Indefinite Graft Survival Research Articles

Rapamycin-Conditioned Dendritic Cells Are Poor Stimulators of Allogeneic CD4+ T Cells, but Enrich for Antigen-Specific Foxp3+ T Regulatory Cells and Promote Organ Transplant Tolerance

The ability of dendritic cells (DC) to regulate Ag-specific immune responses via their influence on T regulatory cells (Treg) may be key to their potential as therapeutic tools or targets for the promotion/restoration of tolerance. In this report, we describe the ability of maturation-resistant, rapamycin (RAPA)-conditioned DC, which are markedly impaired in Foxp3(-) T cell allostimulatory capacity, to favor the stimulation of murine alloantigen-specific CD4(+)CD25(+)Foxp3(+) Treg. This was distinct from control DC, especially following CD40 ligation, which potently expanded non-Treg. RAPA-DC-stimulated Treg were superior alloantigen-specific suppressors of T effector responses compared with those stimulated by control DC. Supporting the ability of RAPA to target effector T and B cells, but permit the proliferation and suppressive function of Treg, an infusion of recipient-derived alloantigen-pulsed RAPA-DC followed by a short postoperative course of low-dose RAPA promoted indefinite (>100 day) heart graft survival. This was associated with graft infiltration by CD4(+)Foxp3(+) Treg and the absence of transplant vasculopathy. The adoptive transfer of CD4(+) T cells from animals with long-surviving grafts conferred resistance to rejection. These novel findings demonstrate that, whereas maturation resistance does not impair the capacity of RAPA-DC to modulate Treg, it profoundly impairs their ability to expand T effector cells. A demonstration of this mechanism endorses their potential as tolerance-promoting cellular vaccines.

Induction of Transplantation Tolerance via Regulatory T Cells

In the last two decades, graft survival has been greatly improved by the introduction of efficient immunosuppressive drugs. On the other hand, late graft loss caused by chronic rejection together with the side effects of long-term immunosuppression, remain major obstacles for successful transplantation. Operational tolerance, which is defined by the lack of acute and chronic rejection and indefinite graft survival with normal graft function in the absence of chronic immunosuppression, represents an attractive alternative. Several approaches have been explored to achieve transplantational tolerance, which is considered the "Holy Grail" of transplantation, including induction of central tolerance by establishing mixed chimerism through hematopoietic stem cell transplantation or induction of peripheral tolerance through modulation of allogeneic immune responses. Graft-specific alloreactive T cells, which largely mediate graft rejection, can be silenced through different mechanisms, including deletion, which may occur within the thymus or in the lymphoid organs; anergy, in which alloreactive T cells cannot adequately respond following restimulation with the specific antigen; and suppression, which may be mediated by direct interactions with regulatory T cells (Tregs) or by soluble factors produced by Tregs. This review attempts to summarize the most novel and successful strategies to achieve operational tolerance via induction of Tregs.

Trafficking of Donor-Derived Bone Marrow Correlates With Chimerism and Extension of Composite Allograft Survival Across MHC Barrier

We proposed to evaluate differences between recipient’s immune response to vascularized skin and combined vascularized skin/bone allografts, under a 7-day αβ-TCR plus cyclosporine (CsA) treatment protocol. Thirty-six transplantations were performed in six groups: group I (isograft control-vascularized skin graft; n = 6); group II (isograft control-combined vascularized skin/bone graft; n = 6); group III (allograft rejection control group-vascularized skin graft; n = 6); group IV (allograft rejection control-combined vascularized skin/bone graft; n = 6); group V (allograft treatment-vascularized skin graft; n = 6); and group VI (allograft treatment-combined vascularized skin/bone graft; n = 6). Isograft transplantations were performed between Lewis rats and allografts were transplanted across the MHC barrier from Brown Norway to Lewis rats. In the allograft treatment group, a combined αβ-TCR+CsA protocol was applied for 7 days. All groups were compared clinically, immunologically and histologically. Statistical significance was determined with two-tailed Student’s t test. Indefinite graft survival was achieved in the isograft control group (>300 days). Allograft rejection controls rejected within 5 to 9 days posttransplant; chimerism levels were undetectable (<.5%). Allografts under the αβ-TCR+CsA protocol had significantly extended survival when skin was combined with bone (61–125 days) compared to vascularized skin allografts (43–61 days). Lymphoid macrochimerism was significantly higher in group VI than group V. Histology confirmed skin and bone viability. Combined vascularized skin/bone allografts had higher and sustained levels of donor-specific chimerism and extended allograft survival.

