Murine Cardiac Allograft Survival Research Articles

CTLA4-Fas Ligand Gene Transfer Mediated by Adenovirus Induce Long-Time Survival of Murine Cardiac Allografts

Background Fas ligand gene transfer to induce peripheral allograft tolerance in animal models has shown controversial results. The immunosuppression effects mediated by engineered FasL depend on whether alloreactive T cells are selectively deleted. In the present study, we tested the feasibility of a strategy to induce long-time survival by fusing CTLA4-FasL gene transfer in vivo. Methods Cardiac allografts from DA(RT-1 a) rats were transplanted heterotopically into the abdomens of LEW(RT-1 1) rats. Plaque units (5×10 9) of either AdCTLA4-FasL, AdCTLA4Ig, or AdEGFP were administered via the portal vein immediately after cardiac transplantation. The frequencies of helper T lymphocyte precursors (HTLp) and cytotoxic T lymphocyte precursors (CTLp) were determined by a combined single limiting dilution assay on days 5 and 20 after transplantation. Results Cardiac allograft survival was significantly prolonged by either AdCTLA4-FasL or AdCTLA4Ig treatment(mean survival times [MST] of 71.0 ± 3.7 and 45.7 ± 2.4, respectively, n = 6) compared with untreated hosts or animals treated with AdEGFP(MST of 5.7 ± 0.5 and 5.2 ± 0.4, respectively, n = 6). In addition, treatment with AdCTLA4-FasL led to significantly prolonged allograft survival compared with AdCTLA4Ig treatment. Furthermore, the frequencies of HTLp and CTLp on day 20 among rats treated with AdCTLA4-FasL was lower than those on day 5, whereas frequencies of HTLp and CTLp on day 20 were similar with those on day 5 in the other groups. Conclusion These results suggest that administration of an adenovirus encoding fusion CTLA4-FasL gene to rat recipients effectively decreased the size of alloreactive T cells and induced long-term survival of cardiac allografts.

Antiproliferative effects of NKH477, a forskolin derivative, on cytokine profile in rat lung allografts

Objective NKH477 was recently identified as a water-soluble forskolin derivative and was reported to prolong survival of murine cardiac allografts. However, the mechanism of the efficacy is not clear in vivo. The aim of this study was to investigate the immunosuppressive effects of NKH477 on acute lung allograft rejection in the rat model and its mechanism of action in vivo. Methods Left lungs were transplanted orthotopically from Brown-Norway donors to Lewis recipients. Recipient rats were untreated or treated daily with different doses of NKH477. Grafts were excised on Day 3 or Day 5 to determine histopathological rejection and expressions of interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ by enzyme-linked immunosorbent assay. The cytokine expression at Day 3 or Day 5 was also evaluated in recipient spleens by immunohistochemistry. Furthermore, mesenteric lymph node cells from recipients at Day 5 were cultured alone or stimulated with donor antigens for 72 hours to determine cell proliferation by means of thymidine incorporation. Results NKH477 significantly extended allograft survival time in a dose-dependent manner and reduced histopathological rejection. Treatment with NKH477 inhibited IFN-γ and IL-10 expression, whereas expression of these cytokines were markedly upregulated in the untreated allografts. Expression of IL-2 and IL-10 also increased in the spleen of untreated allorecipients. NKH477 suppressed expression of both cytokines in the spleen. In addition, lymphocyte proliferation was inhibited in NKH477-treated recipients as compared with untreated recipients. Conclusion These results suggest that NKH477 exerts an antiproliferative effect on lymphocytes in vivo with an altered cytokine profile in rat recipients of lung allografts.

