A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats
J. Clin. Invest. Nora Müller, et al. 118:1405 doi:10.1172/JCI32698 [Go to this article.]

Figure 6
JJ316 induces proadhesive changes and interferes with S1P-mediated migration. (A) Lymph node cells were analyzed before and 10 hours after application of 1.0 mg JJ316 for surface expression of LFA-1 (α_Lβ₂) and CD62L (L selectin) on CD4⁺ T cells by flow cytometry. (B) CD4⁺ T cells were purified from JJ316-treated rats at the indicated time points and analyzed for Edg1 mRNA expression by quantitative PCR. The values were normalized to β-actin (Actb) levels. n = 4; mean ± standard error of the mean. *P < 0.05. (C) The chemotactic response of CD4⁺ T cells isolated 12 hours after JJ316 or PBS injection toward different concentrations of S1P was studied using a transwell migration assay. The index refers to the relative number of cells that had transmigrated after 3 hours as compared with the number of cells that passed the filter in the absence of S1P. n = 4; mean ± standard error of the mean. *P < 0.05. (D) CD4⁺ T cells were purified from JJ316-treated rats at the indicated time points and analyzed for surface expression of CD69 by flow cytometry. Anti-mouse IgG1κ was used as an isotype control. (E) Blood samples from rats i.v. injected with 1.0 mg JJ316 or 0.1 mg FTY720 were taken at the indicated time points and analyzed for the percentages of circulating T cells (left panel) or granulocytes (right panel) by flow cytometry. n = 5; mean ± standard error of the mean. Statistical analysis was performed by comparing the 2 time courses using a 1-way ANOVA.