Suppression Of Graft Rejection Research Articles

Role for CD4 +CD25 + T Cells in Inhibition of Graft Rejection by Extracorporeal Photopheresis

Extracorporeal photopheresis (ECP) is used to treat recurrent severe rejection in clinical heart and lung recipients. The mechanisms of the salutary effects of ECP are poorly understood, but appear to involve regulation of T-cell-mediated alloreactive responses, possibly by induction of regulatory T cells. We created a mouse model of ECP to determine the effects of ECP on T-cell responses in vivo and the contribution of CD4(+)CD25(+) T cells. In this study, 1 x 10(7) splenocytes were treated with 8-methoxypsoralen (8-MOP, 200 ng/ml), followed by ultraviolet A (UVA) irradiation (2 J/cm(2), 350 nm), and then injected intravenously into syngeneic mice. Thirty minutes later, the treated animals received heterotopic cardiac allografts with no immunosuppression. Treated graft recipients were analyzed to determine the effect of ECP on graft survival, deletion of allospecific T cells, and the frequency and in vivo suppressive activity of CD4(+)CD25(+) T cells. ECP extends cardiac allograft survival in at least two different strain combinations. For CBA/Ca recipients of C57BL/6 allografts, median survival time (MST) in ECP-treated animals was 16 days vs 10 days in graft recipients treated with cells exposed only to 8-MOP (p = 0.04). The frequency of splenic CD4(+)CD25(+) cells expressing FoxP3(+) increased 2-fold in ECP-treated CBA/Ca mice (82.6 +/- 5.2%, n = 4) relative to untreated mice (44.9 +/- 4.5%, n = 4, p < 0.001). Adoptive transfer of 3 x 10(5) sorted CD4(+)CD25(+) splenocytes from ECP-treated graft recipients to untreated cardiac allograft recipients 30 minutes after transplantation resulted in extended graft survival compared with animals that received the same number of CD4(+)CD25(+) splenocytes from cardiac allograft recipients not treated with ECP (MST: 24 days vs 13 days, respectively, p = 0.001). Analyses of 5,6-carboxy-fluorescein-succinimidyl-ester (CFSE)-labeled H-2K(b)-specific T cells in the spleen and lymph node showed no evidence of peripheral deletion after ECP treatment. ECP extends graft survival even in fully histoincompatible strain combinations with no immunosuppression. It increases the frequency of FoxP3(+)CD4(+)CD25(+) splenic T cells, and its effects can be transferred to untreated recipients using minimal numbers of CD4(+)CD25(+) T cells, indicating that CD4(+)CD25(+) T cells may play a key role in the immunomodulatory effects of ECP.

Use of dendritic cells in drug selection, development and therapy

Background: Dendritic cells (DC) have the unique ability to induce immunity against tumors and various pathogens or to promote tolerance in autoimmunity and transplantation. Hence, they are central to the regulation of immune responses. Objective/methods: Due to the unique tolerogenic ability of DC, understanding some of the key molecules that regulate DC function may help with targeting the relevant signals in DC as therapeutic options for many disease conditions. DC are also targets of drugs, and many of the anti-inflammatory and pharmaceutical agents used to prevent autoimmunity or inhibit graft rejection interfere with DC function. Results/conclusion: The drug-induced changes in DC may provide information for the selection of drugs and further drug discovery along with the use of DC as adjuvant in the treatment of autoimmunity and prevention of graft rejection in transplantation.

Developmental immunotoxicology (DIT): windows of vulnerability, immune dysfunction and safety assessment

