Biochemical and Biophysical Research Communications
T cell-to-T cell clustering enhances NF-κB activity by a PI3K signal mediated by Cbl-b and Rho
Section snippets
Materials and methods
Materials. NF-κB luciferase reporter and anti-CD3 antibody were gifts from Ron Wange (National Institute on Aging; Baltimore, MD). Cbl-b expression vectors were gifts from Stan Lipkowitz (National Cancer Institute; Bethesda, MD). Anti-CD28 and anti-LFA-1 antibodies were purchased from Pharmingen (San Diego, CA). Poly-lysine coated coverslips were purchased from BD Biosciences (Bedford, MA). PMA, A23187, and wortmannin were purchased from Sigma (St. Louis, MO). The Rho constructs were gifts from
Clustered T cells have greater NF-κB activity than surface-adsorbed unclustered T cells
Once activated, T cells form direct contacts with other T cells to form large T cell aggregates or clusters. In order to determine if the formation of T cells into clusters results in a change in NF-κB activity, Jurkat T cells that were transfected with a luciferase reporter plasmid containing the DNA-binding site for NF-κB were stimulated with anti-CD3 and anti-CD28 antibodies under conditions in which the cells were allowed to form clusters (Fig. 1A, left panel) or where cluster formation was
Discussion
T cell activation has long been known to require co-stimulation of the TCR and CD28 molecules . Here, we show that in addition to these signals, homotypic cell-to-cell clustering significantly enhances T cell activation. We propose that in addition to the co-stimulatory step that occurs between a T cell and an antigen presenting cell, a clustering step significantly enhances NF-κB activation. Furthermore, this clustering-mediated activation appears to involve the downregulation of Cbl-b
Acknowledgments
The authors thank Drs. Ron Wange, Stan Lipkowitz, and Peter Burbelo for their gifts of reagents and Dr. Jit Dave for assistance with photography. This work was supported in part by NIBIB (EB 00262) and NHLBI (HL073305).
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