Defining human xenoglycan-specific B cells

Glycans are a common target of antibody responses. Antibodies against foreign glycans, known as xenoglycans, can protect against highly glycosylated pathogens but can contribute to allergic and inflammatory diseases, such as alpha-gal syndrome, a tickborne disease, and Crohn’s Disease. Therefore, B cells targeting xenoglycans can be harnessed for vaccination and immunotherapy to promote or limit their production of antibodies. However, the B cell subsets and molecular mechanisms underpinning xenoglycan reactivity remain poorly defined. We previously found that humans commonly generate an antibody response against an egg-derived glycan (4S-LacNAc) present in seasonal influenza vaccines. We identified that 4S-LacNAc specific monoclonal antibodies (mAbs) demonstrate some features of natural antibodies produced by B1 cells in mice. However, key distinctions between 4S-LacNAc mAbs and natural antibodies in mice suggest xenoglycan-reactive B cells are a distinct population in humans. Moreover, 4S-LacNAc specific B cell clones could be recalled from year to year, suggesting the establishment of immune memory. To study xenoglycan-reactive B cells, we have generated a blood group antigen A (BGA) specific tetramer. From non-type A human peripheral blood mononuclear cells, we found BGA-specific B cells fell within antigen-experienced B cell subsets. Whether xenoglycan-specific B cells are a distinct B cell subset or are similar to protein-reactive antibodies remains unknown. We hypothesize that xenoglycan-reactive B cells exist within functionally distinct B cell subsets that are primed for antibody secretion relative to protein-reactive B cells and that xenoglycan-specific antibodies utilize restricted repertories, are affinity-matured, and highly specific to a single glycan. First, we will determine if xenoglycan-reactive B cells reside within a distinct memory B cell pool relative to protein-reactive to B cells (Aim 1). Using tetramers for blood group antigens A and B, alpha-gal, tetanus-toxoid, and hemagglutinin, we will perform spectral flow cytometry to determine if there are phenotypic differences between antigen-specificities. Using this information, we will sort these discrete B cell populations to determine if they are enriched for other glycan specificities. We will next determine if these cell populations have increased receptor signaling relative to B cell subsets enriched for protein reactivity. Next, we will define the repertoire and cellular features of B cells targeting common xenoglycans (Aim 2). Using the tetramers described above, we will determine if xenoglycan-reactive and protein-reactive B cells have unique repertoire features and molecular signatures. Next, we will generate mAbs to test if these B cells are undergoing affinity maturation or displaypolyreactivity. Together, the results of this project will provide critical new insight into the nature of glycan-reactive B cells and will inform how these specificities can be targeted by vaccination and immunotherapy, including totreat the tickborne disease alpha-gal syndrome. Project Number: 1R21AI193369-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Jenna Guthmiller | Institution: UNIVERSITY OF COLORADO DENVER, Aurora, CO | Award Amount: $225,953 | Activity Code: R21 | Study Section: Adaptive Immunity Study Section[AI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19336901