Prolonged Cardiac Allograft Survival Research Articles

The Mechanism of Anti-Inflammatory Effects of Prostaglandin E2 Receptor 4 Activation in Murine Cardiac Transplantation

Prostaglandin E2 (PGE2) is a pathogenesis of inflammatory diseases; PGE2 plays a key role in association of anti-inflammation and immune suppression. EP4, which is a PGE2 receptor, is known to suppress the production of inflammatory cytokines and chemokines in vitro. Although it has been reported that EP4 agonists prolonged cardiac allograft survival, little has been elucidated the immunologic mechanism. We injected a selective EP4 agonist (EP4RAG) into recipient mice with heterotopic cardiac transplantation. EP4RAG significantly prolonged the graft survival compared with the vehicle-treated group. Although the vehicle-treated group showed severe myocardial cell infiltration, the EP4RAG-treated group attenuated the development on day 7. EP4RAG suppressed various proinflammatory factors such as cytokines, chemokines, adhesion molecules, and nuclear factor-kappaB (NF-kappaB) compared with the vehicle-treated group. We also demonstrated that EP4RAG suppressed the activation of macrophages, but it did not affect to T lymphocytes in vitro. EP4RAG inhibited the activation of NF-kappaB compared with the control group. Pharmacological selective EP4 activation suppressed the production of proinflammatory factors by inhibition of NF-kappaB activity in cardiac transplantation.

Purified Eicosapentaenoic Acid Induces Prolonged Survival of Cardiac Allografts and Generates Regulatory T Cells

Fish oil, which is rich in eicosapentaenoic acid (EPA), has been found to have immunomodulatory effects. We examined whether administration of purified EPA affected survival of fully mismatched murine cardiac allografts. Hearts from C57BL/10 (H-2(b)) mice were transplanted into CBA (H-2(k)) recipients treated with one intraperitoneal dose of purified EPA the day of transplantation. Untreated CBA recipients and recipients given 0.1 g/kg of EPA rejected C57BL/10 hearts (median survival time [MST], 8 and 13 days, respectively). With a 1.0 g/kg dose of EPA, graft survival was markedly prolonged (MST >100 days). To determine whether regulatory cells were generated, naïve mice (secondary recipients) underwent adoptive transfer of splenocytes from EPA-treated primary recipients and cardiac allograft transplantation. Adoptive transfer of whole, CD4(+) and CD4(+)CD25(+) splenocytes from EPA-treated recipients induced indefinite survival in secondary recipients. Flow cytometry showed that the CD4(+)CD25(+) cells were Foxp3(+). In reverse transcriptase-polymerase chain reaction (RT-PCR) studies, the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA was upregulated by EPA treatment. A PPARgamma antagonist abrogated the prolongation of graft survival induced by EPA treatment (MST, 13 days). Thus, in our model, purified EPA induced prolonged survival of fully mismatched cardiac allografts and generated regulatory T cells dependent on PPARgamma activation.

Quiescent recipient splenic dendritic cells (DC) re-process therapeutic DC into alloantigen for presentation to and down-regulation of anti-donor T cells to prolong allograft survival (141.31)

Abstract Current dogma in transplantation presumes that therapeutic maturation-resistant dendritic cells (MR-DC) prevent allograft rejection by directly interacting with donor-reactive T-cells in vivo. However, this assumption remains untested. Our study therefore aimed to elucidate the mechanism by which donor MR-DC down-regulate the anti-donor response to prolong allograft survival in mice. We employed as prototypic donor MR-DC, bone marrow-derived DC treated with the active form of vitamin D3, 1α,25(OH)2D3. We demonstrate that donor-derived MR-DC prolonged cardiac allograft survival similarly as donor apoptotic or viable splenocytes, suggesting that different donor-derived cellular therapies share a common mechanism of action. We determined that once injected i.v., MR-DC were short-lived and failed to directly tolerize donor-reactive T-cells. Rather, apoptotic MR-DC were internalized and re-processed by quiescent recipient splenic CD11chiCD8α+ and CD11chiCD8- DC, which in turn induced deletion of effector T-cells and outgrowth of regulatory T-cells via the indirect pathway. Importantly, activated recipient DC induced robust T-cell activation via indirect presentation. Our data suggest that therapeutic DC, like other more practical cellular therapies, serve as a source of alloantigen for recipient quiescent DC that in turn down-regulate the anti-donor response to prolong allograft survival.

