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Department of Immunology, The Cleveland Clinic Foundation, Cleveland OH 44195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Regardless of origin, memory T cells specific for donor Ags present a unique challenge in transplantation. The same features of T cell memory that are essential for host protection from pathogens lead to robust immune responses to a transplanted organ, translating into accelerated allograft rejection in experimental animals (5, 6) or increased risk of poor allograft outcome in humans (2, 7). Furthermore, memory T cells appear to be resistant to the effects of currently used graft-prolonging therapies including immunosuppressive drugs, depletional strategies, and conventional costimulatory blockade (8). Our group among others has demonstrated that donor-reactive memory T cells can induce allograft rejection despite administration of standard costimulatory blockade (9, 10, 11).
Using new murine models to study allospecific memory CD4 T cells in cardiac allograft recipients, we have established that memory T cells contribute to allograft rejection through multiple pathways. Upon reactivation, memory CD4 T cells can become effector cells themselves, and can also provide help for the priming of naive T and B lymphocytes that in turn mediate graft destruction. Under these circumstances, eliminating one effector mechanism permits expression of alternative mechanisms of graft rejection, thus making control of memory CD4 T cells a particularly difficult problem (11).
One potential strategy to control memory T cells in the transplant setting is to prevent them from infiltrating the graft. To this end, the immunosuppressive agent FTY720 prolongs survival of solid organ allografts through retention of naive lymphocytes in secondary lymphoid organs (12, 13, 14). Unlike other immunosuppressants, FTY720 does not inhibit the activation and proliferation of T cells or the induction of humoral immune responses (15, 16). Instead, FTY720 is converted in vivo to its phosphate ester metabolite which binds to sphingosine-1-phosphate (S1P)4 receptors subsequently preventing the lymphocytes from leaving the secondary lymphoid organs and from infiltrating sites of inflammation (17). Although FTY720 effectively inhibits the recruitment of newly generated effector T cells to peripheral tissues, it has been reported to have no significant effect on pre-existing activated T cells (15, 16). The direct effect of this agent on resting memory T cells, in particular donor-reactive memory T cells in graft recipients, has not been previously investigated. The present study was performed to specifically address this issue using a model of heterotopic cardiac allograft transplantation in mice.
Using donor-specific TCR transgenic (Tg) CD4 T cells, we showed that FTY720 effectively sequestered circulating memory CD4 T cells in secondary lymphoid organs, a finding associated with prolonged cardiac allograft survival. Nevertheless, the helper functions of the memory CD4 T cells remained unaffected by the lymphoid sequestration resulting in Ab-mediated graft rejection. As humans contain varying levels of allospecific memory T cells and FTY720 is currently being tested in transplant patients, the information gained from this study has important therapeutic implications in clinical transplantation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Female C57BL/6 (H-2b), C57BL/6 µ-chain knockout (KO; µMT–/– B6, H-2b), and congenic C57BL/6 Thy1.1 (H-2b) and male C3H (H-2k) mice, aged 6–8 wk, were purchased from The Jackson Laboratory. Male and female C57BL/10NA;-(Tg)TCR Marilyn-(KO) Rag2 N11, N2 mice (H-2b, Mar), age 6–8 wk, were a gift from P. Matzinger (National Institutes of Health) and O. Lantz (Institut National de la Santé et de la Recherche Médicale, Paris, France). All animals were maintained and bred in the pathogen-free facility at The Cleveland Clinic Foundation. All procedures involving animals were approved by an Institutional Animal Care and Use Committee at The Cleveland Clinic Foundation.
