Rat Heart Allograft Survival Research Articles

Interleukin-5 (IL-5) Therapy Prevents Allograft Rejection by Promoting CD4+CD25+ Ts2 Regulatory Cells That Are Antigen-Specific and Express IL-5 Receptor

CD4+CD25+Foxp3+T cell population is heterogenous and contains three major sub-groups. First, thymus derived T regulatory cells (tTreg) that are naïve/resting. Second, activated/memory Treg that are produced by activation of tTreg by antigen and cytokines. Third, effector lineage CD4+CD25+T cells generated from CD4+CD25- T cells’ activation by antigen to transiently express CD25 and Foxp3. We have shown that freshly isolated CD4+CD25+T cells are activated by specific alloantigen and IL-4, not IL-2, to Ts2 cells that express the IL-5 receptor alpha. Ts2 cells are more potent than naïve/resting tTreg in suppressing specific alloimmunity. Here, we showed rIL-5 promoted further activation of Ts2 cells to Th2-like Treg, that expressed foxp3, irf4, gata3 and il5. In vivo, we studied the effects of rIL-5 treatment on Lewis heart allograft survival in F344 rats. Host CD4+CD25+T cells were assessed by FACS, in mixed lymphocyte culture and by RT-PCR to examine mRNA of Ts2 or Th2-like Treg markers. rIL-5 treatment given 7 days after transplantation reduced the severity of rejection and all grafts survived ≥60d whereas sham treated rats fully rejected by day 31 (p<0.01). Treatment with anti-CD25 or anti-IL-4 monoclonal antibody abolished the benefits of treatment with rIL-5 and accelerated rejection. After 10d treatment with rIL-5, hosts’ CD4+CD25+ cells expressed more Il5ra and responded to specific donor Lewis but not self. Enriched CD4+CD25+ cells from rIL-5 treated rats with allografts surviving >60 days proliferated to specific donor only when rIL-5 was present and did not proliferate to self or third party. These cells had more mRNA for molecules expressed by Th2-like Treg including Irf4, gata3 and Il5. These findings were consistent with IL-5 treatment preventing rejection by activation of Ts2 cells and Th2-like Treg.

Teach the T cells: How learning can shape immunity

Immune responses can be modulated via associative learning paradigms. We have established a model of behaviorally conditioned suppression of immune functions in rodents and healthy humans, employing the immunosuppressant cyclosporine A (CsA) as an unconditioned stimulus, by demonstrating a behaviorally conditioned suppression of T cell proliferation and cytokine production. This learned immune response is mediated centrally via the insular cortex and the amygdala as well as on the efferent arm via the splenic nerve, through noradrenaline and adrenoceptor-dependent mechanisms and is sufficient to prolong heart allograft survival in rats.

Induction of Immunological Tolerance With In Vitro Generated Suppressive Donor Blood Monocytes.

Our previous studies showed that incubation of dendritic cells (DCs) with the chemotherapeutic drug mitomycin C (MMC) renders them immunosuppressive. Donor-derived MMC-DCs injected into the recipient before transplantation prolonged heart allograft survival in rats. Whereas generation of DCs is labour-intensive, peripheral blood mononuclear cells (PBMCs) can be easily harvested. Here we analyse under which conditions DCs can be replaced by PBMCs and study their mode of action. When injected into rats, MMC-incubated donor PBMCs (MICs) strongly prolonged heart allograft survival. Removal of monocytes from PBMCs abrogated their suppressive effect, showing that monocytes are the active cell population. Suppression of rejection was donor-specific. Recipients treated with MICs showed an increased number of CD4+CD25+FoxP3+ Tregs in their peripheral blood and FoxP3-cell infiltration of heart allograft. Most important, tolerance could be transferred to syngeneic recipients with blood cells, whereas depletion of Tregs annihilated the effect. This argues for mediation of suppression by CD4+CD25+FoxP3+ Tregs. Cell culture studies showed that MMC-treated monocytes give rise to myeloid cells, which do neither resemble monocytes, nor DCs but show morphological and phenotypic similarities with monocytic myeloid-derived suppressor cells (MDSCs) occurring in some forms of cancer. Donor-derived MICs also prolonged kidney allograft survival in pigs. MICs were applied for the first time in a patient with therapy-resistant rejection of a haploidentical stem cell transplant, showing that they can be easily generated and used in humans. In conclusion, we describe here a simple method for in vitro generation of monocytic suppressor cells for potential use in clinical organ transplantation.

