ACI Recipients Research Articles

Coinhibition of mTORC1/mTORC2 and RhoA /ROCK pathways prevents chronic rejection of rat cardiac allografts

Chronic rejection of transplanted organs remains an unresolved issue in clinical organ transplantation. The macrophages play a crucial role in the development of chronic rejection. We showed previously that macrophage-specific deletion of RhoA or RhoA/ROCK inhibition prevent macrophage movement to the cardiac allografts and abrogate chronic rejection in rodent transplantation models. Here we assessed the ability of the mTORC1 and mTORC2 inhibitor everolimus, alone or in conjunction with the RhoA/ROCK inhibitor Y27632, to inhibit chronic rejection of Wistar Furth (WF; RT1.Au) rat cardiac allografts heterotopically transplanted into ACI (RT1.Aa) recipients. The transplanted hearts were analyzed for vessel occlusion and tissue fibrosis. T cell and macrophage subsets infiltration was analyzed by immunostaining with T cell and macrophage molecular markers. We found that a combination of the mTOR inhibitor everolimus and the RhoA/ROCK inhibitor Y27632 prolonged allograft survival; decreased vessel occlusion, collagen deposition, and macrophage infiltration; and inhibited the chronic rejection of rat cardiac allografts. These results indicate that coinhibition of the mTORC1/mTORC2 and RhoA pathways in transplant recipients abrogates chronic rejection of rat cardiac allografts, and will aid in the development of clinically applicable anti-chronic rejection therapies.

Mesenchymal stem cells prolong the survival of orthotopic liver transplants by regulating the expression of TGF-β1.

Recent studies have shown that transforming growth factor-β1 (TGF-β1) is prominently associated with acute rejection. This study aimed to explore the role of mesenchymal stem cells (MSCs) in the maintenance of the long-term survival of orthotopic liver transplants (OLTs) via the regulation of TGF-β1 in an experimental rat model. We used Lewis rats as donors and ACI rats as recipients. Hematoxylin and eosin staining was performed to evaluate histomorphological changes, and Western blot was performed to measure protein expression. The expression of TGF-β1 in the liver allografts and spleen and protein levels of forkhead box P3 (FoxP3), interleukin-10 (IL-10), and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) were measured using Western blot. The suppressive capacity of CD4+CD25+ regulatory T cells was evaluated using the MTT assay. Cell-mediated immunotoxicity was evaluated using the mixed lymphocyte reaction of CD4+ T cells and cytotoxic T lymphocyte (CTL) assay of CD8+ T cells. The results showed that MSCs prolonged the survival of the OLT mice by regulating the expression of TGF-β1 at different time points. The administration of MSCs promoted a prolonged survival in the ACI recipients (105±6.6 d) compared with the MSC-untreated recipients (16.2±4.0 d). On the postoperative day (POD) 7, the MSC-treated recipients showed a significantly higher expression of TGF-β1, FoxP3, IL-10, and CTLA-4 than the MSC-untreated recipients. However, on POD 100, the MSC-treated recipients showed a lower expression of TGF-β1 and FOxP3 than that on POD 7. Moreover, on POD 7, CD4+CD25+ regulatory T cells extracted from the MSC-treated recipients showed a higher expression of FoxP3, IL-10, CTLA-4, and suppressive capacity. On POD 7, CD4+ T cells from the MSC-treated recipients showed more significantly diminished proliferative functions than the MSC-untreated recipients; further, a reduced allospecific CTL activity of CD8+ T cells was observed in the MSC-treated recipients. MSCs may represent a promising cell therapeutic approach for inducing immunosuppression or transplant tolerance.

