Mixed chimerism induced without lethal conditioning prevents T cell– and anti-Galα1,3Gal–mediated graft rejection
Hideki Ohdan, … , Kirsten G. Swenson, Megan Sykes
Hideki Ohdan, … , Kirsten G. Swenson, Megan Sykes
Published August 1, 1999
Citation Information: J Clin Invest. 1999;104(3):281-290. https://doi.org/10.1172/JCI6656.
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Article

Mixed chimerism induced without lethal conditioning prevents T cell– and anti-Galα1,3Gal–mediated graft rejection

Abstract

Galα1,3Gal–reactive (Gal-reactive) antibodies are a major impediment to pig-to-human xenotransplantation. We investigated the potential to induce tolerance of anti-Gal–producing cells and prevent rejection of vascularized grafts in the combination of α1,3-galactosyltransferase wild-type (GalT+/+) and deficient (GalT–/–) mice. Allogeneic (H-2 mismatched) GalT+/+ bone marrow transplantation (BMT) to GalT–/– mice conditioned with a nonmyeloablative regimen, consisting of depleting CD4 and CD8 mAb’s and 3 Gy whole-body irradiation and 7 Gy thymic irradiation, led to lasting multilineage H-2bxdGalT+/+ + H-2dGalT–/– mixed chimerism. Induction of mixed chimerism was associated with a rapid reduction of serum anti-Gal naturally occurring antibody levels. Anti-Gal–producing cells were undetectable by 2 weeks after BMT, suggesting that anti-Gal–producing cells preexisting at the time of BMT are rapidly tolerized. Even after immunization with Gal-bearing xenogeneic cells, mixed chimeras were devoid of anti-Gal–producing cells and permanently accepted donor-type GalT+/+ heart grafts (>150 days), whereas non-BMT control animals rejected these hearts within 1–7 days. B cells bearing receptors for Gal were completely absent from the spleens of mixed chimeras, suggesting that clonal deletion and/or receptor editing may maintain B-cell tolerance to Gal. These findings demonstrate the principle that induction of mixed hematopoietic chimerism with a potentially relevant nonmyeloablative regimen can simultaneously lead to tolerance among both T cells and Gal-reactive B cells, thus preventing vascularized xenograft rejection.

Authors

Hideki Ohdan, Yong-Guang Yang, Akira Shimizu, Kirsten G. Swenson, Megan Sykes

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Absence of anti-Gal–producing cells in GalT+/+→GalT–/– mixed chimeras (1...
Absence of anti-Gal–producing cells in GalT+/+→GalT–/– mixed chimeras (19–20 weeks after BMT). (a) ELISPOT detection of anti-Gal–producing (IgM + IgG) cells. Spleen cells (SPL), BMCs (BM), and peritoneal cavity cells (Per C), prepared from normal and conditioned GalT–/– mice, normal GalT+/+ mice, and mixed chimeras 8 days after immunization with rabbit erythrocytes, were used in ELISPOT assay. The frequency of anti-Gal–producing cells was determined as average of red plaque number in triplicate wells of serially diluted cells. The results shown are the average values ± SEM for the individual groups. Number of animals in each group: normal GalT–/– mice, n = 4; normal GalT+/+ mice, n = 4; conditioned GalT–/– mice, n = 5; GalT+/+→GalT–/– mixed chimeras, n = 7 (results are combined for recipients of 20 × 106, 7.5 × 106, and 1 × 106 BMCs). *P < 0.05 compared with normal control GalT–/– mice and similarly conditioned GalT–/– control mice that did not receive BMT. There was no statistically significant difference between mixed chimeras and normal GalT+/+ control mice in any tissue. (b) Serum levels of anti-Gal antibodies after immunization with Gal-bearing xenogeneic cells. Normal and conditioned GalT–/– mice, normal GalT+/+ mice, and mixed chimeras were immunized with rabbit erythrocytes, and serum anti-Gal levels were measured by ELISA assay 8 days after immunization. Average values ± SEM for the individual groups are shown. Number of animals in each group: normal GalT–/– mice, n = 5; conditioned GalT–/– mice, n = 4; mixed chimeras, n = 13 (results are combined for recipients of 20 × 106, 7.5 × 106, and 1 × 106 BMCs); normal GalT+/+ mice, n = 5. *P < 0.05 compared with normal control GalT–/– mice and similarly conditioned GalT–/– control mice that did not receive BMT. There was no statistical difference between mixed chimeras and normal GalT+/+ control mice at any serum dilution in either anti-Gal IgM or IgG levels.