A Combination of Anergic Cells' Adoptive Transfer and Rapamycin Therapy Prolongs Cardiac Allograft Survival in Mice

The in vivo immunoregulatory effect of anergic cells induced by blocking the costimulatory pathway was investigated in this study. Anergic cells were generated in vitro by mixed culture of murine splenic cells from BALB/c and C3H/HeJ under the blockade of anti-CD154 and anti-CD80 monoclonal antibodies, and the in vitro activity of anergic cells were observed. The 3.0 Gy gamma-irradiated BALB/c mice received cardic allografts from C3H/HeJ, and anergic cells were intravenously injected immediately after transplantation. Recipient mice injected with anergic cells also received rapamycin therapy (1 mg/kg/day) for 14 days. On day 7 after transplantation, the subsets of peripheral blood T lymphocytes, the pathology of grafts and the infiltration of lymphocytes in grafts were analysed. Untreated gamma-irradiated animals showed a graft median survival time (MST) of 9 days. Animals injected with anergic cells only or receiving rapamycin therapy alone showed MST of 11 and 17 days, respectively. MST of allograft in mice treated with control cells plus rapamycin therapy was 9 days. Animals injected with anergic cells plus rapamycin therapy, but receiving third-party allografts (C57BL/6J), showed an MST of 15 days. However, anergic cell injection plus rapamycin therapy prolonged allograft survival significantly (MST 28 days, P < 0.01). The rejection was mild and tissue architecture was preserved in recipient mice receiving anergic cell injection plus rapamycin therapy. Furthermore, anergic cells and rapamycin therapy decreased the percentage of peripheral blood CD4+ and CD8+ T cells (including CD25+, CD152+, CD154+ and CD28+ subsets) and greatly reduced the infiltrating lymphocytes in allografts (including CD3+, CD4+, CD8+ and CD25+ T cells). In conclusion, the treatment based on anergic cells' adoptive transfer plus rapamycin therapy demonstrated a significant prolongation of murine cardiac allograft survival in a donor antigen-specific manner. This therapeutic protocol alleviated allograft rejection to solid allograft in vivo.

PRETREATMENT OF DONOR WITH TACROLIMUS PROLONGS MURINE CARDIAC ALLOGRAFT SURVIVAL

Aims: Alloantigens can be presented by donor antigen-presenting cells (APC) or recipient APC. In our previous studies, we have found that immature donor APC decreases recipient APC-mediated immunity. In this study, we want to determine whether pretreatment of donors to suppress donor APC can prolong murine cardiac allograft survival. Methods: In vitro study, donor dendritic cells (DC) were derived from B10 (H-2b; I-Ab) mice bone marrow, and propagated in GM-CSF (1000u/ml) or GM-CSF plus tacrolimus (20ng/ml) to see whether tacrolimus could suppress dendritic cells. Ex vivo study, B10 mice were treated by tacrolimus (2mg/kg) one day, and DC was harvested from spleen to examine the phenotype of the DC and its allostimulatory capacity. In vivo study, B10 mice was treated by tacrolimus (2mg/kg) one day before cardiac harvest, and the cardiac graft was transplanted into C3H (H-2k; I-Ak) mice abdomen heterotopically. Results: Dendritic cells propagated in tacrolimus expressed lower levels of CD80, CD86, and CD40 than the DC propagated without tacrolimus. The allogeneic stimulatory capacity of DC was also suppressed by tacrolimus (0.34±0.001 OD versus 0.45±0.01 OD at stimulator/responder=1/10). When B10 mice were treated by tacrolimus for a day, the DC harvested from spleen expressed lower levels of CD80, CD86, and CD40 than those harvested from naïve mice. The allogeneic stimulatory capacity was also lower than naïve DC (0.29±0.003 versus 0.37±0.01 at stimulator/responder=1/10). While the hearts of tacrolimus-treated B10 mice was transplanted into C3H mice, the cardiac allograft survival could be prolonged from 9.4±0.55 days of naïve hearts to 17.4±5.12 days (p=0.009). Conclusions: Tacrolimus can suppress the maturation and function of DC. Pretreatment of donor with tacrolimus one day before cardiac transplantation can prolong allograft survival.

Intrathymic injection of anti-Fas monoclonal antibody prolongs murine non-vascularized cardiac allograft survival.

