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. 2012 Sep;18(9):1341-52.
doi: 10.1016/j.bbmt.2012.06.007. Epub 2012 Jun 19.

FTY720 markedly increases alloengraftment but does not eliminate host anti-donor T cells that cause graft rejection on its withdrawal

Patricia A Taylor  1 , Ryan M Kelly, Nick D Bade, Michelle J Smith, Heather E Stefanski, Bruce R Blazar
Affiliations

FTY720 markedly increases alloengraftment but does not eliminate host anti-donor T cells that cause graft rejection on its withdrawal

Patricia A Taylor et al. Biol Blood Marrow Transplant. 2012 Sep.

Abstract

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

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Conflict of interest statement

Disclosure of Conflict of Interest:

The authors have nothing to disclose.

Figures

Figure 1
Figure 1
FTY results in transient allogeneic donor engraftment in the B cell and myeloid cell but not the T cell lineages. B6 mice were irradiated with 5.0 Gy TBI on day −1 and infused with 10×106 TCD BALB/c BM on day 0. FTY (3 mg/kg) or water was administered orally for 2 (A) or 4 (A–D) weeks daily from day 0. A) PBLs were phenotyped for percentage of donor class I+ cells at 5 wk and 3 mo. n = 47–52 mice/group, pool of 4 experiments. *P < .05, FTY versus H20. ^P < .05, 5wk versus 3 mo. B) On day 28 after BMT, PBLs were phenotyped for percentage of donor and host CD19+ B, CD11b+ myeloid and CD4+ and CD8+ T cells. n = 7–8 mice/group. ^P < .05, donor H20 versus donor FTY; *P < .05, host H20 versus host FTY. C) Shown are absolute numbers of donor and host cells of each cell lineage from spleen, peripheral LN pool and both tibias and femurs (BM) harvested 28 days after BMT. n = 6 mice/group. ^P < .05, donor H20 versus donor FTY, *P < 05, host H20 versus host FTY. D) Thymuses were harvested 28 days after BMT. Shown are absolute thymocyte number of each of the indicated cell populations. n = 8 mice/group. *P <.05. FTY versus H20. A–D) Shown is mean ± 1 standard error of the mean (SEM).
Figure 2
Figure 2
FTY results in increased stable congenic donor engraftment. B6 CD45.2 mice were irradiated with 1.0 Gy TBI on day −1 and infused with 10×106 TCD congenic B6 CD45.1 BM on day 0. FTY or water was administered from day 0 to day 28. PBLs were phenotyped for percentage of donor CD45.1+ cells at 5 wk and 3 mo. n= 26 mice/group, pool of 3 experiments. *P < .05, FTY versus H20. Shown is mean ± 1 SEM.
Figure 3
Figure 3
FTY increases donor stem cell homing to the host BM cavity. B6 CD45.2 mice were irradiated with 5.0 Gy TBI on day −1 and infused with 10×106 TCD allogeneic BALB/c CD45.1 BM on day 0. To ensure an engrafted control, anti-CD4 and anti-CD8 mAbs were administered on day −2, day 0 and day +4 (designated TCD). FTY was administered day 0 to day 28. Mice determined to have >92% donor PBLs on day 28 were evaluated for donor CD45.1+lineage−Scal-1+ckit+ stem cell and CD45.1+lineage−Sca-1lockitlo CLP numbers in both femurs and tibias on day 29. A) Shown is ckit versus Sca-1 contour plot of donor+ lineage− BM cells in 1 representative mouse of 5 in control versus FTY-treated mice. B) BM, donor stem cell and donor CLP counts are shown on 5 individual mice/group. Mean is indicated by a horizontal bar. *P < .05.
Figure 4
Figure 4
FTY blocks T cell progenitor thymic ingress and mature host T cell thymic egress. A separate cohort of mice as described in Figure 3 were evaluated for total thymocyte and ETP number and thymic phenotype on day 30 (A–D) and thymocyte number and phenotype 3 months after BMT (4E). 4A) Shown is a representative CD4/CD8 plot illustrating that most thymocytes are donor DP in TCD control indicative of normal thymopoiesis while most thymocytes are host SP in FTY-treated, engrafted mice indicative of blocked thymopoiesis on day 28 after BMT. 4B–C) Shown are total thymocyte number (4B) and total donor ETP number (4C) on day 28 after BMT. Note y-axis is a log scale. n = 4–5 mice/group. 4D) Total number of each thymocyte cell population is illustrated. Note y-axis is a log scale for donor cell populations. n = 4–5 mice/group. 4E) Thymocyte numbers 3 months after BMT are shown illustrating that host thymopoiesis has recovered in FTY-treated mice while most thymocytes in TCD controls are of donor origin. n = 5 mice/group. 4B–4E) *P < .001; ^P < .05; NS = not significant (P > .05).