Prolongation of Rat Major Histocompatibility Complex–compatible Cardiac Allograft Survival During Pregnancy

It has been suggested that pregnancy-related hormones play a critical role in mediating selective immune tolerance during pregnancy. An understanding of why a woman's body normally does not reject the fetus is highly relevant to the prevention of transplant rejection. The hearts of female inbred F344 rats (RT-1(lvl)) were transplanted into naive Lewis (RT-1(l); nLewis) or pregnant (pLewis; Day 6, 12 and 18 of pregnancy) rats. The mean survival time (MST) of the cardiac allografts between the nLewis and pLewis rats was compared. We determined the rate of proliferation of the T cells isolated from nLewis and pLewis rats in response to concanavalin A (ConA), anti-CD3 and -CD28 antibody and alloantigen stimulation ex vivo. mRNA expression of several cytokines in these T cells was analyzed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, the effect of estriol on the cardiac allograft was tested. The pLewis rats with transplant on Day 12 of pregnancy had the most significantly prolonged F344 cardiac graft survival (MST 13.3 days) as compared with nLewis recipients (MST 8 days). pLewis T-cell proliferation was stimulated by alloantigen and antibody but ConA was reduced, whereas Th1/Th2 cytokine mRNA profiles in the T cells were similar for nLewis and pLewis rats. Likewise, estriol also significantly prolonged survival of cardiac allografts. The results of this study demonstrate that pregnancy hormones not only appear to play a critical role in maternal acceptance of the fetus, but also have therapeutic potential for prolonging the survival of major histocompatibility complex (MHC)-compatible allografts during pregnancy.

29: Expansion of CD4+CD25+FoxP3+ Regulatory T Lymphocytes with Low-Dose Anti-Thymocyte Globulin Leads to Prolonged Survival of Cardiac Allografts but Not Tolerance

29: Expansion of CD4+CD25+FoxP3+ Regulatory T Lymphocytes with Low-Dose Anti-Thymocyte Globulin Leads to Prolonged Survival of Cardiac Allografts but Not Tolerance

A Combination of Donor Specific Transfusion and Rapamycin Prolonges Cardiac Allograft Survival in Mice

We sought to investigate the effects of donor-specific transfusion (DST) combined with rapamycin (RPM) on engraftment and demonstrate the mechanisms. B6 was treated with DST (on day -8) combined with RPM (from day -7 to -1, 1.5 microL/g per day). On day 0, B6 splenocytes were harvested to coculture with Balb/c splenocytes pretreated with mitomycin C in 1-way mixed lymphocyte cultures (MLC). After 48 hours, the proliferation, we measured apoptosis and the CD4(+)CD25(+) T-cell ratio of cultured cells. The heterotopic cardiac transplantation model was performed from Balb/c to B6 using the recipients pretreated with this protocol. On day 0, RPM was withdrawn and the transplantations performed. The mean survival time (MST) of the grafts evaluated and in vitro assays measured. Both in vitro and in vivo, the protocol suppressed the proliferation of splenocytes as well as enhanced apoptosis and the CD4(+)CD25(+) T-cell ratio. The MST of the grafts was 35 +/- 14.4 days. Our study showed that DST combined with RPM prolonged cardiac allograft survival even if we withdrew immunosuppressants after transplantation. This result may be associated with inhibition of T-cell proliferation. We also demonstrated that RPM expanded CD4(+)CD25(+) T cells and increased splenocyte apoptosis.

Pretransplant Infusion of Mesenchymal Stem Cells Prolongs the Survival of a Semiallogeneic Heart Transplant through the Generation of Regulatory T Cells