Fas is a pro-apoptotic molecule involved in activation-induced cell-death of T lymphocytes, central and peripheral tolerance and immune privilege. We sought to determine the role of Fas in the thymus after organ transplantation. Heterotopic non-vascularized heart transplants from C3H mice were placed in C57Bl/6 recipients. A hamster anti-mouse anti-Fas mAb (JO2) was injected in the thymus of allograft recipients at the time of transplant. Results were compared with intrathymic injections of hamster IgG (HIgG), anti-FasL, as well as surgical thymectomy or intraperitoneal or intravenous injections of JO2 as controls. Intrathymic injection of 5 microg JO2 induced massive thymocyte apoptosis and enhanced allograft survival compared to HIgG (median graft survival 16 days vs 12.5 days, respectively, P=0.01). The effect was receptor and ligand specific. Intraperitoneal or intravenous injections of JO2 did not prolong graft survival. Thymocyte apoptosis was confirmed in vitro, in vivo and in situ. In the thymus, double positive immature CD4+8+ thymocytes were most susceptible to Fas-induced apoptosis. Our data shows that specific modulation of Fas pathways in the thymus at the time of transplant improves modestly but significantly murine heterotopic non-vascularized cardiac allograft survival and is associated with apoptosis of immature CD4+8+ thymocytes.

Microchimerism does not correlate with survival of murine cardiac allografts

The development of microchimerism was evaluated at different time points after infusion of a mixed population of bone marrow and spleen cells from (BALB/c × C57Bl/6)F1 mice in the presence or absence of a cardiac transplant. Microchimerism was observed in the spleen, bone marrow and thymus of transplanted BALB/c mice even after graft rejection. In the absence of transplantation, donor cells persisted especially in the thymus. The results show that despite augmentation of graft survival after donor cell infusion compared to nontreated controls, the development of microchimerism did not sustain cardiac semihistocompatible grafts. Moreover, the persistence of donor cells in the thymus in both situations suggests a role for this organ in the increased graft survival in our model.

Marked prolongation of murine cardiac allograft survival using recipient immature dendritic cells loaded with donor-derived apoptotic cells.

We investigated whether recipient dendritic cells (DCs), pretreated with nuclear factor-kappaB oligodeoxyribonucleotide decoy (NF-kappaB ODN decoy) and loaded with ultraviolet B-irradiated donor apoptotic splenocytes (Apo-SCs), were able to induce murine cardiac allograft tolerance. Heterotopic vascularized heart transplantation was performed from BALB/c to C57BL/6 mice, and recipients (C57BL/6) were given one injection of recipient DCs pretreated with NF-kappaB ODN decoy and loaded with donor (BALB/c) apoptotic SCs (decoy Apo-SCs DCs) through the portal vein at 7 days, before heart transplantation in the absence of immunosuppression. The cardiac allograft survival time and the expressive levels of intragraft cytokine genes [interleukin (IL)-2, IL-10, and interferon-gamma] were evaluated. Our results indicated that injection of decoy Apo-SCs DCs significantly prolonged vascularized heart allograft survival and led to skewing of intragraft cytokine expression towards T helper 2 (IL-10). The mechanisms can be useful for therapy of allograft rejection with minimization of systemic immunosuppression.

Significant prolongation of murine cardiac allograft survival by inhibition of matrix metalloproteinase-2

Significant prolongation of murine cardiac allograft survival by inhibition of matrix metalloproteinase-2

Recipient MHC class II expression is required to achieve long-term survival of murine cardiac allografts after costimulatory blockade.

To study the role of the direct and indirect pathways in achieving tolerance, we used genetically altered mouse strains in two ways: 1) MHC class II-deficient mice were used as donors of skin and cardiac grafts to eliminate the direct CD4(+) T cell response, and 2) B6 II(-)4(+) mice, which are MHC class II-deficient mice expressing an MHC class II transgene only on thymic epithelium, were used as recipients of normal grafts. These mice cannot mount an indirect response. Eliminating the indirect pathway actually made it more difficult to achieve prolonged allograft survival when we used costimulatory blockade than when both pathways were available. Costimulatory blockade was ineffective even when CD4(+) T cells from normal animals were transferred into recipients that lacked MHC class II molecules. These results suggest that an active CD4(+) response through the indirect pathway is necessary for costimulatory blockade to be effective in prolonging allograft survival.

Prolonged survival of murine cardiac allografts by treatment with the 4-amino analog of tetrahydrobiopterin

Prolonged survival of murine cardiac allografts by treatment with the 4-amino analog of tetrahydrobiopterin

Extracellular matrix proteins in organ transplantation.

Extracellular matrix proteins in organ transplantation.

Inducible nitric oxide-mediated myocardial apoptosis contributes to graft failure during acute cardiac allograft rejection in mice.

