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. 2021 Jun 23;4:772. doi: 10.1038/s42003-021-02309-5

Fig. 2. 4-1BB and OX40 agonists induce differential receptor clustering.

Fig. 2

a Jurkat-h4-1BBECD-GFP cells were treated with 10 µg/mL anti-4-1BB mAbs of either h1 or h2 isotype as indicated for 1 h, and then fixed with methanol, nucleus stained with DAPI and imaged using a Leica SP8 confocal microscope. All images are representative of at least 10 images from at least 2 experiments. Scale bar, 4 µm. b Jurkat-NFκB-GFP reporter cells expressing h4-1BB were incubated with serially diluted anti-4-1BB mAbs as indicated for 6 h. The level of NFκB activation was then quantified by GFP expression assessed by flow cytometry. Means ± SEM, n = 3, data representative of 3 independent experiments. c Purified human CD8+ T cells were activated with plate-bound anti-CD3 mAb for one day and then treated with various anti-4-1BB mAbs for 1 or 2 days before 3H thymidine was added at 1 µCi per well for the last 18 h to assess T cell proliferation. d CD4+ CD127lowCD25+ Tregs were purified from human PBMC and cocultured with CFSE-labelled human PBMC at a 1:4 Treg:PBMC ratio in the presence of suboptimal anti-CD3 mAb and various anti-4-1BB mAbs as indicated. Cells were incubated for 4 days and CD8+ T cell proliferation assessed by CFSE dilution using flow cytometry. Representative dot plots are shown. Right panel: mAb activity was normalised against isotype control and 4-1BBL activity was normalised against the average of both isotype controls. Each dot represents one donor. The paired Student t test for comparing h1 and h2 mAbs. One-way ANOVA followed by Tukey’s post hoc test for comparing 4-1BBL with isotype controls. Means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. e Jurkat-hOX40ECD-GFP cells were treated with 10 µg/mL anti-OX40 mAbs of either h1 or h2 isotype as indicated for 1 h, and then fixed with methanol, nucleus stained with DAPI and imaged using a Leica SP8 confocal microscope. All images are representative of at least 10 images from at least 2 independent experiments. Scale bar, 4 µm. f Jurkat-NFκB-GFP reporter cells expressing hOX40 were incubated with serially diluted anti-OX40 mAbs as indicated for 6 h. The level of NFκB activation was then quantified by GFP expression assessed by flow cytometry. Means ± SEM, n = 3, data representative of 3 independent experiments.