Depletion und Suppression humaner HLA-alloreaktiver T-Zellen durch CD95L-exprimierende Zellen

Within the project as presented in this thesis, depletion of alloreactive human T-cells by CD95L-expressing cells was examined, aiming at transfer of donor-lymphocyte-infusions after allogenetic stem-cell-transplantation (SCT). Specifically after HLA-haploidentical SCT with highly purified stem cells, application of donor-lymphocytes could reduce the required time for immune-reconstitution of donor-derived T-cells. Using a mixed-lymphocyte-culture system for stimulation of HLA-alloreactive T-cells, the approach of CD95L expressing cells as stimulator cells for allodepletion was based on the fact that stimulated T-cells are highly sensitive towards CD95L-mediated apoptosis, while unstimulated T-cells are rather resistant. It was investigated, whether stimulation by CD95L-expressing cells could result in efficient apoptosis in simultaneous or sequential culture systems. To this extent, induction of apoptosis by lentivirally transducted CD95L-expressing B-cell-lines or fibroblasts was carried out either simultaneously to – or sequentially after – allogeneic activation of T-cells. It was demonstrated, that allospecific proliferation and cytotoxicity after simultaneous activation and induction of apoptosis were completely abolished by the CD95L-expressing B-cell-line C1R-A1-CD95L. Although we could demonstrate high effectivity of allodepletion, this system was not of clinical practicability because of solely HLA-A1 specific stimulation and apoptosis-induction by C1R-A1-CD95L. As a next step, a sequential culture system was developed for potential clinical use: an EBV-transformed B-cell-line was used for HLA-specific, allogeneic stimulation and apoptosis was induced in a second step by CD95L-expressing cells. In this system, we demonstrated effective inhibition of allogenetic immunoreactions after sequential activation and induction of apoptosis by CD95L: residual alloreactivitiy was abrogated. The advantage of this sequential culture system is the universal applicability of a CD95L-expressing cell-line for depletion of individually, HLA-specific allogeneic stimulated T-cells - thereby clinical applicability is given in principle. CD4+CD25+FoxP3+ regulatory T-cells (Treg) have been proven of great importance for prevention and amelioration of alloreactive immune reactions and Graft-versus-host-Disease after SCT. Therefore, presence and function of Treg was examined after stimulation and induction of apoptosis by CD95L-expressing cells. In both culture systems, simultaneous and sequential, functional CD4+CD25+FoxP3+ Treg were preserved. Treg with intermediate CD25 expression were resistant towards CD95L mediated apoptosis and even enriched after culture with C1R-A1-CD95L. Interestingly, expression of the transcription factor FoxP3 did not necessarily correlate with suppressive function. Function of Treg and expression of FoxP3 was examined after induction of apoptosis for the first time. It was proven that indeed residual CD4+CD25+ cells were regulatory T-cells, and, furthermore, our results indicate antigen-specific function of residual Treg. This is of great importance for potential clinical use to prevent the risk of an otherwise general immune-suppressive function by Treg. In summary, the work presented in this thesis provides a method for effective depletion of alloreactive T-cells without affecting regulatory T-cells by using a CD95L-expressing B-cell-line. The combination of selective induction of apoptosis in stimulated, HLA-alloreactive T-cells and suppression of residual alloreactivity by Treg resulted in a highly efficient system and opens promising options for therapeutic applications.