Effects of human neuraminidase and multivalent glycoconjugates on CD22 organization

Abstract

The human immune system is a network that is crucial to fight against infections and foreign pathogens. It is divided into innate system, which is the first line of defense, and adaptive immune system, which is more specific. The specificity and long-lasting nature of the adaptive immune system is dependent on the clonal expansion of lymphocytes, T cells and B cells, after exposure to pathogens. However, complications can occur when the adaptive immune system is compromised, leading to conditions such as rheumatoid arthritis, lupus, asthma, and immunodeficiency. Therefore, tight control of these systems is necessary, and cellular receptors are partly responsible for this control. Moreover, understanding how to manipulate the immune system may allow us to develop new therapeutics, or overcome immune-incompatibility in transplantation. In this thesis we explore the use of glycoconjugates to alter the organization of immune receptors. We were interested in studying B cell receptors which recognize glycans, such as the well-known negative regulator of BCR, CD22. Although the function of CD22 is well known, its organization and mechanism are poorly understood. CD22 binds to 2,6-sialosides and is basally masked by cis-ligands on the same cell surface, making interactions with trans-ligands more difficult and require high-affinity ligands. We tested the ability of multivalent displays of CD22 ligands along with and specific antigens to BCR to co-cluster these receptors. We selected B cells expressing BCR which recognize human blood group antigen structures. We observed that these ligands could co-cluster CD22 and BCR on cells. We also investigated the role of native enzymes in the organization of CD22 receptors on cultured B cells. We hypothesized that neuraminidase (NEU) enzymes, which cleave terminal sialic acids that may act as cis ligands for CD22, could have a role in regulating receptor organization and dynamics. We used confocal microscopy to visualize and quantitate CD22 clustering on individual cells. We confirmed that NEU enzymes had a role in organizing CD22 receptors on the cell membrane, and both NEU1 and NEU3 were found to influence the size and lateral mobility of CD22 clusters.