In this study, we investigated whether mesenchymal stem cells (MSC) had immunomodulatory properties in solid organ allotransplantation, using a semiallogeneic heart transplant mouse model, and studied the mechanism(s) underlying MSC tolerogenic effects. Either single (portal vein, day -7) or double (portal vein, day -7 and tail vein, day -1) pretransplant infusions of donor-derived B6C3 MSC in B6 recipients induced a profound T cell hyporesponsiveness and prolonged B6C3 cardiac allograft survival. The protolerogenic effect was abrogated when donor-derived MSC were injected together with B6C3 hematopoietic stem cells (HSC), suggesting that HSC negatively impact MSC immunomodulatory properties. Both the induction (pretransplant) and the maintenance phase (>100 days posttransplant) of donor-derived MSC-induced tolerance were associated with CD4(+)CD25(+)Foxp3(+) Treg expansion and impaired anti-donor Th1 activity. MSC-induced regulatory T cells (Treg) were donor-specific since adoptive transfer of splenocytes from tolerant mice prevented the rejection of fully MHC-mismatched donor-specific secondary allografts but not of third-party grafts. In addition, infusion of recipient-derived B6 MSC tolerized a semiallogeneic B6C3 cardiac allograft, but not a fully MHC-mismatched BALB/c graft, and expanded Treg. A double i.v. pretransplant infusion of recipient-derived MSC had the same tolerogenic effect as the combined intraportal/i.v. MSC infusions, which makes the tolerogenic protocol applicable in a clinical setting. In contrast, single MSC infusions given either peritransplant or 1 day after transplant were less effective. Altogether these findings indicate that MSC immunomodulatory properties require HSC removal, partial sharing of MHC Ags between the donor and the recipient and pretransplant infusion, and are associated with expansion of donor-specific Treg.

Prolongation of cardiac allograft survival by syngeneic Hematopoietic stem/progenitor cell transplantation in mice

Organ transplantation is a rapidly developing field, being the only effective treatment for end-stage organ disease. However, the associated immunosuppressant therapy has numerous direct and indirect adverse effects. Hematopoietic stem cell transplantation (HSCT), via immune reconstitution, offers an alternative method of treatment. In this study, we determined the cardiac allograft survival in mice treated with syngeneic HSCT or hematopoietic progenitor cell transplantation (HPCT). BALB/c and C57BL/6 mice were randomly divided into three groups. Mice in Group A (n=7) were untreated while those in Group B (n=8) and C (n=7) were treated with HPCT and HSCT, respectively. Cervical heterotopic cardiac transplantation models were established in all groups and cardiac grafts were observed throughout. Regulatory T (Treg) cell expression in peripheral blood was analyzed by flow cytometry. We recorded the cardiac allograft survival time and constructed Kaplan-Meier survival curves. The number of Treg cells in Group B was significantly higher than that in Group C (P<0.05). The survival time of mice from each group differed significantly according to Kaplan-Meier/log-rank analysis (P<0.01). A total of 62.5% of the grafts in Group B showed long-term survival (>100 days); all the mice in Group A died within 9 days, compared with 59 days in Group C. We conclude that syngeneic HSCT and HPCT can prolong cardiac allograft survival in mice. These two methods could be promising ways to induce immune tolerance in future organ transplantation.

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

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

Lymphocyte changes associated with prolongation of cardiac allograft survival in adult mice using anti-CD4 monoclonal antibody.

This study investigated the effect of anti-CD4 MoAb treatment on lymphocyte phenotype and function and correlated these changes with the prolongation of cardiac allograft survival in adult mice. Indefinite survival of heterotopic cardiac allografts was obtained in several fully allogeneic strain combinations when two doses of the anti-CD4 MoAb, YTS 191.1, were given at the time of transplantation. A dose response analysis in the C57BL/10 to C3H/He strain combination showed that very low doses of YTS 191.1 (25 micrograms/dose) were able to induce prolonged allograft survival when administered perioperatively. At the time of transplantation the immunosuppression induced by administration of the anti-CD4 MoAb is not antigen-specific, as heart grafts from different donor strains, mismatched for both major and minor histocompatibility antigens, showed prolonged survival in treated recipients. Immunocompetence was restored by 6 weeks after MoAb treatment, as recipients regained the ability to reject a cardiac allograft transplanted at this time point. However, while recovery of immunocompetence could be demonstrated in vivo, leucocytes isolated from the peripheral lymphoid organs of treated mice continued to be hyporesponsive in mixed leucocyte culture (MLC). Phenotypic analysis of the peripheral lymphoid tissues showed that C3H/He recipients treated with 25 micrograms/dose of YTS 191.1 had a marked, but not complete, elimination of the CD4+ subset at the time of transplantation, which was gradually restored to 50% of normal by 6 weeks after treatment. Thus, complete elimination of the CD4+ subset was not required to achieve indefinite allograft survival, and immunocompetence, as assessed in vivo, returned even when the CD4+ subset was present at half the normal level. Low doses of anti-CD4 MoAb (25 micrograms) had no effect on the expression of the CD4 molecule by thymocytes, and yet thymocytes were hyporesponsive to alloantigen in vitro. At higher doses of YTS 191.1, immature CD4+8+ thymocytes were selectively depleted. These results suggest that anti-CD4 MoAb therapy may modulate the intrathymic T cell selection process. These studies provide further insight into the mechanism of action of low dose, depleting anti-CD4 MoAb therapy in allograft rejection, and form a basis from which rational modifications to therapeutic protocols in transplantation models can be made.

