Rie Nakai

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.

The possible use of HLA-G1 and G3 in the inhibition of NK cell-mediated swine endothelial cell lysis

The splicing isoform of HLA‐G that is expressed in xenogeneic cells, and its effect on NK‐mediated direct cytotoxicity was examined, using stable Chinese hamster ovary (CHO) cell or swine endothelial cell (SEC) transfectants. cDNAs of HLA‐G (G1 and G3) and human β2‐microglobulin were prepared and subcloned into the expression vector, pCXN. The transfected HLA‐G1 was easily expressed on SEC, and co‐transfection with human β2‐microglobulin led to an enhanced level of HLA‐G1 expression, as evidenced by flow cytometry. The expressed HLA‐G1 significantly suppressed NK‐mediated SEC cell lysis, which is an in vitro delayed‐type rejection model of a xenograft. On the other hand, the swine leucocyte antigen (SLA) class I molecules could be up‐regulated as the result of the transfection of human β2‐microglobulin, but did not down‐regulate human NK‐mediated SEC lysis. The HLA‐G3 was not expressed on CHO and SEC in contrast to HLA‐G1, as the result of the transfection. The gene introduction of HLA‐G3 in SEC showed no protective effect from human NK cells. However, indirect evidence demonstrated that HLA‐G3 transfection resulted in HLA‐E expression, but not itself, when transfected to the human cell line, 721.221, thus providing some insight into its natural function in human cells. The present findings suggest that the expression of HLA‐G1 on the cell surface could serve as a new approach to overcoming NK‐mediated immunity to xenografts.

Co-effect of HLA-G1 and glycosyltransferases in reducing NK cell-mediated pig endothelial cell lysis

Natural killer (NK) cells play an important role in xenograft rejection. The aim of this study was to evaluate the co-effect of human leukocyte antigen (HLA)-G1 expression and the remodeling of glycoantigens such as the alpha-Gal epitope, Galalpha1,3Galbeta1,4GlcNAc-R, by the introduction of glycosyltransferase genes related to NK cell-mediated direct cytotoxicity. Human peripheral blood mononuclear cells or an NK-like cell line, YT cells, was used as an effector and pig endothelial cells (PEC) as the target. A PEC transfectant with HLA-G1 was first prepared by the transfection of HLA-G1 and human beta2 microglobulin. Several new transfectants were then established by the transfection of glycosyltransferase to the HLA-G1 transfectant. The effect of HLA-G1 on NK cell-mediated PEC lysis was lower than that by the glycosyltransferases. Therefore, in the case of the co-transfectants except for HLA-G1+alpha2,6sialyltransferase, such as HLA-G1+N-acetylglucosaminyltransferase-III and HLA-G1+alpha1,2fucosyltransferase, the effect of HLA-G1 expression on NK-mediated killing appeared to be accounted for by the transfected glycosyltransferase activities and the reduced alpha-Gal expression on the cell surface. However, these transfectants showed significant reductions in direct NK cell-mediated cytotoxicity, compared with the single HLA-G1 transfectant. The results herein suggest that a combination of HLA-G1 and glycosyltransferases has considerable potential for the downregulation of NK cell-mediated cytolysis.

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.