The Immunosuppressant Protosappanin A Promotes Dendritic Cell-Mediated Expansion of Alloantigen-Specific Tregs and Prolongs Allograft Survival in Rats

Protosappanin A (PrA), an immunosuppressive ingredient of the medicinal herb Caesalpinia sappan L, prolongs heart allograft survival in rats, possibly by impairing the function of antigen-presenting cells (APCs). We examined the effects of PrA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell–cell and intracellular signaling pathways mediating the immunosuppressive activity of PrA. PrA inhibited LPS-stimulated maturation of Wistar rat DCs in vitro as reflected by reduced expression of costimulatory molecules (CD80 and CD86) and reduced expression of TLR4 and NF-κB, two critical signaling components for antigen recognition. PrA also enhanced the release of IL-10 and decreased the release of IL-12 from DCs, but had no effect on the production of TGF-ß. In mixed cultures, Wistar DCs pretreated with PrA impaired the proliferation of Sprague Dawley (SD) rat T cells while promoting the expansion of SD rat CD4+CD25+ regulatory T cells (Tregs). Both oral PrA treatment and infusion of PrA-pretreated Wistar DCs prolonged cardiac allograft survival (Wistar donor, SD recipient) and expanded recipient CD4+CD25+Foxp3+ Tregs. Donor spleen cells, but not spleen cells from a third rat strain (DA), supported the expansion of recipient CD4+CD25+Foxp3+ Tregs and suppressed recipient T cell proliferation. We conclude that PrA triggers a tolerogenic state in DCs that allows for the induction of alloantigen-specific Tregs and the suppression of allograft rejection in vivo.

Effects of the novel protein kinase C inhibitor AEB071 (Sotrastaurin) on rat cardiac allograft survival using single agent treatment or combination therapy with cyclosporine, everolimus or FTY720

NVP-AEB071 (AEB, sotrastaurin), an oral inhibitor of protein kinase C (PKC), effectively blocks T-cell activation. The immunosuppressive effects of oral AEB were demonstrated in a rat local graft versus host (GvH) reaction and rat cardiac transplantation models. T-cell activation was suppressed by 95% in blood from AEB-treated rats, with a positive correlation between T-cell inhibition and AEB blood concentration. In GvH studies, AEB inhibited lymph node swelling dose-dependently (3-30 mg/kg). BN and DA cardiac allografts were acutely rejected within 6-10 days post-transplantation in untreated LEW rats. AEB at 10 and 30 mg/kg b.i.d. prolonged BN graft survival to a mean survival time of 15 and >28 days, and DA grafts to 6.5 and 17.5 days, respectively. In the DA to LEW model, combining a nonefficacious dose of AEB (10 mg/kg b.i.d.) with a nonefficacious dose of cyclosporine, everolimus or FTY720 led to prolonged median survival times (26 days, >68 days and >68 days, respectively). Pharmacokinetic monitoring excluded drug-drug interactions, suggesting synergy. In conclusion, these studies are the first to demonstrate that AEB prolongs rat heart allograft survival safely as monotherapy and in combination with nonefficacious doses of cyclosporine, everolimus or FTY720. Thus, AEB may have the potential to offer an alternative to calcineurin inhibitor-based therapies.

Nitric Oxide and Indoleamine 2,3-Dioxygenase Mediate CTLA4Ig-Induced Survival in Heart Allografts in Rats

Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) leads to transplantation tolerance in mice depending on indoleamine 2,3-dioxygenase (IDO). We have shown that CTLA4Ig induces indefinite heart allograft survival in rats and that nitric oxide (NO) was implicated in the in vitro active tolerogenic mechanisms mediated by dendritic cells (DCs). Here we studied the in vivo tolerogenic mechanisms by which CTLA4Ig induces graft survival in rats receiving a cardiac allograft. Treatment of recipients with the IDO inhibitor 1-methyltryptophan (1-MT) did not abrogate the indefinite graft survival observed with CTLA4Ig alone. This was also the case after administration of the inducible nitric oxide synthase inhibitor aminoguanidine when again, indefinite allograft survival was maintained. However, administration of both inhibitors led to acute rejection. We show that IDO and NO are responsible for the impaired capacity of DCs from CTLA4Ig-treated rats to stimulate allogeneic T cells. In conclusion, we show that NO and IDO mediate CTLA4Ig-induced tolerance in rat allograft recipients.