Beneficial effect of amrinone on murine cardiac allograft survival.

Amrinone is a non-glycoside positive inotropic agent with an inhibitory effect on a cyclic adenosine monophosphate (AMP) phosphodiesterase isoenzyme. In the present study, we examined the immunosuppressive action of amrinone, since several other cyclic AMP-elevating agents have been shown to suppress T lymphocyte activation. First, the in vivo effects of amrinone were investigated. Oral amrinone treatment, at 40 mg/kg per day, significantly prolonged median cardiac allograft survival compared with non-treated controls (22.0 days versus 10.5 days, P < 0.01) when DBA/2 mouse hearts (H-2d) were heterotopically transplanted into C57B1/6 mice (H-2b). Histopathological examination showed that there was less prominent cellular infiltration in the amrinone-treated than in the non-treated allografts. Plasma amrinone concentrations of mice after a single oral dose of 40 mg/kg were within the range of clinical relevance. To clarify the mechanism of action, in vitro studies were done. The generation of specific cytotoxic T lymphocytes after mixed lymphocyte culture was significantly suppressed by addition of amrinone to the culture medium at 5 micrograms/ml. The production of IL-2 and the interferon-gamma during mixed lymphocyte culture was also suppressed by amrinone at 5 micrograms/ml. However, the level of intracellular cyclic AMP in mouse splenic lymphocytes was not affected significantly by the same dose of amrinone. In conclusion, amrinone has immunosuppressive actions at the therapeutic doses, and it may be a beneficial agent for therapy against acute cardiac allograft rejection.

Adoptive Transfer of Transplantation Tolerance Mediated by CD4 +CD25 + and CD8 +CD28 − Regulatory T Cells Induced by Anti–Donor-Specific T-Cell Vaccination

Previous studies have shown that vaccinating rodents with anti–donor-specific T cells significantly prolonged allograft survival; however, the putative mechanism of the tolerance remains unclear. In this study, we used the model of heterotopic heart transplantation between the C57BL/6 donor mice and BALB/c recipient mice vaccinated with anti-donor (C57BL/6) or anti-third party (C3H)-specific T cells to determine whether T cells prolong survival of mouse heart allografts and which cells were involved in induction of allograft tolerance. We observed that the mean survival time (MST) of C57BL/6 heart grafts in BALB/c mice vaccinated with anti-C57BL/6 specific T cells (43.1 ± 4.7 days) was prolonged from that in untreated BALB/c mice (9.5 ± 1.1 days) or BALB/c mice receiving anti-C3H–specific T cells (10.4 ± 1.9 days). These results suggested that alloantigen-specific T-cell vaccination significantly prolonged cardiac allograft survival. The CD4 +CD25 + or CD8 +CD28 − T cells purified from splenocytes of BALB/c mice vaccinated with anti–donor-specific T cells proliferated markedly in response to irradiated anti-C57BL/6–specific T cells in vitro. Adoptive transfer of these CD4 +CD25 + or CD8 +CD28 − T cells to naïve syngenic mice significantly prolonged the survival of heart allografts. These data suggested that anti–donor-specific T-cell vaccination induced development of CD4 +CD25 + or CD8 +CD28 − regulatory T cells, which in turn mediated allogeneic-specific tolerance.