Developmental immunotoxicity (DIT) is an increasing health concern since DIT outcomes predispose children to those diseases that have been on the rise in recent decades (e.g., childhood asthma, allergic diseases, autoimmune conditions, childhood infections). The enhanced vulnerability of the developing immune system for environmental insult is based on unique immune maturational events that occur during critical windows of vulnerability in early life. The semi-allogeneic pregnancy state, with suppression of graft rejection and associated skewing of the fetal and neonatal immune system, also influences the specific nature of DIT outcomes. In the exposed offspring, targeted immunosuppression can co-exist with an increased risk of allergic and/or autoimmune disease. Because with DIT immune dysfunction rather than profound immunosuppression is the greater concern, testing approaches should emphasize multi-functional assessment. Beyond T-cells, dendritic cells and macrophages are sensitive targets. The last-trimester fetus and the neonate are normally depressed in TH1-dependent functions and postnatal acquisition of needed TH1 capacity is a major concern with DIT. With this in mind, assessment should include a measure of TH1-dependent cell-mediated immunity [cytotoxic T-lymphocyte (CTL) activity or delayed-type hypersensitivity (DTH) response] in conjunction with a multi-isotype T-dependent antibody response (TDAR) and evaluation of innate immunity (e.g., NK activity). Other parameters such as immune histology, immunophenotyping, cytokine responses, and organ weights can be useful when included with immune functional evaluation. A multifunctional DIT protocol using influenza challenge is presented as one example of an approach that permits dysfunction and misregulation to be evaluated.

Regulation of alloimmune Th1 responses by the cyclin-dependent kinase inhibitor p21 following transplantation

The cyclin-dependent kinase (cdk) inhibitor p21 inhibits cellular proliferation of many cell types, including T cells. Autoimmune models, however, have yielded conflicting results regarding the role of cdk inhibitors and T-cell function. The role of p21 in T-cell function after transplantation has not been investigated directly. We hypothesized that p21 plays an important role in alloantigen-driven responses in vitro in mixed lymphocyte cultures (MLC) and in vivo using the heterotopic murine cardiac allograft model. Wild type (WT) and p21-deficient (p21-/-) mice were used as recipients, and the effects of p21 overexpression were assessed by transplanting p21 adenoviral-transfected cardiac allografts. Enzyme-linked immunospot (ELISPOT) and 3H-thymidine incorporation were used to evaluate for T-cell priming and proliferation in vitro, whereas graft histology was evaluated for rejection. When stimulated with alloantigens in vitro, splenocytes from p21-/- mice mounted enhanced proliferative responses and decreased Th2 responses relative to their WT counterparts. No differences in Th1 responses were noted when p21-/- cells were stimulated with alloantigens in vitro; however, after cardiac transplantation, Th1 responses were enhanced in p21-/- recipients relative to WT mice. This enhanced in vivo Th1 response was associated with exacerbated graft rejection in p21-/- recipients. Interestingly, p21 transfection of WT allografts inhibited graft rejection and Th1 priming. p21 controls the intensity of the immune response posttransplantation, with overexpression inhibiting allograft rejection. Our data demonstrate that p21 controls T-cell priming and suggest that p21 and other cdk inhibitors may serve as potential targets for therapeutic manipulation of alloimmune responses.

Administration of granulocyte colony-stimulating factor to recipients followed by intra-bone marrow–bone marrow transplantation accelerates acceptance of allogeneic bone marrow cells in mice

We have recently established a novel method for bone marrow transplantation: intra-bone marrow–bone marrow transplantation (IBM–BMT), by which the rapid recovery of donor-derived hematopoiesis can be expected even when reduced radiation doses are used. In this paper, we examine, using mice, whether the combination of pretreatment of recipients with granulocyte-colony-stimulating factor (G-CSF) and IBM–BMT can induce a more rapid recovery of donor-derived hematopoiesis than IBM–BMT alone. We first pretreated recipients with recombinant human (rh) G-CSF (250 μg/kg/day) for 5 consecutive days (days −6 to −2). On day −1, the recipients were irradiated, and IBM–BMT was carried out on day 0. On day 12, we performed colony-forming units of spleen (CFU-S) assays. The combination of G-CSF pretreatment and IBM–BMT augmented the CFU-S counts, the weight of spleens, and the numbers of donor-derived hematopoietic cells. We next analyzed the mechanisms underlying these effects of G-CSF and found that (i) G-CSF induces Th2 polarization, which can prevent graft rejection, and (ii) G-CSF augments natural suppressor activity, which suppresses graft rejection. The combination of G-CSF pretreatment and IBM–BMT can produce the rapid recovery of donor-derived hematopoiesis and suppress graft rejection. This method would lighten the burden on patients in allogeneic BMT.

