The interplay between Siglecs and their glycan ligands in humoral immunity
Creators: Enterina, Jhon Ralph De La Peña
Publisher: name: University of Alberta Library
Year: 2024

<p>Production of high affinity antibodies following an infection or vaccination requires the formation of germinal centres (GCs). These anatomic microstructures within the secondary lymphoid tissues are sites for B cells to expand and mutate their immunoglobulin genes. GC B cell clones with reasonable affinity to their antigen are positively selected to either differentiate into plasma cells and memory B cells or cycle back in the GC to accumulate more mutations. On the other hand, clones with no or extremely weak affinity to the antigen are eliminated through apoptotic cell death. <br>
Precise detection of GC B cells in humans and mice relies on a unique repertoire of cell surface glycans that are specifically expressed on these cells. However, functional roles for these glycosylation changes in the GC have yet to be determined. In mice, one striking modification relates to the switch in sialic acid monosaccharide structure from Neu5Gc on naïve B cells to Neu5Ac on GC B cells due to the downregulation of the hydroxylase enzyme CMAH. Consequently, this alteration results in the loss of high affinity glycan ligands for both CD22 and Siglec-G, two members of the Siglec protein family and known inhibitory co-receptors on B cells. To dissect the role of glycan remodeling and Siglecs in the GC B cell response, we utilized genetic mouse models as well as functional and transcriptomic assays of B cells to identify whether these receptors and modifications of their glycan ligands play a crucial role in the GC reaction. <br>
In Chapter 3, using a mouse model that maintains high affinity CD22 ligands on GC B cells, we demonstrated that glycan remodeling plays a critical role in the maintenance of B cells in the GC. Sustained expression of CD22 ligands induces higher levels of apoptosis in GC B cells, reduces memory B cell and plasma cell output, and delays affinity maturation of antibodies. These defects are CD22-dependent, demonstrating that downregulation of CD22 ligands on B cells plays a critical function in the GC. <br>
In Chapter 4, we characterized the frequency of high affinity ligands for CD22 on GC B cells using mouse CD22-Fc fusion protein. We discovered that GC B cells express glycans with broad range affinities to CD22 despite significant upregulation of Neu5Ac sialic acid. GC B cells expressing either low affinity or high affinity glycan ligands for CD22 displayed differences in the frequency of high affinity clones and presence of GC B cells with pre-memory B cell (MBC) phenotype. Mechanistically, we identified that these heterogeneity in CD22 ligands was critical for the maintenance of the pre-MBC population within the GC partly by controlling the B cell stimulation following antigen engagement and B cell-T cell interaction. <br>
In Chapter 5, we examined the role that Siglec-G plays in humoral immunity. Our work identified that conditional deletion of Siglec-G on B cells after a T-dependent immunization results in augmented GC and antibody responses. Ex vivo assays revealed Siglec-G expression on GC B cells is not critical for BCR signaling and antigen internalization and processing. However, mice lacking Siglec-G on B cells display a highly activated follicular CD4+ T cell phenotype, as evident by upregulation of transcription factors BCL6 and Nfact1 as well as enhanced expression of key co-stimulatory receptors.<br>
Taken together, my thesis provides compelling evidence that Siglecs and programmed glycosylation changes are crucial in optimizing the GC reaction by modulating the strength of BCR signaling and quality of T cell help.</p>