T cell-to-T cell clustering enhances NF-κB activity by a PI3K signal mediated by Cbl-b and Rho

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Abstract

Full activation of T cells requires the binding of antigen to the T cell receptor and stimulation of the CD28 molecule, a process which typically occurs when T cells bind to an antigen presenting cell. The transcription factor, NF-κB, is an integration point for these two signals and its activation is critical for T cell function. Using antibodies to the TCR and CD28 molecules to activate Jurkat T cells, we show that cells that were permitted to aggregate into multi-cellular clusters increased NF-κB activity compared to unclustered cells. Inhibition of PI3K signaling with wortmannin decreased the clustering-mediated NF-κB signal. Over-expression of a dominant negative form of Cbl-b, an endogenous inhibitor of PI3K, in unclustered cells rescued NF-κB activation to the same levels caused by cell clustering. Inhibiting signaling through Rho with dominant negative RhoA abrogated both clustering-mediated and dominant negative Cbl-b-mediated NF-κB inactivation, but not TCR/CD28 mediated NF-κB activation. Taken together, these results suggest that in addition to pathways stimulated by classical T cell–APC interactions, another signal arising from T cell clustering can enhance activation.

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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