Effect of FTY720 and ICAM-1 mAb mono and combination therapy in cardiac allo-transplantation in rats

Objective To observe the effect of FTY720 and ICAM-1 mAb mono and combination therapy in cardiac allo-transplantation in rats. Methods Rats were randomly assigned to 9 groups, heart allo-transplantation were performed in abdominal site with micro-surgical technique. Recipients with allografts were treated with different doses of FTY720 and (or) ICAM-1 mAb. Graft survival, histopathology and level of serum IL-2, IFN-γ, IL-4, IL-10 were investigated. Results Low doses of FTY720 (1mg/kg) combined with ICAM-1 mAb achieved synergistic effect in the prolongation of cardiac graft survival, combination index(CI)=0.67. Conclusion Concomitant therapy of FTY720 and ICAM-1 mAb achieved a synergistic effect in the prolongation of heart allograft survival in rats.

A novel immunomodulator KRP-203 combined with cyclosporine prolonged graft survival and abrogated transplant vasculopathy in rat heart allografts

To find more effective and less toxic immunosuppressive strategies in long-term treatment for organ transplantation patients, we examined the effects on rat heart allograft survival of a novel sphigosine-1-phosphate receptor agonist, KRP-203, combined with a subtherapeutic dose of cyclosporine (CsA). Rat heart transplantation was performed across a major histocompatibility complex-incompatible (DA to LEW) rat combination. KRP-203 alone showed little or no effect on heart allograft survival. In contrast, KRP-203 combined with a subtherapeutic dose of CsA led to prolonged allograft survival. Histologic analyses showed that the combination completely suppressed acute rejection, as characterized by allograft vasculopathy, mononuclear cell infiltration, and myocardial necrosis in the heart allografts. RT-PCR analysis showed that the allografts treated with CsA or KRP-203 alone showed no suppression of IL-10, IFN-γ, and TNF-α mRNA expression, but when combined with a subtherapeutic dose of CsA it completely suppressed their mRNA expressions. Furthermore, the combination treatment reduced donor-specific antibody production. KRP-203 combined with a subtherapeutic dose of CsA synergistically prolonged rat heart allograft survival. The combination of CsA with KRP-203 may provide an option to prevent allograft rejection and reduce adverse effects.

Combined donor leucocyte administration and immunosuppressive drug treatment for survival of rat heart allografts

Background: Donor leucocytes (DL) play an important role in rat liver transplant tolerance and their postoperative administration can convert rejection to tolerance. They appear to induce early activation, altered patterns of infiltration and death of recipient alloreactive T cells. The ability of immunosuppressive drugs to combine with DL administration was examined in a rat heart transplant model. Methods: Immediately after PVG to DA heterotopic heart transplantation, 6×10 7 spleen DL were injected. Cyclosporine A (CsA), 1.5 mg/kg/day, or methotrexate (MTX), 0.1 or 0.2 mg/kg/day, were given from day (d) 0 to d4 (early) or from d3 to d7 (delayed). Castanospermine (CAST) was administered from d0 to d7 at 100 or 300 mg/kg/day. In a separate experiment, transplanted hearts and recipient spleens were collected from treatment groups for analysis of infiltrate and cytokine mRNA expression. Results: Delayed treatment with CsA or early treatment with MTX but not CAST combined with DL to result in prolonged graft survival. Recipients treated with DL and delayed CsA had a reduced level of intra-graft interleukin (IL)-2, interferon (IFN)-γ, IL-4, and IL-4R mRNA expression and reduced infiltrate compared to DL alone. Early MTX plus DL led to almost complete inhibition of all markers of inflammation during treatment followed by a rapid increase after cessation. In combination with DL, CsA was more effective than MTX for induction of donor-specific tolerance at the dose and administration regimens tested. Conclusions: Delayed CsA or early MTX combine with DL to prolong heart allograft survival. Early and extensive inhibition of rejection by MTX was less effective than delayed and partial inhibition of the response by CsA for induction of transplant tolerance.

