Cardiac Graft Survival Research Articles

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.

Cell-mediated Immunomodulation of Chemokine Receptor 7–expressing Porcine Sertoli Cells in Murine Heterotopic Heart Transplantation

Sertoli cells (SC) have immunomodulative properties, and chemokine receptor 7 (CCR7) can optimize the systemic immunomodulatory effect by guiding SC from the periphery to the secondary lymphoid organs. The effect of immortalized neonatal porcine SC (NPSCi) was evaluated by analysis of cytokine levels. Hyporesponsiveness to donor cells was determined by MLC and analysis of splenocyte phenotypes using a murine allogeneic skin graft model. The effect of CCR7-expressing NPSCi (NPSCi-CCR7) combined with cobra venom factor (CVF) was evaluated using a heterotopically transplanted murine allogeneic heart model. Expression of immune cytokines was markedly modulated by NPSCi. The lymphocyte proliferation and splenocyte phenotypes were significantly suppressed by NPSCi-CCR7. Although pre-transplantation of NPSCi or NPSCi-CCR7 did not prolong graft survival of allogeneic cardiac grafts, CVF treatment facilitated pre-transplantation of NPSCi-CCR7 to prolong survival of allogeneic cardiac grafts (25.5 +/- 7.05 vs 9.5 +/- 0.58 days, p < 0.01). NPSCi may be used as a powerful immunomodulatory tool, and our strategy to traffic NPSCi to lymphoid organs using CCR7 optimizes the systemic immunomodulatory effect in vivo. With the help of initial immunosuppression for humoral mechanisms using CVF, the host immune response against allogeneic cardiac grafts can be effectively ameliorated by immunomodulation of the cellular mechanism with NPSCi-CCR7.

Update: cardiac xenotransplantation

To review the latest development in cardiac xenotransplantation in small and large animal models and related in-vitro studies. With the recent introduction of alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig organs for xenotransplantation, improved cardiac graft survival has been obtained. However, this experience has demonstrated the importance of pig antigens other than Galalpha1,3Gal (Gal) antigens (so-called nonGal antigens) as targets for primate anti-pig antibodies. Several in-vitro studies have confirmed that, although the incidence and levels of anti-nonGal antibodies in nonhuman primates and humans are significantly less when compared with total anti-pig antibodies (i.e., anti-Gal + anti-nonGal), they can result in complement-mediated lysis of GT-KO pig cells. More recently, it has been demonstrated that regulatory T cells suppress the cellular xenogeneic response, thus potentially preventing or reducing T-cell-mediated rejection. The importance of thrombotic microangiopathy as a feature of the immune/inflammatory response and incompatibilities between the coagulation-anticoagulation systems of pig and primate are receiving increasing attention. Development of GT-KO pigs transgenic for one or more 'antithrombotic' genes, for example, CD39 or tissue factor pathway inhibitor, may contribute to overcoming these problems. Although GT-KO pigs have provided an advance over wild-type pigs as a source of organs for transplantation into primates, further genetic modification of GT-KO pigs is required to overcome the remaining immune barriers before a clinical trial of cardiac xenotransplantation can be contemplated.

Critical Role of CD4 Help in CD154 Blockade-Resistant Memory CD8 T Cell Activation and Allograft Rejection in Sensitized Recipients

Allograft rejection in sensitized recipients remains the major problem in clinical organ transplantation. We have developed a donor-type skin-sensitized mouse cardiac allograft model (BALB/c-->C57BL/6) in which both rejection (<5 days) and alloreactive CD8 activation are resistant to CD154 blockade. First, we attempted to elucidate why CD154 blockade fails to protect cardiac grafts in sensitized recipients. The gene array analysis has revealed that treatment with anti-CD154 mAb (MR1) had distinctive impact on host immunity in naive vs sensitized animals. Unlike in naive counterparts, host sensitization mitigated the impact of CD154 blockade on critical immune signaling pathways. Indeed, we identified 3234 genes in cardiac grafts that were down-regulated by MR1 in naive (at least 5-fold), but remained unaffected in sensitized hosts. Moreover, MR1 treatment failed to prevent accumulation of CD4 T cells in cardiac allografts of sensitized recipients. Then, to determine the role of CD4 help in CD154 blockade-resistant immune response, we used CD4-depleting and CD4-blocking Ab, in conjunction with MR1 treatment. Our data revealed that CD154 blockade-resistant CD8 activation in sensitized mice was dependent on CD4 T cells. In the absence of CD4 help, CD154 blockade prevented differentiation of alloreactive CD8 T cells into CTL effector/memory cells and abrogated acute rejection (cardiac graft survival for >30 days), paralleled by selective target gene depression at the graft site. These results provide the rationale to probe potential synergy of adjunctive therapy targeting CD4 and CD154 to overcome graft rejection in sensitized recipients.