Preconditioning With Ozone Abrogates Acute Rejection and Prolongs Cardiac Allograft Survival in Rats

Acute cardiac allograft rejection remains a major cause of morbidity and mortality after heart transplantation and predisposes for the development of graft vasculopathy. The aim of this study was to investigate the immunomodulatory effect of preconditioning of the donor and recipient with medical ozone (O 3/O 2) on acute allograft rejection. Minimizing the initial ischemia-reperfusion injury may result in a reduction of graft vasculopathy and ameliorate long-term outcomes after cardiac transplantation. Lewis rats were challenged with Wistar-Furth cardiac allograft. In donor and recipient animals a medical ozone (O 3/O 2)-pneumoperitoneum was induced by single (1×) or repetitive (5×) insufflation (concentration: 50 μg/mL, 80 mL/kg body weight) of medical ozone intraperitoneally. Without immunomodulatory therapy (n = 11) cardiac allograft survival was 5.9 ± 0.9 days. Preconditioning with medical ozone alone (single bolus as well as repetitive administration, n = 7) of the donor and recipient animals prolonged cardiac allograft survival significantly to 7.6 ± 1.4 days ( P < .05), without any adjunctive immunosuppressive therapy. In this pilot study, the intraperitoneal administration of ozone in donor and recipient animals protected from ischemia-reperfusion injury, reduced the immunogenicity of the graft, and prolonged cardiac allograft survival. Further studies are warranted to elucidate the underlying mechanisms and—more important—to investigate the effect on the development of graft vasculopathy, the major obstacle to long-term graft and patient survivals.

Leukocyte Integrin Mac-1 Promotes Acute Cardiac Allograft Rejection

In allograft rejection, recipient leukocytes and alloantibodies first target donor endothelial cells. Although the leukocyte integrin Mac-1 (alpha(Mbeta2), CD11b/CD18) facilitates cell-cell interactions among leukocytes and interactions between leukocytes and endothelial cells or platelets, its role in allograft survival and vasculopathy is incompletely defined. This study examined parenchymal rejection and graft arterial disease after total allomismatched cardiac transplantation (BALB/c donor heart and B6 recipients) in wild-type (WT) and Mac-1-deficient (Mac-1(-/-)) recipients. Recipient Mac-1 deficiency attenuated parenchymal rejection and significantly prolonged cardiac allograft survival from 8.3+/-1.3 days in WT recipient allografts (n=18) to 13.8+/-2.3 days in Mac-1(-/-) recipient allografts (n=6; P<0.0001). Accumulation of neutrophils and macrophages significantly decreased in Mac-1(-/-) compared with WT recipients. Adoptive transfer of WT but not Mac-1(-/-) macrophages to Mac-1(-/-) recipients exacerbated parenchymal rejection and reduced allograft survival; in contrast, adoptive transfer of WT neutrophils did not affect graft survival. Mac-1(-/-) macrophages expressed significantly lower levels of costimulatory molecules both in vivo and in vitro, and mixed lymphocyte reaction using alloantigen-primed Mac-1(-/-) macrophages resulted in significantly lower antigen-presenting function than for WT macrophages. Tumor necrosis factor-alpha production also fell in cultures with Mac-1(-/-) macrophages. Despite attenuation of acute rejection, recipient Mac-1-deficiency did not prevent late graft arterial disease. These studies demonstrate critical participation of Mac-1 in alloresponses during cellular allograft rejection. These observations establish a molecular target for modulating recipient responses to prolong graft survival.

Tolerance to rat liver allograft after total lymphoid irradiation is mediated by CD4+CD25+ regulatory T cells

We have previously determined that post transplant total lymphoid Irradiation (TLI) induces tolerance in an experimental model of liver transplantation. The purpose of this study is to characterize the mechanism of TLI induced tolerance.TLI significantly prolonged survival in Lewis recipients of fully‐histoincompatible DA liver allografts with 88% of the recipients surviving long‐term (&gt;100 days). CD4+CD25+Foxp3+ T cells were markedly increased in the liver grafts, PBMC, and spleen by day 35 post‐transplant and remained high in the spleen &gt;100 days post‐transplant. Splenocytes from long‐term surviving recipients have a reduced alloresponse in MLR. Moreover, isolated splenic CD4+CD25+ T cells inhibited the MLR of naive Lewis splenocytes to DA splenocytes. Importantly, adoptive transfer of whole splenocytes into naive Lewis recipients of DA heart grafts significantly prolonged cardiac graft survival (MST whole splenocytes 33 days vs. control 18.5 days:p&lt;0.01), whereas depletion of CD4+CD25+ T cells from splenocytes completely abrogated the prolongation of cardiac allograft survival (MST 16 days; p&lt;0.01, vs. whole splenocytes).TLI induces tolerance to liver allografts via a mechanism involving CD4+CD25+Foxp3+ T cells that accumulate in the liver early after transplant and remain in the spleen &gt;100 days. Studies using TLI to induce tolerance in clinical liver transplantation are currently underway.

