*Purpose: Porcine vascular endothelial cells (PECs) play a central role in xeno-recognition and costimulation. The objectives of this study were to characterize the immuno-phenotype of human T cells in response to primary cultured PECs, and to explore the immuno-modulating effects of mTOR blockade of T cells and/or PECs during xenogenic immuno-responses.

*Methods: Primary cultured PECs, isolated from aorta, were used as stimulators. In selected experiments, PECs were pre-treated with 10 ng/mL of rapamycin for 72 hours before stimulation. Human peripheral blood mononuclear cells were obtained from normal individuals and labeled with VPD-450 followed by stimulation with PECs monolayers for 6 days with inhibitors specific for mTOR, B7, and CD11a. Cells were analyzed by flow cytometry to detect cell proliferation. An intracellular cytokine staining (ICCS) assay was performed to detect xeno-specific memory cell responses to PEC following 12 hours stimulation.

*Results: ICCS assay did not detect significant memory T cells responses following PECs stimulation as determined by lack of TNF-α and IFN-γ producing cells. The proliferation of T cells in response to PECs were detected (CD4⁺ 11.6±2.6%, CD8⁺ 24.3±6.06%). Phenotypically, the proliferating CD4⁺ cells composed with a significant higher fraction of CD57⁺PD1⁺ (p=0.01) and CD57^–PD1⁺ (p=0.00377) subsets when compared with non-proliferating cells, and the frequency of effector memory (CD45RA^–CCR7^–) subset in proliferating cells was significantly higher than non-proliferating cells (p=0.0062). Similarly, a dramatic increase of CD57^–PD1⁺ cells was also revealed in proliferating CD8⁺ cells (p=0.006) when compared with non-proliferating cells. These proliferating cells were largely effector memory cells. Belatacept partially inhibited xeno-specific T cell proliferation (CD4⁺ 62.4±17%, CD8⁺ 50±222.4%). The presence of rapamycin during xenogeneic immune responses produced partial inhibitory effects (CD4⁺ 62.96±17%, CD8⁺ 63.8±15.8%). CD11a blockade only achieved minimal inhibition. In contrast, PECs, pre-treated with rapamycin, were unable to induce xenogeneic T cell proliferation with 95% inhibitory effects on both CD4⁺ and CD8⁺ cells.

*Conclusions: Our study did not observe xeno-specific memory T cell-mediated immune responses to PEC. Xenogenic PECs induced primed T cell proliferation characterized by increased fraction of PD1 expressing cells and effector memory subset. Xenogenic T cell proliferation is inhibited by belatacept and rapamycin but not CD11a blockade. Rapamycin pre-treated PECs exhibit a dramatic inhibitory effect in reducing xeno-specific T cell proliferation suggesting a direct role for rapamycin to alter PEC xeno-immunogenicity leading to inhibition of xeno-specific immune response.

« Back to 2020 American Transplant Congress