Background. Natural killer (NK) cells participate in pig-to-primate xenograft rejection both by antibody-dependent and -independent mechanisms. A majority of human NK cells express the inhibitory receptor CD94/NKG2A, which binds specifically to human leukocyte antigen (HLA)-E, a trimeric complex consisting of the HLA-E heavy chain, β2-microglobulin (β2m), and a peptide derived from the leader sequence of some major histocompatibility complex class I molecules. Methods. To use this mechanism for protection of pig tissues against human NK cell-mediated cytotoxicity, we generated transgenic pigs by pronuclear microinjection of genomic fragments of HLA-E with an HLA-B7 signal sequence and of human β2-microglobulin (huβ2m) into zygotes. Results. Three transgenic founder pigs were generated. Northern blot analysis of RNA from peripheral blood mononuclear cells revealed the presence of the expected transcript sizes for both transgenes in two of the three founders. The founder with the highest expression and his offspring were characterized in detail. Fluorescence-activated cell sorting (FACS) and Western blot analyses demonstrated consistent expression of HLA-E and huβ2m in peripheral blood mononuclear cells. Immunohistochemistry revealed the presence of HLA-E and huβ2m on endothelial cells of many organs, including heart and kidney. In vitro studies showed that lymphoblasts and endothelial cells derived from HLA-E/huβ2m transgenic pigs are effectively protected against human NK cell-mediated cytotoxicity, depending on the level of CD94/NKG2A expression on the NK cells. Further, HLA-E/huβ2m expression on porcine endothelial cells inhibited the secretion of interferon (IFN)-γ by co-cultured human NK cells. Conclusions. This novel approach against cell-mediated xenogeneic responses has important implications for the generation of multitransgenic pigs as organ donors for clinical xenotransplantation.

US11 and US2 encode gene products expressed early in the replicative cycle of human cytomegalovirus (HCMV), which cause dislocation of human and murine major histocompatibility complex (MHC) class I molecules from the lumen of the endoplasmic reticulum to the cytosol, where the class I heavy chains are rapidly degraded. Human histocompatibility leukocyte antigens (HLA)-C and HLA-G are uniquely resistant to the effects of both US11 and US2 in a human trophoblast cell line as well as in porcine endothelial cells stably transfected with human class I genes. Dislocation and degradation of MHC class I heavy chains do not appear to involve cell type–specific factors, as US11 and US2 are fully active in this xenogeneic model. Importantly, trophoblasts HLA-G and HLA-C possess unique characteristics that allow their escape from HCMV-associated MHC class I degradation. Trophoblast class I molecules could serve not only to block recognition by natural killer cells, but also to guide virus-specific HLA-C– and possibly HLA-G–restricted cytotoxic T-lymphocytes to their targets.

The appearance of polymorphonuclear and mononuclear leukocytes in the cerebrospinal fluid (csf) is an important hallmark of bacterial meningitis. Chemokines are candidate mediators of cell migration from blood into the subarachnoid space. Therefore, concentrations of C-X-C and C-C chemokines in the csf of patients with pyogenic meningitis were measured by ELISA. Highly significant elevations of chemokine levels in comparison with noninflammatory csf controls were found for IL-8 (median, 21.6 ng/ml; range, < 0.1 to 191.3), growth-related gene product alpha (median, 5.6 ng/ml; range, < 0.1 to 48.2), monocyte chemotactic protein-1 (median, 26.4 ng/ml; range, < 0.2 to 193.8), macrophage inflammatory protein-1 alpha (MIP-1 alpha; median, 1.8 ng/ml; range, < 0.5 to 18.0), MIP-1 beta (median, 10.6 ng/ml; range, < 0.3 to 84.4), but not for RANTES (regulated upon activation, normal T cell expressed and secreted). The csf of bacterial meningitis were chemotactic for neutrophils and mononuclear leukocytes. Correlation analysis demonstrated a strong association between individual chemokine levels and chemotactic activity mediated by csf. A significant reduction of neutrophil chemotaxis was obtained by anti-IL-8 and anti-growth-related gene product alpha Abs, and a reduction of mononuclear cell migration was achieved by a combination of anti-monocyte chemotactic protein-1, anti-MIP-1 alpha, and anti-MIP-1 beta Abs. Since no significant correlation was found between csf leukocyte counts and chemokine concentrations or chemotactic activity mediated by csf, additional factors influence the extent of pleocytosis in vivo.