ICOS-Dependent and -Independent Functions of Memory CD4 T Cells in Allograft Rejection

Donor-reactive memory T cells undermine the survival of transplanted organs through multiple pathways. We have previously reported that memory CD4 T cells resist treatment with anti-CD154 antibody and donor-specific transfusion (DST/MR1) and promote cardiac allograft rejection via generation of effector CD4 T cells and alloantibody. We hypothesized that the helper functions of memory CD4 T cells are independent of T-cell costimulation through CD154 but instead are regulated by alternative costimulatory pathways. This study investigated how blocking ICOS/B7RP-1 interactions affects functions of donor-reactive memory CD4 T cells. Treatment with blocking anti-ICOS mAb synergized with DST/MR1 and prolonged mouse cardiac allograft survival despite the presence of donor-reactive memory CD4 T cells. While blocking ICOS did not diminish the expansion of preexisting memory CD4 T cells or the induction of allospecific effector T cells, it did inhibit recruitment of the activated memory and effector T cells into the graft. In addition, anti-ICOS mAb treatment in combination with DST/MR1 prevented help provided by memory CD4 T cells for production of donor-specific IgG antibody. These results demonstrate the potential efficacy of ICOS blockade in sensitized transplant patients and provide the foundation for rational use of ICOS blockade in combination with other graft-prolonging strategies.

Hydrodynamics‐based delivery of plasmid DNA encoding CTLA4‐Ig prolonged cardiac allograft survival in rats

Although gene therapy using plasmid vectors is thought to be safer compared with viral vectors, poor efficacy of gene transfer is the obstacle preventing wide application of plasmid vectors. However, high levels of foreign gene expression have been achieved by rapid tail vein injection of a large volume of a plasmid DNA solution into rats. Using this technique, we examined the effect of rat CTLA4-Ig gene transfer on prevention of cardiac allograft rejection in this animal model. Recipient Lewis rats were injected with either plasmid pCAGGS-CTLA4-Ig-Glu-tag as a treatment vector or plasmid pCAGGS-signal peptide (SP)-Ig as a control vector by hydrodynamics-based delivery technique on the day before heart transplantation. Hearts from Brown Norway donors were transplanted into the neck of Lewis recipients and graft survival was assessed. The plasma level of CTLA4-Ig reached a peak of nearly 5 microg/mL 1 day after injection, and then slowly decreased but still remained above 0.9 microg/mL until 100 days after injection. The recipient rats treated with the control vector and untreated rats rejected cardiac allografts within 7 days. On the other hand, the median survival time of the grafts treated with pCAGGS-CTLA4Ig-Glu-tag was more than 100 days. Histological examination revealed that long-term survival allografts contained fewer infiltrating lymphocytes. The serum from recipients with long-term survival allograft suppressed allogenic mixed lymphocyte reaction. CTLA4-Ig gene transfer by means of tail vein injection of plasmid DNA into a recipient rat resulted in remarkable prolongation of cardiac allograft survival with persistent plasma level of CTLA4-Ig protein.

Overexpression of program death-1 in T cells has mild impact on allograft survival

Program death-1 (PD-1), an inhibitory receptor upregulated on T cells upon TCR stimulation, has been shown to attenuate a number of immune responses in vivo, including acute allograft rejection. We tested whether constitutive expression of PD-1 would further inhibit allograft rejection. To this end, we generated transgenic mice expressing T-cell-restricted PD-1 under the control of the Lck proximal promoter and CD2 locus control. PD-1 transgenic (PD-1-Tg) mice did not develop gross abnormalities of thymic development and displayed normal numbers of thymocyte subsets and peripheral T cells. In vitro, PD-1-Tg T cells had reduced proliferative and cytokine secretion capacity upon TCR stimulation and cross-linking of PD-1 resulted in diminished phosphorylation of protein kinase C-theta and Akt, as well as increased activation of the phosphate and tensin homolog. However, only T-cell responses to minor but not major mismatches were reduced in vitro. Similarly, PD-1-Tg mice exhibited prolonged survival of cardiac allografts only in mice transplanted with heart allografts expressing multiple minor mismatches and treated with suboptimal doses of cyclosporine A. We conclude that genetic engineering of T cells to express PD-1 constitutively has only a mild impact on allograft survival.