Immunosuppression in phototherapy.

The successful use of phototherapy, especially psoralen plus UVA (PUVA) therapy, in the treatment of a variety of skin diseases is well known. Because the pathology of diseases such as vitiligo, alopecia and lichen planus is thought to involve immune mechanisms, the beneficial effect of PUVA may be due to immunosuppression. PUVA treatment can induce suppression in two ways. In the first (local suppression) psoralen is applied topically, the skin is irradiated with UVA and the contact allergen is applied directly to the irradiated skin. The induction of contact hypersensitivity (CHS) is suppressed and suppressor cells are found in the spleens of treated animals. Systemic suppression results from the injection of psoralen followed by exposure to UVA. The contact allergen is then applied at a distant non-irradiated site. CHS is suppressed and antigen-specific suppressor cells are found in the spleens of treated mice. The ability to induce specific immunosuppression may provide novel methods of inhibiting unwanted immune responses. We have demonstrated that graft rejection and the induction of graft-versus-host disease can be suppressed in an antigen-specific manner by UV radiation. Thus phototherapy may provide promising new treatments for suppressing graft rejection and perhaps may be beneficial in the treatment of autoimmune disease and allergic reactions.

A Key Role for TGF-β Signaling to T Cells in the Long-Term Acceptance of Allografts

TGF-beta is a key immunoregulatory cytokine which supports self-tolerance by signaling to T cells. In this report, we show a crucial role for TGF-beta signaling to T cells in enabling the long-term acceptance of allografts, whether natural or induced therapeutically by coreceptor and costimulation blockade. The requirement for TGF-beta appears most pronounced during the initial exposure to alloantigens. We demonstrate the ability of TGF-beta to direct the development in vitro of regulatory cells that suppress graft rejection in vivo. Such suppression was not affected by anti-TGF-beta treatment of the recipient mice. Despite this, TGF-beta may still have a role in CD4+ cell-mediated suppression of antiallograft responses in vivo, since its neutralization can, in some cases, abrogate suppression. These results show that TGF-beta signaling to T cells is dispensable for mounting destructive responses against skin allografts while appearing to be an essential intermediary in establishing long-term tolerance.

Mice Lacking CD200R1 Show Absence of Suppression of Lipopolysaccharide-Induced Tumor Necrosis Factor-α and Mixed Leukocyte Culture Responses by CD200

CD200:CD200R interactions deliver immunoregulatory signals. A family of CD200Rs (CD200R1-5) has been described, and engagement of CD200R1 by its ligand CD200 suppresses LPS-induced macrophage cytokine production, decreases alloimmune responses in vivo and in vitro, and suppresses collagen-induced arthritis. We generated C57BL/6 mice lacking the genomic exons encoding the extracellular domains of the CD200R1 molecule using transformation of ES cells and explored cell subtypes and immune responses in these mice. Myeloid cells/splenocytes from CD200R1(-/-) mice were not stained in FACS by anti-CD200R1 mAb. Stimulation of splenic tumor necrosis factor-alpha production by lipopolysaccharide was enhanced relative to control (+/+) mice and was not suppressed by addition of exogenous CD200Fc. Modulation of alloreactivity in mixed leukocyte cultures by CD200Fc depended upon CD200R1+ stimulatory cells, although maximal immunoregulation by CD200Fc occurred only when CD200R1+ T responder cells also were used. CD200Fc failed to suppress graft rejection in CD200R1(-/-) mice. CD200:CD200R1 plays an immunoregulatory role in vivo.

Control of Immune Responses by Antigen-Specific Regulatory T Cells Expressing the Folate Receptor

Immune responses can be enhanced or dampened by differential manipulation of Foxp3-expressing CD25(+)CD4(+) natural regulatory T (Treg) cells versus other naive or activated T cells. By searching for a molecule capable of distinguishing these populations, we here found that natural Treg cells constitutively expressed high amounts of folate receptor 4 (FR4). The expression of FR4 and CD25 also separated antigen-stimulated CD4(+) non-Treg cells into the FR4(hi)CD25(-) and FR4(lo)CD25(+) populations, which were different in proliferation and cytokine secretion upon restimulation. These distinctions showed that antigenic stimulation activated and expanded antigen-specific natural Treg cells as well as effector and memory T cells. Accordingly, FR4(hi)CD25(+)CD4(+) T cells enriched from alloantigen-stimulated T cells suppressed graft rejection. Administration of FR4 monoclonal antibody specifically reduced Treg cells, provoking effective tumor immunity in tumor-bearing animals, whereas similar treatment of normal young mice elicited autoimmune disease. Thus, specific manipulation of FR4(hi)CD25(+)CD4(+) Treg cells helps control ongoing immune responses.

