Release of pig leukocytes during pig kidney perfusion and characterization of pig lymphocyte carbohydrate xenoantigens

Abstract

The Galalpha1-3Gal (alphaGal) antigen is considered the main xenoantigen in the pig to human species combination but other porcine antigens have to be considered such as the swine lymphocyte antigen (SLA), the blood group A/O and the Hanganutziu-Deicher (H-D) antigens. The H-D antigens are N-glycolyl-neuraminic acid (NeuGc) terminated gangliosides that are widely distributed in mammalian species but absent in humans. Upon exposure to a vascularized pig organ, the human recipient can be immunized by direct interaction with the pig tissue or/and by transfer of tissue/cells from the organ into the recipient. In the present work, we describe the release of cells from porcine kidneys upon perfusion and the expression of glycolipid based alphaGal, blood group A/O and H-D antigens in pig lymphocytes. Pig kidneys were flushed with 20 ml of NaCl or Lidocain containing 5000 U heparin, and thereafter perfused with 3000-ml perfusion solution and the cells released were counted and examined microscopically. Neutral glycolipid and ganglioside fractions were extracted from purified pig lymphocytes. The extracted components were characterized by thin layer chromatography, degradation and mass spectrometry. The expression of alphaGal and H-D epitopes on cells released from pig kidneys and purified pig lymphocytes were studied by immune electron microscopy. A total amount of about 300 x 106 leukocytes, mainly lymphocytes were released in the perfusate from the kidneys, of which about 100 x 106 cells were eluated in the 600 to 2400 ml perfusate fraction. Immunelectron microscopical analysis with Griffonia simplicifolia isolectin B4 showed staining of pig leukocytes and other cells, morphologically similar to endothelial cells, released in the perfusate. The purified porcine lymphocytes contained 930 microg neutral glycolipid (4.2 microg/mg cell protein) of which 95% was glycolipids with one to four sugar residues. Immunostaining of the neutral glycolipid fractions revealed alphaGal terminated compounds migrating in the five and 10 to 12 sugar regions and blood group A compounds in the six and eight sugar regions. Two major gangliosides NeuGc-GM3 and NeuGc-GD3 were found in the pig lymphocytes. In a patient extracorporeally xenoperfused with a pig kidney, an increased staining of both alphaGal terminated structures as well as the H-D reactive gangliosides were found in the post-perfusion serum samples. In summary, leukocytes, mainly lymphocytes are released from pig kidneys during perfusion which may contribute to immunization of human xenograft recipients.

Studies on Glycolipid Antigens in Small Intestine and Pancreas from α1,3-Galactosyltransferase Knockout Miniature Swine

“…Data from two different laboratories on GalT-KO mice describe unchanged levels of sialic acid in the GalT-KO animals (39,45). The role of NeuGc in xenotransplantation is still unclear and conflicting results regarding the immune response to NeuGccontaining gangliosides have been reported (11,12). Since humans are the only mammals that lack the NeuGc epitope (46), pig-to-non human primate transplantation models are not suitable model systems in this respect.…”

“…The sialic acid-specific reagent resorcinol (33) stained two major components migrating as GM3 and GD3 gangliosides (11) in all small intestine and pancreas fractions (Fig. 5A).…”

“…The ␣1,2FT produces the blood group H determinant present on cells in all human individuals except the rare "Bombay" phenotype which lacks the H-gene (10). The ␣2,3ST produces sialylated N-acetylneuraminic (NeuAc) and N-glycolylneuraminic (NeuGc) acid-containing glycans, of which the latter is a potential xenoantigen (11,12).…”

Recent investigations into pig antigen and anti-pig antibody expression

“…Subsequent studies initially failed to find evidence for a general induced anti-Neu5Gc response (43) but more recently have detected induced anti-Neu5Gc specific antibody in some patients (44). A dominant anti-Gal immune response was also seen in the two patients participating in an ex vivo pig kidney perfusion trial (45) and one individual produced anti-ganglioside antibodies (46). Burn patients treated with whole pig skin showed a moderate increased in anti-Gal antibody but a sustained increase in anti-Neu5Gc antibody reactivity (47).…”

Carbohydrates in xenotransplantation

“…The major components of N ‐acetylneuraminic acid (NeuAc)‐ and NeuGc‐containing glycolipids are GM3 and GD3 gangliosides, the expression of which appears to be different in various cells. NeuGc‐GM3 is reported to be predominant in SEC 63 , whereas NeuGc‐GD3 is predominant in peripheral lymphocytes 64 . As adult pig islets do not express the Gal antigen, their antigenicity is reported to be mainly due to N ‐linked sialic acids (including NeuGc) 65 .…”