Mechanisms of Survival Prolongation of Murine Cardiac Allografts Using the Treatment of CTLA4-Ig and MR1

Backgroud The present study was undertaken to determine the role of costimulatory blockade in a murine cardiac transplant model. Materials and Methods We blocked the CD28/B7 and CD154/CD40 costimulatory pathways by transient administration of CTLA4-Ig and MR1 antibody to study the effects on allograft survival time, deviation of Th1 and Th2 cytokine secretion, and other mechanisms related to prolonged survival. Results Costimulatory blockade prolonged the mean survival time (MST) of cardiac allografts to 43 days for the treated group vs 8 days for the untreated group ( P < .01). The costimulatory blockade down-regulated the expression of 2 Th1 cytokines (interferon-γ [IFN-γ] and interleukin-2 [IL-2]) and 2 Th2 cytokines (IL-4 and IL-10), reduced the numbers of graft-infiltrating CD4 + and CD8 + lymphocytes, and inhibited the expression of both perforin/GrB and FasL in allografts. Conclusions Combined administration of CTLA4-Ig/MR1 inhibited acute rejection reactions in murine cardiac allografts, prolonging the survival of cardiac grafts through several mechanisms, including inhibition of Th1 and Th2 cytokine expression, graft infiltration by CD4 + and CD8 + T lymphocytes, and reduced both perforin/GrB and Fas-FasL.

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.

Induction of peripheral tolerance by local delivery of dendritic cell progenitors to cardiac allografts in a murine heterotopic heart transplantation model

Systemic injection of donor-derived dendritic cell (DC) progenitors is reported to prolong cardiac allograft survival in nonimmunosuppressed hosts. Our purpose was to identify whether tolerance limited to the cardiac allograft is inducible by direct delivery of DCs to the myocardium, thus diminishing the potential for systemic side effects. The donors were 8- to 12-week-old male B10/ H2b mice. The recipients were 8- to 12-week-old male C3H/H2k mice. For DC culture, DCs were propagated from donor bone marrow with granulocytic/macrophage colony stimulating factor (GM-CSF) (GM-CSF DCs) and/or interleukin-4 (IL-4) (GM-CSF+IL-4 DCs). The phenotypes of DCs were analyzed by flow cytometry (FACScan). For DC assay, the JAM test (a DNA fragmentation assay), the mixed lymphocyte reaction (MLR), and flow cytometry were used. For local DC delivery to the myocardium, DCs were injected retrograde into the ascending aorta, and abdominal heterotopic vascularized heart transplantation was performed. Donor heart survival was recorded. JAM assays confirmed the induction of apoptosis in T cells by both DC preparations. Flow cytometry revealed that the GM-CSF DCs expressed diminished co-stimulatory molecules (B7-1 and B7-2) in comparison with the GM-CSF+IL-4 DCs. The mean survival time of cardiac allografts was 13.1 +/- 4.32 days for the controls, 35.0 +/- 25.7 days for DCs cultured with GM-CSF, and 16.9 +/- 7.30 days for DCs cultured with GM-CSF+IL-4. Local delivery of GM-CSF DCs may induce graft tolerance and may prove to be more efficient than systemic delivery of DCs. Local delivery of GM-CSF+IL-4 DCs did not prolong cardiac graft survival, suggesting that the immune response elicited may be immunostimulatory.

In Vivo Optical Bioluminescence Imaging of Collagen-supported Cardiac Cell Grafts

Histology-based survival assessment of cell grafts does not allow for in vivo follow-up. In this study we introduce two new experimental models for longitudinal in vivo survival studies of cardiac cell grafts using optical bioluminescence imaging. H9c2 cardiomyoblasts expressing both firefly luciferase (fluc) and green fluorescent protein (GFP) reporter genes were implanted into Lewis rats. In Model 1, H9c2-fluc-IRES-GFP cells (0.5 x 10(6)) were implanted into a cryoinjured abdominal wall muscle. Cells were injected using either liquid collagen (Matrigel [MG]) or phosphate-buffered saline (PBS) suspension. Cell survival was evaluated in vivo using bioluminescence imaging on days 1, 5 and 10 post-operatively. In model 2, rats underwent ligation of the left anterior descending (LAD) artery. The donor hearts were harvested, and the infarcted region was restored ex situ using 1 x 10(6) H9c2-fluc-IRES-GFP cells seeded in collagen matrix (Gelfoam [GF]) or suspended in PBS (n = 8/group). Hearts were then transplanted into the abdomen of syngeneic recipients. Optical bioluminescence imaging was performed on Days 1, 5, 8 and 14 post-operatively. After 4 weeks, immunohistologic studies were performed. For model 1, at day 5, bioluminescence signals were markedly higher for the H9c2/MG group (449 +/- 129 photons/second x 10(3)) compared with the H9c2/PBS group (137 +/- 82 photons/second x 10(3)) (p < 0.05). For model 2, bioluminescence signals were significantly (p < 0.04) higher in the H9c2/GF group compared with plain cell injection on days 5 (534 +/- 115 vs 219 +/- 34) and 8 (274 +/- 34 vs 180 +/- 23). Data were in accordance with GFP immunohistology. Optical bioluminescence is a powerful method for assessment of cardiac cell graft survival in vivo. Collagen matrices support early survival of cardiomyoblasts after transplantation into injured musculature.

