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Background: "Natural" ABO antibodies (Abs) are produced without known exposure to A/B carbohydrate antigens, posing significant risks for hyperacute rejection during ABO-incompatible transplantation. We investigated anti-A "natural" ABO antibodies versus intentionally induced Abs with regard to the need for T-cell help, the impact of sex, and stimulation by the microbiome., Methods: Anti-A was measured by hemagglutination assay of sera from untreated C57BL/6 wild-type (WT) or T cell-deficient mice of both sexes. Human ABO-A reagent blood cell membranes were injected intraperitoneally to induce anti-A Abs. The gut microbiome was eliminated by maintenance of mice in germ-free housing., Results: Compared with WT mice, CD4 + T-cell knockout (KO), major histocompability complex-II KO, and αβ/γδ T-cell receptor KO mice produced much higher levels of anti-A nAbs; females produced dramatically more anti-A nAbs than males, rising substantially with puberty. Sensitization with human ABO-A reagent blood cell membranes did not induce additional anti-A in KO mice, unlike WT. Sex-matched CD4 + T-cell transfer significantly suppressed anti-A nAbs in KO mice and rendered mice responsive to A-sensitization. Even under germ-free conditions, WT mice of several strains produced anti-A nAbs, with significantly higher anti-A nAbs levels in females than males., Conclusions: Anti-A nAbs were produced without T-cell help, without microbiome stimulation, in a sex- and age-dependent manner, suggestive of a role for sex hormones in regulating anti-A nAbs. Although CD4 + T cells were not required for anti-A nAbs, our findings indicate that T cells regulate anti-A nAb production. In contrast to anti-A nAbs, induced anti-A production was T-cell dependent without a sex bias., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Competing Interests: The authors declare no funding or conflicts of interest.

Heart transplantation is the only long-lasting lifesaving option for patients with terminal cardiac failure. The number of available human organs is however far below the actual need, resulting in substantial mortality of patients while waiting for a human heart. Mechanical assist devices are used to support cardiac function but are associated with a high risk of severe complications and poor quality of life for the patients. Consistent success in orthotopic transplantation of genetically modified pig hearts into baboons indicates that cardiac xenotransplantation may become a clinically applicable option for heart failure patients who cannot get a human heart transplant. In this overview, we project potential paths to clinical cardiac xenotransplantation, including the choice of genetically modified source pigs; associated requirements of microbiological, including virological, safety; optimized matching of source pig and recipient; and specific treatments of the donor heart after explantation and of the recipients. Moreover, selection of patients and the regulatory framework will be discussed., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Elimination of galactose-alpha1,3-galactose (Gal), the major xenoantigen between pig and human, may extend pig-to-human xenograft survival beyond the current barrier of acute vascular rejection. However, it has been suggested that Gal is an essential molecule in the pig and that the generation of a Gal-deleted (Gal KO) pig will not be possible. Should this be the case, understanding the Gal-mediated immune response will be crucial in developing strategies to overcome pig xenograft rejection in humans. There are no existing models of xenograft rejection in which the sole difference between donor and recipient is Gal. We describe a model of exclusively Gal-mismatched skin graft rejection., Methods: The survival of Gal skin grafts on Gal KO mice with the same genetic background was analyzed. To examine innate anti-Gal immunity, Gal KO recipients that were also deficient in T and B cells (RAG-1 KO) were used. To study the role of cognate immunity, recipients were sensitized with a primary Gal allograft before receiving a second Gal graft that was otherwise isogeneic. To test the role of anti-Gal antibodies in this model, recipients were passively immunized with a non-complement-fixing anti-Gal monoclonal antibody., Results: Gal KO mice chronically reject Gal skin grafts by 100 days at a rate of 48% (n=25) on a BALB/c background and 25% (n=8) on a C57BL/6 background. The grafts had an infiltrate that consisted predominantly of CD4 T cells and macrophages, whereas recipients deficient in T and B cells were incapable of rejection and survived for more than 120 days (n=5). Sensitization with a primary Gal allograft increased the incidence and the tempo of rejection of a second Gal-only mismatched skin graft with 99% rejection that ranged from 11 to 45 days (n=26). Passive transfer of mouse IgG anti-Gal monoclonal-antibody-induced rejection in Gal KO and RAG-1/Gal double-KO recipients at a rate of 92% (n=13)., Conclusions: We have established a model to study rejection based solely on a Gal mismatch. Our results indicate that non-complement-fixing anti-Gal antibody can cause rejection in the acute vascular rejection time frame and that T-cell-mediated chronic rejection will be a further barrier to overcome if Gal cannot be deleted from the pig.