HLA-G expression is induced in Epstein-Barr virus–transformed B-cell lines by culture conditions

Human leukocyte antigen-G (HLA-G) belongs to the nonclassical HLA class I family of genes presently designated as class Ib genes. It was found to be expressed mainly in placental tissue and in the thymus. Expression of HLA-G is induced by lymphokines such as interleukin-10 and has been associated with the escape of tumor cells from immune surveillance or with inhibition of graft rejection. In this report, Epstein-Barr virus-transformed B-cell lines established from peripheral blood lymphocytes from healthy volunteers were studied. Our results show that EBV-transformed B-cell lines, but not freshly separated peripheral blood lymphocytes, can be induced to express HLA-G either by subjecting the cultures to nutrient deficiency to hypoxia or to both, however, not all cell lines responded equally to stress conditions. The association of HLA-G with certain cancer transformations may suggest that the resistance to HLA-G expression could be related to susceptibility to the development of malignancy.

Regulatory T Cells and T Cell Depletion

Allogeneic immune responses are modulated by a subset of host T cells with regulatory function (Treg) contained within the CD4(+)CD25(high) subset. Evidence exists that Treg expand after peritransplantation lymphopenia, inhibit graft rejection, and induce and maintain tolerance. Little, however, is known about the role of Treg in the clinical setting. IL-2 and activation by T cell receptor engagement are instrumental to generate and maintain Treg, but the influence of immunosuppressants on Treg homeostasis in humans in vivo has not been investigated. This study monitored Treg phenotype and function during immune reconstitution in renal transplant recipients who underwent profound T cell depletion with Campath-1H and received sirolimus or cyclosporine (CsA) as part of their maintenance immunosuppressive therapy. CD4(+)CD25(high) cells that expressed FOXP3 underwent homeostatic peripheral expansion during immune reconstitution, more intense in patients who received sirolimus than in those who were given CsA. T cells that were isolated from peripheral blood long term after transplantation were hyporesponsive to alloantigens in both groups. In sirolimus- but not CsA-treated patients, hyporesponsiveness was reversed by Treg depletion. T cells from CsA-treated patients were anergic. Thus, lymphopenia and calcineurin-dependent signaling seem to be primary mediators of CD4(+)CD25(high) Treg expansion in renal transplant patients. These findings will be instrumental in developing "tolerance permissive" immunosuppressive regimens in the clinical setting.

Fas ligand exerts its pro‐inflammatory effects via neutrophil recruitment but not activation

Fas ligand (FasL) expression induces apoptosis of activated T cells and has been suggested as a strategy to inhibit graft rejection. Unfortunately, the use of FasL to confer 'immune privilege' in this setting has been hampered by the finding that it may also provoke a destructive granulocytic response. While the Fas/FasL-mediated apoptotic pathways are well defined, the pro-inflammatory effects of FasL are poorly understood. Our aim in this study was to define in vitro the biological effects of FasL on neutrophil recruitment and activation. DAP-3 cells expressing human FasL on the cell membrane (mFasL) potently induced apoptosis in human neutrophils and in activated T lymphocytes. Recombinant human soluble FasL (sFasL), by contrast, was a very weak inducer of apoptosis, even at high concentrations. This latter observation suggests that cleavage of mFasL by naturally occurring matrix metalloproteinases may serve to down-regulate FasL activity in vivo. However, in the presence of a cross-linking antibody, the efficiency of apoptosis-induction by sFasL was greatly increased, suggesting that the lesser pro-apoptotic potency of sFasL reflects an inability to induce trimerization of the Fas receptor. With regard to pro-inflammatory effects, we found that sFasL is a potent neutrophil chemoattractant and, given that it induces little apoptosis, the dominance of sFasL over mFasL may mean that graft-infiltrating neutrophils will survive to mediate inflammation. Neither sFasL nor mFasL produced neutrophil activation as assessed by chemiluminescence assay. This suggests that neutrophils recruited to an inflammatory site by FasL will be activated by mechanisms other than Fas-FasL signalling.