Anergic lymphocytes generated by blocking CD28 and ICOS pathways in vitro prolong rat cardiac graft survival

Regulatory cells may play a pivotal role in inducing and maintaining transplantation tolerance. We investigated the mechanism of anergic lymphocytes with regulatory cell potential generated in vitro by ICOS and CD28 co-stimulatory blockades as a source of cellular therapy for treating allograft rejection. Anergic lymphocytes were generated by a mixed lymphocyte reaction consisting of DA splenocytes as the stimulator and Lewis splenocytes as the responder in the presence of anti-ICOS mAb and rCTLA-4Ig. Immunoregulatory effects of these lymphocytes were evaluated by secondary MLR and using other various stimulations. DA heart was transplanted into 7.5 Gy-irradiated Lewis rat after intravenous administration of these cells and/or Lewis spleen lymphocytes. We observed that these lymphocytes were not only anergic to alloantigen and polyclonal stimulations but also exhibited regulative activity to inhibit the alloreactive T-cell response. Our adoptive transfer studies revealed that irradiated recipients that received both anergic lymphocytes and naïve Lewis lymphocytes had significantly prolonged DA cardiac graft survival (mean 17.5 days) compared with a group that received Lewis lymphocytes alone (mean 10.8 days). Furthermore, some of the recipients accepted the graft indefinitely after receiving anergic lymphocytes alone (> 100 days). These results demonstrated that anergic lymphocytes with regulatory activities can be generated through blocking co-stimulatory signals, CD28 and ICOS, simultaneously in vitro, and may advance a new immunomodulatory strategy for preventing allorejection in organ transplantation.

Long-Term Acceptance of Fully Allogeneic Cardiac Grafts by Cotransplantation of Vascularized Thymus in Miniature Swine

We have previously reported the ability of both thymokidney and vascularized thymic lobe (VTL) allografts to induce transplantation tolerance to renal allografts across a full major histocompatibility complex (MHC) mismatch in thymectomized miniature swine. However, whether vascularized thymus is capable of inducing tolerance to less tolerogeneic organs when it is transplanted simultaneously is not yet known. The present study investigates cardiac allograft survival and the mechanism of long-term acceptance in recipient swine following cotransplantation of VTL and cardiac grafts from fully MHC-mismatched donors. Animals received a heart graft, a heart graft and a VTL, or a heart graft and a donor thymocyte infusion. Immunosuppressive regimens consisted of 12 or 28 days of tacrolimus. All animals that received a VTL maintained their grafts significantly longer than their counterparts that received only a heart graft, and those receiving 28 days of tacrolimus maintained their heart grafts long-term. Recipients of a donor thymocyte infusion demonstrated slightly prolonged cardiac graft survival but all rejected their grafts, highlighting the importance of thymic stroma. Cytotoxic T-lymphocyte responses against third-party antigens by cells from tolerant animals showed restriction by both self and donor MHC, whereas responses of controls were restricted to self MHC only. The presence of donor dendritic cells in the VTL grafts and results of co-culture assays suggest that both central and regulatory mechanisms were involved in achieving long-term acceptance. This is the first demonstration of the long-term acceptance of fully MHC-mismatched cardiac allografts in large animals.

Induction of immune tolerance with heart-thymus composite allotransplantation in rats

Objective To study on the role of thymus transplantation for heart allograft in rats. Methods Vascularized heart-thymus combined transplantation was performed with microsurgical technique. Graft survival, histopathology, level of IL-2, IL-4 and its mRNA expression in serum and cardiac grafts were investigated. Results Heart-thymus combined transplantation achieved effect in the prolongation of cardiac graft survival with short-term administration of cyclosporine. Conclusions Vascularized thymus transplantation induced immune tolerance in thymectomized rats.

