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Novel Insights into the Mechanism of Action of FTY720 in a Transgenic Model of Allograft Rejection: Implications for Therapy of Chronic Rejection¹

Antje Habicht^2,*, Michael R. Clarkson^2,*, Jun Yang^*, Joel Henderson, Volker Brinkmann, Stacey Fernandes^*, Mollie Jurewicz^*, Xueli Yuan^* and Mohamed H. Sayegh^3,*

^* Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital, Boston, MA 02115; Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02130; and Transplantation Research, Novartis Institutes for Biomedical Research, Basel, Switzerland

	Abstract

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FTY720 is a high-affinity agonist at the sphingosine 1-phosphate receptor 1 that prevents lymphocyte egress from lymphoid tissue and prolongs allograft survival in several animal models of solid organ transplantation. In this study we used a recently developed adoptive transfer model of TCR transgenic T cells to track allospecific CD4⁺ T cell expansion and trafficking characteristics, cytokine secretion profiles, and surface phenotype in vivo in the setting of FTY720 administration. We report that FTY720 administration had no effect on alloantigen-driven T cell activation, proliferation, acquisition of effector-memory function, or T cell apoptosis. However, FTY720 caused a reversible sequestration of alloantigen-specific effector-memory T cells in regional lymphoid tissue associated with a decrease in T cell infiltration within the allograft and a subsequent prolongation in allograft survival. Furthermore, delayed administration of FTY720 in a cardiac model of chronic allograft rejection attenuated the progression of vasculopathy and tissue fibrosis consistent with the hypothesis that FTY720 interrupts the trafficking of activated effector-memory T cells. These data have important implications for targeting the sphingosine 1-phosphate receptor 1 in solid organ transplantation.

	Introduction

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The novel immunomodulatory agent FTY720 is already in phase III clinical trials in renal transplantation (1, 2). It is a high-affinity agonist at the sphingosine 1-phosphate receptor 1 and prevents lymphocyte egress from lymphoid tissue (1). In animal models of solid organ transplantation, FTY720 prolongs allograft survival, and promising early clinical data are emerging in renal transplantation (3, 4, 5, 6). However, to date, few studies have focused specifically on the effect of FTY720 on allospecific T cell activation, the relative effect of FTY720 on allospecific naive vs effector-memory T cell trafficking, the consequences of withdrawal of FTY720 in alloimmunity, and its role, if any, in managing chronic allograft rejection.

To address these important questions, we used a recently developed adoptive transfer model, in which naive allospecific CD4⁺ T cells harvested from anti-bm12 (ABM)⁴ TCR transgenic (Tg) mice, specific for the MHC class II molecule I-A^bm12 found on B6.C-H2^bm12 (bm12) are transferred into syngeneic T cell-deficient B6 nude mice that subsequently receive a bm12 skin allograft (7, 8). Using fluorochrome-labeled mAbs directed against the TCR chains V2 and V8, one can track allospecific T cell expansion and trafficking characteristics, cytokine secretion profiles, and surface phenotype in vivo. We used this novel model system to define the mechanisms of action of FTY720 in vivo and its implications for the prevention and therapy of chronic vascularized allograft rejection.

	Materials and Methods

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Mice

C57BL/6 (B6) and B6.C-H2^bm12 (bm12) mice were purchased from The Jackson Laboratory. B6 nude mice were purchased from Taconic Farms. ABM TCR-Tg mice have been described previously and were maintained as a breeding colony in our animal facility in accordance with institutional guidelines. All mice were used at 6–12 wk of age.

Skin transplantation

Full-thickness trunk skin grafts harvested from bm12 donors (1 cm²) were transplanted onto the dorsal thorax of recipient mice, sutured with 4-0 silk, and secured with dry gauze and a bandage for 7 days. Skin graft survival was monitored daily thereafter, and rejection was defined as complete graft necrosis. In the adoptive transfer experiments, bilateral skin grafts (bm12) were placed onto the dorsal thorax of recipient nude mice so as to maximize recovery of alloantigen-specific T cells from lymph nodes and spleen.

