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Hirudin reduces upregulation of CD86 and the human T cell response to thrombin-activated pig endothelial cells

Corin Ezzelarab¹, Tim Sturgeon¹, Tyler Wilhite¹, David Ayares², David K.C. Cooper¹, Mohamed Ezzelarab¹

¹Surgery, University of Pittsburgh, Thomas E. Starzl Institute, Pittsburgh, PA; ²Revivicor, Blacksburg, GA; United States

Background: Activation of coagulation and thrombin formation occur in primate recipients of pig xenografts. Thrombin is known to activate endothelial cells (EC). The human T cell response to thrombin-activated pig EC is yet to be clarified.

Methods: Alpha1,3-galactosyltransferase-gene knockout (GTKO) pig aortic EC (pAEC) were activated by (i) porcine interferon-gamma (pIFN-γ, 40U/ml), (ii) human IFN-γ (hIFN-γ, 200U/ml), or (iii) thrombin (40U/ml or 10U/ml) for 24 hours. Swine leukocyte antigen (SLA) class I and II expression was assessed by flow cytometry. Human PBMC and CD4⁺Tcell responses to activated pAEC were evaluated in mixed lymphocyte reaction (MLR) and stimulation index (SI) was calculated. Pig co-stimulatory molecules CD80 and CD86 mRNA was detected by RT-PCR. The effect of thrombin inhibition by hirudin on the expression of pig CD80 and CD86 mRNA as well as on the human T cell response was evaluated.

Results: Following activation, SLA I expression on pAEC did not change, and only pIFN-γ upregulated SLA II expression on pAEC, while hIFN-γ or thrombin activation did not. In MLR, the human PBMC response to pIFN-γ- and thrombin-activated pAEC at 40U/ml (with SI of 37.4 and 18.3) was significantly higher (p<0.001 and <0.01) than to hIFN-γ- and non-activated pAEC (SI of 5.5 and 6.8). The human CD4⁺T cell response to pIFN-γ- and thrombin-activated pAEC at 40U/ml (SI of 42.1 and 20.1) was significantly higher (p<0.001 and <0.01) than to hIFN-γ- and non-activated pAEC (SI of 3.3 and 5). Thrombin (40U/ml) inhibition by hirudin (20U/ml) significantly reduced the human PBMC response to pAEC (p<0.05). Thrombin activation of pAEC (at 10 and 40U/ml) was associated with upregulation of CD86 mRNA, which was reduced after thrombin inhibition by hirudin.No significant changes was found in CD80 mRNA levels

Conclusions: Thrombin-activated pAEC upregulate CD86 which is associated with a significantly increased human T cell response. Thrombin inhibition by hirudin reduces CD86 mRNA level and the human T cell response to thrombin-activated pAEC. Therapeutic inhibition of thrombin and/or GTKO pigs transgenic for human thrombomodulin may be necessary to reduce the intensity of immunosuppression required in xenograft recipients.