Generation of monoclonal memory CD4 T cells
C57BL/6 animals containing memory Mar CD4 T cells were generated as previously published by our laboratory (11). Briefly, spleen cells from young (4–6 wk) Mar female mice were stimulated in vitro with 3 µM HYDby peptide (NAGFNSNRANSSRSS; Research Genetics) in RPMI 1640 + 5% FCS (HyClone). After 4 days, cells were washed twice, counted with ethidium bromide/acridine orange, and transferred into naive C57BL/6, Thy1.1, or µMT–/– female mice via tail vein injection (5 x 106 live cells/mouse). We previously demonstrated that 4 wk in vivo in the absence of ongoing antigenic stimulation is sufficient to convert primary effector CD4 T cells (CD25highCD44highCD69high) into resting memory CD4 T cells (CD25lowCD44highCD69low) (11). Transferred female mice were rested for at least 4 wk after Mar cell injection and then used in subsequent experiments. In a given experiment, recipients were injected with cells derived from a common pool of activated Mar T cells. Control female B6 mice hosting a population of naive Mar CD4 T cells were generated as previously published (11). Briefly, 5 x 106 spleen cells from young naive Mar TCR Tg females were i.v. injected into naive B6 female mice. Animals were rested for at least 4 wk before use as heart allograft recipients.
FTY720 treatment and costimulatory blockade
FTY720 was provided by Novartis Pharma and was dissolved in distilled water. A dose of 0.3 mg/kg was administered daily by gavage in a volume of 100 µl/10g body weight. In cardiac allograft recipients, treatment with FTY720 was started on day –4 with relation to the surgery and administered throughout the duration of the experiment. Donor-specific transfusion and costimulatory blockade were performed as described by Hancock et al. (18). A total of10 x 106 donor spleen cells plus 0.4 mg of anti-CD154 Ab MR1 (BioExpress) were administered i.v. 1 day before the heart graft placement (donor-specific transfusion (DST)/MR1 treatment).
Placement and evaluation of cardiac allografts
Vascularized heterotopic cardiac allografts were placed in the abdomen using standard microsurgical techniques and palpated daily for evidence of a heartbeat. Rejection was defined as a loss of palpable heartbeat and was confirmed by laparotomy. All grafts were harvested at the time of rejection or at predetermined time points, embedded in paraffin, stained with H&E, and examined by microscopy. A portion of each graft was frozen in OCT compound (Sakura Finetek) for immunohistochemical staining. Frozen sections of cardiac tissue were acetone-fixed, hydrated in PBS for 10 min, blocked with the Biotin Blocking System (DakoCytomation), washed with PBS three times, and incubated for 60 min at room temperature with biotinylated anti-CD4, anti-CD8, or anti-Mac1 Ab (1/50 dilution in PBS-1% BSA; BD Pharmingen). After three additional PBS washes, the sections were incubated with peroxidase-conjugated streptavidin (DakoCytomation) and developed using the Novared Substrate kit (Vector Laboratories). Sections were dehydrated with ethanol and mounted for analysis. For immunofluorescent staining of C3d, 8-µm frozen sections were fixed in acetone for 10 min and air-dried. Slides were immersed in PBS for 10 min and FITC-conjugated rabbit anti-C3d Ab (DakoCytomation), diluted 1/100 in 1% BSA/PBS, was applied for 30 min in a humid chamber. After three washes with PBS, the slides were mounted with Vectashield with 4',6'-diamidino-2-phenylindole (Vector Laboratories) and viewed under a fluorescent microscope.
ELISPOT assay
Assays were performed as outlined previously in detail (19). Briefly, ELISPOT plates (Millipore) were coated overnight with the anti-IFN-
capture Ab (BD Pharmingen) in sterile PBS, blocked with sterile 1% BSA in PBS, and washed three times with sterile PBS. CD8+ T cells were isolated from the spleens of the experimental animals using commercially available murine T cell isolation columns from R&D Systems, following the instructions supplied by the manufacturer. Resultant cells were >95% CD8+ by flow cytometry (data not shown). Purified CD8+ T cells (0.025–0.2 x 106 per well) were plated in HL-1 medium (BioWhittaker) with or without mitomycin C-treated C3H or control BALB/c stimulator cells (0.4 x 106 per well) and then incubated at 37°C, 5% CO2 for 24 h. After washing with PBS followed by PBST (PBS 0.025% Tween 20), biotinylated anti-IFN- detection Ab (BD Pharmingen) was added overnight. After washing with PBST, alkaline phosphatase-conjugated anti-biotin Ab (Vector Laboratories) diluted 1/2000 in PBST was added for 2 h at room temperature. The plates were developed as previously described. The resulting spots were analyzed using an ImmunoSpot Series 1 Analyzer (Cellular Technology).