FTY720 administration

FTY720 was obtained from Novartis Pharmaceuticals and was dissolved in saline. A dose of 1 mg/kg/day was administered by i.p. injection on days 0–13 (14-day treatment group) or on days 0–6 (7-day treatment group).

Adoptive transfer experiments

Adoptive transfer of ABM TCR-Tg T cells was performed as previously described (8). Briefly, spleens and lymph nodes from ABM TCR-Tg mice were harvested, and pooled single-cell leukocyte suspensions were prepared using ACKlysing (BioWhittaker) for erythrocyte depletion. CD4⁺ T cells were purified by negative selection using a magnetic cell separation system (Miltenyi Biotec). CD4⁺ T cells were isolated to >85% purity. An aliquot of cells was stained with anti-CD4, anti-TCR V2.1, and anti-TCR V8.1, and analyzed by flow cytometry to determine the percentage of ABM TCR-Tg CD4⁺ T cells. Typically, >90% of CD4⁺ T cells expressed the Tg TCR. A total of 2 x 10⁶ CD4⁺ V2⁺V8⁺ cells was injected i.v. into B6 nude mice 1 day before bm12 skin transplantation. B6 nude mice were treated with FTY720 for 7 or 14 days and compared with untreated control mice. On days 7, 14, 21, and 30 following transplantation, draining lymph nodes (dLN; axillary, lateral axillary) and non-dLN (ndLN; pooled mesenteric and neck) and spleens were subsequently collected, single-cell leukocyte suspensions were prepared, and ABM TCR-Tg T cells were identified by flow cytometry.

Flow cytometry

Cells were washed in PBS containing 2% FCS (Sigma-Aldrich). Unlabeled anti-CD16/CD31 (anti-FcRIII/anti-FcRII) was used to block unspecific FcR binding. Cells (1 x 10⁶) were stained with PerCP-conjugated anti-CD4 (RM4-5), FITC-conjugated anti-TCR V2.1 (B20.1), and biotinylated anti-TCR V8.1 (MR5-2), followed by allophycocyanin-conjugated streptavidin, to identify the adoptively transferred Tg cells. Subsequently, cells were stained with PE-labeled mAbs against CD25 (PC61) or CD69 (H1.2F3) for detection of activation markers. To determine the percentage of Tg T cells with effector-memory phenotype, cells were stained with FITC-conjugated anti-CD4, PE-conjugated anti-CD44 (IM7), and allophycocyanin-labeled mAb against CD62L (MEL-14), assuming that >90% of CD4⁺ T cells are Tg T cells. All mAbs were purchased from BD Biosciences. Four-color flow cytometry was performed on a FACSCalibur (BD Biosciences), and cells were analyzed using CellQuest software (BD Biosciences).

Intracellular cytokine staining

Cells (1 x 10⁶) were resuspended in HL-1 medium (BioWhittaker); supplemented with 1% L-glutamine (BioWhittaker), 1% penicillin (BioWhittaker), and 10% FCS (BioWhittaker); and restimulated with PMA (5 ng/ml) (Sigma-Aldrich) plus ionomycin (500 ng/ml) (Sigma-Aldrich). Brefeldin A (10 µg/ml) (Sigma-Aldrich) was added. Cells were incubated for 4 h at 37°C. After staining for the surface markers (CD4, V2.1, and V8.1), cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences), according to manufacturer’s instructions, and incubated with PE-conjugated IL-10 (JES5-16E3), IFN- (XMG1.2), or isotype control mAbs for 30 min at 4°C. A gate was set on CD4⁺ V2⁺V8⁺, and the percentage of IL-10/IFN- cells was determined by flow cytometric analysis.

