Allo-specific T-cells encoding for viral IL-10 exert strong immunomodulatory effects in vitro but fail to prevent graft rejection.

Recently, we demonstrated the capacity of allo-specific gene-engineered T lymphocytes as transport vehicle for therapeutic transgenes into allografts. In this study, the influence of viral IL-10 as therapeutic transgene was addressed. Lewis rat T-cell lines specific for DA rat alloantigens were engineered to express vIL-10 by using a retroviral gene expression system. Like T regulatory 1 cells, vIL-10 transgenic T lymphocytes express the phenotype CD4(+)25(+) and secrete, in addition to vIL-10, rat IL-10 and IFN-gamma but no IL-4. First, the capacity of vIL-10 transgenic T-cell lines to modulate alloantigen-specific immune responses was evaluated in vitro. In comparison to control MLR with no transgenic cells or equal numbers of control T(EGFP)-lymphocytes, the proliferation as well as production of IFN-gamma by naive responder cells were significantly diminished. Despite this regulatory capacity in vitro, T(vIL-10)-lymphocytes were not able, either alone or in combination with suboptimal doses of Cyclosporine A, to prolong the survival of either DA rat cardiac or renal allografts in Lewis rat recipients. These data demonstrate that intra-graft IL-10 over-expression is not sufficient to prolong allograft survival in a high-responder strain combination and that the regulatory capacity of T cells in vitro does not predict their in vivo efficiency.

FTY720 prevents anti-CD4 mAb-induced tolerance but cannot reverse established tolerance in a rat kidney transplantation model.

FTY720 is highly effective in various models of transplantation and autoimmunity. In order to find drugs that act synergistically with a tolerance-inducing nondepleting anti-CD4 mAb we studied this combination in a strong DA to LEW kidney transplantation model. Rats were treated with 0.3 mg/kg of FTY720 for 14 days and anti-CD4 mAb RIB5/2, alone or in combination. After kidney transplantation serum creatinine and blood lymphocyte counts were monitored. Immunohistology, ELISPOT and TaqMan trade mark -PCR analysis of biopsies were performed. Short-term application of RIB5/2 but not FTY720 induced long-term survival of kidney transplants. Moreover, the combination of FTY720 + RIB5/2 prevented tolerance induction. In the combination group serum creatinine levels increased 1 week after cessation of therapy and all rats died from uremia within 72 days. Intragraft immunohistology, ELISPOT and real-time RT-PCR analysis at day 21 demonstrated an enhanced T-cell infiltration and activation but a diminished up-regulation of protective genes in the grafts from recipients receiving the combination therapy. In contrast, delayed application of FTY720 to RIB5/2-treated rats did not interact with RIB5/2-induced tolerance. In summary, FTY720 is powerful in preventing intragraft infiltration by naive T cells but this might also affect the early development of graft-protecting regulatory T cells and tolerance induction.