Abstract
Regulatory T Cells (Tregs) constitutively express the inhibitory receptor CTLA-4, which is fundamental to their role in immune suppression. Mechanistically, CTLA-4 on Tregs can attenuate T cell activation by physically removing and internalizing costimulatory ligands CD80 and CD86 from the surface of antigen-presenting cells by transendocytosis. Therefore, the process of transendocytosis can be harnessed as a tool to study the molecular basis of CTLA-4 biology and a key aspect of Treg suppressive function. In this chapter, we describe a method of human Treg isolation and expansion resulting in high CTLA-4 expression. We then detail a transendocytosis assay using artificial antigen-presenting cells (DG-75 B Cell lines) expressing fluorescently tagged ligands mixed with the expanded Tregs. This methodology can be applied to testing of patients carrying CTLA-4 mutations, providing a robust model to assess the degree of functional disruption.
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This research was funded in whole, or in part, by the Wellcome Trust (Grant 204798). For the purpose of Open Access, the author has applied a CC BY public copyright license to any author accepted manuscript version arising from this submission.
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Waters, E., Williams, C., Kennedy, A., Sansom, D.M. (2023). In Vitro Analysis of CTLA-4-Mediated Transendocytosis by Regulatory T Cells. In: Ono, M. (eds) Regulatory T-Cells. Methods in Molecular Biology, vol 2559. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2647-4_12
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DOI: https://doi.org/10.1007/978-1-0716-2647-4_12
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