The mechanism through which nitric oxide (NO) mediates cardiac myocyte death during acute cardiac rejection has not been fully delineated. We sought to determine whether NO promotes myocardial apoptosis and contributes to graft failure during acute cardiac rejection in a murine model. Heterotopic cardiac transplantation was performed from Balb/c (H-2d) to C3H/He mice (H-2k). Recipients were treated with aminoguanidine (AG) at 400 mg/kg every day after surgery. As references, we used isografts in Balb/c mice with and without AG treatment (400 mg/kg/day). Graft survival, histological changes and serum NO levels were assessed. Intra-graft apoptosis was evaluated using a DNA fragmentation detection assay (TUNEL method) and DNA laddering. Significant prolongation of graft survival was observed in allografts treated with AG in comparison with nontreated allografts. Serum NO levels, which peaked on day 7 in nontreated allografts, were significantly decreased in AG-treated allografts. AG treatment decreased the number of apoptotic cells and lowered the ratio of the apoptotic cardiac myocytes in contrast to that of the apoptotic infiltrating cells. DNA laddering was clearly detected in nontreated allografts but was suppressed in AG-treated allografts. Inhibition of NO production by AG prolonged murine cardiac allograft survival. The decrease in intra-graft apoptotic activity paralleled histological improvement. Cardiac myocyte death which occurs through an apoptotic process mediated by NO contributes to graft failure during acute cardiac rejection.

PROLONGATION OF CARDIAC ALLOGRAFT SURVIVAL BY INTRATHYMIC INJECTION OF ANTI-FAS MONOCLONAL ANTIBODY

58 Intrathymic (IT) administration of donor antigen induces deletion of donor reactive thymocytes. In this model of acquired thymic tolerance and in neonatal tolerance, apoptosis (programmed cell death) is thought to play a role in the deletion of donor reactive clones. A major initiator of apoptosis is the interaction of FasL with its receptor Fas. To determine the role of Fas-FasL mediated apoptosis in IT tolerance we studied the effect of IT injection of an apoptosis inducing anti-Fas monoclonal Ab(Jo2) on allograft survival. Methods: Neonatal C3H cardiac grafts were transplanted into a subcutaneous ear pocket of 6-8 wk old C57BL/6 or lpr Fas deficient mice recipients. Groups included: no thymic manipulation, injection of 2.5 or 5µg (in 25µl) Jo2/thymic lobe, 2.5µg/lobe hamster IgG (HIgG, isotype control) or K10 (anti Fas-L) injected IT, surgical thymectomy prior to transplant and 5µg of Jo2 injected intraperitoneally (IP). There were 4 to 6 animals/group. Results: Injection of 5 µg of Jo2 IT significantly improved allograft survival compared to untreated controls. This was Fas-specific (b vs cde), was not the result of a "chemical thymectomy" (b vs f), was not due to a systemic effect of Jo2 (b vs g) and did require precise dosage (b vs h). Naive thymocytes exposed to Jo2 in vitro and thymocytes harvested from Jo2 IT injected animals were assayed for apoptosis by FACS analysis with Hoechst dye 342 and propidium iodide. FACS analysis showed dramatically increased amounts of thymocyte apoptosis compared with controls. TableTableConclusions: IT injection of anti-Fas Ab prolonged murine cardiac allograft survival. This effect may be due to apoptosis of thymocytes and/or activated lymphocytes recirculating through the thymus, or to the systemic release of cytokines and soluble HLA molecules after apoptosis in the thymus.

Adenovirus-mediated gene transfer of viral interleukin-10 inhibits the immune response to both alloantigen and adenoviral antigen.

