Biophysical characterization of molecular mechanisms controlling T cell activation: Role of coreceptor CD8

T cells undergo dynamic changes in their biological sensitivity during their development and differentiation. Consequently, T cells that have not encountered antigen, naive cells, require higher antigenic dose and co-stimulatory interactions to elicit a biological response as compared to immature (thymocytes) and antigen experienced (activated) T cells. The development and differentiation of T cells is accompanied by changes in gene expression partly responsible for the modulation of biological sensitivity, however molecular interactions between the T cell receptor (TCR), coreceptor CD8 and their ligand peptide-major histocompatibility complex (pMHC) are also altered during these different states. To date biophysical characterization of these changes in TCR-pMHC-CD8 interactions and the molecular mechanisms controlling them are not clearly understood.;Since T cell development and differentiation are accompanied by changes in glycosylation, specifically sialylation levels of the coreceptor CD8, to investigate the impact of sialylation on TCR-pMHC-CD8 interactions, quantitative binding assays were performed on untreated naive, activated and desialylated naive T cells using soluble pMHC ligand. Sialic acids were removed from T cells by neuraminidase treatment, a sialidase that removes terminal sialic acids from glycoproteins. Equilibrium binding data showed marked increase in pMHC-Ig binding on desialylated and activated cells compared to naive T cells. Mathematical modeling of the binding data suggested that the increased binding was due to enhanced TCR/CD8 cross-linking on desialylated and activated T cells. We further probed differences between desialylated and activated T cells by performing binding assays with pMHC monomers. These experiments revealed differences in molecular organization of receptors on desialylated and activated T cells. Mathematical modeling of the monomer binding data showed presence of a clustered state of TCR/CD8 on desialylated T cells. This clustered receptor state was proposed to bind to pMHC monomer with high avidity due to the presence of multiple binding sites. Enzymatic resialylation and blocking CD8 interactions supported the role of sialic acids on CD8 for regulating the increased avidity of TCR/CD8 on desialylated T cells. Collectively, this increased receptor avidity upon desialylation translates into enhanced biological response supporting the hypothesis that changes in sialylation could be one of the possible mechanism for regulating biological sensitivity of T cells.;To further characterize TCR-pMHC and CD8 interactions, studies were performed using an anti-CD8 antibody, 53.6.7. Treatment of 53.6.7 antibody resulted in a 3-8 fold increase in the affinity of pMHC ligand to T cells. The ability of 53.6.7 Fab to increase the affinity of pMHC ligand suggests that an antibody stabilized conformation of CD8 is responsible for the increased CD8-pMHC interaction. Infact, treatment with 53.6.7 enhanced early time T cell signaling events, such as Ca2+ upregulation, intracellular phosphorylation and TCR downregulation, correlating well with the increased binding of the pMHC ligand. However, downstream T cell biological responses, such as upregulation of activation markers, cytokine production and cytolytic ability of T cells were actively suppressed by 53.6.7 treatment. These results highlight discordance in early and late T cell signaling events in response to an enhanced CD8-pMHC interaction. These results suggest that alterations in molecular organization of TCR/CD8 and/or flexibility of CD8 molecule are the molecular mechanisms affecting the downstream biological responses upon 53.6.7 treatment.;We also developed a model system to study structural details of ligand induced receptor clustering by small angle neutron scattering (SANS). Fab' molecules were covalently attached to lipid bilayer and were cross-linked by an antibody. Our preliminary results show changes in the SANS spectra after antibody binding indicating presence of Fab' clustering on the membranes. These results validate the use of SANS technique to study structural details of membrane bound receptor-ligand interactions.