Distinct antibody clones detect PD-1 checkpoint expression and block PD-L1 interactions on live murine melanoma cells
Martins C, Silva M, Rasbach E, Singh P, Itoh Y, Williams JB, Statham E, Meurer A, Martinez D, Brandenburg A, Heppt M, Barthel SR, Schatton T (2022)
Publication Type: Journal article
Publication year: 2022
Journal
Book Volume: 12
Article Number: 12491
Journal Issue: 1
DOI: 10.1038/s41598-022-16776-1
Abstract
Monoclonal antibodies (abs) targeting the programmed cell death 1 (PD-1) immune checkpoint pathway have revolutionized tumor therapy. Because T-cell-directed PD-1 blockade boosts tumor immunity, anti-PD-1 abs have been developed for examining T-cell-PD-1 functions. More recently, PD-1 expression has also been reported directly on cancer cells of various etiology, including in melanoma. Nevertheless, there is a paucity of studies validating anti-PD-1 ab clone utility in specific assay types for characterizing tumor cell-intrinsic PD-1. Here, we demonstrate reactivity of several anti-murine PD-1 ab clones and recombinant PD-L1 with live B16-F10 melanoma cells and YUMM lines using multiple independent methodologies, positive and negative PD-1-specific controls, including PD-1-overexpressing and PD-1 knockout cells. Flow cytometric analyses with two separate anti-PD-1 ab clones, 29F.1A12 and RMP1-30, revealed PD-1 surface protein expression on live murine melanoma cells, which was corroborated by marked enrichment in PD-1 gene (Pdcd1) expression. Immunoblotting, immunoprecipitation, and mass spectrometric sequencing confirmed PD-1 protein expression by B16-F10 cells. Recombinant PD-L1 also recognized melanoma cell-expressed PD-1, the blockade of which by 29F.1A12 fully abrogated PD-1:PD-L1 binding. Together, our data provides multiple lines of evidence establishing PD-1 expression by live murine melanoma cells and validates ab clones and assay systems for tumor cell-directed PD-1 pathway investigations.
Involved external institutions
United States (USA) (US)How to cite
APA:
Martins, C., Silva, M., Rasbach, E., Singh, P., Itoh, Y., Williams, J.B.,... Schatton, T. (2022). Distinct antibody clones detect PD-1 checkpoint expression and block PD-L1 interactions on live murine melanoma cells. Scientific Reports, 12(1). https://dx.doi.org/10.1038/s41598-022-16776-1
MLA:
Martins, Christina, et al. "Distinct antibody clones detect PD-1 checkpoint expression and block PD-L1 interactions on live murine melanoma cells." Scientific Reports 12.1 (2022).
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