Although adenoviral vectors are attractive for gene transfer, their effectiveness is limited by host antiviral immune responses. In this study, we determined if host antiallograft and antiviral immunity could be diminished with an adenoviral vector encoding the immunosuppressive cytokine viral interleukin-10 (vIL-10). AdSV40vIL-10, a vIL-10-expressing adenoviral vector with an SV40 promoter, induced significant prolongation of murine cardiac allograft survival to 32.2 +/- 1.7 days compared to 14.2 +/- 1.0 days for controls (p < 0.01). This effect was specific for vIL-10 encoding vector and could be inhibited by anti-vIL-10 monoclonal antibody (mAb). In vivo administration of adenovirus facilitated the generation of adenovirus-specific cytotoxic T lymphocytes (CTL), whereas treatment with AdSV40vIL-10 prevented CTL priming and generation of virus-specific immunity. AdSV40vIL-10 also induced extended expression of a beta-galactosidase reporter from a co-injected LacZ-encoding adenoviral vector. These results demonstrate that adenovirus-mediated gene transfer and expression of vIL-10 prolong allograft survival and inhibit the immune response to adenoviral antigens, thereby improving the persistence of the vector and extending transgene expression. The efficacy of adenoviral vectors can be improved by incorporating immunosuppressive genes into the vector.

Gene transfer of transforming growth factor-beta 1 prolongs murine cardiac allograft survival by inhibiting cell-mediated immunity.

Delivery of immunosuppressants directly to allografts using gene transfer and gene therapy approaches may inhibit immune activation while avoiding the systemic toxicity of conventional immunosuppression. Cardiac grafts from allogeneic (C57BL/6, H-2b) donors were transplanted into CBA/J (H-2k) recipients in a heterotopic, non-vascularized model pSVTGF-beta 1, a plasmid encoding murine transforming growth factor-beta 1 (TGF-beta 1) under the control of an SV40 promoter, was directly injected into grafts at surgery and prolonged survival from 12.0 +/- 0.7 to 25.1 +/- 2.1 days (p < 0.001) in a dose-dependent manner. Plasmid gene transfer-induced immunosuppression was localized to the area of the graft because plasmid injected remote from the graft did not prolong allograft survival and systemic immunity was not influenced by local gene transfer. Limiting dilution analysis of graft-infiltrating cells demonstrated that gene transfer reduced the precursor frequency of donor-specific cytotoxic T lymphocytes (CTL) and activated and total interleukin-2 (IL-2) producing helper T lymphocytes (HTL) in graft-infiltrating cells, whereas CTL generation and HTL precursor frequency in splenic lymphocytes were not altered. Additional data revealed that gene transfer inhibited the priming of TH0 cells and the conversion of primed TH1 cells to activated cells without the participation of TH2 suppressors. These data demonstrate that gene transfer of plasmid DNA encoding TGF-beta 1 in vivo suppresses local T cell immunity, which prolongs allograft survival.

Immunomodulation by an Adenylate Cyclase Activator, NKH477,in Vivoandin Vitro

Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger which modulates T cell function. NKH477 is a direct adenylate cyclase activator derived from forskolin and now under clinical investigation as a positive inotropic agent. While the immunosuppressive effects of forskolin on lymphocytes have been reported, little is known about its effectsin vivo.In this study, we investigated whether NKH477 has immunosuppressive effects in mice, namely on cardiac allograft survival, and on the generation of cytotoxic T lymphocytes (CTL), T cell proliferation in mixed lymphocyte reaction (MLR), and production of interleukin-2 (IL-2) in MLR and in mitogen response. We assessed the effects of standard immunosuppressant cyclosporin A (CsA) on IL-2 production and on allograft survival to estimate the intensity of rejection in this acute rejection model. Saline-treated C57BL/6 (H-2b) mice rejected DBA/2 (H-2d) cardiac allografts with a median graft survival time of 10 days. In contrast, median graft survival was prolonged to 12 and 15 days in mice treated with NKH477 at 1 and 3 mg/kg/day, respectively (P< 0.01 vs control). The equivalent dose of CsA (40 mg/kg/day) to the maintenance dose after clinical cardiac transplantation prolonged median graft survival time to 15.5 days, indicating that high dose of NKH477 was as efficacious as lower dose of CsA. Addition of NKH477 to the culture medium suppressed the generation of CTL, T cell proliferation in MLR, and production of IL-2 in MLR and in mitogen response. These results suggest that NKH477 exerts a beneficial effect on murine cardiac allograft survival by modulating T cell function.

CTLA4-Ig plus bone marrow induces long-term allograft survival and donor specific unresponsiveness in the murine model. Evidence for hematopoietic chimerism.

