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-kappa B, 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-kappa B activity compared to unclustered cells. Inhibition of P13K signaling with wortmannin decreased the clustering-mediated NF-kappa B signal. Over-expression of a dominant negative form of Cbl-b, an endogenous inhibitor of P13K, in unclustered cells rescued NF-kappa B 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-kappa B inactivation, but not TCR/CD28 mediated NF-kappa B 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. (c) 2005 Elsevier Inc. All rights reserved.