Abrogation of Chronic Rejection in Rat Model System Involves Modulation of the mTORC1 and mTORC2 Pathways

Current immunosuppressive regimens fail to avert chronic rejection (CR) of transplanted organs; however, selective targeting of actin-cytoskeletal regulators decreases T-cell motility and abrogates CR in rat model system. Administration of mutated class I major histocompatibility complex molecules or selective targeting of the RhoA pathway, which controls T-cell cytoskeletal activity, using Y27632 (a selective Rock1 inhibitor) resulted in reduced T-cell infiltration and abrogation of CR as judged from the neointimal index (13.9±19.7 vs. 45±37.5; P<0.001) and the number of affected vessels (30% vs. 60%; P<0.01). Here, we examined the role of mammalian target of rapamycin (mTOR) pathway in inhibition of CR. A mutated class I major histocompatibility complex molecule that eliminates CR was delivered into ACI recipients of Wistar-Furth hearts at the time of transplantation with subtherapeutic cyclosporine (10 mg/kg on days 0-2). Controls included untreated and cyclosporine A-treated (10 mg/kg on days 0-2) heart allograft recipients. Western blotting and immunostaining showed that rat heart allografts with abolished CR exhibited down-regulation of the RAPA-sensitive mTORC1 components such as mTOR and Raptor and down-regulation of the RAPA-insensitive mTORC2 elements Rictor and Sin1. The mTOR regulator Deptor and its downstream target Rac1 were also inhibited. Abrogation of CR in rat model system involves modulation of two mTOR pathways: a RAPA-sensitive mTORC1 pathway regulating cellular proliferation and a RAPA-insensitive mTORC2 pathway regulating T-cell motility. Selective targeting of T-cell actin cytoskeletal pathways shows potential for pathway-targeted immunosuppression therapies.

Rock1 Inhibitor Abrogates Chronic Rejection in Rat Cardiac Model System

Background: We have shown previously that the abrogation of acute and chronic rejection of rat cardiac allografts occurs through the down-regulation of RhoA pathway and involves the changes in RhoA kinase (ROCK)-dependent actin cytoskeleton and T cell motility. Here we studied the ability of the Y-27632, a highly selective inhibitor of Rho-associated protein kinase p160ROCK (ROCK1), to abrogate chronic rejection of the allograft and influence T cell infiltration. Methods: Heterotopic cardiac transplants were performed between donor Wistar Furth (WF) and ACI recipient rats. Controls received sub-therapeutic dose of cyclosporine (CsA, 10 mg/kg) for 3 days or 7 days therapeutic dose of cyclosporine. The experimental groups of ACI recipient received one preoperative dose of the Y-27632 inhibitor (2 mg/kg, gavage feed) in conjunction with the sub-therapeutic dose of CsA for 3 days or inhibitor alone for 7 days. The cardiac grafts were harvested at 100 days of post-transplantation for histological and immunohistochemical assessment of chronic rejection, vascular sclerosis, and infiltration by different T cell subtypes. Results: Cardiac allografts from recipients exposed to Y-27632 inhibitor in conjunction with sub-therapeutic dose of CsA showed drastically reduced vascular sclerosis, minimal myocardial total cellular infiltration, and were selectively infiltrated with Foxp3+ T regulatory (Treg) cells. Conclusions: Our novel finding that a single dose of the ROCK1 inhibitor Y-27632 attenuates chronic rejection in rat cardiac model system by promoting development of Treg cells warrants its potential as a novel therapeutic agent specific for the inhibition of chronic rejection.

Soluble donor-like MHC class I proteins induce CD4 +CD25 +CD8 −FoxP3 + cells with potential to ameliorate graft chronic injury

