T lymphocytes recognize antigen as small peptides bound to MHC molecules on the surface of antigen presenting cells (APC). In addition to the T cell antigen receptor and peptide-loaded MHC, dozens of other cell surface molecules play necessary or modulating roles in determining the outcome of the T cell/APC interaction. Antigen recognition is accompanied by large-scale, cytoskeleton-dependent rearrangements of these molecules to form an organized contact interface between the T cell and the APC termed the "immunological synapse". Many laboratories are investigating the ways in which the synapse may provide or regulate the signals necessary for full T cell activation. In this grant application, the focus is on the role of the immunological synapse in the delivery of effector functions from CD4 T cells to APC, using FasL (CD178) and CD40L (CD154) as prototypes of membrane-bound effector cytokines. Since most interactions in vivo may be too short to allow for de novo synthesis of effector cytokines, the proposed experiments examine the possibility that preformed, intracellular stores of FasL and CD40L play important roles in T cell/APC interactions. CD40L was first identified as the mediator of cell contact-dependent CD4 T cell help for B cells, and has since been shown to be a potent activator of macrophages for killing of intracellular pathogens of dendritic cells for priming effector functions and memory in CD4 and CD8 T cells. FasL is a principal mediator of contact-dependent killing by CD4 and CD8 T cells, and regulates T cell responses through T cell suicide or fratricide during activation-induced cell death. Using a combination of state-of-the-art microscopy and functional assays in vitro and in vivo, this proposal will test the hypothesis that pre-formed CD40L and FasL are stored in secretory lysosomes in most primed CD4 effector and memory T cells, are delivered rapidly through the immunological synapse to B cells and other APC in transient antigen-specific interactions, and have important functional consequences distinct from those of newly synthesized CD40L and FasL. It will also investigate structural difference between synapses formed by type 1 and type 2 helper T cells, and test the hypothesis that the function of the ring of adhesion molecules in the mature immunological synapse formed by CD4 T cells is to ensure antigen-specific delivery of effector molecules to the APC and prevent delivery to surrounding bystander cells. PUBLIC HEALTH RELEVANCE: T lymphocytes recognize antigen only on the surface of other cells, called antigen presenting cells (APC), and exert their effects by secreting potent cytokines and other effector molecules. The goal of this application is to understand how various kinds of CD4 T cells deliver effector cytokines to APC. The application is focused on FasL (CD178) and CD40L (CD154), key membrane-bound cytokines which control the adaptive immune response in health and disease. The proposed experiments will contribute to a basic understanding of the regulation CD4 T cell effector functions, which in turn will have public health-related applications in transplantation, autoimmunity, allergy, cancer immunotherapy, and control of infectious diseases.