MITOMYCIN C-TREATED DENDRITIC CELLS SUPPRESS ALLOREACTIVE T CELLS AND INDUCE SPECIFIC UNRESPONSIVENESS TO HEART ALLOGRAFT IN RATS: MEDIATION OF SUPPRESSION BY DOWNREGULATION OF ICAM-1, CD80 AND CD86

P912 Aims: Although dendritic cells (=DCs) strongly stimulate the immune response, it has been shown that, under certain circumstances, these cells can also induce strong immunosuppression. Generation of tolerogenic donor DCs provides an efficient tool for suppression of graft rejection. Mitomycin C (=MMC) is an alkylating agent which partially inhibits protein synthesis and influences the immunogenicity of transplanted cells. Here we use MMC for generation of tolerogenic rat DCs and analyze their mechanism of action on T cells. Methods: Monocyte–derived DCs were generated in the presence of GM–CSF, IL–4, maturated with TNF–α/PGE2, incubated with MMC (20μg/ml) and extensively washed. DC phenotype as well as cell death were analyzed by flow cytometry. The stimulatory capacity of MMC–DCs was tested in an allogeneic T–cell proliferation (BNRT1n [wng]224[/wng] LEWRT1l) assay in-vitro. In a lymph node assay, LEW rats were injected into the footpad with 106 BN- derived DCs and one week later T-cell proliferation was assessed by weighing the popliteal lymph nodes. In an other experiment, 106 MMC–treated BN-DCs were injected intraportaly into LEW rats. One week later the recipients received a heterothopic BN or DART1avl (third party) heart transplant and graft survival was monitored by daily palpation and EKG. Differences with P < 10-2 (rank sum test) were considered statistically significant. Results: MMC-treated allogeneic DCs lose their T-cell stimulatory capacity “in vitro” and this effect can neither be attributed to MMC–induced death of DCs, nor to the T-cell inhibitory action of possible MMC traces in cell culture supernatants. Most importantly, suppressed T cells cannot be restimulated with fresh donor DCs, whereas third party restimulation is possible. FACS analysis showed that MMC-treatment selectively downregulates adhesion (ICAM-1) and costimulatory (CD80, CD86) molecules on the DC cell surface whereas other cell membrane proteins (MHC–II, OX62, ED-2) remain unaffected. Functional blocking of these molecules with specific monoclonal antibodies (= mAbs) confers to DCs the same T-cell suppressive properties in-vitro as treatment with MMC. In-vivo, rats injected with allogeneic DCs pre-treated with MMC or mAbs to ICAM-1, CD80, CD86 showed a suppressed local lymph node reaction. When prospective graft recipients received MMC–treated donor DCs they presented a significantly prolonged heart allograft survival (MST= 30 days) as compared to animals receiving placebo (MST = 9 days) or untreated donor DCs (MST = 6 days) (both P≤10-5). Most notably, a similar prolongation of allograft survival could be induced using DCs pre–incubated with mAbs specific for ICAM–1, CD80, and CD86 (MST = 52 days, P=10-4). Along with our findings in the lymph node assay and the in-vitro data, this result indicates that downregulation of ICAM–1, CD80, and CD86 was responsible for MMC-mediated suppression of allograft rejection. Prolongation of allograft survival by MMC-DCs was donor–specific, since no significant effect on third-party heart allograft survival was noted (untreated: MST = 8 days; MMC: 11 days, P = N.S). Conclusions: Our results show that MMC treatment converts DCs into T-cell suppressive cells able to induce donor–specific prolongation of rat heart allograft survival. This mechanism of suppression is mediated by downregulation of ICAM–1, CD80 and CD86 on DCs. If a similar effect could be defined in man, MMC-DCs might prove to be an efficient tool for immunosuppressive therapy in clinical transplantation.

Early experience with cyclosporine: a historic perspective

Cyclosporine, a new antilymphocyte peptide, was tested in heterotopic heart transplantation between two different strains of rats. Prolongation of rat heart allograft survival was observed in treated hosts as compared with untreated controls ( P < .001). These pioneering studies in pedicle animal grafts proved that cyclosporine is an extremely potent immunosuppressive drug. Its ability to suppress allograft rejection episodes could be applied to other species of animals and to humans.

