Increased death inducing receptors in vivo and activation-induced T-cell death by anti-thymocyte antibody treatment of stable cardiac transplant recipients

Introduction: Specific and unspecific triggering of the immunesystem triggers the CD95 and TNF receptor pathway. We aimed to identify if alloantigen activates T cells to upregulate the apoptosis trigger CD95 and if shedding of sTNF R1 takes place. Moreover, we sought to investigate if drugs which are capable of reducing rejection frequency in heart transplantation are triggering activation induced cell death (AICD) in T-cells. Methods: 20 HTX vs 20 NYHA IV patients were studied. CD95 expression on T lymphocytes (CD3) was determined by FACS analysis. sTNF RI was evaluated by ELISA. CD3 positive cells were stained by annexin V, a protein binding to phosphatidylserine, a phospholipid present on cells undergoing early phases of apoptosis. AICD was quantitated by FACS analysis double positivity of fluorochrome labelled T-helper cells (CD4) and intracellular 7-AAD DNA binding was measured, indicating cell death. AICD potency of induction therapy was evaluated after coculture of HTX PBMCs with IL-2 R-blocker (Daclizumab), OKT-3 and ATG (all 50ug/cc) for 18 hours in a humidified incubator. Unspecific IgG served as control. Results: In vivo, CD3 cells of HTX vs NYHA IV patients presented increased CD95 expression (701/-6.6% vs 331/-4.5%, p,0.001) and bind to annexin V (381/-5% vs 91/-2.5%, p,0.001), indicating that T-cells undergo apoptosis. Less than 0.5% CD3/7-AAD positivity was tested in vivo. Moreover, the death inducing receptor sTNF R1 was significantly increased in HTX vs NYHA IV (3.351/-0.7ng/cc vs 1.11/-0.4ng/cc, p,0.001). The in vitro potency to induce AICD were 71/-1.1 in the OKT3 sample, 111/-4.1 in the ATG sample and 2.41/-0.36 in the IL-2 Rblocking group in relation to unspecific IgG control (all, p,0.05). Conclusion: Allograft exposure is inducing CD95and TNF R1 augmented T lymphocyte apoptosis and renders the immunesystem to become susceptible to AICD. These results suggest that monoand polyclonal therapies are capable of inducing AICD and demonstrate variable agent potencies in vitro.