Modulation of immune responses to porcine xenoantigen by targeting stimulatory and inhibitory signalling pathways

Genetic modification of pigs (e.g. transgenic expression of human complement regulatory molecules or inactivation of α1,3 galactosyltransferase) [1] enabled the development of promising strategies to overcome hyperacute rejection after pig‐to‐primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. Cellular rejection of porcine cells in xenotransplantation models is mediated by monocytes/macrophages, natural killer (NK) cells, and T cells. Research in our laboratory focuses on receptor‐ligand interactions regulating the responses of human cells to porcine tissues and thus could be targets for immunomodulation. NK cell activation is tightly regulated by different inhibitory and activating receptors and their ligands. Transgenic pigs overexpressing HLA‐E have been generated [2]. In vitro experiments revealed that cells from these pigs are only partially protected from NK cell‐mediated lysis. This might be due to the fact that the inhibitory receptor for HLA–E (CD94/NKG2A) is not expressed on all human NK cells. We are currently exploring the concept of downregulating NK cell activity by enhancing CD161/KLRB1‐mediated signalling. CD161 is a C‐type lectin‐like receptor which is expressed on the great majority of human NK cells and can transmit inhibitory signals after binding of its ligand LLT1. The data obtained so far suggest, that porcine cells genetically engineered to overexpress human LLT1 posses reduced potential to activate human NK cells. Activation of T cells by antigen presenting cells (APC) requires interactions between T cell receptor (TCR) and MHC/peptide (“signal one” of T cell activation) as well as interactions between costimulatory receptors and their ligands (“signal two” of T cell activation). T cells receiving “signal one” alone without “signal two” are not activated but achieve a state of unresponsiveness (anergy). In vitro and in vivo data suggest that effective inhibition of human T cell activation can be obtained by blocking the costimulatory CD28‐CD80/CD86 interaction using monoclonal antibodies (mAb) or an antagonistic CTLA‐4.Ig fusion protein [3]. Furthermore, blocking of TCR‐MHC and CD154(CD40L)‐CD40 interactions by mAb to porcine MHC class‐II or CD40 significantly reduced human T cell activation. New approaches for the modulation of anti‐pig immune responses may result from the finding that enhancement of inhibitory signals diminishes T cell activation. We have recently shown that overexpression of the human inhibitory ligand PD‐L1 on porcine APC markedly decreased their capacity to activate human T cells and promotes regulatory T cells [4, 5]. Current studies suggest that enhanced inhibitory signalling does not only downregulate T cell reactivity but also dampens induced antibody responses to porcine xenoantigen. Thus, disruption of stimulatory receptor‐ligand interactions (e.g. by blocking antibodies or “knock‐out/down” technologies) combined with transgenic overexpression of inhibitory ligands in porcine cells and tissues could be an effective approach to downregulate human anti‐pig immunity. References [1] Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout in pigs using zinc‐finger nucleases. Proc. Natl. Acad. Sci. USA 2011; 108: 12013–12017. [2] Weiss EH, Lilienfeld BG, Muller S, et al. HLA‐E/human beta2‐microglobulin transgenic pigs: protection against xenogeneic human anti‐pig natural killer cell cytotoxicity. Transplantation 2009; 87: 35–43. [3] Klymiuk N, van Buerck L, Bahr A, et al. Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice. Diabetes 2012; 61: 1527–1532. [4] Plege A, Schwinzer R. Stimulatory and inhibitory receptor interactions in xenotransplantation. Curr Opin Organ Transplant 2010; 15: 219–223. [5] Plege A, Borns K, Beer L, et al. Downregulation of cytolytic activity of human effector cells by transgenic expression of human PD‐ligand‐1 on porcine target cells. Transpl Int 2010; 23: 1293–1300.