Evelyn Salvaris

Changes In Cell Surface Glycosylation In α1,3-galactosyltransferase Knockout And α1,2-fucosyltransferase Transgenic Mice

BACKGROUND Inactivation of the alpha1,3-galactosyltransferase (GalT) gene by homologous recombination (knockout [KO] mice) and competition for the enzymes N-acetyllactosamine substrate by transgenically expressed alpha1,2-fucosyltransferase (H-transferase) are two genetic approaches to elimination of the Gal alpha1,3Gal (alphaGal) epitope, which is the major xenoantigen in pigs against which humans have preformed antibodies. Such genetic manipulations often have unpredictable results. METHODS A panel of 19 selected lectins was used to characterize the changes in cell surface glycosylation in GalT KO and H-transferase transgenic mice, compared with nontransgenic littermate controls. RESULTS GalT KO mice showed complete elimination of the alphaGal epitope, as reported previously. Surprisingly, however, this was associated with only a modest increase in N-acetyllactosamine residues and had little other effect on the pattern of lectin binding. In contrast, the pattern of lectin binding to H-transferase transgenic mouse cells was more profoundly disturbed and indicated, in addition to the expected expression of H substance and suppression of the alphaGal epitope, that there was a marked reduction in alpha2,3-sialylation and exposure of the normally cryptic antigens, sialylated Tn and Forssman antigens. Similar changes in lectin reactivity with porcine aortic endothelial cells were induced by neuraminidase treatment. CONCLUSIONS Lectins were able to bind underlying carbohydrate structures (sialylated Tn and Forssman antigens) that are normally cryptic antigens on H-transferase transgenic mouse spleen and cardiac endothelial cells, probably as a consequence of the reduction in the electronegativity of the cell surface due to reduced sialylation. As humans have preformed anti-Tn and anti-Forssman antibodies, it is possible that these structures may become targets of the xenograft rejection process, including hyperacute rejection.

Knock out of alpha1,3-galactosyltransferase or expression of alpha1,2-fucosyltransferase further protects CD55- and CD59-expressing mouse hearts in an ex vivo model of xenograft rejection.

BACKGROUND Organs from transgenic animals with high-level endothelial expression of the human complement regulatory factors CD55 and CD59 are significantly protected from human complement-mediated injury. Elimination or reduction of the major xenoepitope alphaGal, achieved by knocking out the alpha1,3-galactosyltransferase gene (Gal KO) or expressing human alpha1,2-fucosyltransferase (H transferase or HTF), also affords protection, although to a lesser degree. In this study, we examined whether the protection provided by strong CD55 and CD59 expression can be augmented by the Gal KO or HTF modifications. METHODS Hearts from four groups of mice (wild type, CD55/CD59, CD55/CD59/HTF, and CD55/CD59/Gal KO) were perfused ex vivo with 40% human plasma. Mean heart work for each group was compared over a 60-min period. RESULTS Wild-type hearts ceased to function effectively within 15 min of plasma addition. CD55/CD59 hearts displayed prolonged survival and maintained approximately 10% maximum work at the end of perfusion. Introduction of Gal KO or HTF onto the CD55/CD59 background resulted in a further prolongation, with work maintained at 20-30% of the maximum level. CONCLUSIONS We used an ex vivo model to demonstrate that eliminating alphaGal expression further prolongs the function of mouse hearts expressing high levels of CD55 and CD59. In addition, we showed that reducing alphaGal by expressing HTF is equally as effective in prolonging CD55/CD59 heart function as knocking out Gal transferase, thus providing a feasible strategy for translating these advances to the pig.

Transgenic expression of human α1,2-fucosyltransferase (H-transferase) prolongs mouse heart survival in an ex vivo model of xenograft rejection

BACKGROUND The expression of human alpha1,2-fucosyltransferase (H-transferase, HT) has been proposed as an alternative strategy to alpha1,3-galactosyltransferase (GT) gene knockout, which is not currently feasible in pigs, to reduce the galactose-alpha1,3-galactose (Gal) epitope expression. HT expression has recently been shown in transgenic mice and pigs to significantly reduce Gal expression on a variety of cells; however, its ability to do so on endothelial cells and its effectiveness at prolonging xenograft survival are yet to be determined. METHODS HT-transgenic, Gal knockout (Gal KO) mice, and mice containing both genetic modifications (HT-transgenic/Gal KO) were tested for H-substance and Gal expression on splenocytes and endothelial cells by flow cytometric analysis. In addition, the hearts of these mice were perfused ex vivo with 6% human plasma, and the effect on cardiac function was determined. RESULTS AND CONCLUSION H-substance expression was detected on both splenocytes and endothelial cells of HT-transgenic mice. The level of H-substance expression was not affected by the presence or absence of GT in the cells, consistent with HT being dominant over GT. The ability of HT expression to reduce Gal expression was highly variable depending on the cell type. Gal expression on splenocytes was almost completely eliminated, whereas on endothelial cells, substantial Gal remained despite a 70% reduction. When perfused ex vivo with human plasma, hearts from HT-transgenic, Gal KO, and HT-transgenic/Gal KO mice demonstrated a similar prolongation in survival, compared with wild-type controls. Therefore, as far as hyperacute rejection is concerned, HT expression may be as effective as Gal KO in protecting against xenoantibody and complement mediated injury. However, the effect of residual Gal on non-hyperacute rejection responses remains to be determined.