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

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

Recombinant dimeric MHC antigens protect cardiac allografts from rejection and visualize alloreactive T cells

Monomeric and dimeric soluble major histocompatibility complex (MHC) molecules down-regulate activated T cells in an antigen-specific manner in vitro. This property could be exploited to modulate alloresponses in vivo but has remained difficult to demonstrate. Here, intraperitoneal infusion of a Lewis-derived rat MHC class I molecule, RT1.A(l)-Fc, in Dark Agouti (RT1.A(a)) recipient rats prolonged cardiac graft survival, which led to permanent engraftment. This effect was mediated by T cell impairment of target cell lysis by CD8+ T cells and down-regulation of interferon-gamma production by CD4+ T cells. The binding of the dimeric MHC allowed ex vivo visualization of alloreactive T cells in peripheral blood, splenocytes, and allografts, revealing low frequency of alloreactive CD8+ T cells after establishment of permanent engraftment of cardiac allografts. Thus, these data show the potential of dimeric MHC molecules to promote graft survival and allow visualization of alloreactive T cells.

CCR4‐deficient mice show prolonged graft survival in a chronic cardiac transplant rejection model

Chronic graft rejection mediated by cellular immune responses still poses a serious clinical problem in transplant surgery. Chemokines coordinate the recruitment of leukocytes in inflammatory and immune responses. Their precise functions in the rejection of allografts are still ill defined. This study investigates the role of chemokine receptor 4 (CCR4) in acute and chronic cardiac allograft rejection in mice. Allogeneic hearts were transplanted into CCR4 deficient (CCR4(-/-)) and control recipients. Reverse transcription-PCR showed transcription of macrophage-derived chemokine and thymus and activation-regulated chemokine, the cognate chemokine ligands of CCR4, within the graft. Compared to wild-type controls, acute allograft rejection in CCR4(-/-) recipients was only slightly prolonged. In contrast, in a gallium nitrate chronic cardiac allograft rejection model, cardiac graft survival was significantly prolonged in CCR4(-/-) recipients. A relative increase in the percentage of graft infiltrating CD8(+) T cells in CCR4(-/-) recipients was observed 30 days after transplantation and was accompanied by a decrease in CD4(+) T cells. Moreover, the percentage of NK1.1(+)CD3(+) graft-infiltrating cells was significantly reduced on day 5 and day 30 post transplantation. These findings indicate that CCR4 is involved in the recruitment of NK1.1(+)CD3(+) cells into cardiac allografts and clearly establish an important and novel role for CCR4 in chronic graft rejection.

The CD28 peptidemimic can induce mixed chimerism and prolong the survival of cardiac allografts

Costimulatory blockade with CD28 peptidemimic (CD28PM, CD28 PM was synthesized by solid phase synthetic methods) prolongs cardiac allograft survival in mice, but has not reliably induced tolerance when used alone. In the current studies, we evaluated the effect of adding B7 blockade to a chimerism inducing nonmyeloablative regimen in mice and observed a significant improvement of donor bone marrow cells (BMC) engraftment, which had been associated with mixed chimerism and long-term survival of cardiac allografts. The mixed lymphocyte reaction (MLR) and the ear pinna cardiac transplantation model were performed to evaluate the effects of CD28PM in induction of specific immune hypo-response and extension of allograft survival. The expressed rates of B7.1 and B7.2 on the C57BL/6 splenocytes were 56.25% and 20.52%, respectively. The specific hypo-response status was established after immunization with CD28PM pre-treated donor splenocytes and the average inhibition rate was only 43% compared with normal control. Subsequently, a total number of 2×10 7 bone marrow cells per mouse were implanted to the recipients. The allogenic chimerism was obviously observed with the rate as high as 8.84% (mean) at the time point of day 14. During the first 50 days post bone marrow transfusion (BMT) the chimerism rate declined stepwise. But from 50 to 100 days, the chimerism rate sustained in a range of 3.35% to 4.6%. The results of transplantation experiments showed the survival of allgenic cardiac grafts were maintained over 100 days in recipients. Thus, donor BMC engraftment with mixed chimerism appears essential for induction of allograft tolerance using this conditioning regimen. Mixed chimerism approach, by the addition of CD28-B7 costimulatory blockade with CD28PM, has been shown to establish mixed chimerism and induce cardiac allograft tolerance in mice.

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.

