Delayed Allograft Rejection Research Articles

Regulatory T Cells in Transplantation

T Regulatory cells (Treg) play an important role in the induction and maintenance of immunological tolerance to self and alloantigens. Recent findings in experimental transplant models have demonstrated that Treg can control acute and delayed allograft rejection. Preclinical attempts to use Treg as a cellular therapy have been successfully undertaken demonstrating the safety and feasibility of such treatment, suggesting that they have therapeutic potential.

In this issue

In this issue

Cover Picture: Eur. J. Immunol. 2/11

The cover image of this issue consists of the immunohistochemical staining of μ-calpain in a healthy human kidney and is taken from Letavernier et al. (pp. 473–484). In this article, the authors analyse the role of calpains in acute allograft rejection. The authors demonstrate that μ-calpain expression is increased in human transplant kidneys undergoing acute or chronic rejection, mainly in infiltrating T cells. The authors further show that calpain inhibition delays allograft rejection. Interestingly, this delay in allograft rejection is associated with a limitation in allograft infiltration by T cells that is due to the slowing down in T-cell migration rather than proliferation.

Critical role of the calpain/calpastatin balance in acute allograft rejection

Rejection of solid organ allograft involves alloreactive T-cell expansion. The importance of NF-κB and NFAT in this process is underscored by the therapeutic efficacy of immunosuppressive agents, which target the two transcription factors. Since calpains, calcium-activated proteases, are involved in the activation of NF-κB and NFAT, we investigated the role of calpains in allograft rejection. In human transplant kidneys undergoing acute or chronic rejection, we show an increased expression of CAPN 1 gene encoding μ-calpain, associated with a marked expression of μ-calpain, mainly in infiltrating T cells. To address the role of calpain in rejection, we used a skin transplant model in transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. We show that calpain inhibition extended skin allograft survival, from 11 to 20 days. This delay was associated with a limitation in allograft infiltration by T cells. In vitro, calpain inhibition by calpastatin transgene expression limited dramatically T-cell migration but, unexpectedly, increased slightly T-cell proliferation. Amplification of IL-2 signaling via the stabilization of IL-2R common γ-chain provided an explanation for the proliferation response. This is the first study establishing that calpain inhibition delays allograft rejection by slowing down T-cell migration rather than proliferation.

Influence of combined treatment of low dose rapamycin and cyclosporin A on corneal allograft survival

To analyze the immune modulatory effect of low-dose systemic treatment with rapamycin (Rapa) alone or in combination with cyclosporin A (CsA) in a high-responder corneal allograft model. A total of 80 C57BL/6 mice received corneal grafts from BALB/c donors. Recipients were treated with either CsA 3 mg/kg/day or Rapa 0.5 mg/kg/day monotherapy or received combined treatment. Immunomodulatory treatment was started on the day of surgery, and continued for 14 days. The frequency of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Treg) in secondary lymphoid organs was measured by flow cytometry. Development of IFN-gamma producing alloreactive T cells was estimated by Elispot. In addition, corneal samples were subjected to real-time RT-PCR analysis for cytokine transcription. Monotherapy with Rapa significantly delayed allograft rejection (13.4 +/- 1.34 days, p = 0.03). However, the combination of both, low-dose Rapa and CsA prolonged corneal allograft survival at a significantly higher level (MST = 17.1 +/- 1.37 days, p = 0.0001) than in the control group (MST = 11.2 +/- 1.91 days). Rapa monotherapy increased the frequency of CD4(+)CD25(+)Foxp3(+)Treg in draining lymph nodes, whereas addition of CsA reduced Tregs. Monotherapy with Rapa as well as combined treatment prevented development of IFN-gamma producing alloreactive T cells in spleen. Combined treatment resulted in down-regulation of intragraft CD3, IL-2, IFN-gamma and IL-10 transcription (p = 0.028, p = 0.027, p = 0.028 and p = 0.027 respectively). Combined treatment with low-dose CsA and Rapa resulted in superior graft survival, and effectively modulated mRNA expression of inflammation and infiltration markers.