Use of the Inhibitory Effect of Apoptotic Cells on Dendritic Cells for Graft Survival Via T-Cell Deletion and Regulatory T Cells

Tolerance induction against donor allo-antigens (allo-Ag) remains one of the most challenging aspects of transplant immunology. The ability of dendritic cells (DC) to participate in immunity and tolerance makes them an excellent tool for tolerance induction. Here, we employed the immunosuppressive properties of apoptotic cells to deliver simultaneously an inhibitory signal and donor allo-Ag to recipient DC for treatment of allograft rejection. DC that captured apoptotic cells remained immature and activated deficiently anti-donor CD4(+) T cells that were unable to upregulate T-cell activation markers, to secrete IL-2 and IFN-gamma and to survive under homeostatic conditions due to low expression of Bcl-X(L), IL-7R and IL-15R. Administration of donor apoptotic cells decreased the systemic anti-donor T- and B-cell response and prolonged cardiac allograft survival in mice. The effect was donor specific and required the interaction of donor apoptotic cells with recipient quiescent CD8alpha(+) DC. When combined with CD40-CD154-blockade, administration of donor apoptotic cells resulted in indefinite graft survival mediated by generation of regulatory T cells. The use of the inhibitory effects of apoptotic cells on the anti-donor response provides a new approach to treat transplant rejection.

Antibody-Induced Transplantation Tolerance That Is Dependent on Thymus-Derived Regulatory T Cells

Targeting of the CD45RB isoform by mAb (anti-CD45RB) effectively induces donor-specific tolerance to allografts. The immunological mechanisms underlying the tolerant state remain unclear although some studies have suggested the involvement of regulatory T cells (T-regs). Although their generative pathway remains undefined, tolerance promoting T-regs induced by systemic anti-CD45RB treatment have been assumed to originate in the peripheral immune system. We demonstrate herein that separable effects on the peripheral and central immune compartments mediate graft survival induced by anti-CD45RB administration. In the absence of the thymus, anti-CD45RB therapy is not tolerogenic though it retains peripheral immunosuppressive activity. The thymus is required for anti-CD45RB to produce indefinite graft survival and donor-specific tolerance, and this effect is accomplished through thymic production of donor-specific T-regs. These data reveal for the first time an Ab-based tolerance regimen that relies on the central tolerance pathway.

Inhibition of Chronic Rejection and Development of Tolerogenic T Cells after ICOS-ICOSL and CD40-CD40L Co-stimulation Blockade

: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation. : The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats. : Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms. : These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.

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.

Prevention of Acute Vascular Rejection by a Functionally Blocking Anti-C5 Monoclonal Antibody Combined with Cyclosporine

