*Purpose: We aim to develop donor-specific immunosuppression by inhibiting T-cell cellular FLICE like inhibitory protein (c-FLIP). Our current work focuses on clinically relevant pharmacological inhibitors of c-FLIP. Utilising Histone Deactylase Inhibitors (HDACis) and also more specific novel small molecule c-FLIP inhibitors, to selectively enhance apoptosis in allo-reactive T-cells.

*Methods: BALB/c T-cells were purified and transfected with siRNA to c-FLIP or scrambled control (SC). These T-cells were used to reconstitute T-cell deficient RAG BALB/c recipient mice. Skin grafts from C57BL/6 donors were then carried out (n=6) and survival of the allograft assessed.

In vitro experiments were designed to ascertain the ability of the HDACis, Vorinostat and Entinostat, to induce apoptosis within activated murine T-cells from total splenocytes. Murine splenocytes and purified T-cells were stimulated using anti-CD3 and anti-CD28 antibodies, in the presence or absence of c-FLIP inhibitor, for 72 hours. Flow cytometry was used, with markers of apoptosis and cell death, to ascertain the incidence of apoptosis within the T cell populations.

*Results:

Figure 1: c-FLIP knock-down in T-cells prolongs allograft survival.

Reconstitution with T-cells transfected with siRNA to c-FLIP resulted in significantly longer allograft survival compared to reconstitution with T-cells transfected with scrambled control.

Figure 2: Populations of lymphocytes treated with Vorinostat have a greater incidence of apoptosis.

Vorinostat and Entinostat induce apoptosis within activated T-cells. A dose-response relationship exists, cells treated with higher doses of Vorinostat or Entinostat have a greater incidence of apoptosis (Fig.2). Preliminary data suggests Entinostat may more selectively induce apoptosis in activated T cells than Vorinostat.

*Conclusions: Vorinostat and Entinostat have potential as immunosuppressive therapies for transplantation. We plan to test these drugs as well as novel small molecule inhibitors in rodent heart transplant recipients shortly.

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