Project description:In collaboration with Professor Anne Dell at the South Kensington Campus of Imperial College London, we have conducted preliminary glycomic surveys of C57BL/6 CD25+ and CD25- CD4+ T cells. Cells selected in serum-containing culture medium demonstrated unusual N-glycan profiles compared to those prepared in phosphate buffered saline (PBS). These profiles were subsequently shown to result from cellular sequestration of glycoproteins present in fetal calf serum (FCS) used to supplement the medium (1). Analysis of the N-glycan profile of murine CD4+CD25+ Tregs prepared in PBS revealed distinct differences from CD4+CD25- T cells – namely, a reduction in sialic acid capping and loss of core fucose. Preliminary studies are underway to ascertain the minimum medium required to sustain general T cell survival, whilst maintaining ‘clean’ glycomic profiles. The effect of glycoprotein withdrawal on CD25+ and CD25- CD4+ T cells in culture was investigated by selecting the cells in FCS-free medium and incubating them for six hours with either FCS-containing medium, or PBS supplemented with FCS, fetuin or fetuin-derived glycans. Early apoptosis was measured using Annexin-V (BD Biosciences, Heidelberg, Germany) and late apoptosis using 7-aminoactinomycin D (7-AAD) (BD Biosciences), in parallel with a complementary apoptosis detection method – measurement of the mitochondrial membrane potential using the BD™ Mitoscreen Kit (BD Biosciences). This apoptosis staining study demonstrated that there was a remarkably higher percentage of death in both CD25+ and CD25- CD4+ T cells cultured in PBS versus cells cultured in RPMI-1640 or medium containing FCS. Both CD25+ and CD25- CD4+ T cells were highly susceptible to apoptosis in glycoprotein-free medium, demonstrating their exquisite sensitivity to the lack of glycoprotein survival factors. Continuing studies are addressing the minimum medium required to sustain cells undergoing polyclonal activation in vitro and the impact of activation on the N-glycan profiles of Tregs versus conventional T cells. To complement this work, we used Core E resources to investigate glycosyltransferase gene expression in freshly isolated versus activated C57BL/6 CD25+ and CD25- CD4+ T cells.

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Glyco-gene expression in naive and activated murine regulatory T-cells vs. conventional T-cells

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