NK Cells Delay Allograft Rejection in Lymphopenic Hosts by Downregulating the Homeostatic Proliferation of CD8+ T Cells

T cells present in lymphopenic environments undergo spontaneous (homeostatic) proliferation resulting in expansion of the memory T cell pool. Homeostatically generated memory T cells protect the host against infection but can cause autoimmunity and allograft rejection. Therefore, understanding the mechanisms that regulate homeostatic T cell proliferation is germane to clinical settings in which lymphodepletion is used. In this study, we asked whether NK cells, which regulate immune responses in lymphocyte-replete hosts, also regulate homeostatic T cell proliferation under lymphopenic conditions. We found that T cells transferred into genetically lymphocyte-deficient RAG-/- mice proliferate faster and generate more CD8+ memory T cells if NK cells were absent. CD8+ T cells that underwent homeostatic proliferation in the presence of NK cells generated mostly effector memory (CD44highCD62Llow) lymphocytes, whereas those that divided in the absence of NK cells were skewed toward central memory (CD44highCD62Lhigh). The latter originated predominantly from proliferation of the "natural" central memory CD8+ T cell pool. Regulation of homeostatic proliferation by NK cells occurred independent of perforin but was reversed by excess IL-15. Importantly, NK depletion enhanced CD8+ T cell recovery in T cell-depleted wild-type mice and accelerated rejection of skin allografts, indicating that regulation of homeostatic proliferation by NK cells is not restricted to genetically lymphocyte-deficient animals. These results demonstrate that NK cells downregulate homeostatic CD8+ T cell proliferation in lymphopenic environments by competing for IL-15. Concomitant NK and T cell depletion may be undesirable in transplant recipients because of enhanced expansion of memory CD8+ T cells that increase the risk of rejection.

Acute Cardiac Allograft Rejection by Directly Cytotoxic CD4 T Cells: Parallel Requirements for Fas and Perforin

CD4 T cells can suffice as effector cells to mediate primary acute cardiac allograft rejection. Although CD4 T cells can readily kill appropriate target cells in vitro, the corresponding role of such cytolytic activity for mediating allograft rejection in vivo is unknown. Therefore, we determined whether the cytolytic effector molecules perforin (PFP) and/or FasL (CD95L) were necessary for CD4 T cell-mediated rejection in vivo. Wild-type C3H(H-2) or Fas (CD95)-deficient C3Hlpr (H-2) hearts were transplanted into immune-deficient C57B6rag (H-2) mice. Then, recipients were reconstituted with naïve purified CD4 T cells from wild-type, PFP-deficient, or FasL (gld)-deficient T-cell donors. In vitro, alloreactive CD4 T cells were competent to lyse donor major histocompatibility complex class II+ target cells, largely by a Fas-dependent mechanism. In vivo, the individual disruption of donor Fas expression (lpr) or CD4 T-cell-derived PFP had no significant impact on acute rejection. However, FasL-deficient (gld) CD4 T cells demonstrated delayed allograft rejection. Importantly, the simultaneous removal of both donor Fas expression and CD4 T-cell PFP completely abrogated acute rejection, despite the persistence of CD4 T cells within the graft. Results demonstrate that the direct rejection of cardiac allografts by CD4 effector T cells requires the alternative contribution of graft Fas expression and T cell PFP expression. To our knowledge, this is the first demonstration that cytolytic activity by CD4 T cells can play an obligate role for primary acute allograft rejection in vivo.

Immune regulatory effect of rapamycin and cyclosporine A on experimental corneal allograft survival

Abstract Purpose To analyze the efficiency of low dose systemic treatment with rapamycin (Rapa) alone or in combination with cyclosporin A (CsA) in preventing corneal allograft rejection in a high responder strain combination. Methods A total of 80 C57BL/6 mice received corneal grafts of BALB/c donors. Recipients were treated with CsA 3mg/kg/day or Rapa 0.5mg/kg/day monotherapy or received combined treatment. Intraperitoneal injections were started on the day of surgery and continued for 14 days. In addition, corneal samples were subjected to real time RT‐PCR analysis for cytokine transcription. The frequency of CD4+CD25+Foxp3+ T regulatory cells (Treg) in secondary lymphoid organs were measured by flow cytometry. Memory T cells were estimated by Elispot. Results Monotherapy with Rapa significantly delayed allograft rejection (p=0.03). However, the combination of low dose Rapa and CsA prolonged corneal allografts survival at a significantly higher level (MST=17.1 ± 1.37 days, p=0.000004). Addition of CsA to Rapa resulted in down‐regulation of intragraft CD3, IL‐2, IFN‐γ and IL‐10 transcription (p=0.028, p= 0.027, p=0.028 and p= 0.027 respectively). Rapa alone increased the frequency of CD4+CD25+Foxp3+ Tregs in draining lymph nodes, whereas addition of CsA reduced Tregs. Rapa monotherapy as well as combined treatment prevented development of alloantigen specific IFN‐γ producing memory T cells in spleen. Conclusion Combined treatment with low dose CsA and Rapa resulted in superior graft survival and effectively modulated mRNA expression of inflammation and infiltration markers. Supported by DFG (150/14‐2) and SFB650 TP14