Figure 4
Figure 4
FTY blocks T cell progenitor thymic ingress and mature host T cell thymic egress. A separate cohort of mice as described in Figure 3 were evaluated for total thymocyte and ETP number and thymic phenotype on day 30 (A–D) and thymocyte number and phenotype 3 months after BMT (4E). 4A) Shown is a representative CD4/CD8 plot illustrating that most thymocytes are donor DP in TCD control indicative of normal thymopoiesis while most thymocytes are host SP in FTY-treated, engrafted mice indicative of blocked thymopoiesis on day 28 after BMT. 4B–C) Shown are total thymocyte number (4B) and total donor ETP number (4C) on day 28 after BMT. Note y-axis is a log scale. n = 4–5 mice/group. 4D) Total number of each thymocyte cell population is illustrated. Note y-axis is a log scale for donor cell populations. n = 4–5 mice/group. 4E) Thymocyte numbers 3 months after BMT are shown illustrating that host thymopoiesis has recovered in FTY-treated mice while most thymocytes in TCD controls are of donor origin. n = 5 mice/group. 4B–4E) *P < .001; ^P < .05; NS = not significant (P > .05).
Figure 5
Figure 5
Donor CLPs from FTY-treated mice can differentiate into T cell progenitors in in vitro cultures. B6 mice were irradiated with 5.0 Gy TBI on day −1 and infused with 10×106 TCD allogeneic BALB/c BM on day 0. To ensure an engrafted BMT control, anti-CD4 and anti-CD8 mAbs were administered on day −2, day 0 and day +4 (designated TCD). FTY was administered day 0 to day 28. A non-BMT BALB/c control is included. On day 29, BM from mice determined to have >92% donor PBLs was harvested from femurs, tibiae and spines (pool of 10 mice/group), depleted of host lineage+ cells and then sorted for donor+lineage−skitloSca-1lo CLP cells. CLPs were cultured on a BM stromal cell line, OP-9DL1, that permits T cell lineage commitment and early T cell differentiation, and phenotyped on the indicated day of culture. Shown is a CD44/CD25 contour plot illustrating maturation from DN1 to DN4 stage of T cell development. No significant differences in cell yield were noted.
Figure 6
Figure 6
Host SP T cells mediate delayed graft rejection in FTY-treated engrafted mice. A) PBLs from FTY-treated mice were phenotyped 4 and 7 weeks after BMT. Shown is decline in engraftment in each mouse from 4 to 7 weeks. n = 16 mice; P < .001, 4 versus 7 weeks. B) On d28 after transplant, 0.5×106 mature B6-type SP thymocytes isolated from FTY-treated allogeneic BMT recipients were adoptively transferred into B6 hosts immediately after lethal irradiation and 1 day prior to infusion of BALB/c BM. Shown is survival. Mice receiving thymocytes died of graft rejection by day 15. Mice not receiving thymocytes survived long-term. n = 8 mice/group; P < .001. C) B6 thy1.1 mice were irradiated with 5.0 Gy TBI on day −1 and infused with 10×106 TCD allogeneic BALB/c thy1.2 BM on day 0. Mice received FTY daily for 4 weeks. High-level chimeras received either no aby, anti-NK1.1 mAb to deplete host NK cells, anti-thy1.1 mAb to deplete host T cells or both anti-NK1.1 and anti-thy1.1 mAbs. Shown is percentage donor engraftment at 4 weeks and 3 months. n = 12–18 mice/group; P < .001, 4 weeks versus 3 months for mice receiving no aby or anti-NK1.1 mAb.
Figure 7
Figure 7
FTY stably increases the anti-CD40L mAb-mediated alloengraftment promoting effect in mice receiving low dose irradiation. B6 mice were irradiated with 2.0 Gy (A,C) or 1.0 Gy (B,D) TBI on d-1 and infused with 40×106 NTCD BALB/c BM on day 0. FTY, anti-CD40L or both was administered for 4 wks. PBLs were phenotyped for percentage of donor cells at 5 wk and 4 mo. C,D) Shown is percentage of donor and host T cells (CD4+ + CD8+) in PBLs at 4 mo. A,C) n=10 mice/group, 1 experiment. B,D) n=20 mice/group, pool of 2 experiments. *P < .05, anti-CD40L versus FTY+anti-CD40L; ^P = .06, anti-CD40L versus FTY+anti-CD40L. Shown is mean ± 1 SEM.
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