Evidence for primordial germinal centers in fish
Creators: Waly, Doaa
Publisher: name: University of Alberta Library
Year: 2022

<p>Affinity maturation is the process to improve antibody affinity for an antigen during an adaptive<br>
immune response; it is mediated by the immunoglobulin mutator enzyme activation-induced<br>
cytidine deaminase (AID). This process is crucial to produce high-affinity memory B-cells and<br>
plasma cells. In homeotherms, affinity maturation and the subsequent selection of B-cells<br>
expressing higher affinity antibodies occur in histologically distinct germinal centers (GCs), which<br>
are composed primarily of B-cells, T follicular helper cells, and follicular dendritic cells. Within<br>
the germinal centers, antigen-specific B-cells proliferate while acquiring AID mediated mutations<br>
in their VDJ exon; these cells then compete for a limited number of antigens trapped on the surface<br>
of follicular dendritic cells. Successful B-cells capture the antigen and present it to T follicular<br>
helper cells, which provide affinity-based help and drive B-cells proliferation and differentiation<br>
into memory B-cells and plasma cells (reviewed in Mesin et al., 2016, De Silva et al., 2015). Fish<br>
were thought to lack affinity maturation, in part because they lack histologically distinct germinal<br>
centers (reviewed in Magor, 2015). However, higher affinity antibodies (approximately 100-fold<br>
increase) were detected in the serum of trout and catfish following immunization (Ye et al., 2011,<br>
Wu et al., 2019). In addition, a functional homologue of AID has been identified in fish, where<br>
AID expressing cells co-exist with a population of pigmented cells called melano-macrophages<br>
(MMΦs) along with IgM+ B-cells and CD4+ T-cells (Saunders et al., 2010).<br>
In my thesis, I tested the hypothesis that melano-macrophage clusters (MMΦCs) are functional<br>
analogues to GCs by doing VDJ repertoires on isolated clusters from the spleen and kidney of<br>
zebrafish. Construction of clonal lineage trees revealed: 1) Each cluster is dominated by a few Biii<br>
cell clonotypes that have generated several hundred daughter cells with acquired mutations. 2)<br>
Proliferation of B-cells within the clusters is inferred to be ongoing, with a few more Ig clones<br>
having smaller lineages. 3) There is evidence for positive selection for replacement mutations in<br>
regions encoding the antigen contact loops but not in regions encoding the framework regions. 4)<br>
Mutation patterns indicate the involvement of AID and error-prone polymerases in the mutation<br>
process. 5) Hill numbers and clonal abundance distribution revealed expansion and diversification<br>
of specific clonotypes, indicating the presence of an effective recruitment mechanism within the<br>
clusters. In addition, zebrafish vaccination with various proteins conjugated to a fluorescent tag<br>
revealed that long-term antigen retention occurs within MMΦCs. Imaging flow cytometry analysis<br>
confirmed the presence of lymphocyte-like cells, and the total number of these cells is consistent<br>
with the number of unique Ig transcripts isolated from each cluster. Identifying a distinct cellular<br>
milieu in which B-cells can undergo antibody affinity modification will provide a better<br>
understanding of how antibody affinity maturation operates in fish and may provide insights into<br>
how autoimmune-associated ectopic germinal centers or tertiary lymphoid structures in mammals<br>
can function.</p>