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Formation of functional selectin ligands on activated T cells and thymic progenitors : the role of core 2 β1,6-N-acetylglucosaminyltransferases in the control of lymphocyte trafficking and thymic progenitor homing Merzaban, Jasmeen Sayed

Abstract

The core 2 β 1,6-N-acetylglucosaminyltransferase (C2GlcNAcT) family of enzymes (C2GlcNAcT-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-I[sup null] mice also roll under shear flow on immobilized Pselectin in a PSGL-l(P-selectin-glycoprotein-ligand- l)-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[sup 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 2- sensitive mAb 1B11. This reveals the well-established C2GlcNAcT-I substrates - CD43 and CD45 - as additional physiological targets of C2GlcNAcT-III. Adoptive transfer of C2GlcNAcT-I[sup null] 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.

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