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 1

Administration of JJ316 induces T lymphopenia and leads to the redistribution of CD4+ T cells.

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Administration of JJ316 induces T lymphopenia and leads to the redistrib...
(A) Blood samples from rats i.v. injected with 1.0 mg, 0.2 mg, or 0.04 mg JJ316 were taken at the indicated time points and analyzed for the percentages of circulating T cells (left panel) and granulocytes (right panel) by flow cytometry. n = 6; mean ± standard error of the mean. Statistical analysis refers to the individual comparison of each time course to untreated control rats by 1-way ANOVA. (B) Leukocytes were isolated from lymph nodes, spleen, lung, and liver 10 and 72 hours after injection of 1.0 mg JJ316 or PBS as a control. Subsequently, the total number CD4+ T cells was determined by microscopic counting followed by flow cytometric analysis of CD4 and TCRβ expression. n = 3; mean ± standard error of the mean. (C) The relative frequencies of CD4+ naive (CD45RC+RT6.1+), activated (CD45RC–RT6.1–), and memory T cells (CD45RC–RT6.1+/CD45RC+RT6.1–) was determined 10 and 72 hours after application of JJ316 or PBS. Thy-1+ RTEs (recent thymic emigrants) were excluded from the analysis. n = 3; mean ± standard error of the mean. (D) CFSE-labeled lymphocytes (4 × 107) were transferred into recipient rats i.v., and 3 days later 0.2 mg JJ316 or PBS were administered. After 10 hours the frequencies of CFSE+CD4+ T cells in blood, liver, lung, and spleen were determined by microscopic counting and flow cytometry. n = 3; mean ± standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001.