Conventional immunosuppressive therapies failed to prevent allograft chronic rejection. New approaches to modulate recipient immune response are needed. Donor-like MHC class I soluble proteins demonstrated therapeutic potential to suppress chronic rejection. The present study was designed to clarify the ability of MHC class I soluble proteins to induce T regulatory cells with true regulatory potential in a fully allogeneic rat cardiac transplant model. Donor-like MHC class I proteins upregulate small population of splenic CD8 − negative CD4 +CD25 +FoxP3 + positive cells. CD4 + splenocytes after MHC therapy suppress lymphocyte proliferation against donor antigens in vitro. ACI recipients of WF hearts treated with CD4 + cells, induced with donor-like MHC class I proteins (CD4-MHC), demonstrated stable survival of the transplanted organ (MST > 120 days; n = 17). Histology revealed that grafts of recipients treated with CD4-MHC had 23.6% vessels affected 100 days postgrafting. On the contrary, hearts obtained from long-term surviving hosts treated with CD4 + cells induced with high-dose CsA (CD4-CsA) had 50–70% of affected vessels. CD4-MHC class I treated transplants were mostly CD3 − negative, had low level of mast and FoxP3 + cell infiltration compared to CD4-CsA treated hearts. Intragraft CD4 + cells were close to mast cells in morphology. The same graft tissues had similar number of CD4 + positive cells and mast cells suggesting existence of CD4 + positive mast cells. On the other hand, a negligible number of FoxP3 + positive cells in the grafts after CD4-MHC treatment supports the idea of CD4 + positive FoxP3 + negative mast cells population. We demonstrate that donor-like MHC class I protein therapy induces population of CD4 +CD25 +CD8 −FoxP3 + cells with potential to ameliorate development of transplant vascular disease and evoke CD4 + positive FoxP3 negative mast cells in the secondary hosts.

Allochimeric MHC I-Conditioned T Cells Attenuate Chronic Rejection in Rat Cardiac Model System

Background: We have shown previously that the mutated class I MHC molecules abrogate acute and chronic rejection and attenuate transplant vascular sclerosis (TVS) through the early changes (1-7 days post-transplantation) in T cell and dendritic cell molecular response. Here we studied a cohort of long-term (100 days) graft survival recipients for changes in T cell molecular response, and the role of regulatory T cells in the abrogation of chronic rejection in adoptive transfer experiments. Methods: Heterotopic cardiac transplants were performed between donor Wistar Furth (WF) and ACI recipient rats. Controls received no treatment or 6 days therapeutic dose of cyclosporine (CsA 10mg/kg). The experimental group of primary ACI recipient received, peri-operatively, the allochimeric [[?]1h l/u]-RT1.Aa MHC I molecule (1mg/kg) in conjunction with the sub-therapeutic dose of CsA for 3 days. Splenic T cells were isolated from ACI recipient at 100 days post-transplantation and either assessed for changes in the expression of chosen protein markers or, in adoptive transfer experiments; they were injected into lightly irradiated secondary ACI recipients grafted with WF hearts. Secondary cardiac grafts were harvested at 100 days of post-transplantation for assessment of chronic rejection, neointimal index (NI) and apoptosis. Results: Secondary cardiac grafts from recipients exposed to allochimeric MHC I-conditioned splenic total T cells or CD4+ T cells showed significantly reduced NI and apoptosis, and were selectively infiltrated with CD4+Foxp3+ (T regulatory, Treg) cells. Conclusion: Adoptive transfer of allochimeric MHC I-conditioned T cells promotes development of Treg cells and attenuates chronic rejection in rat cardiac model system.

Downregulation of RhoA and changes in T cell cytoskeleton correlate with the abrogation of allograft rejection

Proper actin cytoskeleton architecture and dynamics are indispensable for events in the immunological response such as T cell migration, redistribution of T cell receptors, and interaction with antigen presenting cells. Thus, T cell activation, downstream signaling events and effector functions are all actin-dependent. Actin cytoskeleton architecture and dynamics are regulated by proteins belonging to the superfamily of small GTP-binding proteins, such as RhoA GTPase. We previously showed that the administration of an MHC class I allochimeric molecule [α1h1/u]-RT1.Aa, which contains donor-type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient-type (ACI, RT1a) sequences, to the ACI recipient of heterotopic WF heart resulted in the restriction of the TCR repertoire, inhibition of T cell infiltration into the heterotopic cardiac allografts, abrogation of acute and chronic rejection, and induction of indefinite survival of the allograft. Here we show that the allochimeric molecule treatment caused downregulation of RhoA GTPase in T cells. This resulted in dramatic changes in the distribution of actin and the actin-binding protein, Hip55, in these cells, which in turn, inhibited T cell infiltration into the graft. This indicates that the immunosuppressive activity of the allochimeric molecule is achieved via downregulation of the RhoA pathway and disruption of the proper organization of T cell actin cytoskeleton to inhibit T cell functions such as motility and/or TCR signaling events.

