A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats
Nora Müller, Jens van den Brandt, Francesca Odoardi, Denise Tischner, Judith Herath, Alexander Flügel, Holger M. Reichardt
Nora Müller, Jens van den Brandt, Francesca Odoardi, Denise Tischner, Judith Herath, Alexander Flügel, Holger M. Reichardt
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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 4

Analysis of cytoskeletal rearrangements after stimulation by JJ316 in vitro by confocal microscopy.

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Analysis of cytoskeletal rearrangements after stimulation by JJ316 in vi...
(A) CD4+ T cells, CD4+CD25+ T cells, CD8+ cells, and B cells were purified from naive rats and stimulated with JJ316 on microscopic slides for the indicated time periods. F-actin polymerization was visualized using phalloidin–Alexa Fluor 594, and CD4+ T cell populations were additionally stained for FoxP3 expression. Scale bar: 5 μm. (B) The diameters of at least 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 CD4+FoxP3+ and the CD4+CD25+FoxP3+ Tregs and the pound signs to the CD4+FoxP3– Th and the CD8+ cells (both shown on the top of the graph). Changes in the size of B cells are not significant (shown below the graph). (C) Magnetically purified CD4+CD25– and CD4+CD25+ T cells were stimulated as in A. The percentage of FoxP3+ cells in both cell populations was determined before (0 hours) and 7 hours after JJ316 stimulation in vitro. Mean ± standard error of the mean. Statistical analysis by Student’s t test. (D) CD4+ T cells were stimulated with JJ316 (JJ) for 30 minutes in the absence or presence of the PI3K inhibitor LY294002 (LY) or the MAPK inhibitor U0126 (U) and stained with phalloidin and an anti-FoxP3 antibody as in A. The relative number of polarized cells is depicted in each case as a measure for activation by JJ316. Treatment and 150 individual cells per subtype were quantified by a computer-aided method; mean ± standard error of the mean. ***P < 0.001; ###P < 0.001.