Evidence for primordial germinal centers in fish

• Author / Creator

  Waly, Doaa

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

• Subjects / Keywords

  • Immunology
  • B-cells
  • fish
  • evolution
  • germinal centers
  • AID
  • Aicda.

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-4xr8-cx10

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.