Keratinocyte Growth Factor Increases the CD4+Foxp3+ Regulatory T Cell-Pool by an Early Thymus-Independent and a Late Thymus-Dependent Mechanism.

Administration of keratinocyte growth factor (KGF) before and shortly after experimental allogeneic bone marrow transplantation (allo-BMT) has been shown to reduce the incidence and severity of graft versus host disease (GVHD) and to inhibit graft rejection. As naturally occurring CD4+Foxp3+ regulatory T cells (Treg) play an important role in the prevention of GVHD and graft rejection, we evaluated the effect of KGF on peripheral and thymic CD4+Foxp3+ regulatory T cell numbers in normal mice and BMT recipients. KGF (5mg/kg/day) was administered subcutaneously to adult B6 mice for 3 consecutive days. KGF enhanced the frequency of CD4+Foxp3+ Treg in peripheral blood and spleen twofold, to more than 15 % of CD4+ T cells, resulting in an increase in absolute numbers of CD4+Foxp3+ Treg within one week. From 2 weeks onwards, the frequency of CD4+Foxp3+ Treg gradually normalized, but the absolute numbers of Treg remained elevated (> 10 weeks) due to an increase of both CD4 and CD8 T cell numbers. In order to assess the relative contribution of thymic output versus peripherally expanded Treg, we next determined the frequency of T-cell receptor excision circles (TREC) in peripheral blood T cells. Analysis of sorted CD4+Foxp3+ Treg and CD4+Foxp3− conventional T cells showed a decline in TREC frequency in both subsets 1 week after KGF administration, suggesting peripheral expansion. In contrast, the TREC frequency in peripheral T cells was significantly higher at 5 and 10 weeks after KGF, indicating a late thymic dependent effect. Normal B6 mice that were adoptively transferred with congenic T cells showed an enhanced, but transient, increase of the frequency of CD4+Foxp3+ Treg within one week, also suggesting that the early effect of KGF on peripheral Treg is independent of thymic output. In addition, we assessed the effects of KGF on Foxp3+ thymocytes. Thymic weight and thymic cellularity of all subsets, including CD4+Foxp3+ thymocytes, were 2 to 3-fold increased one week after KGF administration as compared to control mice. In addition, we found that KGF transiently affected the thymic architecture. The medullary epithelial compartment was virtually absent one week after KGF administration. Normal thymic architecture gradually reappeared after 2–3 weeks. To determine whether KGF-enhanced Treg numbers in BMT recipients would inhibit graft rejection, KGF was administered from day -3 until day -1 to Rag-1−/− mice. Subsequently, the mice were 3Gy irradiated, supplied with 105 B6 CD45.1 congenic T cells and transplanted with a MHC-matched minor-Ag mismatched T cell depleted 129Sv bone marrow graft. As in normal B6 mice, KGF-treatment of BMT-recipients enhanced peripheral CD45.1+CD4+Foxp3+ Treg numbers. This was associated with a reduced rate of bone marrow graft rejection (2 out of 8 KGF-treated mice as compared to 5 out of 7 PBS-treated mice). Taken together, our data show that KGF enhances the peripheral Treg pool by an early, transient and selective thymus-independent mechanism and thereafter by a non-selective thymus-dependent mechanism. The KGF-mediated expansion of the pool of peripheral Treg may contribute to the inhibitory effects of KGF on graft rejection.

Two-signal blockade with anti-CD45RB and anti-CD154 monoclonal antibodies inhibits graft rejection via CD4-dependent mechanisms in allogeneic skin transplantation

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low) and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low) T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two- signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.

