The mTOR inhibitor rapamycin (Rapa) is known to suppress T cell function. However, many of its in vivo effects, particularly its synergistic effects with costimulation blockade, cannot be explained through its known effects on T cell activation. Vascular endothelial cells (ECs) are the first barrier between host immunity and the allograft and thus play a central role in allo-recognition and costimulation. We hypothesized that Rapa might directly alter the alloimmunogenicity of ECs.

Human ECs were stimulated by TNF-α or IFN-γ for 72 hours with or without Rapa or vehicle solution (VS). The expression of HLA-class I and II, adhesion molecules, costimulatory molecules, and inhibitory molecules PD-L1 and PD-L2 were assessed using flow cytometry. Peripheral blood mononuclear cells labeled with VPD-450 were stimulated by resting, Rapa-conditioned, and VS-conditioned ECs and the allo-specific T cell responses were assessed by flow cytometry to detect cell proliferation.

Rapa/IFN-γ treated ECs showed significant reduction in CD40, HLA-DR, and HLA-DQ expression (p<0.05) when compared with VS/IFN-γ treated-ECs. Rapa/TNF-α treated ECs demonstrated higher OX40L (p=0.0051), CD58 (p=0.0001), and HLA-ABC (p=0.007) expression after stimulation when compared to VS/TNF-α treated-ECs.There was significantly higher PD-L1 (p=0.0001) and PD-L2 (p=0.0001) expression on Rapa-treated ECs compared to untreated.ECs treated with Rapa/IFN-γ also showed higher PD-L1 (p=0.001) and PD-L2 (p=0.0001) expression when compared to VS/IFN-γ treated. Furthermore, Rapa/TNF-α conditioned ECs significantly upregulated PD-L-2 (p=0.002) but not PD-L1 as compared to VS-/TNF-α treated ECs.ECs induced allo-specific T cell proliferation with significant higher levels of PD1 expression on proliferating CD4⁺ (72±6.2%, p=0.0001) and CD8⁺ cells (53.75±6.35%, p=0.002) than non-proliferating cells. Importantly, Rapa-treated ECs significantly inhibited allo-specific CD4⁺ (81.32±9.36%, p=0.001) and CD8⁺ (60.56±13.8%, p=0.007) cell proliferation when compared with VS treated ECs.

Rapamycin alters the surface phenotype of ECs such that they show enhanced expression of the inhibitory signals PD-L1 and PD-L2. Allo-specific proliferating T cells upregulate PD1 expression, and Rapa treated ECs exhibit inhibitory effects in reducing allo-specific T cell proliferation. This finding implies a direct role for Rapa in altering donor EC alloimmunogenicity, leading to inhibition of the alloimmune response.

CITATION INFORMATION: Bendersky V, Xu H, Kirk A. Rapamycin Directly Alters Human Endothelial Cell Alloimmunogenicity. Am J Transplant. 2017;17 (suppl 3).

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