Inhibition of the complement cascade at C5 prevents formation of pro-inflammatory molecules C5a and C5b-9, which play a key role in allograft rejection. The present study was undertaken to determine whether blocking terminal complement with anti-C5 monoclonal antibody (mAb) alone or combined with cyclosporine (CsA) would prevent acute vascular rejection (AVR) in a mouse cardiac allograft model. C3H mouse hearts were transplanted into BALB/c mice and randomized into five groups with the following treatments: (1) no treatment; (2) CsA alone; (3) control mAb; (4) anti-C5 mAb alone; and (5) anti-C5 mAb and CsA. Allografts in untreated or control mAb-treated recipients were rapidly rejected at 8.0+/-0.6 and 8.2+/-0.8 days, respectively. These grafts exhibited typical AVR, characterized by vasculitis, hemorrhage, and thrombosis. A high level of complement activity was also demonstrated in these animals. High-dose CsA was not able to inhibit complement activation or AVR, and grafts were rejected in 15.5+/-1.1 days. Anti-C5 monotherapy completely inhibited complement activation and attenuated AVR, but grafts were rejected in 8.3+/-0.5 days by acute cellular rejection. In contrast, a combination of anti-C5 mAb and CsA successfully achieved indefinite graft survival (>100 days). This combined therapy completely inhibited terminal complement activation and prevented both humoral- and cellular-mediated rejection. Combination therapy of anti-C5 mAb and CsA achieves indefinite graft survival in a mouse cardiac allograft model. These data suggest that inhibition of terminal complement using anti-C5 mAb may be an effective therapeutic adjunct to prevent AVR in clinical transplantation.

Early Accumulation of Interferon-?? in Grafts Tolerized by Donor-Specific Blood Transfusion: Friend or Enemy?

We previously documented an early (day-2) interferon (IFN)-gamma accumulation in cardiac allografts of rats made tolerant by donor-specific blood transfusion (DSBT) but not in rejecting controls. This contrasted with the IFN-gamma peak seen later (day 5) in rejecting but not in tolerant rats. To further examine the role of early intragraft IFN-gamma in DSBT-induced tolerance, we studied whether IFN-gamma up-regulation correlates with the magnitude of the DSBT effect and how IFN-gamma is influenced by interventions abrogating tolerance. The protective effect of DSBT depended upon the timing of administration: day-12 DSBT induced indefinite graft survival; day-6 DSBT gave a moderate, and day-0 DSBT, no graft prolongation. IFN-gamma up-regulation correlated with the DSBT effect: it was maximal after day-12 DSBT, intermediate after day-6 DSBT, and absent after day-0 DSBT. Tolerant splenocytes transferred tolerance into naive rats in a donor-specific manner, indicating that alloantigen-specific regulatory cells operate. Thymectomy prevented regulatory cells development, caused further amplification of intragraft IFN-gamma, and led to rejection, although graft survival was still prolonged. Day 2 intragraft IFN-gamma correlates with the DSBT protective effect. Thymectomy abrogates DSBT-induced tolerance, prevents regulatory cell development, and paradoxically causes further accumulation of intragraft IFN-gamma. These data indicate that DSBT has a stimulatory and a (thymus-dependent) inhibitory effect on early intragraft IFN-gamma. Intragraft IFN-gamma is beneficial, providing it occurs early and remains moderate. The role of intragraft IFN-gamma in tolerance and rejection depends upon the timing and the degree of production and perhaps the type of IFN-gamma producing cells (regulatory or effector).