C4d Deposition and Clearance in Cardiac Transplants Correlates With Alloantibody Levels and Rejection in Rats

Antibody-mediated rejection of human cardiac transplants is correlated with C4d deposits and macrophage infiltrates in capillaries of endomyocardial biopsies. We produced an antibody to rat C4d to study C4d deposition and clearance in Lewis rats that were sensitized with a blood transfusion from DA rats 7, 14 or 21 days before cardiac transplantation. Cyclosporin A (CsA) immunosuppression was initiated after transplantation at a dose that inhibited graft rejection, antibody production and C4d deposition in unsensitized recipients. Blood transfusion elicited high levels of circulating IgG alloantibodies, predominantly of the complement-activating IgG2b subclass, that peaked 14 days after transplantation. At this time, macrophages accumulated in capillaries, and C4d deposits were diffuse and intense on arteries, capillaries and veins. Grafts that survived 90 days in sensitized recipients still had deposits of C4d that were associated with increased interstitial fibrosis and vasculopathy in arteries. Clearance of C4d was determined by retransplanting DA cardiac allografts from Lewis recipients back to DA recipients. C4d deposits were decreased to minimal levels within 5 days after retransplantation. Thus, C4d deposition is not limited to the capillaries, but extends throughout the arterial tree, and despite formation of a covalent bond, C4d is cleared within days.

Targeting CD45RB alters T cell migration and delays viral clearance

CD45 is a receptor tyrosine phosphatase essential for TCR signaling. One isoform, CD45RB, is down-regulated in memory cells and targeting CD45RB with a specific antibody has been shown to inhibit graft rejection. Its role in immunity to infection, however, has not been tested. Here, we report the effect of anti-CD45RB antibody treatment on the induction of anti-influenza CD8+ T cells and viral clearance. Anti-CD45RB-treated mice had delayed pulmonary viral clearance compared with untreated mice whose infection was completely cleared by day 8 post-infection. In anti-CD45RB-treated mice, the total CD4+ and CD8+ T cell numbers in both the lungs and mediastinal nodes were substantially reduced at days 5 and 8; this effect was less marked for the spleen. CD8+ T cells specific for influenza virus were also reduced compared with the control group in all three organs at day 8. By day 11, when both treated and control groups showed no virus remaining in the lungs, specific CD8+ T cell numbers were at similar low levels. Homing to lymph nodes and lung of dye-labeled T cells was greatly inhibited (by >80%) by anti-CD45RB treatment. This reduced homing corresponded with reduced CD62L and beta1-integrin expression in both uninfected and infected mice. Since CD62L plays a critical role in homing lymphocytes to lymph nodes, and high levels of CD62L and alpha4beta1-integrin are expressed by lymphocytes that home to bronchus-associated lymphoid tissue, we suggest that reduced expression of these molecules is a key explanation for the delay in immune responses.

Splenectomy and renal allograft survival in the rat.

The effect of splenectomy on renal allograft survival is not clear. In the rat, spleens isolated from recipients with functioning grafts have been shown to be a major source of cells that are capable of suppressing the rejection response (suppressor T lymphocytes). Thus the removal of the spleen in these allograft recipients could be detrimental to renal allograft survival. This study investigates this hypothesis, and looks for the presence of suppressor cells in other lymphoid organs apart from the spleen. In the rat renal allograft model, donor Lewis spleen cells given to DA recipients intravenously 1 week before transplantation of a Lewis kidney leads to indefinite allograft survival (median survival time (MST) greater than 100 days). Splenectomy before or after pretreatment with donor spleen cells failed to abrogate this effect (MST greater than 100 days). Experiments were performed in which cells or serum were prepared from long-term surviving splenectomized animals which had already been pretreated and transplanted, and then were injected into untreated recipients (adoptive transfer experiments). This was done to determine if cells capable of suppressing graft rejection were present in lymphoid organs outside the spleen in these splenectomized recipients. Thus the IV transfer of 10(8) lymph node cells harvested from splenectomized DA recipients with a long-term surviving LEW graft (LTS), into untreated but lightly irradiated (200 rad) DA recipients resulted in indefinite survival of a fresh Lewis kidney (MST greater than 100 days). In contrast, adoptive transfer of normal DA lymph node cells was ineffective (MST 13 days). Thus splenectomy is not necessarily detrimental to graft survival, as cells capable of preventing graft rejection are found in other lymphoid organs, such as lymph nodes, in splenectomized recipients.

