Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Ravetch Laboratory

Abstract

Antibodies are a fundamental component of the adaptive arm of the immune system. Interactions of the antibody’s fragment crystallizable (Fc) domain with Fc receptors (FcR) on effector leukocytes is essential for proper immunological function and bridges the humoral with the cellular components of immunity. While the Fc domain of immunoglobulin G (IgG) has historically been considered to be relatively constant, recent studies have demonstrated that in conjunction with IgG subclasses, the single conserved N-linked glycan present on the CH2 domain imparts a considerable amount of heterogeneity in the molecule. Given the stepwise construction of this N-glycan structures, families of highly related, but non-equivalent, glycoproteins (known as glycoforms), are produced which can elicit vastly different effector functions. In the first part of this thesis, I examine the expression of FcγRs on T cells in a variety of immunological settings. Molecular studies between mouse and human FcγRs and accessory molecules (Fc common gamma chain and CD3ζ), demonstrate considerable interspecies differences in heterooligomer pairing which may account for the differences observed between species in FcγR expression patterns. Furthermore, using a FcγRhumanized model, I demonstrate that FcγRIIIa is induced on CD8+ T cells in the context of viral infection, and functions as a novel co-stimulatory molecule which can lower the threshold of TCR stimulation required for cellular activation. In the context of viral infections, host outcome is often not dictated by viral cytopathicity, but instead by the quality and the magnitude of the immune response. The abundance of IgG glycoforms produced during an infection can vary greatly, modulating the immune effector repertoire throughout the course of infection. Notably, several studies have demonstrated the increase of afucosylated IgG glycoforms, which exhibit enhanced FcγRIIIa binding, during viral infections and inflammation. Despite the importance of studying these glycoforms and the potential role that they may play in signaling through FcγRIIIa on T cells, there is a clear scarcity of tools to selectively manipulate these molecules. To address this, in the second part of this thesis, I highlight the discovery of a novel class of engineered nanobodies which can discriminate between IgG glycoforms. Using this platform, I define two classes of nanobodies, one which can recognize IgG lacking its core-fucose and one which can recognize IgG bearing terminal sialic acid residues. Structural studies of these nanobodies reveal that these molecules have a novel mode of recognition, utilizing protein-protein and protein-carbohydrate contacts for binding. Lastly, I demonstrate that these nanobodies can be used as powerful prognostic tools in the settings of dengue virus and SARS-CoV2 infection, as novel therapeutics for the selective disruption of Fc-FcγR interactions, and as research tools for interrogating the B cell receptor (BCR) glycan structure.

Comments

A Dissertation Presented to the Faculty of the David Rockefeller Graduate Program in Biosciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

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