Harvard and Massachusetts Institute of TechnologyCambridgeUSA
  7. 7.Department of MedicineBrigham and Women’s Hospital and Harvard Medical SchoolBostonUSA
  8. 8.Department of Medical Oncology and Cancer Vaccine CenterDana Farber Cancer InstituteBostonUSA
  9. 9.Ludwig Institute for CancerBostonUSA
  10. 10.Belfer Institute for Applied Cancer ScienceBostonUSA
  11. 11.Novartis Institutes for Biomedical ResearchCambridgeUSA

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