M Yamada

Remodeling of the Major Pig Xenoantigen by N-Acetylglucosaminyltransferase III in Transgenic Pig

We have been successful in generating several lines of transgenic mice and pigs that contain the human β-d-mannoside β-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galα1–3Galβ1–4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as α1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.

Modulation of the leader peptide sequence of the HLA-E gene up-regulates its expression and down-regulates natural killer cell-mediated swine endothelial cell lysis.

Background. The inhibitory function of HLA class I molecules, HLA-G1 and HLA-E, on natural killer (NK) cell-mediated cytolysis has previously been reported. In this study, we report on a study of the effects of the co-expression of these molecules on the inhibition of NK cell-mediated cytolysis, using a newly constructed gene. Methods. Complementary DNA (cDNA) of HLA-G (G1 and G3), HLA-E, and human &bgr;2-microglobulin (h&bgr;2m) were prepared and transfected into swine endothelial cell (SEC) and Chinese hamster ovarian tumor (CHO) cell. The leader peptide sequences of HLA-G1 and HLA -E genes were changed to VMAPRTLFL or VMAPRTLVL, which corresponds to the original HLA-G1 and HLA-A2. The cell surface expression of the modified genes was evaluated by flow cytometry, and NK cell-mediated cytolysis by human peripheral blood mononuclear cells (PBMC) was assessed. Results. The transfectant with the h&bgr;2m and HLA-G1 genes showed a clear expression of the HLA-G1 molecule and had an inhibitory effect on NK cell-mediated SEC lysis. Whereas neither the transfectant with the h&bgr;2m and HLA-E genes, nor that with the h&bgr;2m and HLA-G3 genes, expressed the HLA molecule on SEC, the transfectant with triple genes, h&bgr;2m, HLA-E, and HLA-G3, expressed the HLA-E molecule and also inhibited NK-mediated SEC lysis. Conversely, the modification of the leader sequence of the HLA-E gene successfully induced the expression of the HLA-E molecule on the SEC surface. Furthermore, the transfectant expressed both HLA-G1 and HLA-E molecules, thus efficiently enhancing the inhibition of NK-mediated SEC lysis. Conclusion. The co-expression of HLA-G1 and HLA-E molecules with the modified genes has potential for use in preventing xenograft rejection, as mediated by human NK cells.

A surface-bound form of human C1 esterase inhibitor improves xenograft rejection.

BACKGROUND The purpose of the present study was to investigate the effect of the C1 esterase inhibitor (C1-INH) molecule against human complement attack on a swine endothelial cell (SEC) membrane. Human C1-INH functions as an inhibitor for complement reaction in the first step of the classical pathway in the fluid phase. METHODS A surface-bound form of human C1-INH (C1-INH-PI) consisting of a full-length coding sequence of C1-INH and a glycosylphosphatidylinositol (GPI) anchor of the decay-accelerating factor (CD55) was constructed, and stable Chinese hamster ovarian tumor (CHO) cell lines and SEC lines expressing C1-INH-PI were then prepared by transfection of the constructed cDNA. The basic function of the transfected molecules on the xenosurface was investigated using CHO transfectants for the sake of convenience. The efficacy of C1-INH-mediated protection of SEC from human complement was then assessed as an in vitro hyperacute rejection model of a swine-to-human discordant xenograft. RESULTS Flowcytometric profiles of the stable CHO and SEC transfectants with C1-INH-PI showed a medium level of expression of these molecules. The C1-INH levels were significantly reduced as a result of phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, suggesting that the molecules were present as the PI-anchor form. Approximately 51.3 x 10(4) and 13.3 x 10(4) molecules of C1-INH-PI blocked human complement-mediated cell lysis by approximately 75% on the CHO cell and by 60-65% on the SEC cell, respectively. In addition, the complement-inhibiting activity of human C1-INH molecules is not homologously restricted. CONCLUSIONS The results suggest that the surface-bound form of C1-INH represents a good candidate as a safeguard against hyperacute rejection of xenografts.

Effect of various forms of the C1 esterase inhibitor (C1-INH) and DAF on complement mediated xenogeneic cell lysis.

Fukuta D, Miyagawa S, Yamada M, Matsunami K, Kurihara T, Shirasu A, Hattori H, Shirakura R. Effect of various forms of the C1 esterase inhibitor (C1‐INH) and DAF on complement mediated xenogeneic cell lysis. Xenotransplantation 2003; 10: 132–141.

A surface-bound form of human C1 esterase inhibitor on xenografts: the complement regulatory function.

THE PURPOSE of the present study was to investigate the effect of the C1 esterase inhibitor (C1-INH), on a swine endothelial cell (SEC) membrane against human complement attack. Human C1-INH functions as an inhibitor for the complement reaction at the first step of the classical pathway in the fluid phase. The basic function of the transfected molecules on the xenosurface was investigated using Chinese hamster ovary (CHO) transfectants for the sake of convenience. The efficacy of C1-INH-mediated protection of SEC from human complement was then assessed as an in vitro hyperacute rejection model of a swine to human discordant xenograft.