Immunisation with vimentin causes rejection of syngeneic cardiac grafts

The role of non-HLA antibodies following solid organ transplantation is unclear. In patients, antibodies to the autoantigen vimentin are associated with transplant associated coronary artery disease after cardiac transplantation. However, whether these antibodies are directly damaging or a bystander effect is unknown. Here we have investigated survival of cardiac grafts in mice undergoing and an immune response to vimentin. C57BL/6 mice received two subcutaneous injections (7 days apart) of recombinant vimentin in Freunds incomplete adjuvant, controls received urea in saline. Two weeks after the first injection they received heterotopic heart transplants from syngeneic or allogeneic (129/sv) donors. Hearts were palpated every day to estimate graft survival and rejected hearts were subjected to histological and immunocytochemical analysis. Antibodies to vimentin were quantitated by ELISA. Immunised mice (n = 7) produced signficant IgG anti-vimentin titres at two weeks after immunisation (mean titre 516+/−365, compared to controls 5.75+/−6.3, p = 0.001). The survival of syngeneic hearts in saline/urea immunised mice was 100% at 32 days (n = 7) whereas the mean survival time of the syngeneic heart in vimentin immunised mice (n = 7) was 14.1+/−4.3 days (p = 0.0007). Histology of the mice’s own hearts demonstrated no effects of vimentin immunisation. The mean duration of allogeneic grafts in saline immunised and vimentin immunised mice was not different (at 7.4 and 7.0 days respectively). Histology of the rejected hearts showed myocyte necrosis, vascular thrombosis and a mixed cellular infiltrate. Immunocytochemistry showed no T cells in the syngeneic grafts. Immunoglobulin from vimentin immunised but not control mice bound to cultured C57BL/6 mouse endothelial cells treated with hydrogen peroxide (91.7% vs 22.8% ) but not to normal endothelial cells (3.5% vs 3.5%). In conclusion, the results suggest that the autoimmune response to vimentin is potentially damaging and that surgical manipulation maybe sufficient to sensitise the grafts to autoimmune damage.

Allogeneic chimerism induced by B7 antisense peptide pre-treated splenocytes prolongs the survival of allograft in mice

To investigate the roles of B7 antisense peptide (B7AP) in blocking the CD28-B7 pathway and inducing the allogeneic chimerism. B7 antisense peptide was synthesized by solid phase synthetic methods and purified with HPLC. The C57BL/6 splenocytes of mice were pre-treated by B7AP, and subsequently injected in travenously to BALB/c mice. Three days later the mice were injected with fresh-made bone marrow cells derived from C57BL/6 mice. The B7 expression and allogeneic chimerism were analyzed with FACS. The lymphocyte proliferation reaction and the mice pinna cardiac transplantation model were exerted to study the relation between chimerism and prolongation of allograft in vitro and in vivo. Lymphoproliferation of the splenocytes derived from BALB/c mice immunized with the B7AP pretreated C57BL/6 splenocytes versus splenocytes from C57BL/6 mice was inhibited dramatically with a inhibition rates up to 43%. Under this condition, the allogeneic chimerism was successfully induced after BMT. Both the chimerism and the survival of allogeneic cardiac grafts were prolonged over 100 days (n = 6). Synthetic B7 antisense peptide can induce allogeneic chimerism in mice and consequently prolong the survival of allogeneic cardiac grafts.

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.

Infusion of in vitro–generated DN T regulatory cells induces permanent cardiac allograft survival in mice

Previously, we have demonstrated that pretransplant donor lymphocyte infusion (DLI) can activate recipient-derived CD3 +CD4 −CD8 − double negative T regulatory (DN Tr) cells which have a potent immune regulatory function in vitro and in vivo. Here we studied the regulatory ability of DN T cell clones generated from the spleens of naı̈ve anti-L d transgenic TCR + (2C×dm2) F1 mice. We were able to identify subsets of DN T cell clones that were able to kill anti-L d CD8 + T cells, and therefore had regulatory properties, and DN T cells with no regulatory properties. Next, we investigated the ability of these in vitro generated DN T cell clones to enhance cardiac allograft survival. (2C×dm2) F1 transgenic mice were infused with either regulatory or non-regulatory DN T cell clones, or left untreated one day before receiving an L d-mismatched cardiac grafts from (C57BL/6×Balb/c) F1 mice. Injection of non-regulatory DN T clone cells did not prolong cardiac graft survival in (2C×dm2) F1 mice when compare to untreated controls. In contrast, all of the cardiac grafts survived more than 100 days in mice that received DN Tr clone cells prior to transplantation. These results demonstrate that DN Tr cells can be generated in vitro and protect cardiac allograft from rejection when infused into recipients prior to transplantation. They also suggest that DN Tr cells may provide a novel therapy for the treatment of allograft rejection.