Immunosuppressive treatment and progression of histologic lesions in kidney allografts

Renal transplantation is the best therapeutic option for patients with end-stage renal disease. Although short-term results are excellent, long-term graft survival has not improved substantially in recent times. Chronic allograft nephropathy (CAN) and death with a functioning graft are the most important causes of graft loss. Recent evidence shows that nephrotoxicity of calcineurin inhibitors contributes to CAN, and the introduction of non-nephrotoxic drugs such as mycophenolate mofetil (MMF) and mammalian target of rapamycin inhibitors may provide new immunosuppressive strategies to improve long-term results after renal transplantation. MMF decreases the risk of developing chronic allograft failure and is useful for treating established CAN, because it has a beneficial effect on allograft fibrosis. Treatment with sirolimus (SRL), a basic immunosuppressive drug given in association with MMF, may offer better renal function, decrease the prevalence of CAN, and downregulate expression of genes responsible for the progression of CAN than treatment with cyclosporine A (CsA). SRL also permits an early elimination of CsA from SRL-CsA-steroid regimens and shows better renal function and improved renal histology without risk of rejection. Notably, this approach improves graft survival at 4 years. Further multicenter studies are needed to determine whether both approaches produce similar results by comparing immunosuppression caused by SRL-based and tacrolimus (TAC)-based treatments. Because TAC is the most commonly used anticalcineurin drug, it is important to compare the effects of steroid-TAC-SRL treatment with and without elimination of TAC. Finally, although caution is needed, the use of non-nephrotoxic immunosuppressive treatment may change the natural history of CAN.

Thiol Redox and Immune Regulation in Corneal Transplantation

Penetrating keratoplasty (PKP) is the most common type of clinical grafting performed in humans. Although PKP has emerged as the most successful form of transplantation, PKP in "high-risk" eyes shows high incidence of allograft rejection. The incidence of epithelial rejection after limbal transplantation (LT) is extremely higher and swifter than PKP rejection, and even intensive systemic immunosuppressive therapy is often of no avail. Because failure of corneal grafts is an important cause of blindness, developing new strategies for suppressing graft rejection is a worthy goal for research. Corneal allograft rejection is mainly mediated by the TH1-type immune response, which leads to a delayed-type hypersensitivity reaction. Because the TH2-type immune response regulates the TH1-type immune response, we have successfully elicited allograft survival after both PKP and LT by inducing systemic TH2-type immune responses. Because intracellular thiol redox status of antigen-presenting cells (APC) reportedly regulates TH1/TH2 balance via distinctive cytokine production by APC, we also investigated the effect of modulating macrophage intracellular thiol redox status on corneal allograft survival. These strategies are quite effective in major histocompatibility complex (MHC) matching in mice, although it is believed that MHC matching has no effect on corneal allograft survival according to many rodent studies. Recently, many laboratories are reconsidering HLA matching for allograft survival in human corneal transplantation. It may be possible that MHC matching improves corneal allograft survival in the context of TH1 suppression. We propose that the suppression of the TH1-type immune response and MHC matching together may promote allograft survival in humans.

Promotion of Long-Term Heart Allograft Survival by Combination of Mobilized Donor Plasmacytoid Dendritic Cells and Anti-CD154 Monoclonal Antibody

Infusion of donor immature dendritic cells (DC) can significantly prolong survival of organ allografts, and this is believed to be due to antigen recognition by T cells in the absence of co-stimulation. In this study we report that a single pre-operative infusion of donor-mobilized immature plasmacytoid dendritic cells (pDCs) is superior to that of other DC sub-sets in suppressing allograft rejection. The combination of pDC infusion with injection of anti-CD154 monoclonal antibody further inhibited graft rejection and, in 50% of the mice, led to indefinite graft survival. This finding suggests a role for the plasmacytoid DC sub-set in facilitating organ transplant survival and also in the treatment of autoimmune disorders.