APMIS 111: 1019–26, 2003 Copyright C APMIS 2003 Printed in Denmark . All rights reserved ISSN 0903-4641 The resistance of delayed xenograft rejection to a(1,3)-galactosyltransferase gene inactivation and CD4 depletion in a mouse-to-rat model ALASTAIR B. HANSEN, 1 SVEND KIRKEBY, 2 BENT AASTED, 3 KIRSTEN DAHL, 4 AXEL KORNERUP HANSEN, 4 HANS DIEPERINK, 5 METTE SVENDSEN, 5 EJVIND KEMP, 1 KARSTEN BUSCHARD 6 and ANTHONY J.F. D’APICE 7 1 Department of Pathology, Herlev Hospital, University of Copenhagen, Herlev, 2 Department of Oral Function and Physiology, Dental School, Copenhagen, 3 Department of Veterinary Microbiology and 4 Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, Copenhagen, 5 Department of Nephrology, Odense University Hospital, Odense, 6 Bartholin Instituttet, Kommunehospitalet, Denmark and 7 Immunology Research Centre, St. Vincent’s Hospital, Victoria, Australia Hansen AB, Kirkeby S, Aasted B, Dahl K, Hansen AK, Dieperink H, Svendsen M, Kemp E, Busch- ard K, d’Apice AJF. The resistance of delayed xenograft rejection to a(1,3)-galactosyltransferase gene inactivation and CD4 depletion in a mouse-to-rat model. APMIS 2003;111:1019–26. Critical to the prevention of xenograft loss is the prevention of delayed xenograft rejection (DXR), due to its resistance to conventional immunosuppression. The role of the carbohydrate galactose-a1,3-galac- tose (a1,3Gal) has been a matter of great debate and it has been proposed that the reaction between a1,3Gal epitopes on donor endothelial cells and recipient anti-a1,3Gal antibodies (Abs) may damage the graft during DXR. Recipient anti-a1,3Gal Abs are produced by CD4-dependent B cells. To test the above-mentioned hypothesis, hearts from a1,3Gal-free mice (GT-Ko mice), generated by a1,3-galacto- syltransferase gene disruption, were transplanted to anti-a1,3Gal antibody-free Lew/Mol rats. This model consists of an a1,3Gal/a1,3Gal-antibody-free environment, eliminating a possible influence of this specific system on DXR. A subgroup of recipients were furthermore CD4 depleted in order to inhibit CD4-dependent B-cell antibody production. Rejected hearts were evaluated by light- and immunofluor- escence microscopy. Treatment effects on recipient T-cell subsets and cytokine expression were analyzed by flow cytometry, while antibody production was measured by ELISA. All recipients developed DXR with no differences among the groups. DXR was related to thrombosis with IgG and IgM desposition in vessel walls, as well as macrophage and granulocyte accumulation in the myocardium. No complement C3, CD4 cells or NK cells were found. Flow cytometric analysis confirmed peripheral blood CD4 de- pletion and IFN-g suppression in CD4 Ab-treated recipients. Finally, ELISA showed that specific anti- a1,3Gal Ab production was absent. However, Ab(s) against an unidentified Gala 1 were found among recipients. In our model, DXR is resistant to a1,3-galactosyltransferase gene inactivation and CD4 de- pletion. However, other Gala 1 epitopes and antibodies may play a role during DXR. Further studies are needed to elucidate the precise pathways leading to DXR. Key words: Xenotransplantation; rejection; a(1,3)-galactosyltransferase; knockout mouse; CD4. Alastair Hansen, Department of Pathology, Herlev Hospital, University of Copenhagen, DK-2730 Herlev, Denmark. e-mail: alha/herlevhosp.kbhamt.dk Delayed xenograft rejection (DXR), also known as acute vascular rejection, can occur after inhi- Received April 1, 2003. Accepted July 28, 2003. 1019 bition of hyperacute rejection (HAR) during disconcordant xenotransplantation or as an ini- tial rejection form during concordant xeno- transplantation (1). DXR is characterized by endothelial activation, thrombosis, graft infil-