SYNERGISTIC EFFECT OF ICOS/B7RP-1 AND CD40/CD40L COSTIMULATION BLOCKADE IN THE INDUCTION OF ALLOGENEIC TOLERANCE

O461 Aims: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. Therefore new immunotherapies are needed to obtain tolerance. ICOS and its receptor, B7RP-1, are members of the CD28-B7 families which play an important role in T cell activation and survival. Methods: We used an adenovirus coding for CD40Ig (which blocks CD40-CD40L interactions) in a rat heart allograft transplantation model. We analyzed the effect of co-treatment with an anti-ICOS MAb and CD40Ig on chronic rejection. Results: CD40Ig and anti-ICOS treatment led to indefinite graft survival in all recipients (>120 days). Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival (17±1.5 vs 7.6±1.6, p=0.002, n=4). Analysis of chronic rejection lesions at day 120 in the CD40Ig+anti-ICOS group (n=7) vs. the CD40Ig group (n=17) showed that the percentage of vessels occluded (15.2±6.6 vs. 33.7±4.4, p=0.017), degree of vessel occlusion (1.1±0.5 vs. 2.4±0.2, p=0.007) and vessel lesions (1±0.3 vs. 2.3±0.1, p<0.001) but not fibrosis were significantly lower. Importantly, 4 of the 7 CD40Ig+anti-ICOS-treated recipients showed complete absence of chronic rejection lesions whereas all CD40Ig-treated recipients showed signs of chronic rejection. The CD40Ig+anti-ICOS group showed decreased graft infiltration by TCRαβ+, CD4+ and CD8α+ cells as well as macrophages and mast cells. Recipients in the CD40Ig+anti-ICOS group displayed significantly inhibited anti-donor CTL activity. Alloantigenic proliferative responses of splenocytes in the CD40Ig+anti-ICOS group were strongly inhibited and were reversed by IL-2. CD40Ig treatment strongly but incompletely inhibited total IgG, Th2 and Th1-dependant alloantibody production. The residual total IgG and Th1 but not Th2 alloantibody were significantly reduced in the sera of recipients of CD40Ig+anti-ICOS treated allografts at day 120, suggesting an inhibition of Th1 responses. Conclusions: These data indicate that the chronic rejection mechanisms that are CD40-CD40L-independent are ICOS/B7RP-1-dependent. Operational tolerance can be obtained by simultaneous blockade of both costimulatory pathways.

SEROTONIN RECEPTOR ANTAGONIST INDUCES INDEFINITE SURVIVAL OF FULLY-ALLOGENEIC CARDIAC GRAFTS.

P1118 Aims: Sarpogrelate hydrochloride, a serotonin receptor antagonist, is used clinically as an antithrombotic agent for peripheral arterial disease. It has also been reported that sarpogrelate hydrochloride possesses a potential to inhibit O2- production by neutrophils in vitro models. We investigated whether sarpogrelate hydrochloride could induce unresponsiveness to fully-mismatched cardiac graft in mice. Methods: CBA (H2k) mice underwent heterotopic cardiac transplantation on day 0 from C57BL/10 (H2b) donors using microsurgical technique. The CBA recipients were left untreated or treated with 10mg/kg of sarpogrelate hydrochloride from day 0 through day 7. To examine the mechanisms, adoptve transfer study was performed. Hundred days after heterotopic cardiac transplantation, 50x106 splenocytes of the treated recipients with functioning graft were adoptively transferred into naive CBA mice (secondary recipients), and then C57BL/10 hearts were transplanted into the secondary recipients on the same day. Histological examination of the allografts and mixed leukocyte culture analysis were also performed. Results: Naive CBA mouse rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 8 days). The treatment with sarpogrelate hydrochloride induced indefinite graft survival (MST, >100 days). Histological examinations of grafts at 30 days after transplantation did not show any signs of acute rejection in the recipients treated with sarpogrelate hydrochloride. In the adoptive transfer study, majority of the grafts in the secondary recipients enjoyed remarkably prolonged survival (MST, >40 days), suggesting that regulatory cells were generated. In vitro, sarpogrelate hydrochloride suppressed cellular proliferation in mixed leukocyte reaction. Conclusion: The treatment with sarpogrelate hydrochloride induced indefinite survival of cardiac grafts and generated regulatory cells. Sarpogrelate hydrochloride can be not only an antithrombotic agent but also a strong immunomodulating agent.

INHIBITION OF TERMINAL COMPLEMENT WITH A FUNCTIONALLY BLOCKING ANTI-C5 MONOCLONAL ANTIBODY PREVENTS ACUTE HUMORAL REJECTION AND ACCELERATED HUMORAL REJECTION IN A MOUSE CARDIAC ALLOGRAFT TRANSPLANTATION MODEL