Intensity of transplant chronic rejection correlates with level of graft-infiltrating regulatory cells

The understanding of chronic rejection (transplant vascular sclerosis, or TVS) mechanisms is a major goal of transplantation. In this study we tested a cardiac transplant model for TVS development in connection with emerging T-regulatory cells (T-regs). We used 40-mer peptides derived from the donor MHC Class I alpha1 helix of the alpha1-domain to make recipients tolerant. ACI recipients were transplanted with either RT1.A(u) (WF), RT1.A(l) (LEW), RT1.A(c) (PVG), or RT1.A(b) (BUF) cardiac grafts. The grafts were analyzed 120 days later for TVS and development of T-regs. Donor MHC peptides were injected through the portal vein (0.1 mg) into ACI recipients of WF hearts in addition to sub-therapeutic cyclosporine (CsA, 10 mg/kg for 3 days post-operatively). Peptide treatment specifically prolonged graft survival for >100 days (n = 31). ACI recipients of WF or LEW hearts treated with PVG peptides promptly rejected the transplanted grafts (15 +/- 4 and 20 +/- 1 days, respectively). Presence of T-regs in tolerant recipients was confirmed by the adoptive transfer of T cells into a new cohort of syngeneic recipients (mean survival time [MST] >100 days, n = 3). CD4(+) and FoxP3(+) cells were detected in 70% of the chronically rejected grafts vs 38% (CD4) and 22% (FoxP3) in the well-preserved transplants. IgG and IgM deposits were found in only half of surviving cardiac grafts with a high level of TVS. Blood vessels in grafts with attenuated TVS were 80% IgG and IgM positive. Interleukin (IL)-4 and IL-2 were markedly down-regulated in the hearts with high TVS compared with well-preserved grafts. Long-term-surviving hearts demonstrated increased IL-10 expression. Interferon-gamma (IFN-gamma) was more evident in the grafts with a high TVS. Donor MHC Class I peptides can specifically prolong transplant survival and generate T-regs. The level of intragraft T-regs correlates with severity of TVS and IL-2/IL-4 down-regulation.

Intragraft gene expression profile associated with the induction of tolerance by allochimeric MHC I in the rat heart transplantation model

The MHC class I allochimeric protein containing donor-type epitopes on recipient-type heavy chains induces indefinite survival of heterotopic cardiac allografts in rats. We analyzed gene expression profile of heart allograft tissue. Mutated peptide [alpha1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient-type (ACI, RT1a) was delivered into ACI recipients of WF hearts at the time of transplantation in addition to a 3 days course of oral cyclosporine. Microarray analysis was performed using Affymetrix Rat 230 2.0 Microarray. Allochimeric molecule treatment caused upregulation of genes involved in structural integrity of heart muscle, downregulation of IL-1beta a key modulator of the immune response, and downregulation of partitioning defective six homolog gamma PAR6, which is involved in T cell polarity, motility, and ability to scan dendritic cells (DC). These indicate that the immunosuppressive function of allochimeric molecule and/or the establishment of allograft tolerance depend on the induction of genes responsible for the heart tissue integrity, the suppression of cytokine pathway(s), and possibly the impairment of T cells mobility and their DC scanning ability. These novel findings may have important clinical implications for inhibition of chronic rejection in transplant recipients.

Intragraft selection of the T cell receptor repertoire by class I MHC sequences in tolerant recipients.