A Novel Alloantigen-Specific CD8+PD1+ Regulatory T Cell Induced by ICOS-B7h Blockade In Vivo

Delayed ICOS-B7h signal blockade promotes significant prolongation of cardiac allograft survival in wild-type but not in CD8-deficient C57BL/6 recipients of fully MHC-mismatched BALB/c heart allografts, suggesting the possible generation of CD8(+) regulatory T cells in vivo. We now show that the administration of a blocking anti-ICOS mAb results in the generation of regulatory CD8(+) T cells. These cells can transfer protection and prolong the survival of donor-specific BALB/c, but not third party C3H, heart grafts in CD8-deficient C57BL/6 recipients. This is unique to ICOS-B7h blockade, because B7 blockade by CTLA4-Ig prolongs graft survival in CD8-deficient mice and does not result in the generation of regulatory CD8(+) T cells. Those cells localize to the graft, produce both IFN-gamma and IL-4 after allostimulation in vitro, prohibit the expansion of alloreactive CD4(+) T cells, and appear to mediate a Th2 switch of recipient CD4(+) T cells after adoptive transfer in vivo. Finally, these cells are not confined to the CD28-negative population but express programmed death 1, a molecule required for their regulatory function in vivo. CD8(+)PD1(+) T cells suppress alloreactive CD4(+) T cells but do not inhibit the functions by alloreactive CD8(+) T cells in vitro. These results describe a novel allospecific regulatory CD8(+)PD1(+) T cell induced by ICOS-B7h blockade in vivo.

Recipient Conditioning with Mesenchymal Stem Cells and Interleukin-10 Prolonged Cardiac Allograft Survival (102.1)

Abstract Mesenchymal Stem Cells (MSC) and viral interleukin-10 (vIL-10) have immunosuppressive properties. In this study, we tested their ability to prevent cardiac allograft rejection. Bone marrow derived MSC from Lewis rat were expanded ex vivo and transduced with rvIL-10-retrovirus. Autologous MSC or vIL-10 transduced MSC were injected (~25 x 106; i.v.) into irradiated (4 Gy) rat (RT1.Al). Six weeks later heterotopic heart (RT1.An) transplantation (Tx) was performed. MSC therapy prolonged (P&amp;lt;0.05) cardiac allograft survival (14±1 days; n= 4) compared to untreated controls (7±1 days; n=4). Also, vIL-10-MSC treatment prolonged (P&amp;lt;0.01) graft survival (38±1 days; n=6) compared to empty vector treated group (15±1 days; n=4). In vIL-10-MSC preconditioned animals that received donor bone marrow after heart Tx, graft survival was 22±2 days (n=8). Intragraft expression of co-stimulatory molecule (CD80) and cytokines (IL-2, IFN-γ) as determined by RT-PCR was lower (P&amp;lt;0.03) in vIL-10-MSC treated grafts compared to untreated control grafts. Ex vivo expanded MSC were CD34−, CD45+(5 %), CD29+(90%), CD80 (0%), CD 86 (8%), CD90+(93%), MHC Class I+(23–57%), and MHC Class II- as determined by FACS. Both vIL-10 and empty vector engineered MSC expressed CD29 (&amp;gt;95%) but not other molecules. The vIL-10-MSC produced ~ 6 ng/ml of vIL-10 ex vivo. vIL-10-MSC addition in MLR cultures inhibited lymphoproliferative response (P&amp;lt;0.05). TGF-β expression in stimulated (TNF-α or IL1-β) and unstimulated MSC was 46 – 65% of β-actin. IL-2, IL-4, IL-10, IFN-γ, and TNF-α expression was negligible in MSC (0–7% of β-actin). VEGF and HGF mRNA expression in MSC was 58% and 5% of β-actin, respectively. TgGFP+ MSC were demonstrable in various tissues for &amp;gt;28 days. Both MSC and vIL-10 conditioning to promote allograft survival seems promising.