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. 2020 Jun 24;11:3196. doi: 10.1038/s41467-020-16838-w

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — T-cell/Jurkat NFAT activation is dependent on crosslinking of mα-CD3. a Monovalent anti-CD3 IgG is able to activate luciferase in a Jurkat NFAT luciferase reporter cell line after crosslinking by plate-coated anti-human Fc (red curve) whereas in the absence of crosslinking no luciferase activity can be detected (blue curve). The dotted line indicates the luminescence for Jurkat NFAT-cells without mα-CD3 IgG on plate-coated anti-human Fc. Each value represents the mean of triplicates, standard deviation is indicated by error bars (representative experiment of n = 3). b Monovalent α-CD3 IgG is able to activate CD8-positive T cells, measured by quantification of the early activation marker CD69, after crosslinking by plate-coated anti-human Fc (red curve) whereas in the absence of crosslinking no CD8-positive T-cell activation can be detected (blue curve). The median fluorescence intensity (MFI) for CD69 of CD8 T cells is shown. Each value represents the mean of triplicates, standard deviation is indicated by error bars (representative experiment of n = 3). c, d mαCD3 IgGs were bound to plate-coated anti-human Fc antibody before Jurkat NFAT reporter cells or PBMCs were added. Jurkat NFAT activation is measured in relative luminescence units (RLU) and T-cell activation was assessed by quantification of CD69 by FACS analysis. Cleavage of the Prot-mαCD3 IgG containing a matriptase cleavable linker was performed by incubation of Prot-mαCD3 IgG with purified recombinant human matriptase for 24 h at 37 °C. c Jurkat NFAT activation mediated by Prot-mαCD3 IgG. Cleaved Prot-mαCD3 IgG, blocked Prot-mαCD3 IgG, mαCD3 IgG and mαCD3 IgG with an N-terminal fusion of a nonspecific fusion (anti-CEA Fab) are shown. The dotted line indicates the luminescence for Jurkat NFAT-cells without any CD3 IgG. Each value represents the mean value of triplicates, standard deviation is indicated by error bars (representative experiment of n = 3). d The median fluorescence intensity (MFI) for CD69 of CD8 T cells is shown. Each value represents the mean value of triplicates, standard deviation is indicated by error bars (representative experiment for three different human PBMC donors).