Functional studies of stable L cell transfectants expressing intercellular adhesion molecule 1 [ICAM-1]

High molecular weight DNA isolated from human tonsil was transfected into mouse L cells to produce transfectants expressing the human intercellular adhesion molecule [ICAM‐1]. The transfected ICAM‐1 molecule was highly expressed on the cell membrane and our studies show that the transfected ICAM‐1 molecule was a fully functional adhesion protein. All leucocyte subtypes showed specific binding to the ICAM‐1 transfectants, their adhesion being inhibited by Fab 1 fragments of W‐CAM‐1 antibody and LFA‐1 MoAb (leucocyte function antigen). B cell lines(RAJI, Nalm1) showed the highest degree of ICAM‐1 mediated adhesion. Normal lymphoblasts showed comparable levels of binding whilst normal neutrophils (both resting and activated by (N‐formyl‐methionyl‐leucyl‐phenylalanine) fMLP) showed the least ICAM‐1‐mediated adhesion. Despite a significant level of adhesion to ICAM‐1 transfectants shown by T lymphoblasts generated in a two way MLR there was no evidence of cytolysis of ICAM‐1 transfectants. These studies demonstrate the potential of ICAM‐1 transfectants as tools for analysis of the role of ICAM‐1 in lymphoid adhesion.

Long-Term Function of Genetically Modified Porcine Neonatal Islet Xenografts in Baboons

Introduction In xenotransplantation the stringent pig-to-baboon islet xenograft preclinical model is a difficult one with the maximum period of normoglycemia reported to only 1 day. Previously we have shown that neonatal islet cell clusters (NICC) from GTKO/CD55-CD59-HT pigs prevent both hyper acute rejection (HAR) and the instant blood mediated inflammatory response (IBMIR) in immunosuppressed baboons. However, the maturation and function of the NICC were not examined in detail in the initial study, because (i) the recipients were not diabetic, and (ii) the standard clinical-based immunosuppression used was insufficient to prevent biopsy-proven rejection within 1 month. In the current study, therefore, we used streptozotocin-induced diabetic recipients and changed to a costimulation blockade-based immunosuppressive regimen. Aims To achieve long-term normoglycemia in diabetic baboons transplanted with neonatal pig islets, and to investigate the effect of ceasing immunosuppression. Materials and Methods: Five diabetic baboons received intraportal infusion of NICC (10,000-50,000 IEQ/kg) from 1-5 day old GTKO/CD55-CD59-HT piglets. From day -3 recipients were treated with anti-CD2 induction and subsequently maintenance with oral tacrolimus, anti-CD154 and belatacept, which were progressively ceased. Graft survival and function was followed by daily blood sugar levels (BSL), IVGTT, OGTT and immunohistochemical analysis of liver biopsies taken at various time points over the study period. Results and Discussion None of the baboons exhibited signs of thrombosis associated with IBMIR, with no change to platelet counts, vWF, fibrinogen or D-dimer levels from baseline. Recipients developed normal fasting BSL and had normal IVGTT and OGTT, with porcine insulin and C-peptide secreted in response to glucose bolus stimulus. All animals have become normoglycaemic off all exogenous insulin. Liver biopsies revealed strong positive staining for insulin, glucagon and somatostatin in xenografts. One recipient receiving 50,000 IEQ/kg was insulin-independent for >7 months, including 7 weeks after the last drug (belatacept) was ceased. A second recipient receiving 10,000 IEQ/kg took longer to achieve insulin independence, but remained insulin independent >18 months, including 6-months off all immunosuppression. The fourth and fifth animals are being followed out past 6-months and 2-months post transplant. Conclusion We have confirmed that GTKO/CD55-CD59-HT neonatal islets are protected from IBMIR in the baboon model. More importantly, we have demonstrated for the first time long-term survival and function of porcine islets in baboons. The costimulation blockade-based immunosuppression permitted maturation of the islets such that the dose required to achieve normoglycemia (10,000 IEQ/kg) was lower than that reported in any NHP model. Remarkably, this recipient remained normoglycemic for 6 months after immunosuppression was ceased. Bristol-Myers Squibb Company for donation of Belatacept,. Keith Reimann at NIH Nonhuman Primate Reagent Resource for supply of Anti CD154 antibody.