Isolation of peripheral blood and organ-infiltrating lymphocytes
Peripheral blood samples were diluted 1/10 with heparin (250 U/ml in HBSS; American Pharmaceutical Partners). Cells were pelleted by centrifugation followed by RBC lysis and two washes with PBS. To isolate organ-infiltrating lymphocytes, animals were anesthetized and injected i.v. with 10–15 ml of sterile PBS until all organs were visibly blanched. Livers and lungs were individually harvested, cut into pieces with a sterile razor, and incubated with 25 mg of collagenase V (Sigma-Aldrich) in 25 ml of sterile HBSS at 37°C for 30 min with gentle intermittent vortexing. After incubation, the suspensions were filtered through a 40-mm cell strainer to remove larger pieces of residual tissue. RBC were lysed from the filtrate; cells were washed once with HBSS and counted with trypan blue. Resultant cells were stained with Abs against various cell surface markers as described below.
Flow cytometry
PE-conjugated anti-mouse CD4, FITC-conjugated anti-mouse CD25, FITC-conjugated anti-mouse CD44, FITC-conjugated anti-mouse CD62L, FITC-conjugated anti-mouse CD69, FITC-conjugated anti-Thy1.1 Abs, streptavidin-PE, and streptavidin-PerCP were purchased from BD Pharmingen. Cells isolated from spleens, lymph nodes, peripheral blood, lungs, and livers of experimental animals were incubated on ice for 30 min with the appropriate Ab (1 µg of Ab per 1 x 106 cells in 100 µl of PBS/0.1% BSA), followed by three washes in PBS/0.1% BSA. When biotin-conjugated Abs were used, cells were additionally incubated with streptavidin-PE or streptavidin-PerCP, followed by three more washes. The labeled cells were resuspended in 1% paraformaldehyde and analyzed on a BD Biosciences FACScan using CellQuest software (BD Biosciences). A total of 1,000,000 events was acquired and analyzed for each sample.
Ab detection by flow cytometry
Serum samples were collected from the experimental animals. Donor C3H or third-party BALB/c thymocytes were isolated, and live cells were counted by trypan blue exclusion and divided into aliquots (1 x 106 cells per sample). Cells were pelleted by centrifugation and resuspended in 100 µl of diluted serum (serial dilutions were made from 1/10 to 1/2430 in PBS/5% FCS/0.02% NaN3) followed by a 1-h incubation on ice and three washes with PBS/5% FCS/0.02% NaN3. Detecting FITC-conjugated goat anti-mouse IgG Ab (BD Pharmingen) was diluted 1/100 in PBS/5% goat serum/0.02% NaN3 and added to the pelleted cells (100 µl/sample). Cells were incubated 1 h on ice in the dark, washed three times, fixed in 1% paraformaldehyde in PBS, and analyzed by flow cytometry. The data are presented as the percentage of positively stained thymocytes.
Statistical analysis
Statistical analysis to determine differences between groups for recall immune responses was performed using the Student t test for equal or unequal variances. A value of p < 0.05 was considered statistically significant. Kaplan Meier survival analysis was performed to determine the difference in median graft survival between groups.