Gene expression profiles of rat heart allografts with immunosuppression

TRANSPLANTATION biology has entered the era of the genome. Genome analysis will provide new insights in development, differentiation, and homeostasis of host immune or nonimmune responses resulting in destruction of allograft tissues. Genome analysis also has the potential to reveal new therapeutic pathways in the treatment of organ transplantation rejection with minimal side effects. Our previous studies of remarkable cellular surface markers and cytokine gene expression in the rat heart transplantation model strongly indicated that day 5 after transplantation is an optimal time for evaluation of gene expression involved in acute allograft rejection. In addition, the studies indicated that the predetermined equivalent effective doses (optimal doses) of tacrolimus and cyclosporin (CyA), which can induce similar survival of rat heart allografts with strong histocompatibility differences, were suitable for the further comparative study of these two drugs at the genome level. In the current study, we identify candidate genes involved in host immune or nonimmune responses against allografts. Our strategy is to identify genes that are affected during graft rejection and that respond to treatment with the immunosuppressive agents tacrolimus and cyclosporin, by measuring expression levels of a large number of genes in rat heart allograft samples using microarray-based technology with cDNA clones as gene-specific hybridization targets. These gene expression profiles also allow the recognition of similarities and differences between tacrolimusand CyA-mediated immunosuppression in organ transplantation.

Rescuing acute rejection of rat cardiac allografts with FTY720

FTY720 is a novel immunosuppressant isolated and chemically modified from the culture filtrate of the ascomycete Isaria sinclarii, and it has been shown to effectively prolong the survival of rat skin, heart and liver allografts, and the survival of renal allografts in canine and subhuman primates. Moreover, FTY720 induces tolerance in rat cardiac allografts when combined with allochimeric class I MHC antigen, and potentiates the immunosuppressive effects of cyclosporine (CsA) and/or sirolimus. This study aimed to evaluate the effect of FTY720 in rescuing ongoing acute rejection of solid organ transplants in a rat heterotopic cardiac transplantation model.

Anti-TCR Vbeta-specific DNA vaccination prolongs heart allograft survival in adult rats.

Allospecific T cells are known to play a central role in the process of allograft rejection. Recently, it has been shown that T cell function could be specifically targeted using DNA vaccination. In our model, PCR analysis of the TCR-beta chain repertoire of T cells infiltrating rejected allografts showed specific expansions of the Vbeta13 and Vbeta2 families. In this study, we tested the effect on allograft survival of DNA vaccination against a specific TCR Vbeta, in a model of heart allograft rejection in adult rats. Our results showed that anti-TCR Vbeta13 DNA vaccination lead to a significant prolongation of allograft survival compared to vaccination against other Vbeta families or untreated recipients. The prolongation of allograft survival correlated in vitro with a decrease in anti-donor reactivity of spleen cells from Vbeta13-vaccinated rats. These results show that, in a transplantation model, DNA vaccination could be used as a method to specifically manipulate a T cell response and thus prolong allograft survival.

15AU81, a prostacyclin analog, enhances donor-specific hepatocytes to prolong the survival of rat heart but not small bowel allografts.

Prostacyclin analogs have previously been shown to have not only cytoprotective but also independent immunosuppressive effects. The effect of one such analog, 15AU81, to enhance the immunosuppressive effects of liver was investigated. We have previously demonstrated that cyclosporine (CsA) in conjunction with rapamycin (RAPA) potentiates class I+, class II- donor-specific hepatocytes to prolong rat cardiac and small bowel allograft survival. Brown Norway (BN; RT1n) hepatocytes alone (5 x 10(7)/kg, administered intrasplenically) failed to prolong the survival of BN heart allografts in Wistar Furth (WFu; RT1u) recipients, beyond that of untreated controls (MST = 7.2 +/- 0.8 days). Survival of BN hearts was increased to 11.4 +/- 1.7 days in WFu recipients treated with BN hepatocytes and 50 microg/kg/day 15AU81 administered by continuous s.c. infusion for 14 days using osmotic pumps (p < 0.05). The further addition of RAPA 0.0075 mg/kg/day and CsA 0.375 mg/kg/day delivered for 14 days by continuous i.v. infusion (CIVI) using osmotic pumps (a combination that alone prolonged BN heart allografts in WFu hosts to 18.4 +/- 1.3 days and in conjunction with BN hepatocytes prolonged survival to 27.2 +/- 1.9 days) prolonged allograft survival to 35.2 +/- 5.2 days. In contrast, the survival of small bowel allografts was not enhanced by 15AU81 administration. Survival of BN small bowel transplants in LEW recipients treated with hepatocytes alone (MST = 11.6 +/- 1.5 days) or hepatocytes plus 15AU81 (MST = 10.0 +/- 1.0 days) was similar to controls (MST = 10.2 +/- 1.9 days). Treatment with hepatocytes and RAPA/CsA increased survival to 21.2 +/- 1.5 days. The further addition of 15AU81 failed to augment this (MST = 17.0 +/- 1.9 days). In vitro WFu lymphocyte proliferative responses from animals pretreated with BN hepatocytes, 15AU81, or both treatments, for 2 weeks prior to harvesting, exhibited a reduction of at least 50%, compared to untreated controls upon allostimulation with irradiated BN or ACI spleen cells. These findings demonstrate that 15AU81 interacts favorably with hepatocytes either alone or in conjunction with RAPA and CsA to enhance their immunosuppressive effects on rat heart allograft survival. The failure to enhance small bowel allograft survival may be explained by the inability at this low dosage of 15AU81 to influence the intense graft versus host reaction elicited by small bowel transplants.

