BADERC- Gerald L Waneck

Gerald L Waneck, Ph.D.

Inhibition of Human Anti-pig NK Cells by HLA Expression

Diabetes resulting from destruction of islets may one day be cured by xenotransplantation of pig islet cells. The primary barrier in pig to primate xenotransplantation is hyperacute rejection (HAR), mediated by preformed xenoreactive natural antibodies (XNA) that bind to pig cell surface molecules, followed by activation of complement. Recent progress in animal cloning has allowed the production of “knockout” pigs which are deficient in both chromosomal copies of alpha-galactosyltransferase, thereby reducing the major epitope recognized by XNA. However, previous studies have shown that even when HAR is prevented, human anti-pig cellular responses may lead to delayed xenograft rejection (DXR). DXR is characterized by progressive infiltration of activated monocytes and natural killer (NK) cells, in contrast to allograft rejection, which involves CD8⁺ T cell infiltration. CD8⁺ cytotoxic T lymphocytes (CTLs) are activated to lyse cells that express foreign major histocompatibility complex class I (MHC-1) molecules, while NK cells are inhibited from lysing cells that express self MHC-1 molecules. The role of NK cells in tissue transplantation is controversial, although NK cells play a critical role in resisting bone marrow grafts in a number of animal models. Recent results from our group and others suggest that human NK cells are likely to play a major role in the DXR of pig tissues, because pig MHC-1 is not recognized by human NK cell inhibitory receptors. Moreover, the FcgRIIIA receptor (CD16), a major activation molecule for NK cell cytotolytic activity on human NK cells, can recognize anti-pig XNA bound to pig tissues.

NK cells freshly isolated from human blood can kill pig target cells in serum-free medium. Killing is enhanced when the cultures contain human serum (a source of IgG), or when NK cells are first activated by allogeneic or xenogenic stimulators. NK cells cloned from these cultures, which have KIRs specific for a given HLA ligand (e.g., HLA-Cw3), can be completely inhibited from killing transfected pig cells that express this ligand, even in the presence of XNA. However, expression of HLA in pig cells has the unwanted effect of amplifying the CD8⁺ CTL response, thus solving one problem by creating another. By genetically engineering HLA ligands that are defective in interaction with CD8, this unwanted amplification can be minimized. The long term goal of this project is to make pigs transgenic for genetically modified HLA ligands, not only to block NK cell-mediated DXR of xenografts such as Islets of Langerhans, but also to increase the likelihood of making pig-human mixed bone marrow chimeras, without stimulating the CTL response. Mixed xenogeneic bone marrow chimerism, which is being explored by Drs. David Sachs and Megan Sykes, would make patients immunotolerant to subsequent pig islet xenografts.

References:

1. Seebach JD, Waneck GL. Natural killer cells in xenotransplantation. Xenotransplantation. 1997; 4:201-211.

2. Manilay JO, Waneck GL, Sykes M. Altered expression of Ly-49 receptors on natural killer cells developing in mixed bone marrow chimeras. International Immunol. 1998; 10:1943-1955.

3. Seebach JD, Pazmany L, Waneck GL, Minja F, Germana S, LeGuern C, Sachs DH. HLA-G expression on porcine endothelial cells protects partially against direct human NK cytotoxicity but not against ADCC. Transpl Proc. 1999; 31:1864-1865.

4. Manilay JO, Waneck GL, Sykes M. Levels of Ly-49 receptor expression are determined by the frequency of interactions with MHC ligands: Evidence against receptor calibration to a “useful” level. J Immunol. 1999; 163:2628-2633.

5. Sharland A, Patel A, Lee JH, Cestra AE, Saidman S, Waneck GL. Genetically modified HLA class I molecules able to inhibit human NK cells without provoking alloreactive CD8⁺ CTL. J Immunol. 2002; 168:3266-3274.

6. Sharland A, Lee JH, SaidmanS, Waneck GL. CD8-interaction mutant HLA-Cw3 molecules protect porcine cells from human NK cell-mediated antibody-dependent cellular cytotoxicity without stimulating CTLs. Transplantation. 2003; 76:1615-1622.






