Aim 1. The mechanisms utilized by T-cells to signal a positive or negative response are central to their function. Strategies have been developed to dissect the consequences of the earliest tyrosine phosphorylation events in signaling using antisense RNA in stably transfected T-cell clones. The consequences on T-cell signaling by altered peptide ligands will be characterized in the 2C CTL response. Anti-sense transfectants will be generated and effects on signaling pathways determined. Comparison of the effects on signaling in antisense transfectants of Th1 and Th2 clones will also be made. In the CD4 clones chosen for study the mechanism by which dissociation between cytokine secretion and other effector functions will be studied.
Aim 2 will utilize the highly developed 2C CTL experimental system to define our understanding of T-cell recognition. The affinity of the 2C TCR for its cognate allopeptide, p2Ca, as well as mutants of 2C has been determined and can now be utilized to explore the effect of affinity on well defined biological processes, including positive selection and antagonism of the 2C TCR.
Aim 3 will focus on the importance of the CD8 co-receptor which has been shown to strengthen the functional response where mutant peptides have been utilized that show very low or undetectable affinity. Methods will be developed to evaluate low affinity interactions in the 2C system between the TCR and MHC/peptide complex. This will include the development of a system to examine the contribution of CD8-plus TCR binding to wildtype and mutant peptide MHC complexes.
