FTY720 Promotes Engraftment of Allogeneic Donor Bone Marrow Stem Cells but Impairs Thymopoiesis Resulting in Delayed Graft Rejection.

Abstract 3530

Poster Board III-467

FTY720 (FTY), a sphingosine-1-phosphate receptor agonist, inhibits lymphocyte egress from lymphoid tissues although the complete mechanism of its immunomodulatory effects is not fully understood. We previously published that FTY inhibited but did not prevent graft-versus-host disease by multiple mechanisms. Using the same dose and schedule (3 mg/kg orally d0-28) we evaluated FTY for its effect on allogeneic bone marrow (BM) engraftment in sublethally-irradiated mice. C57BL/6 mice were irradiated with 5.0 Gy total body irradiation (TBI) on day -1, and received 10⁷ T-cell depleted BALB/c BM cells on day 0. At 5 wks, FTY-treated mice had a mean 84% ± 4% (mean ± SEM, n=47) donor chimerism in peripheral blood leukocytes (PBL) versus 5% ± 2% in water-treated controls (n=38, p<0.001). However, engraftment promotion was transient in most mice. PBL phenotyping at 3 months revealed that mean donor chimerism decreased to 22% ± 6%. Of the 32 mice that were >90% donor at 5 wks, only 6 were >50% donor at 3 months indicating that even high level donor chimeras were subject to delayed graft rejection. We found that although FTY promoted robust donor engraftment in the NK, myeloid and B cell lineages in BM, spleen, and lymph nodes by the first week after transplantation, thymopoiesis was severely impaired at 1 month resulting in near absent donor (and also host) thymic T cell production. FTY-treated mice had very low thymocyte cellularity (<7×10⁶, n=10). Most thymocytes (65-85%) were host CD4 or CD8 single positive T cells. We hypothesized that upon cessation of FTY, which prevents thymocyte egress, the mature host single positive T cells were released into the periphery and mediated delayed graft rejection. Consistent with this hypothesis, the in vivo depletion of host T cells but not host NK cells, at the time of cessation of FTY treatment, abrogated the loss of the donor graft indicating that host T cells were responsible for delayed graft rejection. Also consistent with our hypothesis, and demonstrating the immune competence of the host T cells retained in the thymus, the adoptive transfer of thymocytes from FTY-treated engrafted mice into lethally-irradiated C57BL/6 recipients mediated donor BALB/c BM rejection. To further examine the mechanism of early and robust albeit transient engraftment promotion in some cell lineages, but near absent thymopoiesis, we evaluated the absolute number of donor lin⁻Sca-1⁺cKit⁺ stem cells in the BM at 1 month. For these experiments, an engrafted control was deemed to be a more useful comparator than water-treated mice that rejected their graft. To ensure an engrafted control using the same TBI and allogeneic cell dose parameters, control mice were given peri-transplant injections of anti-CD4 and anti-CD8, a strategy that depletes host T cells and results in durable high level donor chimeras. Consistent with reports that FTY supports migration and bone marrow homing of stem cells, FTY-treated mice had a 4.9-fold increase in the absolute number of donor lin⁻Sca-1⁺cKit⁺ stem cells in the BM compartment compared to anti-CD4/8-treated mice. We hypothesized that the lack of donor thymopoiesis was the result of common lymphoid progenitors being trapped in the BM compartment and unable to migrate to and/or enter the thymus. Consistent with this hypothesis, FTY-treated mice had 125-fold fewer donor-type lin^locKit^hiCD25⁻ early thymic progenitors (ETPs) compared to anti-CD4/8-treated control mice. In contrast to FTY-treated mice, anti-CD4/8-treated mice had evidence of vigorous donor thymopoiesis. Collectively these data indicate that although FTY supports donor stem cell migration/homing to the BM and early donor NK, myeloid and B cell engraftment, the block in donor thymopoiesis and retention of thymic host T cells result in only transient engraftment in most sublethally-irradiated mice. These data have important implications in the use of FTY in BMT and further warrant examination of thymopoiesis in patients receiving FTY for immune suppression.