			Gerald L Waneck, Ph.D. Inhibition of Human Anti-pig NK Cells by HLA Expression Diabetes resulting from destruction of islets may one day be cured by xenotransplantation of pig islet cells. The primary barrier in pig to primate xenotransplantation is hyperacute rejection (HAR), mediated by preformed xenoreactive natural antibodies (XNA) that bind to pig cell surface molecules, followed by activation of complement. Recent progress in animal cloning has allowed the production of “knockout” pigs which are deficient in both chromosomal copies of alpha-galactosyltransferase, thereby reducing the major epitope recognized by XNA. However, previous studies have shown that even when HAR is prevented, human anti-pig cellular responses may lead to delayed xenograft rejection (DXR). DXR is characterized by progressive infiltration of activated monocytes and natural killer (NK) cells, in contrast to allograft rejection, which involves CD8⁺ T cell infiltration. CD8⁺ cytotoxic T lymphocytes (CTLs) are activated to lyse cells that express foreign major histocompatibility complex class I (MHC-1) molecules, while NK cells are inhibited from lysing cells that express self MHC-1 molecules. The role of NK cells in tissue transplantation is controversial, although NK cells play a critical role in resisting bone marrow grafts in a number of animal models. Recent results from our group and others suggest that human NK cells are likely to play a major role in the DXR of pig tissues, because pig MHC-1 is not recognized by human NK cell inhibitory receptors. Moreover, the FcgRIIIA receptor (CD16), a major activation molecule for NK cell cytotolytic activity on human NK cells, can recognize anti-pig XNA bound to pig tissues. NK cells freshly isolated from human blood can kill pig target cells in serum-free medium. Killing is enhanced when the cultures contain human serum (a source of IgG), or when NK cells are first activated by allogeneic or xenogenic stimulators. NK cells cloned from these cultures, which have KIRs specific for a given HLA ligand (e.g., HLA-Cw3), can be completely inhibited from killing transfected pig cells that express this ligand, even in the presence of XNA. However, expression of HLA in pig cells has the unwanted effect of amplifying the CD8⁺ CTL response, thus solving one problem by creating another. By genetically engineering HLA ligands that are defective in interaction with CD8, this unwanted amplification can be minimized. The long term goal of this project is to make pigs transgenic for genetically modified HLA ligands, not only to block NK cell-mediated DXR of xenografts such as Islets of Langerhans, but also to increase the likelihood of making pig-human mixed bone marrow chimeras, without stimulating the CTL response. Mixed xenogeneic bone marrow chimerism, which is being explored by Drs. David Sachs and Megan Sykes, would make patients immunotolerant to subsequent pig islet xenografts. References: 1. Seebach JD, Waneck GL. Natural killer cells in xenotransplantation. Xenotransplantation. 1997; 4:201-211. 2. Manilay JO, Waneck GL, Sykes M. Altered expression of Ly-49 receptors on natural killer cells developing in mixed bone marrow chimeras. International Immunol. 1998; 10:1943-1955. 3. Seebach JD, Pazmany L, Waneck GL, Minja F, Germana S, LeGuern C, Sachs DH. HLA-G expression on porcine endothelial cells protects partially against direct human NK cytotoxicity but not against ADCC. Transpl Proc. 1999; 31:1864-1865. 4. Manilay JO, Waneck GL, Sykes M. Levels of Ly-49 receptor expression are determined by the frequency of interactions with MHC ligands: Evidence against receptor calibration to a “useful” level. J Immunol. 1999; 163:2628-2633. 5. Sharland A, Patel A, Lee JH, Cestra AE, Saidman S, Waneck GL. Genetically modified HLA class I molecules able to inhibit human NK cells without provoking alloreactive CD8⁺ CTL. J Immunol. 2002; 168:3266-3274. 6. Sharland A, Lee JH, SaidmanS, Waneck GL. CD8-interaction mutant HLA-Cw3 molecules protect porcine cells from human NK cell-mediated antibody-dependent cellular cytotoxicity without stimulating CTLs. Transplantation. 2003; 76:1615-1622.