My laboratory is focussed in the area of T cell immunity. Specifically, our goal is to uncover mechanisms used by T cells that uncouple death pathways and permit these cells to survive for long periods of time in vivo. Recently, we have found that T cell costimulation provides an important growth signal, but does not confer optimal T cell survival. Also, it has been shown that bacterial lipopolysaccharide (LPS) prevents peripheral T cell deletion. In the last 3 years we have combined these observations and found that these stimuli synergize to promote profound T cell growth and survival. Based on these data it has been suggested that a T cell benefit's from having three signals in vivo as opposed to two. This raises the question of what is the nature of the third signal? To study these issues we have developed three different T cell costimulation models (CD40/CD28, OX40, and 4-1BB), or alternatively, manipulate three different costimulatory-ligand pairs in the same model of T cell activation (superantigen or peptide stimulation). In the early 90's a method was developed that allowed T cells to be observed in vivo during a response to superantigen (SAg). Basically, T cells are activated in vivo with superantigens and the responding T cells are tracked over time in vivo using T cell receptor specific mAb. Additionally, we have used the DO11.10 transfer model developed by the Jenkins' laboratory. Both systems allow us to follow superantigen or peptide-specific T cells without the problem of discerning between bystanding T cells and Ag-specific ones. These systems allow the immunobiology of T cell activation to be analyzed in vivo. In the first project we are examining the effects of CD40 ligation on T cell immunity. Our data show that even though CD40 activation significantly enhances T cell clonal expansion, the SAg-specific T cells are still destined to die and do not permanently increase the frequency of specific T cells. We found that the observed enhanced clonal expansion was a function of CD28 ligation, also demonstrating that ligation of CD28 does not block deletion in vivo. The question raised was; could these T cells be protected from deletion? We found that profound clonal expansion and long-term survival was substantially enhanced when CD40 activation was combined with a small dose of bacterial LPS (Maxwell et al, JI In Press). Our current studies are designed to uncover how synergism is manifested in this model. Our approach is to analyze the behavior of the rescued T cells before they are exposed to death stimuli in vivo which is around days 2 and 3 after SEA treatment (1). Recently, we found that the LPS-rescued cells spend a greater amount of time bound to APCs suggesting that this interaction may be important for long-term survival.In the second project the goal is to understand how OX40 functions as a T cell costimulatory signal. Just as with CD40, OX40 activation enhances T cell clonal expansion but only moderately prevents deletion. When OX40 ligation is combined with LPS stimulation, expansion and rescue from deletion are substantially increased (2). This is particularly intriguing because CD28 is the critical T cell costimulatory signal in the CD40 model and OX40 is key in the other, and yet the results are the same even though these molecules are in separate receptor families. This suggests that survival induction may be funneled down the same intracellular signaling pathway. Nevertheless, these results beg the question; can CD40 and OX40 stimulation synergize? The answer is no, only in the presence of LPS can rescue be observed with activation of either, or both, of these molecules. These data provide in vivo evidence for the existence of a third signal that functions differently than costimulation. Our current efforts are to understand when OX40 stimulated T cells are susceptible to rescue from apoptosis. The third project is centered on understanding the nature of 4-1BB T cell costimulation. 4-1BB, like OX40 and CD40, is a member of the TNF/NGF superfamily of receptors and is expressed on activated T cells. Using our SAg model we have investigated how ligation of this molecule will influence T cell activation in vivo. As with the others, clonal expansion was increased, but in contrast, T cell deletion was inhibited. Specifically, CD8 T cells were rescued, but not CD4 T cells (3). This result was further complicated by our recent findings that LPS or Poly IC, in combination with 4-1BB ligation, dramatically enhanced survival of both T cell subpopulations. As with the other models, we are teasing apart which cytokines are involved and the responsive ability of the rescued T cells. Perhaps, most interestingly, we have found that the 4-1BB stimulated T cells behave as regulatory cells in that they profoundly inhibit T cell proliferation and cytokine production. We are currently uncovering whether the tolerance effect the CD8 T cells exert on the CD4 T cells is through a death or suppressive mechanism.