Introduction: Transgenic expression of human modulatory molecules in pigs is a promising approach to prevent human anti-porcine immune responses and thus, an important step on the way to clinical xenotransplantation. We have previously shown that porcine cells overexpressing the human inhibitory ligand PD-L1 (CD274) trigger inhibitory signals in PD-1+ (CD279) human T, B, and NK cells thereby attenuating their activation (Buermann A, et al. Xenotransplantation. 2016: 23: 347). Furthermore, blocking of CD28-CD80/86-mediated co-stimulation by transgenic expression of CTLA4.Ig (CD152.Ig) in pigs decreases human T cell activation (Baehr A, et al. PLoS One. 2016: 11: e0155676). It is not yet clear, which modulatory effects could be expected when the two concepts are combined. Thus, it was the aim of this study to establish and characterize porcine model cell lines which overexpress hPD-L1 together with hCTLA4.Ig.
Methods: The porcine B cell line L23 as an antigen presenting cell (APC) was used as the model cell line. L23 cells were transfected with either hPD-L1, LEA29Y (a more potent derivate of human CTLA4.Ig, provided by Dr. N. Klymiuk, Munich), or both. L23-hPD-L1, -LEA29Y, and -hPD-L1/LEA29Y transfectants were tested for their potential to stimulate human immune cells in different in vitro cell culture assays.
Results: The different transgenic cell lines were characterized using flow cytometry analyses. L23-hPD-L1 cells strongly expressed the PD-L1 molecule on their cell surface. L23-LEA29Y cells produced LEA29Y which was detectable in the supernatant as well as bound to CD80/86 on the cell surface of transgenic L23 cells. Measurement of cell surface-bound-LEA29Y over time showed that in fresh medium not all CD80/CD86 epitopes of L23 transfectants were occupied by LEA29Y but after five days in culture complete saturation of epitope binding was obtained. Characterization of double transgenic L23-hPD-L1/LEA29Y cells revealed both, strong expression of hPD-L1 and production of LEA29Y. Co-culturing of L23-hPD-L1 or L23-LEA29Y cells with human T cells showed a reduced potential to stimulate proliferation for about 30-50% and 20-30% respectively, compared to stimulation with mock-transfected control L23 cells. No further reduction of proliferation was observed when the double transgenic L23-hPD-L1/LEA29Y cells were used as stimulators for human T cells.
Conclusion: Porcine APC genetically engineered to provide strong inhibitory signals (overexpression of hPD-L1) or prevent CD28-CD80/86 costimulatory interactions (production of hCTLA4.Ig) are weak stimulators of human T cell proliferation. So far, no synergistic inhibition of proliferation was observed by the combination of both approaches. It remains to be determined whether other effector functions of human T cells are modified by simultaneous enhancement of inhibition and blockade of co-stimulation.