We have been successful in generating several lines of transgenic mice and pigs that contain the human ␤-Dmannoside ␤-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 Gal␣1-3Gal␤1-4Glc-NAc-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 ␣1,3-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.

SUMMARYThe 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 b2-microglobulin were prepared and subcloned into the expression vector, pCXN. The transfected HLA-G1 was easily expressed on SEC, and co-transfection with human b2-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 b2-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.

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Objective Systemic juvenile idiopathic arthritis (sJIA) is characterized by fever, arthritis, rash, hepatosplenomegaly, and macrophage activation syndrome; however, its pathogenesis is still unclear. Elevated serum interleukin (IL)‐18 concentrations and decreased natural killer (NK) cell activity are characteristic of active disease; thus, we examined IL‐18 signaling in NK cells from sJIA. Methods We analyzed mitogen‐activated protein kinase (MAPK) p38 and nuclear factor κ light chain enhancer of activated B cells (NFκB) p65 phosphorylation in NK cells after in vitro recombinant IL‐18 (rIL‐18) stimulation in 31 patients with sJIA. Associations between clinical features, serum IL‐18, and phosphorylation intensity were analyzed. Furthermore, we investigated the effects of high IL‐18 concentrations on phosphorylation in NK cells. Results Patients were divided according to their disease activity: systemic features (n = 8), chronic arthritis (n = 7), remission on medication (n = 10), and remission off medication (n = 6). MAPK p38 and NFκB p65 phosphorylation intensity were the highest in healthy controls, followed by remission off medication, remission on medication (vs. control; MAPK p38, P < 0.01; NFκB p65, P < 0.05), chronic arthritis (P < 0.001, P < 0.001), and systemic features (P < 0.001, P < 0.001). The systemic features group showed a complete defect in phosphorylation. Serum IL‐18 was the highest in the systemic features group followed by chronic arthritis, remission on medication (P < 0.01), remission off medication (P < 0.01), and healthy controls (P < 0.01). Phosphorylation intensity was negatively correlated with serum IL‐18 (MAPK p38, r2 = 0.42; NFκB p65, r2 = 0.54). Furthermore, healthy control NK cells were cultured with rIL‐18; impaired phosphorylation was reproduced in vitro. Conclusion Impaired IL‐18 signaling in NK cells correlated with disease activity in sJIA. High serum IL‐18 exposure induces impaired MAPK and NFκB phosphorylation in NK cells.