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Martins, Christina [Author]; Silva, Mariana [Author]; Rasbach, Erik [Author]; Singh, Praveen [Author]; Itoh, Yuta [Author]; Williams, Jason B. [Author]; Statham, Edith [Author]; Meurer, Anna [Author]; Martinez, Daniela V. [Author]; Brandenburg, Anne [Author]; Heppt, Markus V. [Author]; Barthel, Steven R. [Author]; Schatton, Tobias [Author]

Distinct antibody clones detect PD-1 checkpoint expression and block PD-L1 interactions on live murine melanoma cells

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  • Media type: E-Article
  • Title: Distinct antibody clones detect PD-1 checkpoint expression and block PD-L1 interactions on live murine melanoma cells
  • Contributor: Martins, Christina [Author]; Silva, Mariana [Author]; Rasbach, Erik [Author]; Singh, Praveen [Author]; Itoh, Yuta [Author]; Williams, Jason B. [Author]; Statham, Edith [Author]; Meurer, Anna [Author]; Martinez, Daniela V. [Author]; Brandenburg, Anne [Author]; Heppt, Markus V. [Author]; Barthel, Steven R. [Author]; Schatton, Tobias [Author]
  • Published: 21 July 2022
  • Published in: Scientific reports ; 12(2022), Artikel-ID 12491, Seite 1-14
  • Language: English
  • DOI: 10.1038/s41598-022-16776-1
  • Identifier:
  • Keywords: Immunotherapy ; Melanoma
  • Origination:
  • Footnote:
  • Description: 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.
  • Access State: Open Access