Engagement of CD28 outside of the immunological synapse results in up-regulation of IL-2 mRNA stability but not IL-2 transcription.

During T cell activation by APC, CD28 is colocalized with TCR in the central supramolecular activation cluster (cSMAC) region of the immunological synapse. CD28 signaling through PI3K results in the recruitment of protein kinase C (PKC)theta to the cSMAC, activation of NF-kappaB, and induction of IL-2 transcription. These results suggest that localized engagement of CD28 within the cSMAC may be required for CD28 activation and/or signal integration with TCR signals. To test this model we have examined the mechanism of CD28-mediated induction of IL-2 secretion when CD28 is engaged outside of the immunological synapse. CD4 T cells were stimulated with Ag presented by B7-negative APC and CD28 costimulation was provided in trans by anti-CD28-coated beads or by class II-negative, B7-positive cells. We show that induction of IL-2 secretion under these conditions did not require expression of PKCtheta and did not induce NF-kappaB activation or IL-2 transcription. In contrast, CD28 costimulation in trans did induce IL-2 mRNA stability, accounting for the up-regulation of IL-2 secretion. These data indicate that the ability of CD28 to up-regulate IL-2 transcription requires colocalization of TCR and CD28 at the plasma membrane, possibly within the cSMAC of the immunological synapse. In contrast, the ability of CD28 to promote IL-2 mRNA stability can be transduced from a distal site from the TCR, suggesting that signal integration occurs downstream from the plasma membrane. These data support the potential role of trans costimulation in tumor and allograft rejection, but limit the potential functional impact that trans costimulation may have on T cell activation.