Quantification of apoptotic alloantigen-specific cells

Cells (1 x 10⁶) recovered from adoptively transferred mice were stained for surface expression of V2.1 and V8.1, resuspended in apoptosis buffer (BD Biosciences), according to manufacturer’s instructions, and incubated with 7-aminoactinomycin D and PE-conjugated annexin V for 15 min at room temperature. A gate was set on V2⁺V8⁺ 7-aminoactinomycin D^– lymphocytes, and the percentage of annexin V⁺ cells was determined by flow cytometry.

Immunohistochemistry

Acetone-fixed, cryopreserved skin sections (5 µm) were incubated with purified rat anti-mouse CD4 mAb (H129.19) (BD Pharmingen). After incubation with an HRP-conjugated anti-rat mAb, staining was developed by incubation of the tissue sections in aminoethylcarbazole (DakoCytomation). Sections were counterstained with hematoxylin solution (Sigma-Aldrich). The number of CD4⁺ cells in three high-powered fields of a representative area of inflammation was counted and expressed as mean ± SD.

Chronic allograft rejection model

Heterotopic vascularized cardiac transplants from bm12 donors into B6 recipients were performed by standard microvascular techniques (9). FTY720 (1 mg/kg/day) was administered i.p., either from days 0 to 13 in the prevention group or from days 14 to 28 posttransplantation in the interruption group. Graft function was monitored by daily palpation of the transplanted hearts. All heart grafts were routinely harvested on day 60.

Morphometric and histologic analysis

Cardiac graft samples fixed in 10% Formalin were embedded in paraffin, sectioned, and stained with H&E for evaluation of cellular infiltration. The sections were also stained with elastic Van Giesen (elastic fibers) for arteriosclerosis scoring and with Mason’s trichrome (collagen) for fibrosis and infiltrate scoring. Arteriosclerosis was assessed by light microscopy. The severity of arteriosclerosis was graded according to the percentage of luminal occlusion by intimal thickening using a scoring system previously described (10). Briefly, a vessel score of 0 indicated a normal artery; 1, <10% luminal occlusion; 2, 20–50% luminal occlusion; 3, >50% luminal occlusion. Only vessels that were cut orthogonally and displayed a clear internal elastic lamina were scored. A blinded examiner read all samples.

Statistics

Comparisons between experimental groups were performed using Student’s t test. A p value <0.05 was considered statistically significant.

	Results

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FTY720 prolongs skin allograft survival in a MHC class II mismatch model

We have previously reported that the higher precursor frequency of alloreactive T cells in ABM mice (>90%) is associated with more rapid rejection of skin allografts as compared with wild-type (WT) B6 mice (precursor frequency <5%) (8, 11). We therefore sought to use this model to determine whether the alloreactive T cell clone size affected the immunosuppressive efficacy of FTY720 in skin allograft rejection. FTY720, administered for 14 days, significantly prolonged skin allograft survival in WT B6 mice from a median graft survival (MST) of 14 days in untreated controls up to a MST of 44 days in treated animals (p = 0.001; Fig. 1A). As expected, untreated ABM Tg mice had an accelerated time course to rejection compared with WT B6 recipients (MST = 11 vs 14 days, p < 0.05). However, even in the setting of an enlarged clone size, FTY720 significantly delayed graft rejection compared with nontreated animals, albeit that the prolongation was not as striking as that observed in WT B6 recipients (MST = 33 days; p = 0.003 vs control; Fig. 1B).

View larger version (13K): [in this window] [in a new window]   FIGURE 1. FTY720 prolongs skin allograft survival. B6 and ABM mice were transplanted with bm12 skin allografts on day 0. Graft survival is displayed as a Kaplan-Meier plot. FTY720 led to a significant prolongation in allograft survival in both WT B6 (n = 5) (A) and ABM (n = 5) (B) recipients compared with controls. Note the prolongation in allograft survival is less marked in the ABM recipients (MST = 33 days) as compared with WT (MST = 44 days) due to the higher precursor frequency of alloantigen-specific T cells in ABM mice.