Trichosanthin, an Extract of Trichosanthes kirilowii, Effectively Prevents Acute Rejection of Major Histocompatibility Complex–Mismatched Mouse Skin Allograft

Trichosanthin is an active component extracted from the root tuber of the Chinese medicinal herb Trichosanthes kirilowii. Trichosanthin has abortifacient, anti-tumor, anti-HIV, and immunoregulatory functions. In the current study, we explored its potential effect on allograft rejection in a murine skin transplantation model across a fully mismatched major histocompatibility complex. It was found that treatment of recipient mice with trichosanthin (0.25 or 1 mg/kg, IP) significantly delayed allograft rejection. T cells that originated from recipients treated with trichosanthin were restimulated with donor-specific splenocytes showed a significantly reduced response compared with that of control recipients. In line with these results, the mRNA levels for interleukin (IL)-2 and interferon-γ were decreased and the levels of IL-4 and IL-10 were increased in splenic T cells originating from trichosanthin-treated recipients. These results indicated that trichosanthin may have potential therapeutic value for transplantation rejection and other inflammatory diseases.

Prolongation of Cardiac Allograft Survival by Rapamycin and the Invariant Natural Killer T Cell Glycolipid Agonist OCH

Invariant natural killer T (iNKT) cells are glycolipid-responsive cells with potent immunomodulatory properties. Although iNKT cells have been implicated in cardiac allograft tolerance, whether in vivo triggering of iNKT cells with Th2-promoting glycolipids offers a therapeutic benefit in heart transplantation remains unexplored. C3H (H-2k) hearts were transplanted into C57BL/6 (H-2b) mice. The recipients were left untreated or received the Th2-promoting iNKT cell agonist OCH, the antirejection agent rapamycin, or both. Allografts were recovered on postoperative day 8 or at endpoint, stained with hematoxylin-eosin, and analyzed for intragraft transcript levels of effector cytokines and iNKT cells' invariant T-cell receptor segment Valpha14-Jalpha18. The presence of circulating alloantibodies was assessed in recipients' sera at similar time points. A second fully mismatched cardiac allograft model (BALB/c-to-C57BL/6) was used to further validate the efficacy of our treatment regimens. Combination immunotherapy with OCH and rapamycin significantly enhanced C3H allograft survival and led to nearly normal graft histology with minimal vascular changes and mononuclear cell infiltration, and an almost normal IgG1:IgG2a ratio in recipients' sera. These were accompanied by elevated intragraft mRNA levels of interleukin (IL)-4, and to a lesser extent IL-10 and IL-13, and high transcript levels of Valpha14-Jalpha18 T-cell receptor gene segment. Furthermore, when used alone or together with rapamycin, OCH delayed allograft rejection in our BALB/c-to-C57BL/6 model. In vivo administration of OCH may deviate alloimmune responses towards a Th2 phenotype and prolong allograft survival. Select iNKT cell glycolipid agonists can therefore be used in monotherapy or combination immunotherapy of transplant rejection.

Suppressor of cytokine signalling-3 expression inhibits cytokine-mediated destruction of primary mouse and rat pancreatic islets and delays allograft rejection

