Effect of galectin-9 on Immunoglobulin D surface binding and internalization in pre-basophilic and T cell lines

Student thesis: Doctoral ThesisDoctor of Philosophy


Immunoglobulin D remains the only antibody class with unknown specific function and structure. It was long thought to simply be expressed as a B cell receptor alongside IgM on B cell surfaces. The discovery of soluble IgD, as well as its elevated presence in certain inflammation-driven human conditions such as asthma and rheumatoid arthritis, has shifted this paradigm and has raised many questions regarding its exact role in health and disease. In recent years, IgD has been ascribed both an innate-like function, being able to bind bacterial superantigens, as well as involvement in hypersensitivity reactions to common antigens, through the action of a yet uncharacterized receptor (IgDR) on effector cell types. Thus, IgD may facilitate the link between the microbiome and asthma development, which has been observed in the past. Recent evidence has suggested the hyaluronan receptor CD44 and its ligand – the immunomodulator galectin-9 – as parts of the putative IgDR, engagement of which leads to immune response enhancement.

In this project I examined the IgD-binding and internalization capabilities of the human pre-basophilic KU812 cell line (CD44+) and the human T cell line Jurkat (CD44-). A custom IgD fluorescent probe was developed and used in conjunction with flow cytometry and confocal fluorescent microscopy. Flow cytometry showed relatively weak, temperature-dependent binding of IgD to the two cell types, which was markedly enhanced by galectin-9 addition. It is here demonstrated for the first time, via confocal microscopy, that labelled-IgD is internalized and accumulated into early and/or recycling endosomes in the perinuclear region of these cells. This internalization is inhibited by exogenous galectin-9, which retained IgD to the surface. Proteins from cell lysates captured via IgD-pulldown were analyzed by LC/MS for candidate receptor identification. Although speculative, according to the LC/MS data, the glycoproteins CD59 and CD82 seem to be the most promising putative IgD-binding partners in untreated and galectin-9-treated cells, respectively. Taken together, these findings point to a modal function of IgD in effector cells, which is dependent on the presence of inflammation-inducible proteins such as galectin-9.
Date of Award1 Aug 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorAndrew Beavil (Supervisor) & James McDonnell (Supervisor)

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