To determine the effect of FTY720 on alloreactive T cell expansion and trafficking within secondary lymphoid organs, we used a previously reported adoptive transfer model as outlined in Materials and Methods (7, 8). Adoptively transferred recipients were sacrificed on days 7, 14, 21, and 30 following engraftment, and the percentage of TCR-Tg T cells in the dLN, ndLN, and spleen was determined by flow cytometric analysis. The Tg CD4⁺ T cell population in the regional dLN peaked in size on day 14 consistent with our previous report (7). As shown in Fig. 2A, FTY720 significantly increased the number of Tg CD4⁺ T cells expressed as a percentage of all cells in the dLN (p = 0.01) and ndLN (p = 0.01) after 14 days of therapy, while simultaneously decreasing the percentage of alloantigen-specific CD4⁺ T cells within the spleen (p = 0.009). To measure the direct effect of FTY720 on alloantigen-specific T cell expansion, we determined the percentage of CD4⁺ cells positive for the Tg TCR (V2⁺V8⁺ cells) in the different lymphoid compartments, which followed a similar pattern of distribution with a significantly higher percentage in lymph nodes and simultaneously reduced percentage in the spleen (Fig. 2B). The interruption of normal T cell trafficking was accompanied by a reduction in graft-infiltrating CD4⁺ T cells as assessed by immunohistochemistry, consistent with the prolongation in allograft survival observed in the skin allograft survival model (54 ± 4 vs 20 ± 7 cells per high-power field; p = 0.01) (Fig. 3). Importantly, FTY720-induced lymphocyte sequestration within the lymph nodes was reversible. The percentage of Tg CD4⁺ T cells in the lymph nodes of FTY720-treated animals compared with control animals equalized within 7 days after cessation of therapy, as determined on day 21 after the 14-day course of FTY720 therapy (Fig. 2, C and D).

View larger version (50K): [in this window] [in a new window]   FIGURE 2. FTY720 triggers reversible alloantigen-specific T cell trapping in lymph nodes. Nude B6 recipients were reconstituted with 2 x 106 CD4+ V2+V8+ T cells and transplanted with two bm12 skin grafts. dLN, ndLN, and spleen were harvested on indicated time points; leukocytes were isolated and stained for the expression of CD4, V2, and V8; and the percentage of alloantigen-specific Tg T cells in each lymphoid compartment was determined by flow cytometry. A and B, Fourteen days following engraftment, administration of FTY720 led to a significant increase in Tg T cells in both lymph node compartments, while there were significantly fewer Tg T cells in the spleen. C and D, The FTY720-induced lymphocyte sequestration within the lymph node was reversible given that by day 21, 7 days following discontinuation of therapy, the percentage of Tg CD4+ T cells in all three compartments of FTY720-treated animals compared with control animals had equalized. Results are expressed as mean ± SD for values derived from at least four individual mice at each time point.

View larger version (68K): [in this window] [in a new window]   FIGURE 3. FTY720 reduces CD4+ T cell infiltration in skin allografts. Representative photomicrograph of immunohistochemical staining for CD4 demonstrating reduced CD4+ T cell infiltration in skin allografts of nude B6 recipients treated with FTY720 (A) as compared with control animals (B) 14 days after transplantation.

FTY720 does not affect alloantigen-specific CD4⁺ T cell activation, cytokine the generation, or apoptosis

