Effective Depletion of Alloreactive T Cells by CD95l-Transduced Cells with Preservation of FoxP3 Regulatory T-Cells.

Adoptive transfer of donor T-cells after stem cell transplantation across HLA barriers could bridge the period of prolonged immunodeficiency until new thymic derived T-cells develop. In such an approach, however, donor T-cells reactive to alloantigens of the recipient induce GvHD, necessitating their selective elimination, inactivation or modulation. Depletion of alloreactive cells has been attempted by monoclonal antibodies, but prevention of GvHD was insufficient in clinical application so far. We developed a system allowing efficient selective depletion of alloreactive cells by CD95L-mediated apoptosis, which in addition has the advantage of preserving FoxP3 regulatory T-cells. An apoptosis-resistant B-cell line (C1RA1) was transduced with lentiviral CD95L (C1RA1-CD95L). CD95L was stably expressed by 65–80% of the cells and was fully functional to induce apoptosis in Jurkat cells. MNC of donor A were stimulated with APC of donor B, and, after 5 days of culture, viable responder cells were enriched and cultured with C1RA1-CD95L for another 3 days. T cells obtained by this culture system had lost proliferative activity towards cells of donor B, while proliferation against 3rd-party cells was fully preserved. Likewise, cytotoxic activity against donor B was markedly reduced. Since CD4+CD25+ T-cells were still detectable at levels of 7 to 10% of MNC at the end of the culture despite effective depletion of alloactivated cells, we analyzed for the presence of FoxP3+ regulatory T-cells (Treg). While gene expression of FoxP3 was almost undetectable in MNC before culture, it was present after activation of MNC and was strongly expressed after induction of apoptosis with C1RA1-CD95L at the end of our culture system. Suppression of proliferation of about 50% by these cells was noted in a suppressor assay using fresh MNC from donor A stimulated with cells of donor B. When suppression was analyzed in a stimulation system with 3rd-party stimulatory cells, reduction of proliferation was less prominent, indicating the possibility that the suppressive activity of Treg preserved in our culture system could be alloantigen-specific. In summary, we have developed a culture system, allowing alloantigen-specific depletion of T cells while preserving FoxP3+ regulatory T-cells. This sequential system of alloantigen-specific stimulation followed by CD95L-mediated apoptosis of activated cells by an unrelated, modified cell line offers the advantage of broad applicability in clinical transplantation settings.