I-2190A is a potent immunosuppressive drug for vascularized heart transplantation in rats.

The effect of a new immunosuppressant-I-2190A was tested in a rodent heart allograft model. Grafts were transplanted to recipients heterotopically. There were 5 groups: group 1 received no immunosuppressive agents; group 2 was given CsA (2.0 mg/kg, i.p.); group 3 was administered I-2190A (0.1 mg/kg, i.p.) in carboxymethyl cellulose (CMC); group 4 received injection of I-2190A (0.5 mg/kg, i.p. in CMC); group 5 received the combination treatment of I-2190A (0.1 mg/kg) and CsA (2.0 mg/kg). Immunosuppressants were discontinued 14 days after operation. No statistically significant difference in grafts median survival time (MST) was found between group 2 (9.5 days) and group 1 (9 days). The MSTs of grafts in group 3 (22 days, P < 0.05), group 4 (> 100 days, P < 0.01) and group 5 (> 100 days, P < 0.01) were significantly prolonged compared with control group 1 (9 days). Our results suggest that I-2190A is a potent immunosuppressant able to significantly prolong heart allograft survival in rats after a short time treatment, Low-dose I-2190A could potentiate the effect of sub-therapeutic dose of CsA as well.

Modulation of the CD2 Receptor and Not Disruption of the CD2/CD48 Interaction Is the Principal Action of CD2-Mediated Immunosuppression in the Rat

CD48, the murine homolog of human CD58, binds to CD2 in rats and mice. Whereas inhibition of CD2 signaling leads to profound immunosuppression, no information is available on CD48-targeted therapy in the rat. We could show that anti-CD2 treatment (OX34) efficiently inhibited TCR-driven as well as CD2-mediated proliferation, whereas blocking of ligand binding (OX45) remained completely uneffective. Inhibition of allogeneic MLR by OX45 turned out to be due to induction of unspecific suppressive mechanisms.In vivo,OX45 failed to prolong rat heart allograft survival in contrast to that seen with OX34. Grafts were rejected despite persistent and complete downmodulation of CD48 on lymphocytes without any cell depleting effect, rendering receptor/ligand interactions physically impossible. Combined application of CD2 and CD48 mAb did not enhance immunosuppression induced by CD2 mAb alone. Provided that there is no alternative CD2 ligand in the rat, we conclude that CD2-directed immunotherapy is mediated by suppressive events induced by modulation of the CD2 receptor (“negative signaling”) rather than by mere disruption of the CD2–CD48 interaction.

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats

The inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation. The net effect of such inhibition is reduction in the membrane expression of certain glycoproteins. Hence cellcell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of N-linked glycoproteins including adhesion molecules is inhibited. The aims of this study were to measure the immunosuppressive ability of castanospermine (CAST) in a rat heart allograft model, to measure its effect on membrane expression of adhesion molecules (LFA-1α, LFA-1β, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W 3 13 ), to test its tolerogenic potential and its toxicity. Membrane expression of these molecules was measured by flow cytometry for single cells and by immunoperoxidase staining for the allograft. In grafted rats CAST significantly reduced the expression of LFA-1α on lymphoid cells in the thymus, lymph node, spleen and heart allografts, ICAM-1 expression on endothelial cells of the allograft vasculature, class I and class II MHC expression on lymphoid cells in the thymus, class II MHC expression on lymphoid cells in the allograft; and CD4, CD8, CD45 and W 3 13 expression on lymphoid cells in some organs. By contrast, in non-grafted rats CAST significantly upregulated expression of class I MHC and CD45 in the thymus, lymph node and spleen, ICAM-1 and CD4 on lymphoid cells in the spleen, but reduced expression of LFA-1α on lymphoid cells in the thymus. It also prolonged rat heart allograft survival in a dose-dependent manner and with limited testing was relatively non-toxic. In conclusion, CAST is an immunosuppressive molecules which may work by downregulation of the ligand-receptor adhesion molecule pair, LFA-1α-ICAM-1 although subtle downregulation of class I and II MHC, CD4 and CD8 molecules could also contribute to its immunosuppressive activity. Hence, both lymphocyte-endothelial cell binding and lymphocyte activation may be inhibited by CAST. This work suggests that CAST may hold significant potential as a transplant immunosuppressant probably as an adjuvant agent to inhibitors of interleukin 2 secretion.