We then wanted to test whether FTY720 has additional effects on alloreactive T cells beyond lymph node sequestration. Upon recognition of Ag presented by APCs within the lymph node, lymphocytes become activated. In the adoptive transfer model, FTY720 did not alter this process, as evidenced by equivalent Ag-driven T cell activation in control and FTY720-treated recipients. The expression of the activation markers CD25 and CD69 on the surface of adoptively transferred Tg CD4⁺ T cells was similar at all time points in the three lymphoid compartments assessed (Fig. 4A). Furthermore, FTY720 did not suppress either Th1 or Th2 cytokine production by CD4⁺ T cells, as measured by intracellular cytokine staining for IFN- and IL-10 at the same time points in any of the three compartments (Fig. 4B). Taken together, these data indicate that although the distribution of alloantigen-specific T cells in the secondary lymphoid organs is altered by FTY720, alloantigen-specific CD4⁺ T cell activation is unaffected by FTY720 treatment and the activated cells retain a similar effector cytokine profile.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. FTY720 does not affect alloantigen-specific CD4+ T cell activation, cytokine generation, or apoptosis. Reconstituted nude B6 recipients were transplanted with two bm12 skin grafts. At the indicated time points, spleen and lymph node cells were stained for expression of CD4, V2, and V8. A, Further staining of Tg T cells for the marker CD25 demonstrated no difference in expression between the FTY720-treated and control animals in lymph nodes and spleen at any of the indicated time points. B, Restimulation of 1 x 106 cells for 4 h with PMA and ionomycin showed no difference in the percentage of IFN--secreting alloantigen-specific T cells in the three lymphoid compartments on days 7, 14, 21, and 30. C, Furthermore, the frequency of Tg T cells undergoing apoptosis, as evaluated by annexin V staining of CD4+ V2+V8+ T cells, did not alter at the indicated time points in spleen and lymph nodes. The values shown represent the mean ± SD obtained from at least four individual mice at each time point.

FTY720 does not affect the generation of CD4⁺ T cells with effector-memory phenotype, but alters their distribution

Acquisition of an effector-memory function is associated with enhanced expression CD44 and concomitant loss of CD62L expression. We sought to evaluate the effect of FTY720 not only on alloantigen-induced acquisition of an effector-memory phenotype, but also on the distribution of naive vs effector-memory T cells within the various secondary lymphoid compartments. In the adoptive transfer model, FTY720 had no effect on the percentage of CD4⁺ cells, demonstrating the CD44^highCD62L^low phenotype in the dLN, ndLN, or spleen at day 14, the point of maximal T cell expansion (Fig. 5A). We then examined the distribution of effector-memory cells in secondary lymphoid tissue. As shown in Fig. 5B, the percentage of CD4⁺CD44^highCD62L^low adjusted to the number of all cells in the different compartments was significantly higher in both dLN and ndLN in FTY720-treated recipients as compared with control, while being simultaneously reduced in the spleen, suggesting that FTY720 sequesters effector-memory T cells within secondary lymphoid tissue.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. FTY720 does not affect the generation of effector-memory T cells in the adoptive transfer model, but after their distribution. Spleen and lymph node cells from nude B6 recipients reconstituted with Tg T cells and transplanted with two bm12 skin grafts were harvested on day 14 after engraftment. Cells were stained for CD4, CD44, and CD62L, and the percentage of cells expressing the effector-memory phenotype CD44highCD62Llow was determined, assuming that >90% of CD4+ T cells are Tg. As shown in A, there was no significant difference in the percentage of CD4 cells with an effector-memory phenotype between control and FTY720-treated animals, suggesting equivalent effector-memory cell generation. B, However, when adjusted to the number of all cells in the lymphoid compartments, the number of CD4+CD44highCD62Llow T cells significantly increased in the lymph nodes of FTY720-treated recipients. This reflects trapping of sensitized CD4 effector-memory cells in lymphoid tissue. The values shown represent the mean ± SD obtained from four individual mice.

Given the results of the adoptive transfer model above, we performed vascularized cardiac transplants in the same MHC class II mismatch model (bm12 into B6) to evaluate the immunomodulatory effects of FTY720 in chronic rejection. In this model, cardiac allografts survive >60 days, but develop the morphologic features of progressive chronic rejection manifested by allograft vasculopathy, interstitial inflammation, and fibrosis (11). As outlined in Materials and Methods, FTY720 was given for 2 wk starting on the day of transplant (prevention model) or from days 14 to 29 (interruption model). All heart allografts continued to beat until time of harvest. In the prevention model, administration of FTY720 significantly attenuated the development of chronic allograft rejection as manifested by the prevention of myocardial fibrosis and attenuation of allograft vasculopathy. The mean vasculopathy score in the FTY720 early treatment group was 0.3 ± 0.8 vs 2.1 ± 1.3 in control animals (p < 0.0001) (Fig. 6). Interestingly, FTY720 also interrupted the progression of chronic rejection when given starting 14 days after engraftment (chronic allograft vasulopathy score 0.7 ± 1; p < 0.001 vs control) (Fig. 6). These data indicate that, by targeting the trafficking of effector-memory T cells, FTY720 administration is effective in preventing progression of established chronic allograft rejection.

