Negative costimulation to prevent human anti‐pig T cell responses

Genetic modification of pigs [e.g. transgenic expression of human complement regulatory molecules or inactivation of α(1,3)‐galactosyltransferase] 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. Our group focuses on the inhibition of human anti‐pig T cell responses by targeting ‘‘negative’’ costimulatory pathways. We tested the hypothesis that over‐expression of the human negative costimulatory ligands PD‐L1 and PD‐L2 on pig cells will result in reduced human anti‐pig T cell responses. To this end, pig cell lines stably over‐expressing human PD‐Ligands (L23‐PD‐L1/L2 cells) and appropriate controls (L23‐GFP cells) were generated and used for in vitro stimulation of purified human CD4 + T cells. Furthermore, the effect of PD‐L expression on cellular cytotoxicity was tested by 51 Cr‐release assays using L23‐PD‐L1/L2 and L23‐GFP cells as targets and IL‐2‐activated human CD8 + T cells and NK cells as effector. CD4 + T cells responded with significantly reduced proliferation to L23‐PD‐L1 or L23‐PD‐L2 cells and produced less IL‐2, IFNγ, TNFα, IL‐4, and IL‐5 than cells stimulated with mock‐transfected B cells. The concentration of IL‐10, however, was increased in CD4 + T cells responding to stimulation with PD‐L1 or PD‐L2 transfectants. Furthermore, in cultures of CD4 + T cells stimulated for 3 weeks with PD‐L1 or PD‐L2 transfectants a CD4 + CD25 high Foxp3 + subset showed up that effectively suppressed the activation of conventional CD4 + T cells. CD8 + T cells and NK cells acquired cytolytic potential and up‐regulated the PD‐1 receptor during cultivation with IL‐2. Both cell types lysed L23‐GFP cells very effectively but were less potent on L23‐PD‐L1 targets (reduced cytotoxicity of about 50%). When cytotoxic effector cells were used that do not express PD‐1 (293‐95L cells), L23‐GFP and L23‐PD‐L1 cells were lysed with similar intensity suggesting that PD‐1/PD‐L1 interactions are required for PD‐L1 mediated protection. Flow cytometric studies revealed that a proportion of activated CD8 + PD‐1 + T cells underwent apoptosis when exposed to PD‐L1 expressing L23 cells. Together these observations support the assumption that PD‐1/PD‐Ligand pathways are interesting targets to prevent human anti‐pig cellular immune responses after xenotransplantation [1]. PD‐L over‐expression might not only empede the initiation of a human anti‐pig T cell response by suppressing CD4 + T cells but may also protect pig cells from destruction by human cytotoxic effectors. Supported by the Deutsche Forschungsgemeinschaft (Transregio Forscher‐gruppe ‘‘Xenotransplantation’’, FOR 535). Reference 1. P lege A, B orns K, B aars W, S chwinzer R. Suppression of human T‐cell activation and expansion of regulatory T cells by pig cells overexpressing PD‐ligands. Transplantation 2009; 87: 975–982.