Prolonged allograft survival by the inhibition of costimulatory CD2 signals but not by modulation of CD48 (CD2 ligand) in the rat.

The CD2 receptor is an important costimulatory molecule in T cell activation. Its ligand CD48 in rodents is supposed to be a homologue of human CD58, because of its similarities in structure and distribution. We evaluated the immunosuppressive activity of CD2/CD48-directed therapy in vitro and in vivo for the efficacy in prolonging rat heart allograft survival in a high responder transplant model. CD2-directed monoclonal antibody (mAb) therapy significantly prolonged median survival time to 45 days (P < 0.001). Suppression was mediated by down-modulation of CD2 below 20% on lymph node cells without considerable cell depletion. In contrast, CD48 mAb could not prolong graft survival. Rejection occurred in the presence of complete CD48 modulation and, therefore, despite disruption of the CD2-CD48 interaction. CD48 mAb failed to inhibit lymphocyte activation via a mitogenic pair of CD2 mAbs and inhibited mixed lymphocyte reaction (MLR) only by an unspecific mechanism. In conclusion, our results suggest a negative regulatory signal transduction by inhibitory CD2 mAbs and argue against a pivotal role of mere disruption of the CD2-CD48 interaction in CD2-mediated immunosuppression.

Synergistic interaction of 3 M KCl-extracted donor antigens (e-HAg) with cyclosporine or cyclosporine/sirolimus for prolongation of rat heart allograft survival

Extracted donor histocompatibility antigens (e-HAg) may potentiate the effects of drugs to protect organ allografts from rejection. We examined the capacity of e-HAg when combined with cyclosporine (CsA) alone, sirolimus (rapamycin, RAPA) alone, or CsA/RAPA combinations to prolong heart allograft survival in rats. Wistar-Furth (WF: RT1 u) rats that received CsA (10 mg/kg/day) by oral gavage for 3 (days 0, 1 and 2) or 7 (days 0, 1, 2, 3, 4, 5 and 6) consecutive days displayed modest prolongation of Brown Norway (BN; RT1 n) heart allograft survival from a mean survival time of 7.2 ± 0.8 days in untreated controls to 12.2 ± 1.1 days and 18.6 ± 2.7 days, respectively ( p < 0.01). Although administration on the day of transplantation (day 0) of a single intravenous (i.v.) dose of BN e-HAg (5 mg/kg) failed to affect allograft survival, both three (days 0, 1 and 2) and five (days 0, 1, 2, 3 and 4) injections significantly potentiated the effect of a 3-day course of oral CsA (18.6 ± 1.3 days ( p < 0.01) and 20.0 ± 1.4 days ( p < 0.01), respectively) and of a 7-day course of oral CsA (25.3 ± 4.4 days ( p < 0.05) and 33.5 ± 9.3 days ( p < 0.01), respectively). Median-effect analysis confirmed a synergistic interaction between CsA (0.5 mg/kg × 7 days, i.v.) and e-HAg with combination index (CI) values less than 0.7 (CI = 1 shows additive interactions, CI < 1 synergistic, and CI > 1 antagonistic, interactions). In contrast, e-HAg failed to affect the immunosuppressive effect of RAPA. However, e-HAg (5.0 mg/kg × 3 days) significantly potentiated the effects of a 7-day or 14-day course of RAPA (0.01 mg/kg)/CsA (0.5 mg/kg) combination therapy, namely from 26.0 ± 4.8 days with a 7-day treatment of CsA/RAPA alone to 32.6 ± 3.6 days ( p < 0.01) and from 28.2 ± 2.7 days with a 14-day course of CsA/RAPA alone to 42.0 ± 4.9 days ( p < 0.05), respectively (CI = 0.2–0.5). Thus, e-HAg potentiates the immunosuppressive effects of CsA alone and of the CsA/RAPA combination, but not of sirolimus alone.