Downregulation of cytolytic activity of human effector cells by transgenic expression of human PD-ligand-1 on porcine target cells.

Cellular rejection is a relevant hurdle for successful pig-to-primate xenotransplantation. We have shown previously that the induction of a human anti-pig T cell response (in vitro activation of CD4(+) T cells) can be suppressed by the overexpression of human negative costimulatory ligands (e.g. programmed death receptor ligand, PD-L1) on pig antigen presenting cells. Here, we asked whether PD-L1 mediated enhancement of negative signaling might also be efficient during the effector phase of human anti-pig cellular immune responses. The porcine B-cell line L23 was transfected with human PD-L1, and clones were selected stably expressing PD-L1 with low, medium, or high density. Mock-transfected L23 cells were effectively lysed by human cytotoxic effector cells (IL-2 activated CD8(+) T cells and CD56(+) cells). The lytic potential of the effectors decreased with increasing levels of PD-L1 and was reduced by about 50% in L23-PD-L(high) targets. A proportion of activated CD8(+) effector cells underwent apoptosis when exposed to PD-L1 expressing L23 cells. These data suggest that the overexpression of PD-L1 on target cells may (a) trigger negative signals in effector cells that prevent the release of cytolytic molecules and/or (b) induce apoptosis in the attacking effector cells thereby protecting targets from destruction.

Stimulatory and inhibitory receptor interactions in xenotransplantation.

PURPOSE OF REVIEW: Cellular human antipig immune responses are increasingly recognized as an important barrier to successful clinical xenotransplantation. This review addresses the role of monocytes/macrophages, natural killer (NK) cells, and T cells in xenograft rejection. We focus on the receptor-ligand interactions that regulate the responses of these cells to porcine tissues and thus could be targets for immunomodulation. RECENT FINDINGS: Activation of human monocytes by pig cells is partly due to the incapacity of porcine ligands to bind to inhibitory receptors such as signal regulatory protein alpha. Porcine UL16-binding protein 1 can functionally interact with human NK group 2D protein, thereby contributing to human NK cell activity. Transgenic pigs overexpressing human leukocyte antigen class E were generated. Cells from these pigs induced diminished NK-cell lysis, suggesting that human leukocyte antigen class E expression compensates for the inability of porcine ligands to bind to the inhibitory CD94/NK group 2A receptor on human NK cells. A new concept for the modulation of antipig T-cell reactivity may result from the finding that porcine antigen-presenting cells that overexpress human negative costimulatory PD ligands also induce diminished responses of human T cells. SUMMARY: Disruption of stimulatory receptor-ligand interactions (e.g. by blocking antibodies or 'knockout/down' technologies) combined with transgenic overexpression of inhibitory ligands in porcine cells and tissues could be an effective approach to downregulate human antipig cellular immune responses.

Suppression of human T-cell activation and expansion of regulatory T cells by pig cells overexpressing PD-ligands.

BACKGROUND: Genetic modification of pigs (e.g., transgenic expression of human complement regulatory molecules or inactivation of alpha1,3galactosyltransferase) 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. This report tested the hypothesis that overexpression of human negative costimulatory PD-Ligands (PD-L) in pig antigen presenting cells might be an approach to prevent human anti-pig T-cell responses. METHODS: The pig B-cell line L23 was transfected with the pIRES-AcGFP vector containing human PD-L1 or PD-L2. Stable transfectants (L23-PD-L1, L23-PD-L2 cells) were established and used for in vitro stimulation of purified human CD4+ T cells. RESULTS: Human CD4+ T cells responded with significantly reduced proliferation to L23-PD-L1 or L23-PD-L2 cells and produced less IL-2, IFNgamma, TNFalpha, 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. CONCLUSIONS: These findings imply that PD-1/PD-Ligand pathways are interesting targets to prevent human anti-pig T-cell responses after xenotransplantation, and also suggests that PD-1/PD-Ligand interactions may play a role in the control of the activity and/or homeostasis of regulatory T cells.