O190* Aims: Antibody-mediated rejection including hyperacute rejection (HAR) or accelerated humoral rejection (ACHR) in presensitized recipients and de novo acute humoral rejection (AHR) in non-presensitized recipients cannot be prevented by anti-T cell therapy, leading to early graft loss. Complement has been shown to play an important role in antibody-mediated rejection. We previously reported that a functionally blocking anti-C5 monoclonal antibody (mAb) inhibited terminal complement activation and prevented HAR in a rat-to-presensitized mouse heart transplant model. The present study was undertaken to determine whether blocking terminal complement with anti-C5 mAb would prevent antibody-mediated rejection in mouse allograft models of AHR and ACHR. Methods: In the AHR model, C3H mouse hearts were heterotopically transplanted into BALB/c mice. In the ACHR model, BALB/c recipients were presensitized with C3H skin grafts one week prior to heart transplantation from same donor, a model designed to mimic HAR or ACHR in presensitized patients. Graft survival was determined by daily palpation. Complement activity and circulating anti-donor antibody levels were measured by a presensitized hemolytic assay and flow cytometry, respectively. In addition, antibody and complement deposition, as well as CD4 and CD8 cell infiltration in heart grafts were detected by immunohistochemistry. Results: AHR model: Allografts in untreated recipients were rapidly rejected at 8.0 ± 0.6 days. These grafts exhibited typical AHR, characterized by elevation of circulating anti-donor antibodies and antibody deposition, vasculitis, hemorrhage, fibrin deposition and thrombosis in the grafts. Administration of cyclosporine A (CsA, 15mg/kg, daily, S.C.) did not inhibit AHR, and grafts were rejected in 15.5 ± 1.1 days. Anti-C5 mAb monotherapy (40mg/kg/day, twice a week, I.P.) completely inhibited terminal complement activity and attenuated pathological changes of AHR, but did not prolong graft survival. In contrast, the combination of anti-C5 mAb and CsA successfully prevented AHR and achieved indefinite graft survival in all recipients (>100 days). This therapy completely inhibited terminal complement activity and prevented both cellular- and humoral-mediated rejection. Evaluation of the long-term surviving allografts revealed no pathology. ACHR model: After presensitization, anti-donor antibody levels, predominantly total IgG and IgG1, were rapidly elevated in BALB/c recipients. In untreated recipients, C3H heart grafts were rejected in 3 days by ACHR, characterized by severe hemorrhage, infarction and thrombosis. Immunohistochemistry showed massive antibody and complement deposition but minimal CD4+ and CD8+ cell infiltration in heart grafts. Treatment with CsA (15mg/kg, daily, S.C.) and/or cyclophosphamide (CyP, 40mg/kg, I.V., days 0 and 1) did not prevent ACHR and grafts were rejected in 3-4 days. Remarkably, the addition of anti-C5 mAb to the same dosing regimen of CsA and CyP effectively inhibited antibody-mediated rejection. These grafts have survived for more than 70 days and continue to demonstrate normal function and histology. Conclusions: The combination therapy of anti-C5 mAb and CsA achieves indefinite graft survival in a mouse allograft model of AHR and triple therapy of anti-C5 mAb, CsA and CyP effectively prevents ACHR in presensitized mouse recipients. These data suggest that inhibition of terminal complement using a novel anti-C5 mAb may be of value in the prevention of humoral rejection in clinical transplantation including presensitized recipients.

Fate of alphaGal +/+ pancreatic islet grafts after transplantation into alphaGal knockout mice.

Important phylogenetic differences between pig and human tissues prevent xenotransplantation from becoming a clinically feasible option. Humans lack the galactose-alpha1,3-galactose (alphaGal) epitope on endothelial cell surfaces and therefore have preformed anti-alphaGal antibodies. The role of these antibodies in rejection of non-vascular xenografts remains controversial. This study investigated the role of anti-alphaGal antibodies in rejection of non-vascularized alphaGal+/+ grafts in alphaGal -/- mice. alphaGal +/+ and alphaGal -/- pancreatic islets were transplanted under the renal capsule of streptozotocin-induced diabetic (1) alphaGal -/- mice and (2) alphaGal +/+ mice. alphaGal -/- recepients were immunized with rabbit red blood cell membranes (RRBCs) to produce elevated anti-alphaGal antibody levels. Six of the 18 alphaGal -/- mice rejected the alphaGal +/+ grafts within 68 days whereas indefinite graft survival was achieved in the control groups. Animals with surviving islet grafts were challenged with alphaGal +/+ skin grafts. Although all alphaGal +/+ skin grafts were rejected within 58 days, the islet grafts remained intact. This observation correlated with the level of alphaGal expression (which was very low on islets compared to skin) rather than the actual titre of anti-alphaGal antibody. The results suggest that the level of alphaGal expression plays an important role in graft survival. Therefore, its removal is important in the development of a pig islet donor for future clinical therapy.

