Identification and characterization of an alternative cancer-derived PD-L1 splice variant
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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.
KeywordsHPV PD-L1 Secreted PD-L1 Cancer immunology
American type culture collection
Cancer cell line encyclopedia
Cell signaling technology
Enzyme linked immunosorbent assay
Fragments per kilobase of transcript per million mapped reads
Head and neck squamous cell carcinoma
National Cancer Institute
The Cancer Genome Atlas
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.
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.
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 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  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.