View larger version (78K): [in this window] [in a new window]   FIGURE 6. FTY720 interrupts the progression of chronic allograft rejection. B6 mice received vascularized bm12 cardiac allografts. FTY720 (1 mg/kg/day) was administered i.p. either on days 0–13 (prevention group) or from days 14 to 28 (interruption group) posttransplantation. Representative photomicrographs of elastic Van Giesen staining, demonstrating advanced allograft vasculopathy in a control animal with surrounding inflammatory infiltrate (A) as compared with a normal vessel in an early treatment recipient (B) and a vessel demonstrating only minimal disruption of the basement membrane in a delayed treatment recipient (C). The percentage of myocardial fibrosis was significantly decreased in both treatment groups (D) (p < 0.05 for both vs control), and the chronic vasculopathy score was significantly lower in the treated groups compared with controls (E) (p < 0.0001 for both groups vs control). The values shown represent the mean vessel score ± SD obtained from three individual mice.

	Discussion

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Naive T cells continuously enter secondary lymphoid organs, traffic to the T cell zone, and survey the resident APCs (15). Under normal circumstances, the T cells fail to identify their specific Ag, and after a period of hours exit the lymphoid organ and circulate to other secondary lymphoid tissue to repeat the process. However, if a T cell encounters its Ag, as would occur in the dLN of a skin allograft, then it is activated, divides, and acquires effector functions that include the ability to enter nonlymphoid tissue (16). The data from our study suggest that while FTY720 sequesters T cells within lymph node tissue, it does not alter alloantigen-driven T cell activation or cytokine secretion profiles.

Apoptosis of alloactivated T cells is reported to be central to the development of peripheral tolerance (12). FTY720 induces apoptosis of T cells in vitro, and several reports have suggested that its in vivo immunomodulatory effects are mediated, at least in part, by T cell apoptosis (17, 18, 19, 20). Interestingly, we observed no increase in apoptosis of alloantigen-activated Tg T cells in our model, which is in keeping with emerging clinical data suggesting that apoptosis may not play a key role in the immunosuppressive activity of FTY720 (21). Our results suggest that the observed prolongation in skin allograft survival is solely due to interruption of lymphocyte trafficking from the lymph node rather than modulation of T cell activation or function.

Following alloantigen encounter in secondary lymphoid tissue, the T cell undergoes a series of changes in the expression of cell surface adhesion molecules that alter its trafficking characteristics (16). The acquisition of an effector-memory phenotype (CD44^highCD62L^low) confers an ability to enter nonlymphoid tissue and a diminished, but not absent capacity to enter lymph node tissue (16). The changes in the expression of cell surface adhesion molecules may also affect the ability of FTY720 to trap effector-memory lymphocytes within the lymph node. If circulating effector-memory T cells were resistant to the effects of FTY720, this would have significant implications for its use in clinical transplantation. Sensitized transplant candidates and patients with chronic rejection have a higher precursor frequency of effector T cells directed against donor alloantigen (22, 23). Prevention of rejection in the former group or interruption of alloimmune-driven responses in the latter by FTY720 would require inactivation or trapping of effector-memory cells within lymph node tissue. In our study, we observed no effect of FTY720 on the generation of effector-memory T cells, but equivalent trapping as compared with naive T cells. A previous report by Xie et al. (22, 23, 24) in a nonalloimmune model suggested that FTY720 did not trap T cells preactivated in vitro within lymphoid tissue following adoptive transfer. However, preactivation in vitro may not reliably reproduce alloantigen-specific activation in vivo. Furthermore, in our in vivo model examining the progression of chronic alloimmune-mediated allograft injury, delayed administration led to interrupted progression of allograft vasculopathy, as evidenced by diminished intramural inflammation and intimal proliferation. In aggregate, these results strongly support the contention that FTY720 modulates the trafficking characteristics of both naive and effector-memory T cells. The clinical relevance of this observation is that FTY720 is likely to be an effective immunomodulatory agent in sensitized allograft recipients, i.e., those with pre-existing effector-memory T cells against donor alloantigen, and it may also act to attenuate the immune-dependent mechanisms mediating the progression of chronic allograft dysfunction.

