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T cell-directed immunosuppression is associated with downregulation of thrombin activation, but upregulation of inflammatory responses in xenograft recipients

Mohamed Ezzelarab¹, Corin Ezzelarab¹, Kumiko Isse¹, Burcin Ekser^1,2, Gabriel Echeverri^3,4, David Ayares⁵, David Cooper¹

¹University of Pittsburgh, Thomas E. Starzl Institute, Pittsburgh, PA, United States; ²Transplantation and Advanced Technologies, Vascular Surgery and Organ Transplant Unit, University Hospital of Catania, Catania, Italy; ³Mediterranean Institute for Transplantation and Advanced Specialized Therapies, Palermo, Italy; ⁴Transplant Unit, Fundacion Valle del Lili, Cali, Colombia; ⁵Revivicor, Blacksburg, VA, United States

Background: Thrombotic microangiopathy remains a major problem in xenotransplantation. It is known that inflammation induces activation of coagulation. We evaluated the inflammatory responses and activation of coagulation in GTKO pig aortic patch xenograft recipients receiving costimulation-based immunosuppressive therapy (IS).

Methods: Baboons received either no IS (n=2), minimal IS (CTLA4-Ig or antiCD154mAb alone; n=4), or full IS (ATG + CTLA4-Ig or antiCD154mAb + MMF; n=3). Mean values of absolute CD3⁺T cell numbers were determined (CD3-mean; cells/µl). The T cell response was evaluated and mean stimulation index was calculated (SI-mean). Mean values of thrombin-antithrombin complex (TAT), D-dimers, fibrinogen, and IL-6 levels were calculated. Fold increase of C-reactive protein (CRP) level (compared to pre-Tx) was determined, and mean values were calculated (FiCRP-mean). CRP deposition in xenografts was assessed histologically.

Results: In comparisonto minimal and no IS, full IS was associated with (1) significant reduction in CD3⁺cells (CD3-mean of 234 with full ISvs 1524 with minimal IS and 1343 with no IS [p<0.05]), (2) suppression of the T cell response (SI-mean of 4 vs 15 and 32), (3) reduction in TAT (µg/l) to 12 vs 66 and 272, (4) no change in D-dimers (1-4µg/ml), (5) a higher mean fibrinogen level (267µg/dl vs 166 and 157), (6) a higher mean IL-6 level (38pg/ml vs 17 and 4),and (7) a higher FiCRP-mean (9.2 vs 2.9 and 1). There were significant positive correlations between (i) CD3⁺cells and TAT levels (r²=0.922; p<0.01), (ii) CRP and fibrinogen levels (r²=0.8517; p<0.01), and (iii) CRP and IL-6 levels (r²=0.927; p<0.001). A significant negative correlation was found between CRP levels and the number of CD3⁺cells (r²=-0.762; p<0.01). With full IS, CRP was detected in the graft capillaries only, in contrast to no IS where CRP-positive macrophages were detected.

Conclusions: Costimulation-based IS inhibited adaptive T cell responses, reduced thrombin activation, and prevented CRP-positive macrophage infiltration, but was associated with upregulation of systemic CRP and IL-6 levels. Prevention of the adaptive immune response and reduction of activation of coagulation by full IS is countered by the increase in the innate systemic inflammatory response. Prevention of the innate inflammatory response might be needed to control coagulation dysfunction.