| Cytotoxic CD8+ T lymphocytes mediate an immune response by inducing cytolytic killing of cells expressing antigenic peptides presented on Class I MHC molecules. Recognition of MHCI ligand by the clonotypic T cell receptor (TCR) is aided by the coreceptor, CD8, a heterodimeric glycoprotein whose extracellular region consists of an Ig-like head domain and a heavily glycosylated mucin-like stalk. The coordinate (cognate) binding of both TCR and CD8 to the same MHCI molecule is vital for T cell activation. In addition, CD8 can also bind to MHCI independently of the TCR (i.e. non-cognate binding). The avidity of this non-cognate binding is highly regulated during development and differentiation, in part due to changes in sialylation of glycan appendages on CD8. These changes can be monitored on the cell surface by the use of multimeric MHCI ligands. We show here that MHCI binding to CD8 on T cell surfaces is highly dependent on metabolic activity, and that staining conditions can be modified so that robust and specific CD8 binding can be seen on T cells at all stages. We then use MHCI multimers to show that CD8 binding is dramatically altered during the course of T cell activation, which can also be attributed, at least in part, to changes in surface sialylation. The specific sialyltransferase, ST3Gal-I, was reexamined for its impact on non-cognate binding. In contradistinction to previous reports, the ST3Gal-I knockouts displayed similar non-cognate binding compared to the wild type and influences CD8 sensitivity through an alternative mechanism. |
