Insights into the mechanism of FTY720 and compatibility with regulatory T cells for the inhibition of graft-versus-host disease (GVHD)

Abstract

The immunomodulator FTY720 (FTY) has been shown to be beneficial in experimental models of organ transplantation and autoimmunity. We show that FTY significantly inhibited but did not prevent graft-versus-host disease (GVHD) in lethally irradiated or nonirradiated allogeneic recipients. Although most studies implicate prevention of lymphocyte egress from lymphoid organs as the primary mechanism of action, our data indicate that FTY effects on the host are more likely to be responsible for GVHD inhibition. FTY reduced splenic CD11c+ cells by 50%, and similarly reduced CD4+ and CD8+ T-cell responder frequencies in the spleen early after transplantation. Imaging of GFP+ effectors indicated that FTY modified donor effector T-cell migration to secondary lymphoid organs, but did not uniformly trap T cells in lymph nodes or prevent early effector migration to GVHD parenchymal target organs. Administration of FTY only prior to transplantation inhibited GVHD, indicating that the primary function of FTY may be targeted to host cells. FTY was additive with regulatory T cells for GVHD inhibition. FTY slightly impaired but did not abrogate a graft-versus-leukemia (GVL) effect against C1498, a myeloid leukemia. Our data further define the mechanisms of action and provide insight as to the potential clinical uses of FTY in allogeneic bone marrow transplant recipients.

Figure 1

FTY inhibits GVHD; irradiation does not limit efficacy. (A) Lethally irradiated B6 mice were infused with BALB/c BM. Cohorts received 15 × 10⁶ (15S) or 25 × 10⁶ (25S) BALB/c splenocytes. Mice were administered sterile water or FTY (3.0 mg/kg) by oral gavage daily from days −1 to 28. Survival is indicated (n = 3 experiments pooled for a total of 24 mice per group for 15S; n = 8 per group for 25S; P < .001 for 15S, P = .006 for 25S, FTY vs water). (B) B6 RAG2-deficient mice were lethally irradiated or not irradiated and given 3 × 10⁶ BALB/c purified CD25-depleted T cells. Lethally irradiated mice received BALB/c BM. FTY or water was given as for panel A. Survival is indicated (n = 7-8 per group; P = .003 for no TBI, P = .006 for TBI, FTY vs water).

Figure 2

FTY modulates effector T-cell migration to lymphoid organs but does not uniformly trap effectors in LNs or prevent effector migration to GVHD target parenchymal organs. (A-I) Lethally irradiated BALB/c mice received B6 wild-type BM and 2 × 10⁶ B6 GFP⁺ purified CD25-depleted T cells. Mice were administered sterile water or FTY by oral gavage daily from day −1 to day of imaging. A total of 3 mice per group per time point was imaged. Data were reproduced in a second experiment in the same strain combination and in a third experiment in a different strain combination; findings were similar. A representative image is shown of the indicated organ on day 4 and day 7. See “Materials and methods” for imaging details.

Figure 3

Consistent with imaging data, FTY decreases GVHD scores in spleen and colon, but not in liver or lung. Lethally irradiated B6 mice were given BALB/c BM and splenocytes (25 × 10⁶) and administered sterile water or FTY by oral gavage from day −1 to time of tissue harvest. Control mice received only BM. On day 7 after transplantation, 5 per group were electively killed, and indicated GVHD target tissues were harvested, sectioned, and stained with hematoxylin and eosin and scored for GVHD histopathology. Shown is average score (± 1 SD). *P ≤ .01 versus BM-only control; ^P ≤ .006, water- versus FTY-treated mice.

Figure 4

FTY administered only prior to transplantation inhibits GVHD. (A) Lethally irradiated B6 mice were given BALB/c BM and splenocytes (15 × 10⁶) and administered sterile water or FTY by oral gavage at the indicated schedule. Survival is indicated (Control and FTY: day −10 to −2, pool of 2 experiments, n = 16 per group; FTY day −10 to day 28 and FTY day −1 to day 28 in 1 experiment, n = 8 per group; P < .004, control vs any FTY group; P > .13 for any FTY schedule comparison). (B-D) Lethally irradiated B6 mice were given BALB/c BM and CFSE-stained splenocytes (50 × 10⁶). FTY was administered daily from day −10 to day −3 (FTY pre) or day −1 to day 4 (FTY post) by oral gavage. Controls received sterile water. Spleens were harvested on days 3 and 5, and CFSE divisions were evaluated on gated donor (H2^d) CD4⁺ and CD8⁺ cells. (B) A representative donor CD4⁺ CFSE histogram from each group for both days is shown. CFSE-stained splenocytes placed in culture served as control to illustrate no divisions. (C,D) Shown is average CD4 (C) and CD8 (D) responder frequency plus or minus 1 SD calculated from CFSE data according to reference in “Materials and methods.” (n = 3 per group per time point; *P ≤ .014 FTY pre or post vs Control; ^P < .05 FTY pre vs FTY post). Day-3 data were reproduced in a second experiment.

Figure 5

FTY and T_reg cells are additive for GVHD inhibition. (A) Lethally irradiated B6 mice were given BALB/c BM and splenocytes (15 × 10⁶). Cohorts received 1 infusion of BALB/c L Sel⁺ ex vivo activated and expanded T_reg cells (3.5 × 10⁶) on day 0. FTY or water was administered daily from day −1 to day 28. Shown is survival (pool of 2 experiments, n = 16 per group; P < .001, control vs any treatment group; P < .03, T_reg cells or FTY vs FTY + T_reg cells). (B-E) Lethally irradiated B6 mice were given BALB/c BM and CFSE-stained splenocytes (50 × 10⁶). Cohorts received 1 infusion of BALB/c L Sel⁺ ex vivo activated and expanded T_reg cells (10 × 10⁶) on day 0. Sterile water or FTY was administered daily from day −1 to day 4 by oral gavage. Spleens were harvested on day 5, and CFSE divisions were evaluated on gated donor (H2^d) CD4⁺ and CD8⁺ cells. Shown is average CD4⁺ and CD8⁺ responder frequency and proliferative capacity as indicated plus or minus 1 SD calculated from CFSE data according to “Materials and methods” (n = 4 per group per time point; *P ≤ .018 vs control; ^P ≤ .037, FTY vs T_reg cells or FTY + T_reg cells).

Figure 6

FTY does not abrogate a GVL effect. Lethally irradiated B6 mice were given BALB/c BM and C1498ff (3 × 10⁴ cells) on day 0. Cohorts received splenocytes (15 × 10⁶) on day 0 and were administered water or FTY daily by oral gavage from day −1 to day 28. Mice were imaged on days 14 and 28 (n = 5 per group) as indicated in “Materials and methods” after injection with luciferin. Identical exposure times were taken for all pictures. Green indicates a greater tumor load than blue. C indicates a control mouse that did not receive C1498ff. A cross indicates that a mouse died. All mice receiving BM and tumor but no spleen died of tumor by day 28. Although spleen infusions protected most mice from widely metastic tumor growth, several mice died of GVHD. Overall tumor incidence for water-treated mice was 1 of 5 versus 3 of 5 for FTY-treated mice.

Figure 7

FTY impairs but does not abrogate GVL. A separate cohort of mice as described in Figure 6 were evaluated for survival (n = 8 per group). Both FTY-treated and control mice receiving splenocytes survived longer than mice not receiving splenocytes, indicative of a GVL effect (P ≤ .001). However, FTY-treated mice died before controls due to a greater tumor relapse, indicating that FTY impaired GVL effect under these experimental conditions (P = .01).