Purpose: ABOi HTx can be performed safely in infants due to lack of natural anti-A/B antibodies (Ab), in contrast to adults. Following ABOi HTx, B cell tolerance to donor A/B blood group antigen(s) develops by mechanisms not well understood. For detailed study we developed a model using mice transgenic for expression of human A-antigen as donors (A-Tg, C57BL/6 (B6) background) and B6 wild-type (WT) mice as recipients. We showed that A-Tg heart grafts undergo antibody-mediated rejection (AMR) in adult WT mice with anti-A Ab, whereas A-Tg HTx into juvenile WT mice (4 weeks old) results in tolerance to A-antigen. The present study investigated whether tolerance can be induced in WT mice following administration of A-antigen in a form other than a transplant.

Methods: Neonatal WT mice (<1 day) were injected i.v. with A-antigen expressing splenocytes/bone marrow cells (n=17) from adult A-Tg mice, or were untreated (n=15). As adults (7 weeks), mice were injected i.p. with human A erythrocytes (RBC) in an attempt to induce anti-A Ab. Subsequently mice (neonatal-treated n=9; untreated n=6) received heterotopic A-Tg HTx. Grafts were monitored for function (beating) and assessed for AMR by histology at the experimental endpoint 14-21 days post-Tx or when beating ceased. Serum anti-A and 3^rd party Abs were assessed by hemagglutination (with A-Tg and reagent human A/O RBC).

Results: Anti-A Abs were induced to high levels in all mice not treated as neonates (median titre 1:1024, range 1:256-2048) and were undetectable-low in neonatal-treated mice (median titre ≤1:2); anti-human non-A RBC Ab (3^rd party) were comparable in both groups. In untreated mice, 5/6 grafts survived to day 14-21 post-Tx despite abundant anti-A Ab, however C4d deposition in capillaries (3/6) and morphologic features of AMR (4/6) were present. In contrast, in neonatal-treated mice, all grafts (9/9) survived to day 14-21 with no C4d deposition; 6/9 grafts had distorted morphology that may be artifact or related to ischemia.

Conclusion: The inability to elicit abundant anti-A Abs in adult neonatal-treated mice by either injection of A-RBC or Tx of A-Tg heart grafts, together with 3^rd party Ab production, suggests that robust A-antigen-specific tolerance was induced by neonatal exposure to A-expressing cells.This model will prove useful for addressing mechanisms of ABO-tolerance.

CITATION INFORMATION: Motyka B, Lamarche B, Labonte K, Wang S, Pearcey J, Tao K, Mengel M, Sis B, Cowan P, West L. Neonatal Tolerance to Blood Group A-Antigen in a Mouse Model of ABO-Incompatible Heart Transplantation (ABOi HTx). Am J Transplant. 2016;16 (suppl 3).

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