It is becoming increasingly evident that along with the level of cognate peptide-MHC complexes (pMHC) their distribution on the cell surface as well as association with other membrane proteins also influences the antigen presentation. We have shown that MHC class I molecules (MHC-I) are co-clustered with adhesion molecules (ICAM-1) in both membrane rafts and soluble membrane fraction and that productive engagement of these molecules leads to their additional recruitment into rafts and increased association with Scr kinases. Disruption of the raft integrity on target cells decreases potency of viral peptides presentation to CTL. We believe that productive engagement of raft-included MHC-I and ICAM-1 during CTL-target cell encounter results in a signaling that augment presentation of cognate pMHC complexes by target cells to CTL. Based on this, we are set up to investigate functional significance of molecular assemblies on target cells and CTL. We want to identify membrane and intracellular proteins involving in these assemblies and to define mechanisms of "cross-talk" between these molecules. Our efforts are supported by 5 RO1 AI52812-01 Grant from NIAID. One important aspect of the proposal has been missing thus far. Namely, it is essential to determine the level and distribution of cognate pMHC complexes on target cells. This requires a reagent that can distinguish MHC molecules loaded with a peptide of interest from other pMHC complexes. A soluble TCR analog specific for a given pMHC may serve as an elegant tool to probe the pMHC on the surface of target cells. Because of the relatively low affinity of the TCR-pMHC reaction, soluble TCR should be oligomerized to increase the avidity of the binding. We have shown that tetrameric TCR from CTL clone D3 specific for p17 Gag epitope SLYNTVATL (SL9) can specifically detect the presence of SL9-HLA-A2 complexes on target cells. However, at lower epitope density the distance between the pMHC complexes on the cell surface may be too long for the tetramer to engage all binding sites at the same time resulting in a loss of its avidity. With this in mind, we propose here to produce other forms of oligomeric D3 TCR by increasing the valency of the tetramer and/or the distance between tetramer binding sites. We will use these reagents to characterize the distribution of SL9-HLA-A2 complexes on target cells infected with recombinant Vaccinia virus carrying HIV Gag and will determine how this parameter influences sensitivity and magnitude of CTL responses. In the future, these new reagents will be used to detect HIV infected cells in samples derived from infected individuals.