Abstract

FTY720 (FTY), a sphingosine-1-phosphate receptor agonist, inhibits lymphocyte egress from lymphoid tissues although the complete mechanism of its immunomodulatory effects is not well understood. We studied FTY in murine GVHD and engraftment models as a single agent and in combination with known beneficial strategies. FTY (3 mg/kg orally d0-28) inhibited GVHD, increasing the median survival time (MST) of lethally irradiated C57BL/6 (B6) mice given BALB/c BM and a moderate T cell dose from 30d to 71d (p<.001) or a higher T cell dose from 7d to 32d (p<.006). FTY was additive with ex vivo activated and expanded CD4+CD25+ regulatory T cells (Tregs) for GVHD inhibition. In contrast to control mice which all died of GVHD by d70 (MST = 37d), mice receiving FTY or Tregs or both had 100 day survival rates of 50%, 71% and 100%, respectively. The additive effects of FTY and Tregs were reproduced in a second strain combination. Although we hypothesized that FTY trapped Tregs in the lymph nodes allowing for increased contact time for Tregs to inhibit GVHD effector T cells, imaging studies of green fluorescent protein (GFP)+ Tregs did not support this hypothesis. Moreover, imaging studies of GFP+ effectors did not indicate that FTY inhibited donor T cell egress from lymphoid tissues. To study alloantigen-specific responses, B6 TCR Tg CD8+ and CD4+ T cells that are reactive against BALB/c alloantigen were adoptively transferred into B6 rag mice prior to sublethal irradiation and infusion with BALB/c BM. FTY reduced the expansion of Tg CD8+ and CD4+ T cells in the spleen 10d after BMT by 80% and 78%, respectively. In addition to GVHD inhibition, FTY also promoted donor BALB/c BM engraftment in sublethally (5.0 Gy) irradiated B6 mice although donor chimerism was not stable in all mice. In contrast to 4 of 26 water-treated controls, all 29 FTY-treated mice (3 mg/kg d0-13) were engrafted at 1 month as assessed by PBL phenotyping (ave of 9% vs 68% donor. p<.001). However, by 3 months, the engraftment rate in FTY-treated mice had decreased to 12 of 28 mice. All 9 mice receiving FTY for an additional 2 weeks (d0-28) had high donor chimerism levels (>96%) at 1 month but chimerism was lost in 3 mice by 6 months after BMT. In a different strain combination, even a 2 wk FTY course increased donor chimerism that remained stable for 6 months. Imaging studies revealed that FTY did not prevent the egress of adoptively transferred host-type GFP T cells in allogeneic BM recipients. FTY was also tested in combination with anti-CD40L mAb, a potent engraftment-promoting agent in mice. In contrast to anti-CD40L mAb, FTY did not promote engraftment in mice conditioned with very low levels of irradiation (0.5–2.0 Gy) although the combination of anti-CD40L mAb and FTY resulted in superior stable engraftment rates and levels compared to anti-CD40L mAb as a single agent. Interestingly, FTY increased donor chimerism in syngeneic, as well as, allogeneic recipients. These data indicate that FTY is efficacious as a single or adjunct agent for the prevention of GVHD and graft rejection and further indicate that inhibition of lymphocyte egress was not essential for the beneficial effects of FTY in BMT recipients.

Disclosures: Volker Brinkmann is an employee of Novartis which produces FTY.

Author notes

*

Corresponding author