Allograft rejection is dependent on T cell activation, which requires both the engagement of the T cell receptor by antigen in the context of the MHC molecules and costimulatory signals delivered by cell surface molecules such as B7-CD28/CTLA4 pathway. CTLA4-Ig is a fusion protein that blocks this pathway and has previously been shown to prolong both allograft and xenograft survival. The current study demonstrates markedly prolonged murine cardiac allograft survival and specific prolongation of secondary skin grafts using a combination of CTLA4-Ig plus donor bone marrow. A role for hematopoietic chimerism in the establishment of CTLA4-Ig-induced transplantation tolerance was investigated using reverse transcriptase polymerase chain reaction analysis of recipient tissues. Expression of donor-specific MHC class II transcripts in both peripheral and lymphoid tissues was demonstrated at greater than 200 days after transplant. To investigate the functional significance of this observation, heart donors, and donor bone marrow were irradiated before transplantation in CTLA4-Ig-treated recipients. A reduction in allograft survival was associated with irradiation of both the donor heart and the bone marrow. These results suggest that there may be a donor-derived radiosensitive element that enhances allograft survival in this model. Reverse transcriptase polymerase chain reaction analysis of allografts of tolerant and control animals at days 5, 8, and 12 after transplantation failed to demonstrate a dramatic difference in the expression of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma message. Cytotoxicity effector transcripts were largely intact in CTLA4-Ig + bone marrow-treated recipients as they showed no decrease in intragraft granzyme, perforin, Fas, or Fas ligand transcripts during thr first 8 days after transplant. These results imply that complex mechanisms may be important for the induction and maintenance of transplantation tolerance in the CTLA4-Ig plus bone marrow murine cardiac allograft model.

Prolongation of murine cardiac allograft survival by microspheres containing TNF alpha and IL1-beta neutralizing antibodies.

Efficient delivery of therapeutic agents to a critical microenvironment may increase the efficacy of drugs used to modulate the allograft rejection response. This study demonstrates the ability of the combination of microspheres containing neutralizing anti-TNF alpha and anti-IL1-beta antibodies to significantly prolong murine cardiac allograft survival. These results suggest that the microsphere technique is an efficacious method to target antibody delivery to prolong allograft survival.

Major histocompatibility complex-specific prolongation of murine skin and cardiac allograft survival after in vivo depletion of V beta+ T cells.

The preferential usage of certain T cell receptor (TCR) V beta genes has been well established in several major histocompatibility complex (MHC)-restricted immune responses. However, V beta usage among allogeneic responses remains unclear. Because recent findings of ours and others indicate that V beta 8 predominates in certain Ld-restricted, peptide-specific responses, we examined the V beta 8 usage in allogeneic responses to Ld. To selectively recognize the Ld molecule, cells from BALB/c-H-2dm2 (dm2), the Ld-loss mutant mouse, were stimulated in vitro or in vivo with wild-type BALB/c cells. We report here that after the intraperitoneal administration of the anti-V beta 8 monoclonal antibody (mAb) F23.1, peripheral V beta 8 T cells were depleted from dm2 mice. This in vivo depletion abrogated the ability of dm2 splenocytes to mount a primary response to Ld molecules. This abrogation was specific, since the response of V beta 8-depleted dm2 cells to Kb/Db antigens was the same as that of control nondepleted dm2 cells. Furthermore, in vivo depletion of V beta 8 cells was found to cause a dramatic prolongation of Ld-disparate skin grafts (mean survival time [MST] 22.1 +/- 2.1 vs. 10.3 +/- 1.1 d for saline-treated controls, or 10.9 +/- 1.7 d for controls treated with mAb KJ23 to V beta 17). By contrast, V beta 8 depletion had no effect on recipients grafted with haplotype-mismatched skin or single Dk-locus-disparate skin. These findings demonstrate that V beta 8+ T cells predominate in allogeneic response to Ld but not other alloantigens. The effect of V beta 8 depletion was found to be even more dramatic on recipients grafted with Ld-disparate vascularized heart transplants (MST > 100 vs. 8.6 +/- 0.5 d for controls). In total, these findings establish the efficacy of using mAb to the V beta gene family to specifically and significantly enhance the survival of allografts. The implications of detecting V beta 8 usage in both alloreactive or MHC-restricted TCR responses to the same class I molecule are discussed.