BackgroundAllograft tolerance of ACI (RT1a) recipients to WF (RT1u) hearts can be induced by allochimeric class I MHC molecules containing donor-type (RT1Au) immunogenic epitopes displayed on recipient-type (RT1Aa) sequences. Here, we sought the mechanisms by which allochimeric sequences may affect responding T cells through T cell receptor (TCA) repertoire restriction.Methodology/Principal FindingsThe soluble [α1h u]-RT1.Aa allochimeric molecule was delivered into ACI recipients of WF hearts in the presence of sub-therapeutic dose of cyclosporine (CsA). The TCR Vβ spectrotyping of the splenocytes and cardiac allografts showed that the Vβ gene families were differentially expressed within the TCR repertoire in allochimeric- or high-dose CsA-treated tolerant recipients at day +5 and +7 of post-transplantation. However, at day 30 of post-transplantation the allochimeric molecule-treated rats showed the restriction of TCR repertoire with altered dominant size peaks representing preferential clonal expansion of Vβ7, Vβ11, Vβ13, Vβ 14, and Vβ15 genes. Moreover, we found a positive correlation between the alteration of Vβ profile, restriction of TCR repertoire, and the establishment of allograft tolerance.ConclusionsOur findings indicate that presentation of allochimeric MHC class I sequences that partially mimic donor and recipient epitopes may induce unique tolerant state by selecting alloresponsive Vβ genes.

Microvascular Thrombosis and Cardiac Allograft Vasculopathy in Rat Heart Transplantation

The role of a hypercoagulable microvasculature in the development of cardiac allograft vasculopathy (CAV) after heart transplantation in humans is not well understood. The aim of this study was to identify an animal model by which to further evaluate the role of coagulation in the pathogenesis of CAV. Adult male PVG (RT-1(c)) rats were transplanted into ACI (RT-1(av1)) recipients (n = 29). ACI donors into ACI recipients (n = 31) and rats with a sham operation (n = 33) served as controls. All rats received cyclosporine (10 mg/kg/day) on Days 0 to 9 after surgery. Grafts and native hearts were harvested at 10 days to 3 months after surgery. Hearts were processed for immunohistochemistry and light microscopy. A hypercoagulable microvasculature was defined as presence of microvascular fibrin and capillary antithrombin. CAV was defined as the presence of concentric intimal proliferation and chronic inflammatory infiltrate in the arterial intima, and assessed by computer-assisted image analysis. Donor and recipient hearts from PVG-ACI rats showed high levels of fibrin (donors 7.5% to 21.9%, recipients 5.1% to 20.2%) and antithrombin (donors 5.2% to 27.9%, recipients 3.3% to 20.8%) at 10 days to 3 months post-transplant. ACI-ACI donor and recipient hearts had lower deposition of fibrin (donors 0.9% to 9.9%, recipients 0% to 4.0%) and antithrombin (donors 1.4% to 15.2%, recipients 0.8% to 4.5%). Hearts from sham-operated rats had negligible amounts of fibrin (0% to 1.5%) and antithrombin (0% to 2.8%). There was a strong association (p < 0.001) between presence of fibrin and capillary antithrombin and development of CAV. A hypercoagulable microvasculature in a rat model of heart transplantation was associated with development of CAV, as found in humans.

Identification of Early Tolerance Regulator Genes Induced by Allochimeric Therapy Using Microarray-Based Genomewide Scan

We have demonstrated that peri- or postoperative delivery of allochimeric [a 1h u]-RT1.A a class I major histocompatibility complex molecules with donor-type (RT1A u) immunogenic epitopes presented in recipient-type (RT1A a) sequences induced donor-specific tolerance in ACI (RT1 a) recipients of WF (RT1 u) heart allografts. A genomic scan during the early posttransplant period was performed to elucidate the underlying operative mechanisms. A rat genome study after transplantation was carefully designed using Affymetrix Rat Genome 230 2.0 Array. The allochimeric treatment group is 3-day cyclosporine (CsA)-treated ACI recipients that accepted Wistar Furth RT1 u cardiac allografts with postoperative dosage of allochimeric molecules, while the control is 3-day CsA-treated ACI recipients of WF cardiac allografts. All the samples were harvested 5 days after heart transplant as the early stage of tolerance detection. Following array data normalization and modeling, we compared the above two treatment groups and identified a total of 250 tolerance regulator genes induced by allochimeric molecules only.

