The purpose of the present proposal is to provide the candidate, a clinical fellow in the UCSF division of Rheumatology, with training for a career in the study of the basic mechanisms of autoimmune disease. The candidate will participate in a didactic program which will provide training in molecular biology, cell biology, and the biochemistry of signal transduction. Laboratory experience will focus on an investigation of the activation of T lymphocytes, cells which play a central role in the initiation of an immune response. Current models of T cell activation suggest that at least two types of activation signals are required, an antigen-specific signal as a result of antigen recognition by the T cell receptor (TCR), and an antigen-independent 'costimulatory' signal delivered by antigen presenting cells (APC). The absence of costimulatory activity may protect self tissues from an autoimmune response to tissue-specific antigens, and the delivery of a TCR stimulus in the absence of costimulatory signal(s) may result in inactivation of the T cell by a process known as clonal anergy. Recent work has demonstrated that stimulation of the T cell surface molecule CD28 synergistically activates T cells in combination with TCR stimulation, and activated APC express a ligand for CD28. The present proposal will focus on two related investigations of the role of CD28 as a costimulatory molecule for human T cells. First, the nature of the structural requirements of signalling via CD28 will be investigated using a PCR-directed mutagenesis strategy to define the region(s) of the CD28 molecule which are necessary for activation of signalling machinery. The signalling function of mutated CD28 molecules will be investigated by reconstituting CD28-negative leukemic T cells in order to functionally define whether intracellular, transmembrane, or extracellular regions of the molecule activate signal transduction. Polypeptides from the relevant region of CD28 will then be used in attempts to affinity purify and/or identify the CD28-associated signal transduction molecules, information which may lead to rational strategies for pharmacologic inhibition of T cell activation. A second project will investigate the role of CD28 in activation of IL2 secretion by antigen specific human T cell clones. The ability of CD28 and other signalling molecules to provide costimulatory signals and prevent induction of clonal anergy in antigen-specific responses will be investigated using both functional assays (IL2 secretion and proliferation) and IL2 gene enhancer constructs capable of detecting the activity of the recently identified CD28-induced transcriptonal activator complex. Anergy may be mediated by interruption of the TCR signalling pathway, interruption of the CD28 signalling pathway, or induction of an inhibitory signal which interferes with IL2 gene transcription. The lab's previous experience in analyzing the biochemical events in TCR signalling combined with further knowledge of the biochemical events in CD28 signalling will allow investigation of these possible mechanisms of anergy induction.
| Stein, P H; Fraser, J D; Weiss, A (1994) The cytoplasmic domain of CD28 is both necessary and sufficient for costimulation of interleukin-2 secretion and association with phosphatidylinositol 3'-kinase. Mol Cell Biol 14:3392-402 |