AdCTLA-4lg combined with donor splenocytes, bone marrow cells and anti-ICOS antibody treatment induce tolerance in a rat heart transplantation model

Abstract It is difficult to induce rat cardiac allograft tolerance by co-stimulator blockade of the B7-CD28 pathway with CTLA-4Ig monotherapy alone. However, combined therapies of AdCTLA-4Ig plus donor-specific spleen-cell infusion, bone marrow cell infusion, and anti–ICOS antibody have been demonstrated to effectively induce indefinite acceptance of rat cardiac allografts. In this report, we compared the tolerance of cardiac allograft tolerant recipients induced by the above three strategies. The results show that treating Lewis recipients of a DA cardiac allograft with a combination of AdCTLA4-Ig and anti-ICOS antibody, donor splenocytes or bone marrow cells produced indefinite graft survival. Second transplantation of donor type skin or heart grafts could not affect the survival of primary heart graft in anti-ICOS treated groups, but results in rejection of primary heart grafts in other two groups, and that co-stimulator blockade, CD28 and ICOS simultaneously with CTLA-4Ig and anti-ICOS antibody, facilitates the development of CD25 + CD4 + regulatory T cells and induces stable transplantation tolerance in the rat cardiac allograft model. This also provides an effective therapy in clinical transplantation for promoting permanent graft survival by generating regulatory T cells.

Induction of operational tolerance and generation of regulatory cells after intratracheal delivery of alloantigen combined with nondepleting anti-CD4 monoclonal antibody.

We previously showed that intratracheal delivery of alloantigen induced prolonged survival of fully allogeneic cardiac grafts in mice. Here, this treatment protocol was combined with nondepleting anti-CD4 monoclonal antibody (mAb) to induce operational tolerance. CBA (H-2k) mice were pretreated with intratracheal delivery of whole splenocytes from C57BL/10 (H-2b) mice or a 15-mer Kb peptide, with or without intraperitoneal administration of nondepleting anti-CD4 mAb (YTS177). Seven days later, C57BL/10 hearts were transplanted into the pretreated CBA mice. In addition, some naive CBA mice underwent adoptive transfer of splenocytes from pretreated CBA mice and transplantation of a C57BL/10 heart on the same day. Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time, 12 days). Mice given intratracheal delivery of whole splenocytes or Kb peptide demonstrated prolonged graft survival (median survival time, 84 and 76 days, respectively). Concurrent administration of YTS177 and intratracheal delivery of splenocytes or Kb peptide resulted in indefinite graft survival. Mice with long-surviving C57BL/10 cardiac grafts showed acceptance of skin grafts from C57BL/10 mice but not BALB/c mice, demonstrating that operational tolerance had been induced. Adoptive transfer of splenocytes from mice pretreated with intratracheal delivery of splenocytes or Kb peptide plus YTS177 induced indefinite survival of cardiac grafts in secondary recipients, indicating that regulatory cells had been generated. In a murine model, intratracheal delivery of donor splenocytes or Kb peptide combined with YTS177 induced operational tolerance and generated regulatory cells.

AdCTLA-4Ig combined with donor splenocytes, bone marrow cells and anti-ICOS antibody treatment induce tolerance in a rat heart transplantation model.

