The two signal model for antigen-specific T lymphocyte activation has now been widely accepted. In this model the first signal is provided by antigen-specific activation of the TCR. The provision of a second co- stimulatory signal which is not mediated via the antigen-specific T cell receptor is necessary for activation of resting naive T cells and T cell clones leading to IL-2 production. The T cell molecule CD28 is mediating co-stimulatory signals for T cells. CD28 therefore plays a role in development of T cell unresponsiveness and development of antigen- specific tolerance. We have recently made several important observations concerning CD28 mediated signals. The first is that the CD28CYT contains the SH2 binding motif for PI3 kinase and that CD28 ligation results in binding of- PI3 kinase to CD28CYT and activation of PI3 kinase. Secondly, we made the observation that CD28 crosslinking results in tyrosine phosphorylation and activation of the tec tyrosine kinase EMT/ITK and thirdly, that the guanine nucleotide exchange factor vav becomes tyrosine phosphorylated shortly after CD28 ligation. We have also demonstrated that the human leukemic NK cell line YT, which is CD28+ can be used for evaluating CD28 mediated activation of cytolytic effector mechanisms. Our preliminary data demonstrate that specific Pi3 kinase inhibitors have a dramatic effect on CD28 mediated cytolytic effector function. furthermore, CD28 mediated signals upregulate and activate integrins (LFA-1 and VLA-4) on the Jurkat cell line, and changing tyrosine in position 173 to phenylalanine in the CD28CYT abolishes the CD28 mediated upregulation of integrin function. These studies will be further extended in the proposal.
The specific aims are: (1) To determine the minimal requirements of CD28CYT for transmission of CD28 signals following CD28 crosslinking. These studies will focus on tyrosine phosphorylation patterns, IL-2 production and LFA-1 activation. The analysis will be performed using CD28CYT deletion mutant and substitution mutations of tyrosines in the CD28CYT. (2) To determine if PI3-kinase binding to CD28CYT is essential for CD28 mediated signal transduction leading to cytolytic effector function. (3) To determine the structural interaction sites between EMT kinase and CD28CYT and the effects of EMT- dominant negative mutations on CD28 mediated activation signals. These studies will delineate the downstream effects of CD28 activation.
