Formation of functional selectin ligands on activated T cells and thymic progenitors: The role of core 2 beta1,6-N-acetylglucosaminyltransferases in the control of lymphocyte trafficking and thymic progenitor homing

The core 2 beta 1,6-N-acetylglucosaminyltransferase (C2G1cNAcT) family of enzymes (C2G1cNAcT-I,-II,-III) synthesize branched O-glycans. A significant body of work highlights the importance of C2GlcNAcT-I in controlling selectin-ligand-mediated cell trafficking while little is known about the role of the two other C2GlcNAcT isoenzymes.; 1. The first objective of this thesis is to determine in vitro and in vivo T cell stimulation conditions that guide P-selectin ligand expression in absence of C2GlcNAcT-I. Mitogen stimulation of splenocytes maintained under very-high-density culture conditions uncovers C2GlcNAcT-I-independent P-selectin ligand formation in CD8 + T cells, but not CD4+ T cells. CD8+ T cells of C2GlcNAcT-Inull mice also roll under shear flow on immobilized P-selectin in a PSGL-1 (P-selectin-glycoprotein-ligand-1)-specific manner. Using RT-PCR analysis, we identify C2GlcNAcT-III as the likely source of core 2 activity. Up regulation of P-selectin ligand in C2GlcNAcT-I null CD8+ T cells correlates with higher core 2 enzyme activity, as measured by a standard enzymatic assay and cell-surface binding of the core 2sensitive mAb 1B11. This reveals the well-established C2GlcNAcT-I substrates---CD43 and CD45---as additional physiological targets of C2GlcNAcT-III. Adoptive transfer of C2GlcNAcT-Inull T cells from mice transgenic for the male antigen (HY) T cell receptor shows that C2GlcNAcT-I-independent P-selectin ligand formation occurs on CD8+ T cells under in vivo stimulation conditions. In consequence, C2GlcNAcT-III emerges as a contributor to P-selectin ligand formation that may co-operate with C2GlcNAcT-I to control CD8+ T cell trafficking.; 2. A second objective of this thesis is to explore whether C2GlcNAcT-I plays a role in controlling early T cell progenitor migration to the thymus. PSGL-1 is expressed by these thymic progenitors. In order for PSGL-1 to be recognized by P-selectin which is constitutively expressed at low levels on the thymic endothelium, it must be modified by C2GlcNAcT-I. Using mice with deficiencies in PSGL-1, C2GlcNAcT-I and P-selectin, we employ parabiosis and competitive repopulation to show that C2GlcNAcT-I modification of PSGL-1 expressed on thymic progenitors is a functionally imperative component of the thymic homing process.