Anti-Carbohydrate Antibodies Associated With Hyperacute Rejection in a Vascularized Mouse Heart-to-Rat Xenotransplantation Model

Abstract

Specificity of immune reactions has always been sought, because it facilitates intervention with unwanted mechanisms. Specific carbohydrate antigens have been proposed to be targets of antibodies in early immune responses in pig-to-man xenografts. This work was undertaken to determine carbohydrate structure for antibody response in the experimental xenograft model mouse-to-rat. Glycolipids were prepared from nine different mouse organs and separated for carbohydrate size on thin layer plates. Sera taken from normal untreated rats showed only weak or absent IgM antibody-binding to the separated mouse glycolipids. This is in accordance with the observation that mouse heart grafts are not hyperacutely rejected by the rat. However, sera taken from mouse heart xenografted rats show clear IgG and IgM antibody binding to neutral glycolipids migrating in the five-sugar region of the thin-layer plate. These rats have previously been reported to hyperacutely reject a second xenograft. Glycolipids with this particular mobility and immunostaining properties are the dominant ones in the mouse caval vein preparation, which probably represents a rather pure vascular structure. The target antigen structure was identified, by mass spectrometry and proton nuclear magnetic resonance spectroscopy, to be the Forssman pentaglycosylceramide. A commercial monoclonal antibody directed toward the Forssman antigen bound the same biochemical structure as the antibodies derived from the mouse heart-xenografted rats. Most of the IgM activity, but very little of the IgG activity was adsorbed using the Forssman terminal disaccharide solid phase.

Characterization of Forssman and Other Antigen/Antibody Systems in Vascularized Mouse Heart to Rat Xenotransplantation

“…In accordance with earlier studies on the glycolipid expression in the mouse [5], the four-sugar structure is probably globoside. In addition, it is known that the mouse generally expresses much gangliotetraosylceramide.…”

“…In addition, it is known that the mouse generally expresses much gangliotetraosylceramide. Despite being a four‐sugar glycolipid and thus one sugar shorter than the Forssman pentaglycosylceramide, gangliotetraosylceramide migrates in the six‐sugar region, below the Forssman antigen, in thin‐layer chromatography [5,24]. In this region of the thin‐layer plate, we could not see any anisaldehyde glycolipid staining, however.…”

“…The globoside staining on the thin‐layer plate is sufficiently strong to account for most, and perhaps all, the tetraglycosylceramide fragmentation in the electron ionization mass spectra. Based on this and the absence of discernible peaks in the 1 H‐NMR spectra we conclude that gangliotetraosylceramide was not a major component of the mouse aorta, in contrast to the mouse small intestine [5].…”

“…Naive rat serum contains only small amounts of IgM, and no IgG anti-Forssman antibodies [5]. Serum from transplanted rats as well as precipitated antibodies and IgG and IgM subfractions all agglutinate sheep erythrocytes (known to express large amounts of the Forssman antigen) [6].…”

IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens

“…The Forssman antigen is a heterophile glycoprotein expressed by animals and enteric bacteria that is a target of natural IgM antibodies associated with hyperacute rejection in xenotransplantation. 47 The significance of this finding has yet to be elucidated.…”