The resistance of delayed xenograft rejection to α(1,3)‐galactosyltransferase gene inactivation and CD4 depletion in a mouse‐to‐rat model

Abstract

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-alpha1,3-galactose (alpha1,3Gal) has been a matter of great debate and it has been proposed that the reaction between alpha1,3Gal epitopes on donor endothelial cells and recipient anti-alpha1,3Gal antibodies (Abs) may damage the graft during DXR. Recipient anti-alpha1,3Gal Abs are produced by CD4-dependent B cells. To test the above-mentioned hypothesis, hearts from alpha1,3Gal-free mice (GT-Ko mice), generated by alpha1,3-galacto-syltransferase gene disruption, were transplanted to anti-alpha1,3Gal antibody-free Lew/Mol rats. This model consists of an alpha1,3Gal/alpha1,3Gal-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 immunofluorescence 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 depletion and IFN-gamma suppression in CD4 Ab-treated recipients. Finally, ELISA showed that specific anti-alpha1,3Gal Ab production was absent. However, Ab(s) against an unidentified Galalpha 1 were found among recipients. In our model, DXR is resistant to alpha1,3-galactosyltransferase gene inactivation and CD4 depletion. However, other Galalpha 1 epitopes and antibodies may play a role during DXR. Further studies are needed to elucidate the precise pathways leading to DXR.

FK778 in Experimental Xenotransplantation: A Detailed Analysis of Drug Efficacy

“…Plasmapheresis was performed before heart perfusion to eliminate circulating antibodies and complement in the negative control group, as reported by Bach et al 12 The hearts were snap frozen and 5-m sections were stained on human IgM, IgG, C5c and C5b (membraneattack complex, MAC) with fluorescein-isothiocyanateconjugated antibodies (Serotec, Germany) using a technique described elsewhere. 13 All sections analyzed were stained simultaneously to eliminate procedure-related variances. Fluorescence emission was quantified using QWIN software (Leica, Germany) to measure myocardial immunoglobulin and complement deposition.…”

Dietary Gluten Reduces the Number of Intestinal Regulatory T Cells in Mice

“…Single‐cell spleen cells from 32 BALB/c mice (see Experimental design under the heading Materials and methods for details) were prepared in PBSA by squeezing the spleen manually against a stainless steel mesh, and single cell Peyer′s patch cells from the same mice were prepared in a Medimachine (DAKO A/S, Glostrup, Denmark). Extracellular and intracellular staining procedures were performed as published previously [28].…”