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Results |
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We first investigated how FTY720 affects the localization of memory CD4 T cells. To assess distribution of memory CD4 T cells of known specificity, we used TCR Tg Mar CD4 T cells specific for the male Ag-derived peptide HYDby plus I-Ab (20, 21). We have previously reported that memory Mar CD4 T cells develop following adoptive transfer of in vitro-activated effector cells into naive female hosts (11). After 4 days of in vitro culture with I-Ab peptide, the vast majority of Mar CD4 T cells acquired an effector phenotype (95% cells were CD25high, 70% were CD44high, and 70% expressed CD69). To distinguish between transferred and endogenous CD4 T cells, in vitro-primed Thy1.2+ Mar cells were transferred into congenic female Thy1.1 B6 recipients as outlined in Materials and Methods. Four weeks later, Thy1.2+ Mar cells could be readily detected in spleen, lymph nodes, lung, liver, and peripheral blood (data not shown). At this point, 
40% of Mar cells retrieved from the spleen were CD44high, while <2% of Mar cells expressed markers of acute T cell activation, CD25 and CD69, consistent with memory phenotype. Six weeks later, adoptive transfer groups of animals were fed FTY720 (0.3 mg/kg/day) or water as a control. After 4 days of treatment, animals were sacrificed and the presence of Thy1.2+ memory CD4 T cells in various lymphoid and nonlymphoid compartments was determined by flow cytometry. In water-treated animals, Thy1.2+ memory CD4 T cells were easily detectable in the blood, and were found in the spleen and lymph nodes. Treatment with FTY720 almost completely eliminated the circulating memory CD4 T cells in the blood (Fig. 1A) with a simultaneous 2- to 3-fold increase in these memory cell numbers in the spleen and lymph nodes (Fig. 1B). In contrast, the numbers of memory CD4 T cells in the lung and liver remained unaltered by FTY720 (Fig. 1B). These results indicate that short-term treatment with FTY720 induces relocation of circulating memory CD4 T cells into secondary lymphoid organs but has no detectable effect on memory CD4 T cells residing in the peripheral tissues.
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We next tested whether treatment with FTY720 would prolong heart allograft survival in recipients containing donor-specific memory CD4 T cells. We used a well-characterized model system that allowed identification of donor-reactive memory CD4 T cells of a defined specificity within the context of the wild-type alloresponse. TCR Tg Mar T cells do not cross-react with H-2k alloantigens in vitro or in vivo (20, 21). Upon adoptive transfer into wild-type B6 female recipients of C3H male heart allografts, Mar T cells can only respond to the donor-derived HYDby peptide processed by recipient APC and presented in the context of I-Ab through the indirect pathway (21). Our previous studies revealed that similar to polyclonal CD4 T cells responding through the indirect pathway, TCR Tg Mar cells provide help for donor-specific CD8 T cells and B cells and can thus serve as representative cells for studying memory CD4 T cell responses (11).
B6 mice containing memory or naive Mar CD4 T cells were treated with either FTY720 (0.3 mg/kg/day orally starting on day –4 and throughout the duration of the experiment) or with water as a control and received heart transplants from C3H male donors. Treatment with FTY720 significantly prolonged cardiac allograft survival in the recipients containing naive Mar T cells with four of six grafts surviving longer than 30 days vs a median survival time (MST) of 7.3 ± 0.3 days (n = 8) in water-treated mice (Fig. 2A). In recipients containing memory Mar T cells, treatment with FTY720 resulted in a modest prolongation of heart graft survival (MST of 19.3 ± 1.5 days, n = 6, vs 5.5 ± 0.3 days, n = 6, in nontreated recipients), with all grafts rejected by day 22 (Fig. 2A).
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Notably, regardless of DST/MR1 therapy, the delayed rejection in the recipients treated with FTY720 was characterized by a distinct pathology, with epicardial mononuclear cell infiltration and central areas of necrosis (Fig. 3, B, D, and F). In addition, we found that many of the large blood vessels (>80%) in the grafts from FTY720-treated mice were occluded typical of Ab-mediated injury (Fig. 3, insets). At the same time, elastin staining of the grafts revealed no signs of neointimal hyperplasia (data not shown).
