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Abstract
Trogocytosis, the process whereby lymphocytes capture membrane components from the cells they interact with, is classically evidenced by the transfer of fluorescent lipophilic compounds or biotinylated proteins from target cells to T or B cells. A particular class of molecules, not studied explicitly so far in the context of trogocytosis is glycoconjugates. Here, we used a method to metabolically install chemical labels in target cell glycoconjugates. Working with those target cells, we describe the conditions allowing CTL to be detected based on glycoconjugate trogocytosis triggered by antigen or stimulatory antibodies. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this approach to the detection of CD4+ T and B cells. Overall, glycoconjugates were transferred between target cells and lymphocytes during trogocytosis with efficiencies comparable or higher than measured for biotinylated proteins or lipophilic dyes incorporated into general membrane lipids. From a technological point of view, our approach can be employed to detect reactive lymphocytes via glycoconjugate trogocytosis. More generally, we believe that the ever-growing ability to employ chemistry in living systems to label particular compounds will be powerful in unraveling the contributions of glycosylation to various aspects of T and B cells biology.
| ABBREVIATIONS | ||
| Ac4ManLev: | = | Peracetylated N-levulinoyl mannosamine |
| Ag: | = | Antigen |
| APC: | = | Allophycocyanin |
| ARP: | = | Aldehyde reactive probe |
| BCR: | = | B cell receptor |
| Biotin-LC-sulfo-NHS: | = | sulfo-N-hydroxy-succinimidyl ester-long chain biotin |
| CTL: | = | Cytotoxic T lymphocytes |
| CyaA: | = | Adenylate cyclase from Bordetella pertussis |
| DiO: | = | 3,3′-dioctadecyl-oxacarbocyanine perchlorate |
| E:T: | = | Effector-to-target |
| EDTA: | = | Ethylene diamine tetraacetic acid |
| FcR: | = | Fc receptor |
| FCS: | = | Fetal calf serum |
| GalNAz: | = | N-azidoacetylgalactosamine |
| HEL: | = | Hen egg lysozyme |
| IS: | = | Immunological synapse |
| ManLev: | = | N-levulinoyl mannosamine |
| mAb: | = | Monoclonal antibody |
| ManNAz: | = | N-azidoacetylmannosamine |
| MHC: | = | Major Histocompatibility Complex |
| MOE: | = | Metabolic oligosaccharide engineering |
| OVA: | = | Ovalbumin |
| PBS: | = | Phosphate-buffered saline |
| SiaLev: | = | N-levulinoyl sialic acid |
| TCR: | = | T cell receptor |
| TL: | = | Thymic Leukemia |
| TRAP: | = | TRogocytosis Analysis Protocol |
Notes
*S.D. and A.A. contributed equally to this work.