Donor MHC Class I Peptides in Conjunction With Self-Epitopes Induce Donor-Specific Tolerance in a Dose-Dependent Manner But Unable to Abrogate Chronic Rejection

Understanding specific tolerance mechanisms is a primary goal of transplantation science. We have previously shown that hosts treated with MHC class I protein have donor sequences in the α 1-helix of the α 1 domain on a background of self-epitopes, resulting in the development of donor-specific tolerance. However, the nature of class I alloantigenic determinants that regulate the alloimmune response remains unclear. The α 1-helical sequence of RT1.A, l which shares RT1.A u sequences, was substituted in the RT1.A a molecule to produce the composite [α 1h l/u]-RT1.A a MHC class I allochimeric molecule. Immunodominant epitopes were identified within the hypervariable region of the α 1 domain of RT1.A a (ACI), RT1.A l (Lewis, LEW), and RT1.A u (Wistar Furth [WF]). To clarify the mechanisms of tolerance development through presentation of donor-type immunogenic epitopes and cryptic self-epitopes we used synthetic peptides corresponding to donor immunogenic determinants with peptides derived from recipient self-sequences (RT1.A a—aa 10 to 49 P1 and 91 to 120 P3; and P2 RT1.A l/u 50 to 90). ACI recipients of LEW and WF cardiac allografts were injected through the portal vein (PV) at day 0 with four doses (2, 0.5, 0.25, and 0.125 mg/rat) of three peptide mixtures in conjunction with subtherapeutic CsA (10 mg/kg for 3 days). Allograft survival was strongly dose-dependent. Only low-dose regimens were consistent in tolerance induction, but such therapy did not abrogate development of chronic rejection (CR), unlike allochimeric therapy with soluble MHC class I protein. Different effects of protein or synthetic peptide therapies on development of CR suggest that development of specific tolerance is an active immunologic process and it depends on the form of allogeneic epitopes presented.

Development and Maintenance of Donor-Specific Chimerism in Semi-Allogenic and Fully Major Histocompatibility Complex Mismatched Facial Allograft Transplants

The clinical application of composite tissue allograft transplants opened the discussion on the restoration of facial deformities by allotransplantation. We introduce a hemifacial allograft transplant model to investigate the rationale for the development of operational tolerance across a major histocompatibility complex (MHC) barrier. Thirty rats were studied in five groups of six animals each. The composite hemiface isograft transplantations were performed in group 1. Allograft rejection controls included semi-allogenic transplantations from LBN (RT1(1+n) donors (group 2) and fully allogenic transplantations from ACI (RT1a) donors (group 3) to LEW (RT1(1)) recipients. In the allograft treatment groups, recipients of LBN (group 4) and ACI donors (group 5) were treated with cyclosporine A monotherapy (16 mg/kg/day, tapered to 2 mg/kg/day). Face allografts were evaluated clinically and histologically. Donor-specific chimerism for MHC class I RT1n and RT1a antigens was assessed by flow cytometry. Mixed lymphocyte reaction for donor-specific tolerance in vitro was tested at day 160 posttransplant. Isograft controls survived indefinitely. All nontreated allografts rejected within 5 to 8 days posttransplant. Long-term survival was achieved in 100% of LBN (up to 400 days) and ACI (up to 330 days) recipients. At day 160, posttransplant donor-specific chimerism was present in recipients of LBN (10.14% CD4/RT1n, 6.38% CD8/RT1n, 10.02% CD45RA/RT1n) and ACI (17.54% CD4/RT1a, 9.28% CD8/RT1a) transplants, and mixed lymphocyte reaction confirmed tolerance in recipients of LBN transplants and moderate reactivity in recipients of ACI allografts. Operational tolerance was induced in hemiface allograft transplants across an MHC barrier under cyclosporine A monotherapy protocol. It was associated directly with the presence of multilineage donor-specific chimerism.