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We next tested whether helper functions of naive or memory CD4 T cells were altered by lymphoid sequestration with FTY720. To assess activation of endogenous CD8 T cells under these conditions, we purified CD8 T cells from the spleens of heart graft recipients on day 7 posttransplant and tested them in a recall IFN- ELISPOT assay against donor C3H or third-party BALB/c stimulators. In accordance with previously published data (15), treatment with FTY720 did not inhibit but instead slightly increased frequency of donor-specific IFN--secreting CD8 T cells compared with nontreated recipients in the groups injected with naive Mar CD4 T cells (1420 ± 205 vs 890 ± 112/200,000 CD8 T cells). Similarly, the antidonor CD8 T cell response was not diminished in the recipients injected with memory CD4 T cells and treated with FTY720 compared with untreated mice containing memory CD4 T cells (1888 ± 201 vs 1480 ± 49/200,000 CD8 T cells).
Consistent with our previous results (11), treatment with DST/MR1 inhibited activation of endogenous IFN--producing CD8 T cells in mice injected with naive Mar T cells (50 ± 8/200,000 CD8 T cells, Fig. 4A) to the level observed in naive B6 mice (48 ± 6/200,000 CD8 T cells, data not shown). In the presence of memory CD4 T cells, treatment with DST/MR1 also resulted in decreased frequency of IFN--producing CD8 T cells (562 ± 40/200,000 CD8 T cells) presumably through preventing help provided by endogenous CD4 T cells. However, the number of activated antidonor CD8 effector T cells was still 10-fold higher than that observed in naive B6 mice or in recipients injected with naive Mar T cells and treated with DST/MR1. Regardless of the beneficial effect on allograft survival, addition of FTY720 to the DST/MR1 treatment did not significantly alter the generation of IFN--producing donor-reactive CD8 T cells (710 ± 152/200,000 CD8 T cells) in the recipients injected with memory CD4 T cells. These results indicate that memory CD4 T cells remain capable of providing help for donor-reactive CD8 T cells despite lymphoid sequestration with FTY720.
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We next analyzed antidonor humoral immunity in the various groups of recipients. Serum samples were collected at the time of graft rejection and tested for reactivity to donor C3H or third-party BALB/c thymocytes. Fig. 4B shows that monotherapy with FTY720 did not abrogate the development of donor-reactive IgG alloantibody in the presence of either naive or memory Mar CD4 T cells. Although DST/MR1 therapy significantly inhibited alloantibody production in mice injected with naive Mar T cells, further addition of FTY720 to the treatment protocol did not alter the level of C3H-reactive alloantibody. In contrast, recipients containing Mar memory CD4 T cells developed anti-donor IgG alloantibody regardless of DST/MR1 treatment (Fig. 4B). Furthermore, serum from the recipients injected with memory CD4 T cells and treated with both DST/MR1 and FTY720 also had high levels of IgG alloantibody specific for donor C3H (Fig. 4B) but not third-party BALB/c (not shown) alloantigens. These results demonstrate that lymphoid sequestration of naive or memory CD4 T cells did not prevent their ability to provide help for IgG alloantibody production. Immunofluorescent staining of graft sections from mice treated with FTY720 alone or with the combination of FTY720 and DST/MR1 revealed heavy deposition of the C3d complement component (Fig. 6), further suggesting an alloantibody-mediated rejection mechanism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Recent studies demonstrated that FTY720 prolongs the survival of solid organ allografts through retention of naive lymphocytes in secondary lymphoid organs (12, 13, 14). Despite increasing interest in this immunosuppressant, the effect of FTY720 on the migration and function of alloreactive memory CD4 T cells has not been previously evaluated. Our data clearly show that treatment with FTY720 results in the disappearance of memory CD4 T cells from the circulation with simultaneous accumulation of these cells in secondary lymphoid organs. These findings suggest that memory CD4 T cells behave similarly to the alloreactive effector T cells that have been activated under conditions of FTY720 treatment and are efficiently sequestered by this agent (A. Habicht, M. Clarkson, and M. Sayegh, manuscript in preparation). In contrast, the trafficking of pre-existing effector CD4 T cells is not significantly affected by FTY720 (16). This differential susceptibility to FTY720-induced sequestration may be determined by several factors. The ability of FTY720 to trap T cells in lymphoid organs largely depends on the homing properties of these cells. It has been previously shown that effector CD4 T cells have a limited ability to migrate into lymphoid organs presumably due to the low expression of the lymph node homing receptors CD62L and CCR7 (23). In contrast, a significant proportion of memory T cells re-expresses these molecules and can re-enter lymphoid tissues becoming susceptible to the effects of FTY720. In addition, expression of S1P receptor-1 appears to be associated with the activation status of T cells (17, 24). Although S1P receptor-1 expression is rapidly reduced after T cell activation, the expression and function of this molecule still needs to be evaluated on resting memory T cells.