It is difficult to induce rat cardiac allograft tolerance by co-stimulator blockade of the B7-CD28 pathway with CTLA-4Ig monotherapy alone. However, combined therapies of AdCTLA-4Ig plus donor-specific spleen-cell infusion, bone marrow cell infusion, and anti-ICOS antibody have been demonstrated to effectively induce indefinite acceptance of rat cardiac allografts. In this report, we compared the tolerance of cardiac allograft tolerant recipients induced by the above three strategies. The results show that treating Lewis recipients of a DA cardiac allograft with a combination of AdCTLA4-Ig and anti-ICOS antibody, donor splenocytes or bone marrow cells produced indefinite graft survival. Second transplantation of donor type skin or heart grafts could not affect the survival of primary heart graft in anti-ICOS treated groups, but results in rejection of primary heart grafts in other two groups, and that co-stimulator blockade, CD28 and ICOS simultaneously with CTLA-4Ig and anti-ICOS antibody, facilitates the development of CD25+ CD4+ regulatory T cells and induces stable transplantation tolerance in the rat cardiac allograft model. This also provides an effective therapy in clinical transplantation for promoting permanent graft survival by generating regulatory T cells.

Display of Fas ligand protein on cardiac vasculature as a novel means of regulating allograft rejection.

Fas ligand (FasL) is a potent death-inducing molecule with important functions in immune homeostasis and tolerance to self-antigens. The complex biological activities of FasL and its inefficient expression using conventional gene transfer approaches limit its use for immunomodulation to prevent allograft rejection. We have recently generated a chimeric FasL with core streptavidin (SA-FasL) with potent apoptotic activity and designed a novel approach to display it on the surface of several cell types via biotinylation. We herein tested whether SA-FasL can also be displayed on vascular endothelial cells in the heart and examined its effect on graft survival after transplantation into syngeneic and allogeneic hosts. SA-FasL was efficiently displayed on the vasculature of BALB/c hearts with a half-life of 9 days in vivo. Transplantation of hearts displaying SA-FasL into syngeneic hosts resulted in indefinite graft survival without detectable toxicity to the grafts and hosts. In contrast, transplantation of allogeneic C57BL/10 hearts displaying SA-FasL into BALB/c recipients resulted in graft rejection, but in a delayed fashion as compared with control hearts (mean survival time=17.4+/-5 versus 9.6+/-1 days). Allograft survival was further extended to 21+/-2.6 and 24+/-3 days (P<0.05) by intravenous treatment of graft recipients with 1 dose of SA-FasL-decorated donor splenocytes on days 2 and 6 after transplantation, respectively. This study shows for the first time that exogenous proteins can be displayed on the endothelium of solid organs for therapeutic purposes. This approach provides a convenient and rapid means of displaying exogenous proteins on the surface of cells, tissues, and solid organs, with broad research and therapeutic implications.

Molecular profiling of the role of the NF-κB family of transcription factors during alloimmunity

Allograft rejection involves a complex network of multiple immune regulators and effector mechanisms. In the current study, we focused on the role of nuclear factor (NF)-kappaB/Rel. Previous studies had established that deficiency of the p50 NF-kappaB family member prolonged allograft survival only modestly. However, because of its crucial role in signal transduction in inflammatory and immune responses, we hypothesized that other NF-kappaB/Rel family members may produce more profound effects on alloimmunity. Therefore, in addition to p50, we analyzed the role of c-Rel, which is expressed predominantly in lymphocytes. Also, to investigate NF-kappaB activation in T cells, we examined transgenic mice that express a transdominant inhibitor of NF-kappaB [IkappaB(DeltaN)] regulated by a T cell-restricted promoter. Allograft survival was prolonged indefinitely in the c-Rel-deficient and IkappaB(DeltaN)-transgenic recipients. To determine the molecular basis of NF-kappaB modulation of rejection, we analyzed a panel of 58 parameters including effector molecules, chemokines, cytokines, receptors, and cellular markers using hierarchical clustering algorithms and self-organizing maps in p50(-/-), c-Rel(-/-), and IkappaB(DeltaN)-transgenic, experimental groups plus allogeneic-, syngeneic-, and lymphocyte-deficient (alymphoid) control groups. Surprisingly, profiles of gene expression in the c-Rel recipients (which have indefinite graft survival) were similar to the p50(-/-) and allogeneic recipients (which rapidly reject grafts). As expected, gene expression in the IkappaB(DeltaN) recipients (which also have indefinite graft survival) was similar to profiles of nonrejecting syngeneic and alymphoid recipients. Importantly, self-organizing maps identified a small subset of genes including several chemokine receptors and cytokines with expression profiles that correlate with graft survival. Thus, our results demonstrate a crucial role for NF-kappaB in acute allograft rejection, identify different molecular mechanisms of rejection by distinct NF-kappaB family members, and identify a small subset of inducible genes whose inhibition is linked to graft acceptance.