Infectious tolerance mediated by CD8 + T-Suppresor Cells After UV-B–irradiated donor-specific transfusion and rat heart transplantation

Aims CD8+CD28− human T-suppressor cells (Ts), which can be generated in vitro, act directly on APC rendering them tolerogenic to unprimed and primed CD4+ T cells. The aim of this study was to investigate the possibility that CD8+ T cells mediate the induction of tolerance in a heart transplantation model in rodents. Materials and methods Blood from Lewis rats was UV-B–irradiated and transfused into ACI recipients on days -21, -14, and -7 before heart allograft transplantation on day 0. CD4 + and CD8 + T cells were positively selected from ACI rats, which had tolerated Lewis heart allografts for more than 100 days and were adoptively transferred to naïve ACI rats pretreated (day -1) with gamma irradiation. These ACI rats underwent transplantation with Lewis hearts 24 hours after adoptive transfer of putative T-suppressor cells. Results Adoptive transfer of CD8 + T cells from tolerant ACI to naïve ACI rats significantly prolonged Lewis heart mean allograft survival time (MST ± SD) to 69 ± 13 days as compared with 15 ± 1 and 14 ± 1 days in animals adoptively transferred with CD4+ T cells or untreated controls, respectively ( P < .001). Similarly, adoptive transfer of CD8 + T cells from secondary ACI recipients to naïve syngeneic animals also significantly prolonged survival of heart allografts to MST ± SD of 72 ± 4 for CD8 + and 15 ± 4 days for CD4 + T cells ( P < .001). Conclusions These data demonstrate that allogeneic tolerance induced in ACI recipients by treatment with UV-B–irradiated blood from Lewis donors is mediated by CD8+ T-suppressor cells.

Facial tissue allograft transplantation

A hemifacial allograft transplant model was used to investigate the rationale for development of functional tolerance across an MHC barrier. Thirty hemiface transplantations were performed in five groups of six Lewis (RT1 1) rat recipients each. Isografts were performed in group 1. Transplants were obtained from semiallogenic LBN(RT1 1+n) in group 2 and from fully allogenic ACI(RT1 a) in group 3 donors, which served as allograft rejection controls. Group 4 grafts using LBN donors and group 5 using ACI donors in addition received CsA monotherapy (16 mg/kg/d for 1 week) and maintained at 2 mg/kg/d. Signs of graft rejection were sought daily. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 8 days posttransplant. Eighty-three percent of face-transplant recipients from LBN donors and 67% from ACI donors did not show any signs of rejection up to 270 days and 200 days, respectively. Flow cytometry at day 63 in LBN recipients showed the presence of donor-specific chimerism for MHC class I RT1 n antigens, namely 3.39% CD4/RT1 n; 1.01% CD8/RT1 n T-lymphocytes; and 3.54% CD45RA/RT1 n B-lymphocytes. In ACI recipients the chimerism test revealed 10.55% CD4/RT1 a and 4.59% of CD8/RT1 a T-lymphocytes. MLR assay at day 160 posttransplant revealed suppressed responses against LBN donor antigens in group 4, but moderate reactivity to ACI donor antigens in group 5. Functional tolerance toward hemifacial allograft transplants induced across MHC barrier using a CsA monotherapy protocol was associated with the presence of donor-specific chimerism in T- and B-cell subpopulations.

Rat CD8 + FOXP3 + T suppressor cells mediate tolerance to allogeneic heart transplants, inducing PIR-B in APC and rendering the graft invulnerable to rejection

Human CD8 + FOXP3 + T suppressor cells (T S) were previously shown to induce the expression of the inhibitory receptors, Immunoglobulin-like transcript (ILT) 3 and ILT4 on dendritic and endothelial cells, rendering them tolerogenic to allogeneic T cells. We have demonstrated the importance of CD8 + T S in a rat model of heart allo-transplantation. Tolerance was induced in ACI recipients by multiple transfusions of UVB-irradiated blood from Lewis heart donors. CD8 + T cells from tolerant ACI rats expressed FOXP3, transferred tolerance to naïve secondary hosts and induced the upregulation of the inhibitory receptor, paired immunoglobulin-like receptor (PIR)-B, an ILT4 orthologue, in Lewis dendritic cells (DC) and heart endothelial cells (EC). When long-term surviving Lewis heart allografts with PIR-B + EC were retransplanted from a primary to a secondary ACI recipient they did not elicit rejection. This study focuses attention on the need to develop agents that act directly on graft EC in order to achieve tolerance.