Another important conclusion from our results is that not all memory CD4 T cells are equally affected by FTY720. This is consistent with the current view that memory CD4 T cells are a heterogenous population based on recirculation patterns and functional properties. Although memory CD4 T cells expressing high levels of the lymph node homing receptors CD62L (L-selectin) and CCR7 reside in secondary lymphoid organs, CD62LlowCCR7low memory CD4 T cells preferentially circulate through nonlymphoid peripheral tissues and perform pathogen surveillance. These subsets are generally referred to as "central" and "effector" memory T cells, respectively. As susceptibility to the effects of FTY720 depends on the T cell migration pattern, it is possible that FTY720 selectively affects central but not effector memory CD4 T cells. Supporting this hypothesis, our data show that effector memory CD4 T cells do not leave peripheral tissue during short-term treatment with FTY720 (Fig. 1).
Despite these findings, it is possible that after transplantation memory CD4 T cells migrate out of the periphery, reach secondary lymphoid organs, and provide help for alloantibody production thus influencing rejection of an allograft during FTY720 therapy. Also, these memory CD4 T cells may encounter donor Ags and become reactivated in the periphery. However, the functions of activated peripheral donor-specific memory CD4 T cells, their contribution to allograft rejection, and susceptibility to the existing therapies are poorly understood. These clinically relevant issues are currently a subject of intensive study by this and other laboratories.
The central finding of the current study is that efficient sequestration of circulating memory CD4 T cells delayed heart allograft rejection and synergized with DST/MR1 to significantly prolong heart allograft survival. We have previously reported that donor-specific memory CD4 T cells can contribute to allograft rejection through multiple pathways. After re-exposure to donor Ags, memory CD4 T cells may expand, infiltrate the graft, and directly mediate tissue destruction. In our model, memory CD4 T cell can recognize donor male Ag solely through the indirect pathway as there is no cross-reactivity to H-2k alloantigens. Therefore, Mar cells reaching the graft interact exclusively with infiltrating recipient APCs presenting donor male Ag. Our previously published data show that such interactions are sufficient to induce chronic vascular injury in the graft (25). However, we did not detect any CD4+ T cells in the heart grafts from recipients treated either with FTY720 alone (data not shown) or with the combination of FTY720 and DST/MR1 (Fig. 5), and no neointimal hyperplasia was found in any of the studied grafts (data not shown). These results demonstrate that FTY720 efficiently retains memory CD4 T cells in lymphoid organs and prevents them from causing tissue injury within the graft site.
In addition to direct effector mechanisms within the graft, memory CD4 T cells provide help for the activation of donor-specific CD8 T cells and for alloantibody production. To specifically evaluate the effect of FTY720 on helper functions of donor-reactive memory CD4 T cells, we focused on recipients treated with a combination of FTY720 and DST/MR1. In these recipients, the responses of endogenous naive CD4 T cells are inhibited by costimulatory blockade, and the only help for inducing antidonor immune responses is provided by transferred memory CD4 T cells. We found that despite lymphoid sequestration, memory CD4 T cells remain capable of providing help for the generation of antidonor effector CD8 T cells and alloantibody production. These results are consistent with previous findings in a viral infection model in which FTY720 did not impair the humoral response or induction of virus-specific cytotoxic CD8 T cells (15). Regardless of the robust priming of donor-specific CD8 effector T cells, the T cells were not detectable in the graft due to continuous FTY720 treatment. Therefore, even though FTY720 does not abrogate helper functions of memory CD4 T cells, it indirectly inhibits one of the effector mechanisms by which memory CD4 T cells contribute to allograft destruction thus leading to prolonged graft survival.
