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ABO Antibody Production in Mice

  • Author / Creator
    Adam, Ibrahim S. Y.
  • Introduction: ABO-incompatible organ transplants (ABOi-Tx) recipients are at high-risk of rapid rejection mediated by ABO antibodies (ABO Abs). To manage and expand ABOi-Tx, it is necessary to understand the mechanism(s) by which natural and induced ABO Abs develop. Prior studies have used chemically-synthesized carbohydrate (CHO) structures or mouse models with various limitations to study induction of ABO Abs production. ‘Natural’ (spontaneous) ABO Abs are thought to be produced in humans and mice without exposure to A/B-Ags by mechanisms that remain poorly understood, although early studies suggested a role for the participation of bacterial flora. West and colleagues found that infant recipients of ABOi heart Tx (HTx) develop B cell tolerance to donor A/B-Ags and that ABO Abs are mainly produced by CD27+ IgM+ B cells. CD22 participates in B cell tolerance and West research group found that CD27+ IgM+ B cells express high-levels of CD22 in human and this expression decreases with age. Chemically-synthesized CHO-Ags have also been used to study Ab response in CD22 knock-out (KO) mice, but various outcomes have been reported that could be attributed to the nature of these Ags and/or lack of consideration for natural Ab production. Herein, I investigated the role of CD4+ T cell participation in induction of ABO Abs production, the impact of CD4+ T cell, sex, and bacterial flora on development of natural ABO Abs, and the impact of CD22 modulatory molecules in natural and induced ABO Abs production.
    Results: Wild-type (WT) mice were stimulated by A-Ag in the context of syngeneic, allogeneic, and xenogeneic stimulation. Injection of syngeneic A-transgenic blood cell membranes (A-Tg BCM) did not induce anti-A Ab production, but human blood group A BCM (Hu A-BCM) and allogeneic A-Tg BCM induced abundant anti-A Abs. However, Hu A-BCM failed to elicit anti-A Ab following CD4+ T cell depletion. In CD4KO mice, injection of Hu A-BCM did not induce anti-A Ab production, but reconstitution with sex-matched WT CD4+ T cells restored the ability of the CD4KO mice to produce anti-A Ab to Hu A-BCM. In CD4, MHC-II, and αβ/γδ T cell receptor KO mice, females produced significantly higher natural anti-A Ab than males and Hu A-RBC injection did not induce more anti-A Ab beyond that naturally produced. Treatment of CD4KO mice with broad-spectrum antibiotics significantly reduced natural anti-A Ab production, but did not abolish it. Importantly, germ-free mice produced natural ABO Abs without exposure to A/B-Ags. CD22KO mice developed higher natural anti-A Ab than WT mice, with higher Abs production in female CD22KO than male mice. Furthermore, injection of Hu A-BCM induced massive amount of anti-A Ab production. In contrast to WT mice, anti-A Ab was elicited by syngeneic A-Tg BCM in CD22KO mice, or following CD4+ T cell depletion and Hu A-BCM injection.
    Conclusion: Induction of ABO Abs production requires exposure to non-self A/B-Ags and foreign protein, consistent with T-dependent Ab response. In contrast, natural ABO Abs developed in the absence of CD4+ T cells, suggesting a T-independent Abs response. Production of dramatically higher natural ABO Abs in CD4, MHC-II, and TCR KO female mice than in males suggests a sex-linked role in natural ABO Abs production. In contrast to the common paradigm, bacterial flora is not absolutely required for natural ABO Abs production, suggesting that nAbs are spontaneously produced. In CD22KO mice, occurrence of higher natural and induced ABO Abs than WT mice and induction of ABO Abs production without CD4+ T cell participation suggests a modulatory role for CD22 signaling in ABO Abs production. In summary, my findings indicate that: A) Induction of ABO Abs production requires CD4+ T cell participation (T-dependent), B) Natural ABO Abs production occurs in complete absence of bacterial flora or CD4+ T cells (T-independent), C) Without CD22, induction of ABO Abs production no longer requires CD4+ T cells (T-independent), but CD4+ T cell participation result in more ABO Abs (T-dependent), and D) Sex hugely impacts natural ABO Abs level.

  • Subjects / Keywords
  • Graduation date
    Fall 2020
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-fa9g-cq76
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.