Given that FTY720 does not appear to prevent T cell activation, differentiation, and acquisition of an effector-memory phenotype, and cessation of therapy results in egress of alloreactive T cells from the lymph node tissue, prompt alloimmune-mediated injury would be the expected consequence of discontinuation of therapy. This hypothesis is consistent with the findings of the skin allograft survival data that demonstrate enhanced prolongation in mice treated for 14 as opposed to 7 days, and long-term acceptance or tolerance to donor alloantigen was not observed upon withdrawal of FTY720 in either group. The clinical implication of these findings may be that withdrawal of FTY720 might be associated with a higher rate of allograft rejection due to release of allospecific effector T cells previously activated within the lymph node either directly by donor APCs or indirectly by self APCs expressing donor alloantigen. This would argue against using FTY720 as an induction immunosuppressive agent, but rather using it as part of a maintenance regimen. In contrast, a failure to prevent T cell activation and effector-memory cell generation might be expected to lead to beneficial effects such as the retention of viral immunity leading to fewer posttransplant infections, less reactivation of latent viral infections, and a lower frequency of lymphoproliferative disorders in comparison with conventional immunosuppressants, as suggested by previous studies (25).

An important concern in interpreting the effects of an immunomodulatory agent in a lymphopenic environment is the extent, if any, to which homeostatic proliferation may affect the experimental outcome. The transfer of naive T cells into a lymphopenic environment initiates a proliferative (homeostatic) response, which involves TCR recognition of self-peptide/MHC complexes, and the acquisition of phenotypic and functional characteristics of memory-like T cells (26, 27, 28). However, our adoptive transfer model system relies on specific identification of the V2⁺ and V8⁺ T cell population that expands in response to alloantigen. We have previously reported the time course of alloreactive vs homeostatic T cell expansion and showed that the percentage of Tg cells that undergo homeostatic proliferation remains relatively small, and that the expansion observed predominantly represents that of alloantigen-specific T cells (8). In addition, and relevant to this study, FTY720 has not previously been demonstrated to alter lymphopenia-induced proliferation and did not prevent either the acquisition of an effector-memory phenotype or T cell activation/function in our study.

In summary, our results provide valuable mechanistic insights into the use of FTY720 in allograft rejection. FTY720 efficiently traps alloantigen-specific effector-memory cells in lymphoid tissue without affecting APC-T cell priming interactions or enhancing T cell apoptosis. Our data suggest the use of this novel immunomodulatory as a component of a multifaceted maintenance immunosuppressive strategy in combination with other agents that modulate T cell activation and/or promote the development of tolerance.

	Disclosures

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V. Brinkmann is an employee of Novartis Pharmaceuticals Corporation, which provides FTY720.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants RO1-AI51559, R21-HL079450, R01-AI37691, and PO1-AI50157 from the National Institutes of Health.

² A.H. and M.R.C. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Mohamed H. Sayegh, Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital, 221 Longwood Avenue, Boston, MA 02115. E-mail address: msayegh{at}rics.bwh.harvard.edu

⁴ Abbreviations used in this paper: ABM, anti-bm12; dLN, draining lymph node; MST, median graft survival; ndLN, non-dLN; Tg, transgenic; WT, wild type.

Received for publication July 7, 2005. Accepted for publication September 30, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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