The pro-inflammatory cytokines IL-1 and IFNgamma are critical molecules in immune-mediated beta cell destruction leading to type 1 diabetes mellitus. Suppressor of cytokine signalling (SOCS)-3 inhibits the cytokine-mediated destruction of insulinoma-1 cells. Here we investigate the effect of SOCS3 in primary rodent beta cells and diabetic animal models. Using mice with beta cell-specific Socs3 expression and a Socs3-encoding adenovirus construct, we characterised the protective effect of SOCS3 in mouse and rat islets subjected to cytokine stimulation. In transplantation studies of NOD mice and alloxan-treated mice the survival of Socs3 transgenic islets was investigated. Socs3 transgenic islets showed significant resistance to cytokine-induced apoptosis and impaired insulin release. Neither glucose-stimulated insulin release, insulin content or glucose oxidation were affected by SOCS3. Rat islet cultures transduced with Socs3-adenovirus displayed reduced cytokine-induced nitric oxide and apoptosis associated with inhibition of the IL-1-induced nuclear factor-kappaB and mitogen-activated protein kinase (MAPK) pathways. Transplanted Socs3 transgenic islets were not protected in diabetic NOD mice, but showed a prolonged graft survival when transplanted into diabetic allogenic BALB/c mice. SOCS3 inhibits IL-1-induced signalling through the nuclear factor-kappaB and MAPK pathways and apoptosis induced by cytokines in primary beta cells. Moreover, Socs3 transgenic islets are protected in an allogenic transplantation model. SOCS3 may represent a target for pharmacological or genetic engineering in islet transplantation for treatment of type 1 diabetes mellitus.

Stopping the Primal RAGE Reaction in Myocardial Infarction

The report of Andrassy and colleagues1 in the current issue of Circulation adds to the growing body of evidence that the receptor for advanced glycation end products (RAGE) and its ligands, particularly high mobility group box-1 (HMGB1), are central mediators of ischemia/reperfusion (I/R) injury in the heart.1–4 A major cause of injury, especially in the reperfusion phase, is the influx of inflammatory cells into the stressed heart. Andrassy and colleagues show that infiltrating leukocytes express proinflammatory HMGB1 and that HMGB1 plays fundamental roles in injury responses in the I/R heart. In homeostasis, HMGB1 is largely a nuclear protein. In stress conditions, HMGB1 may be released from injured cells, particularly on necrosis. The chief receptor for HMGB1, RAGE, is expressed in multiple cell types in the I/R heart, such as inflammatory cells, and also in cardiomyocytes and vascular cells (endothelial cells and smooth muscle cells).2 Although HMGB1 may interact with distinct receptors beyond RAGE (for example, toll receptors),5 the current study of Andrassy and colleagues reveals major roles for RAGE in transducing the effects of HMGB1 in the heart, as administration of recombinant HMGB1 or antagonists of this molecule had significant proinjury effects in the wild-type mouse heart, but no additive effects were noted in mice devoid of RAGE. The biology of RAGE, however, is complex and extends beyond HMGB1 in the injured heart. Article p 3216 In addition to HMGB1, other ligands of RAGE likely play pivotal roles in the response to ischemia or I/R in the heart. For example, rapid generation of pre–advanced glycation end products (AGE) species (methylglyoxal) and AGEs in transient occlusion/reperfusion of the LAD coronary artery has been illustrated.2 In hypoxia, in both the murine heart and in isolated primary murine endothelial cells, AGE-immunoreactive species are central mediators of regulation of …

Farnesyl transferase inhibitors delay primary skin allograft rejection in class II MHC‐mismatched mice

We are investigating the possibility that farnesyl transferase inhibitors (FTIs) may be useful anti‐rejection drugs. We have shown that the FTI, L‐744,832 can significantly delay allograft rejection in mice using MHC class II mismatched skin allografts. When treatment with this FTI begins two days prior to the allograft procedure and extends for 5 days, we found that rejection was delayed in all treated mice, 26±14 days, compared to untreated mice, 13±4 days. Interestingly, this delay in rejection can extend for greater than 30 days in 30% of the treated mice. To determine whether FTI treatment can delay alloreactive effector responses, we examined the effect of L‐744,832 treatment on allograft rejection in mice that had previously been sensitized to the allo‐antigens. We found that treatment with this FTI did not significantly delay second‐set allograft rejection. This observation, coupled with the long delay in some of the first set allograft treatments, suggests that FTIs may be able to induce tolerance of the alloreactive CD4+ T cells. To further investigate the action of FTIs on graft rejection we are measuring the effects of treatment using mixed lymphocyte cultures and using in vivo tolerance assays. We suggest that FTIs may modulate acute immune responses without systemically suppressing the immune system.

