A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats
Nora Müller, … , Alexander Flügel, Holger M. Reichardt
Nora Müller, … , Alexander Flügel, Holger M. Reichardt
Published March 20, 2008
Citation Information: J Clin Invest. 2008;118(4):1405-1416. https://doi.org/10.1172/JCI32698.
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Research Article

A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats

Abstract

Administration of the CD28 superagonistic antibody JJ316 is an efficient means to treat autoimmune diseases in rats, but the humanized antibody TGN1412 caused devastating side effects in healthy volunteers during a clinical trial. Here we show that JJ316 treatment of rats induced a dramatic redistribution of T lymphocytes from the periphery to the secondary lymphoid organs, resulting in severe T lymphopenia. Live imaging of secondary lymphoid organs revealed that JJ316 administration almost instantaneously (<2 minutes) arrested T cells in situ. This reduction in T cell motility was accompanied by profound cytoskeletal rearrangements and increased cell size. In addition, surface expression of lymphocyte function–associated antigen-1 was enhanced, endothelial differentiation sphingolipid G protein–coupled receptor 1 and L selectin levels were downregulated, and the cells lost their responsiveness to sphingosine 1–phosphate–directed migration. These proadhesive alterations were accompanied by signs of strong activation, including upregulation of CD25, CD69, CD134, and proinflammatory mediators. However, this did not lead to a cytokine storm similar to the clinical trial. While most of the early changes disappeared within 48 hours, we observed that CD4+CD25+FoxP3+ regulatory T cells experienced a second phase of activation, which resulted in massive cell enlargement, extensive polarization, and increased motility. These data suggest that CD28 superagonists elicit 2 qualitatively distinct waves of activation.

Authors

Nora Müller, Jens van den Brandt, Francesca Odoardi, Denise Tischner, Judith Herath, Alexander Flügel, Holger M. Reichardt

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Figure 3

Analysis of JJ316-induced cytoskeletal rearrangements ex vivo by confocal microscopy and SEM.

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Analysis of JJ316-induced cytoskeletal rearrangements ex vivo by confoca...
(A) CD4+ T cells, CD8+ cells, and B cells were isolated at the indicated time points after treatment with 1.0 mg JJ316 and stained with phalloidin–Alexa Fluor 594 (red) to visualize F-actin polymerization. Double staining with an anti-FoxP3 antibody in combination with a secondary anti-rat Alexa Fluor 488 antibody (green) allowed distinguishing between CD4+ Th and Tregs. Scale bar: 5 μm. (B) The diameters of 150 individual cells per subtype and the time point were quantified by a computer-aided method; mean ± standard error of the mean. Statistical analysis by 2-way ANOVA. The asterisks refers to the Tregs (shown on top of the graph), the pound signs to the 3 other cell types (shown below the graph). ***P < 0.001; ###P < 0.001. (C) CD4+ T cells were purified before and 12 hours after JJ316 administration and analyzed by SEM. Scale bar: 5 μm. (D) Western blot analysis of cofilin-phosphorylation in CD4+CD25– Th and CD4+CD25+ Tregs magnetically purified 72 hours after PBS or JJ316 injection. Staining with an anti-cofilin antibody served as a loading control. One representative experiment out of 3 is shown. (E) CD4+CD25– Th and CD4+CD25+ Tregs were purified 72 hours after JJ316 administration and analyzed by SEM (left panel). Scale bar: 5 μm. To visualize microtubules and to study the localization of the microtubule-organizing center, Th and Tregs were stained with an anti–β-tubulin antibody (right panel). Scale bar: 5 μm.