Unlike lymphocytes, soluble donor-specific IgG alloantibodies are not influenced by S1P receptor-1 agonist. Taken together, the high serum levels of donor-reactive Ab, the absence of T cells in the graft and the infiltration of Mac1+ cells with intense complement deposition are consistent with Ab-mediated rejection. This mechanism was further confirmed by the finding that the combination of FTY720 and DST/MR1 leads to long-term graft survival in the absence of mature B cells and Ab (Fig. 7A).
Notably, withdrawal of FTY720 resulted in rapid rejection characterized by intense infiltration of CD8 T cells into the graft. These results emphasize that due to the short half-life of FTY720, the effective lymphoid sequestration is rapidly reversed upon drug withdrawal. As FTY720 does not impair priming of donor-specific T cells, generated effector CD8 T cells can leave the lymphoid organs and mediate rejection shortly after the treatment has been discontinued. Recently published data suggest that the combination of FTY720 and DST/MR1 promotes induction of CD4+CD25+ regulatory T cells leading to long-term cardiac allograft survival in mice with interrupted CD62L-mediated lymph node homing (26). Our data indicate that if regulatory T cells are indeed induced under these conditions, they are still unable to control the deleterious effect of memory CD4 T cells on allograft outcome even in the absence of alloantibody-mediated rejection. Therefore, compliance with continuous administration of FTY720 is critical for graft survival even when B cell responses are controlled.
In summary, our data clearly indicate that sequestration of both memory CD4 T cells along with newly generated donor-reactive effector T cells improves cardiac allograft survival. Therefore, FTY720 is likely to be beneficial in T cell-sensitized allograft recipients, especially in combination with costimulatory blockade. However, our findings emphasize that memory CD4 T cells can influence antidonor immune responses despite lymphoid sequestration and that additional strategies will be required to control alloantibody-mediated graft injury under these conditions.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by a Scientist Development grant from the American Heart Association and a Women and Minority Faculty grant from the American Society of Transplantation (to A.V.). 
2 Q.Z. and Y.C. contributed equally.
3 Address correspondence and reprint requests to Dr. Anna Valujskikh, Department of Immunology, NB-30, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: valujsa{at}ccf.org
4 Abbreviations used in this paper: S1P, sphingosine-1-phosphate; KO, knockout; Tg, transgenic; DST, donor-specific transfusion; PBST, PBS 0.025% Tween 20; MST, median survival time.
Received for publication July 28, 2005. Accepted for publication October 26, 2005.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes. J. Immunol. 163: 2267-2275. This article has been cited by other articles:
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  V. Pavlov, H. Raedler, S. Yuan, S. Leisman, W.-h. Kwan, P. N. Lalli, M. E. Medof, and P. S. Heeger Donor Deficiency of Decay-Accelerating Factor Accelerates Murine T Cell-Mediated Cardiac Allograft Rejection J. Immunol., October 1, 2008; 181(7): 4580 - 4589. [Abstract] [Full Text] [PDF]
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) or water (
) as outlined in Materials and Methods. After 4 days of treatment, animals were sacrificed and peripheral blood and organ-infiltrating lymphocytes were isolated. The percentage from total peripheral blood cells (A) or absolute numbers (B) of Thy1.2+ Mar memory CD4 T cells in various compartments were evaluated by flow cytometry. The results represent mean values for five mice per group. *, p < 0.05 vs water-treated mice.