A limited course of soluble CD83 delays acute cellular rejection of MHC-mismatched mouse skin allografts

CD83 is a surface marker expressed on matured dendritic cells (DCs). It plays a pivotal role in the mediation of DC/T cell interaction and induction of T-cell activation. Previous studies have suggested that a soluble form of CD83 could suppress DC maturation and inhibit T-cell activation and, as a result, it can prevent paralysis associated with experimental autoimmune encephalomyelitis. Here, we explored its potential effect on allograft rejection in a fully major histocompatibility complex-mismatched murine skin transplantation model. A form of mouse soluble CD83 (CD83-Ig) fused the extracellular domain of murine CD83 with human IgG1alpha Fc tail was purified from transfected COS-7 cell. It was found that the treatment of recipient mice with CD83-Ig significantly delayed allograft rejection. Especially, when T cells originated from recipients treated with CD83-Ig re-stimulated with donor-specific splenocytes, they showed a significant reduced responding capability as compared with that of originated from control recipients. In line with these results, a reduction for serum IFN-gamma and IL-2 and a decreased mRNA expression of IFN-gamma and IL-2 in allograft infiltrated immune cells were also observed. Our results suggest that CD83-Ig could be useful for the treatment of allograft rejection in combination with other therapeutic strategies.

Delayed allograft rejection by the suppression of class II transactivator

(CIITA) down-modulation on allograft rejection. To inhibit the function of CIITA, we constructed a series of CIITA mutants and found one exhibiting the dominant-negative effect on the regulation of major histocompatibility complex (MHC) class II expression. To test whether the CIITA dominant-negative mutant reduces immunogenicity, CIITA-transfected melanoma cells were injected into allogeneic host and assessed for immune evading activity against host immune cells. We demonstrated that the CIITA dominant-negative mutant allowed tumor nodules to develop earlier in the lung than control by this tumor challenge study. Furthermore, skin grafts deficient for CIITA also survived longer than wild-type in allogeneic hosts. Both the tumor challenge and skin graft studies suggest the inhibition of CIITA molecules in donor tissue would be beneficial to the control of allo-response.

Prolongation of alloskin graft survival by catalytic scavengers of reactive oxygen species

We tested the effects of salen manganese (Salen–Mn) complexes, which are scavengers of reactive oxygen species exhibiting superoxide dismutase and catalase activities on the rejection of and alloresponse to fully allogeneic skin grafts in mice. We showed that pre-transplant treatment of C57Bl/6 donor skin or of BALB/c recipients with Salen–Mn complexes significantly delayed allograft rejection. ELISPOT analysis of alloimmune response of treated mice revealed a significant reduction of the frequency of type 1 cytokine (pro-inflammatory) producing T-cells, while the number of activated T-cells producing type 2 cytokines was elevated. In addition, anti-oxidative treatment of graft recipients resulted in a profound inhibition of their donor-specific cytotoxic T-cell response. Our results indicate that salen manganese complexes mediate their effect on graft rejection both by reducing the susceptibility of graft tissue to ROS-mediated injury and by exerting an anti-inflammatory effect in recipients.

Lentiviral Gene Transfer of the Chemokine Antagonist RANTES 9-68 Prolongs Heart Graft Survival

Allograft tolerance might be achieved by expressing immunomodulatory proteins through gene therapy. We have evaluated the possibility of promoting significantly allograft survival in a vascularized cardiac allograft model by performing ex vivo gene transfer. We used a lentiviral vector encoding the chemokine antagonist RANTES 9-68 that is capable of competing with native RANTES. The Fisher donor/Lewis recipient rat strain combinations were used and all animals received for the first 5 days posttransplantation a subtherapeutic dose of cyclosporine A (1.5 mg/kg). Ex vivo gene transfer into heart allograft was performed by multiple injections of the SIN.cPPT lentiviral vector, which corresponds to the multiply attenuated, self-inactivating lentivector derived from the human immunodeficiency virus (HIV)-1. About 6% of the cardiac tissue had integrated lentiviral vector, which closely matches the mean in vivo RANTES antagonist expression of 5% obtained by immunohistochemistry. In vivo RANTES 9-68 expression has significantly prolonged graft survival (median [25%-75%]: 20 [17-26] days), compared to the control 15 ([14-15] days; P=0.0007). Furthermore, hearts transduced with RANTES 9-68 showed a significant (P<0.05) reduction in cell infiltration and intragraft expression of TNF-alpha, IFN-gamma, endogenous RANTES, and TGF-beta. Lentiviral gene transfer of RANTES 9-68 antagonist attenuates significantly the inflammatory response and delays allograft rejection, despite low levels of transduction. Future improvement of heart transduction by lentiviral vectors, as it has been achieved with other vectors, might become an attractive alternative therapy for treating allografts that require sustained gene expression for better organ preservation.

