*Purpose: Porcine endothelial cells (PECs) are the first barrier between a pig’s organ and circulating immune system. Unlike allogeneic immune responses, PECs fail to induce rapid memory cell-mediated responses in humans. The objectives of this study were to investigate and characterize the initial immune-responses of human CD14⁺ monocytes to PECs, and to explore whether PECs with pre-mTOR inhibition alter human monocyte-mediated xeno-recognition and xeno-responses.

*Methods: PEC membranes were pre-labeled with PKH26 or SLA-I-FITC. In selected experiments, PECs were pre-treated with rapamycin. Human PBMCs or purified CD14⁺ monocytes were co-cultured with PECs. In additional experiments, PEC-PBMC co-cultures were separated by a transwell-insert. A VPD-450 based T-cell proliferation assay was performed using a transwell system. PEC-stimulated cells were analyzed by FACS focusing specifically on CD14⁺ monocytes to detect uptake of PKH26⁺-membranes and microvesicles, and CD40 and CD80 expression. An intracellular cytokine staining (ICCS) assay was performed to detect cytokine production following PEC stimulation.

*Results: Unlike T cells, CD14⁺ cells showed a significant increase (p<0.05) of cytokine producers including IL-6 (9.3±7%), IL-12 p40/p70 (11.1±7.2%), and TNF-a (5.7±3.2%). PEC-stimulated monocytes upregulated CD40 (102.5±38.2%) and CD80 (135.6±57.9%) expression when compared with resting cells. CD14⁺ cells became positive for PKH26 following incubation of PBMCs or purified monocytes with PKH26-labeled PECs (56.7±1.8.2%, 52.1±11%). CD14⁺ cells but not CD3⁺ cells in transwell-insert demonstrated a significant increase of MFI of PKH26 (p=0.0085) over baseline levels during coculture with PKH26⁺-PECs. These monocytes also upregulated CD40 (57.4.5±21.1%) and CD80 (36.5±34.7%) expression. Furthermore, CD14⁺ cells in PBMC-PEC co-cultures became SLA-I-FITC positive. PBMCs in transwell-insert demonstrated proliferation without direct contact to PECs. Rapamycin-pretreated PECs did not prevent cytokine production in PEC-stimulated monocytes, and the nibbling of PEC membrane or extracellular microvesicles by monocytes was not inhibited. However, rapamycin-pretreated PECs significantly altered monocyte-derived CD40 upregulation (p=0.007) following their interaction.

*Conclusions: Our study demonstrates xenogeneic monocyte-mediated immune responses to PECs characterized as nibbling PEC membrane, microvesicles, and SLA-I, production of cytokines, and upregulation of costimulatory molecules. Monocytes may play a critical role in initiating indirect xeno-specific T cell proliferative responses. Rapamycin-conditioned PECs do not exhibit significant inhibition of monocyte-mediated xeno-recognition and activation but significantly altering CD40 expression. Our findings warrant a comprehensive therapeutic approach directly targeting monocytes and preventing xeno-specific immunity.

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