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. 2022 Jul 20;10(7):e004122. doi: 10.1136/jitc-2021-004122

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© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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TGF-β1 is internalized by BA in HEK293-PD-L1 cells and degraded in the lysosomes. (A) Biotinylated TGF-β1 binding to BA/PD-L1 complex or binding to TGF-βR on the cells surface was measured via MFI of SA-APC by flow cytometry. (A) Left panel: biotinylated TGF-β1 binding to TGF-βR was blocked by pre-incubation of cells with unlabeled TGF-β1 (130 ng/mL). Right panel: the binding of biotinylated TGF-β1 to TGF-βR was measured without pre-blocking TGF-βR. The box highlights the biotinylated TGF-β1 binding to the endogenous TGF-βR in the control groups, in which neither isotype control (inactive anti-PD-L1) nor TGF-β trap antibody binds to PD-L1. (B) Internalization of TGF-β1 by BA measured by flow cytometry at 20 hours (n=2) compared with 4 hours (n=3). The internalization of BA serves as a positive control. (C) Live-cell imaging for internalization/colocalization of biotinylated TGF-β1/SA-AF488 by BA in LE and lysosomes measured at time 0 hour and time 10 hours. Cells treated with the combination of anti-PD-L1 and TGF-β trap served as a control. The BA and anti-PD-L1+TGF-β trap combination were labeled with pHrodo conjugated goat anti-human IgG to track the internalization of BA or anti-PD-L1 to low pH organelles such as LE and lysosomes. (D) Imaging for internalization/colocalization of TGF-β1 by BA in lysosomes measured at 8 hours post treatment. AF488 conjugated anti-TGF-β1 antibody and AF594 conjugated anti-LAMP2 antibody were used to recognize TGF-β1 and LAMP2, respectively. Individual channel and merged image are shown. (E) MFI of Bio-TGF-β1/SA-AF488 with or without chloroquine (a lysosomal inhibitor) and sodium azide (an inhibitor for receptor internalization) was measured every 12 hours for 3 days. Data represent 3 independent assays (n=3). (F) The extracellular biotinylated TGF-β1 in the culture supernatant at 48 hours was measured by a sandwich ELISA. The HEK293-PD-L1 cells were pre-treated with or without anti-PD-L1. Treatment with BA or TGF-β trap was compared. (G) Model of simultaneous binding/internalization/degradation of active TGF-β by BA. Controls for figure 7 are located in online supplemental figure S7. APC, antigen-presenting cell; BA, bintrafusp alfa; IgG, immunoglobulin G; LE, late endosomes; MFI, median fluorescence intensity; PD-L1, programmed death-ligand 1; SA, streptavidin; TGF-β, transforming growth factor-β.