Prevention of Acute Graft-Versus-Host Disease by CD4+CD25+ Regulatory T Cells Is Dependent on the CD30/CD153 Pathway.

CD4+CD25+ regulatory T (Treg) cells suppress acute graft versus host disease (aGVHD), prevent autoimmunity and delay allograft rejection. CD30 and other TNF-R family members have been demonstrated to be expressed by Treg and to function as alternative costimulatory pathways for T cell activation. In this study we assessed the significance of the CD30/CD153 pathway in Tregs suppression of aGVHD in a murine major MHC mismatch BMT model. Using bioluminescence imaging proliferation of donor derived luciferase-labeled CD4+ and CD8+ T cells was quantified at serial time points after transplantation. Treg suppressed the early expansion of alloreactive T-cells. Immunofluorescence microscopy revealed a predominant infiltration of donor derived Treg in CD153 positive regions of secondary lymphoid organs, namely parafollicular T cell zones of lymph nodes and the subepithelial dome regions of Peyers Patches. In vivo blockade of the CD30/CD153 pathway with anti CD153 Ab did not alter Treg migration to secondary lymphoid organs but reduced their suppressive effect. Proliferation of donor T cells as measured in photons/second/mouse was significantly higher in animals receiving Treg and CD153 blocking antibodies as compared to recipients of Treg only (p=0.0038). Gene expression profiling of Treg with DNA microarrays indicated a Treg signature that was consistently found in different mouse strains. This Treg signature was altered after CD153 blockade in vitro. Importantly, aGVHD lethality was significantly increased (p=0.021) when CD30-CD153 interaction was blocked during Treg transfer. This study provides direct evidence that the TNF-R family member CD30 is critical for Treg cell function in the regulation of pathological T cell responses that lead to aGVHD.

Modulation of acute inflammation by targeting glycosaminoglycan–cytokine interactions

Glycosaminoglycans (GAGs) located on cellular membranes and the extracellular matrix (ECM) are able to interact with chemokines and pro-inflammatory cytokines, leading to local cytokine/chemokine accumulation. The tissue-bound cytokines/chemokines function in promoting leukocyte migration and activation, contributing to local inflammation. Hence, targeting of GAG–cytokine interactions may provide an avenue for the attenuation of inflammatory responses. A cationic peptide (MC2) derived from the heparin-binding sequence of mouse IFN-γ was previously shown by our laboratory to delay allograft rejection in an animal model. In order to further investigate potential anti-inflammatory properties of the MC2 peptide, we have studied its activity in an acute peritoneal inflammation model. Groups of C57Bl/6 mice were injected intraperitoneally with either ConA or thioglycollate and treated with saline (control), the MC2 peptide or two control cationic peptides, poly- l-lysine (PLL) and poly- l-arginine (PLA). Treatment with the MC2 peptide, but not PLA or PLL, resulted in statistically significant reductions in total cell numbers, concentration of total proteins and concentrations of pro-inflammatory cytokines (TNFα, IL-6 or IL-1β) in peritoneal lavage fluids, without alterations to the qualitative cellular composition of the exudate. These results suggest that targeting GAG–cytokine interaction is a viable approach to reduce inflammation.

CD4+CD25+ regulatory T cells suppress allograft rejection mediated by memory CD8+ T cells via a CD30-dependent mechanism.

CD4(+)CD25(+) regulatory T (Treg) cells suppress naive T cell responses, prevent autoimmunity, and delay allograft rejection. It is not known, however, whether Treg cells suppress allograft rejection mediated by memory T cells, as the latter mount faster and stronger immune responses than their naive counterparts. Here we show that antigen-induced, but not naive, Treg cells suppress allograft rejection mediated by memory CD8(+) T cells. Suppression was allospecific, as Treg cells induced by third-party antigens did not delay allograft rejection. In vivo and in vitro analyses revealed that the apoptosis of allospecific memory CD8(+) T cells is significantly increased in the presence of antigen-induced Treg cells, while their proliferation remains unaffected. Importantly, neither suppression of allograft rejection nor enhanced apoptosis of memory CD8(+) T cells was observed when Treg cells lacked CD30 or when CD30 ligand-CD30 interaction was blocked with anti-CD30 ligand Ab. This study therefore provides direct evidence that pathogenic memory T cells are amenable to suppression in an antigen-specific manner and identifies CD30 as a molecule that is critical for the regulation of memory T cell responses.