Induction of donor-specific tolerance by adenovirus-mediated CD40Ig gene therapy in rat liver transplantation.

Blockade of CD40-CD40 ligand (CD154) costimulatory pathway with anti-CD154 antibody (Ab) prolongs allograft survival in experimental organ transplantations; however, repeated agent administration is needed to provide an adequate immunosuppression. Seeking for simple and effective approach to interfere this signaling, we applied adenovirus-mediated gene therapy by encoding CD40Ig gene (AdCD40Ig). Liver graft from ACI (RT1av1) rat was transplanted orthotopically into LEW (RT1l) rat, and AdCD40Ig was given to animals via the penile vein immediately after grafting (n=6). A single treatment with AdCD40Ig at 1x10(9) plaque forming units induced specific expression of CD40Ig gene on allograft liver, produced substantial amount of the protein in the sera, and allowed indefinite graft survival. Whereas, LEW recipients given no treatment or control adenovirus vector (AdLacZ) promptly rejected ACI liver. In addition, AdCD40Ig-treated, long-term survivors accepted skin graft from the donor strain but not the third party graft. Histopathology revealed that liver structure of the long-term surviving animals was completely preserved in normal with no infiltration of mononuclear cells. Blockade of CD40-CD154 pathway by CD40Ig gene therapy is a potent alloantigen-specific immunosuppressive strategy to induce permanent acceptance of liver allograft and would be a new therapeutic candidate in a clinical liver transplantation.

Inhibition of graft arteriosclerosis development in rat aortas following heme oxygenase-1 gene transfer

Heme oxygenase 1 (HO-1) is an enzyme which degrades heme into three end products: biliverdin, free iron and carbon monoxide. This enzyme has recently been shown to have anti-inflammatory and tissue protective effects. HO-1 expression is involved in organ protection in pathological situations, and immunosuppressive treatments resulting in indefinite graft survival without chronic rejection have been associated with HO-1 expression by cells of the vessel wall. The aim of this study was to analyze the effect of specific HO-1 overexpression. We used a recombinant adenovirus coding for human HO-1 cDNA in a rat aorta chronic rejection model, 30 days after transplantation. Control groups included rats non treated or treated with a non-coding adenovirus Addl324. We first demonstrated that AdHO-1 was efficiently expressed in endothelial cells in vitro, and in rat aortas ex vivo after adenovirus gene transfer. We found that intimal thickening in AdHO-1 treated aortas (10.8±3.8%, n=5) was significantly decreased compared to untreated (21.2±5.6%, n=5) or Addl324-treated (21.1±1.2%, n=4) aortas. Immunohistology showed that treatment with AdHO-1 resulted in a significant reduction in leukocyte infiltration and a decreasing number of VSMC in the intima, compared to Addl324-treated aortas. However, this effect of HO-1 on chronic rejection did not imply modifications on numbers of apoptotic cells in the graft or of alloantibody levels. We have demonstrated, for the first time, that specific HO-1 overexpression following gene transfer of HO-1 inhibited chronic rejection by reducing leukocyte and VSMC infiltration of the aorta intima.