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
Book Volume: 12
Article Number: 12491
Journal Issue: 1
DOI: 10.1038/s41598-022-16776-1
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.
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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