Superoxide Dismutase Mimetic M40401 Reduces Ischemia-Reperfusion Injury and Graft Coronary Artery Disease in Rodent Cardiac Allografts

The oxidative stress associated with ischemia-reperfusion (I/R) of cardiac allografts leads to production of injurious cytokines and expression of proinflammatory adhesion molecules. This is one of the most important alloantigen-independent factors associated with graft coronary artery disease (GCAD). M40401 is a newly developed cell permeable superoxide dismutase mimetic, which has been shown to scavenge superoxide anion with highly specific and enhanced catalytic activity. We hypothesized that M40401 would exert a protective effect in I/R injury of cardiac allografts and ameliorate the progression of GCAD. Recipient ACI rats were pretreated with M40401 or vehicle control. PVG donor hearts were heterotopically transplanted into the abdomen of ACI recipients. Cardiac allografts were analyzed for adhesion molecule mRNA expression and tumor necrosis factor-alpha expression after 4 hr of reperfusion. Neutrophil infiltration was detected by myeloperoxidase activity. Intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1 mRNA were detected by reverse-transcriptase polymerase chain reaction. Immunohistochemical analysis of adhesion molecule expression was also performed. Additional grafts were procured 90 days after transplantation and assessed for the development of GCAD by computer-assisted image analysis. In the M40401-treated group, adhesion molecule expression was significantly less than in the vehicle control group. Treated grafts also had lower myeloperoxidase activity and tumor necrosis factor-alpha concentration compared with controls. Neointimal proliferation and intima to media ratios in M40401-treated allografts were significantly decreased compared with controls. Selective removal of superoxide anion by M40401 results in inhibition of I/R injury. Furthermore, M40401 treatment decreases the development of oxidative stress-associated GCAD. This treatment strategy may have broad cardioprotective applications for all cardiac operations in addition to cardiac transplantation.

FUNCTIONAL TOLERANCE IN FACIAL TISSUE ALLOGRAFT TRANSPLANTS

O133* Aims: We introduce a hemi-facial allograft transplant model to investigate the rationale for development of functional tolerance across MHC barrier. Materials: Thirty composite hemiface transplantations were performed in 5 groups of 6 rats each. Lewis (RT1l) rats served as recipients in all groups. Isograft transplants were performed in Group 1. Transplants from semi-allogenic LBN(RT1l+n) (Group 2) and fully-allogenic ACI(RT1a) (Group 3) donors served as allograft rejection controls. Group 4 (LBN donors) and Group 5 (ACI donors) received CsA monotherapy of 16 mg/kg/day for 1 week, tapered to 2 mg/kg/day over 4 weeks and continued at this dose level thereafter. Signs of graft rejection were evaluated daily. Flow cytometry evaluated donor specific chimerism for MHC class I RT1n and RT1a antigens at days 21 and 63 post-transplant. Mixed lymphocyte reaction (MLR) for donor specific tolerance was tested at day 160 post-transplant. Histological grading of graft rejection was evaluated after H-E staining. Results: Isograft controls survived indefinitely. All non-treated allografts were rejected within 5 to 9 days post-transplant. 83% of face-transplant recipients from LBN donors and 67% from ACI donors did not show any signs of rejection up to 270 days and 200 days respectively. The rejection signs were reversed by CsA dose adjustments in remaining allografts. Donor specific chimerism at day 63 in LBN recipients showed 3.39% CD4/RT1n, 1.01% CD8/RT1n T-lymphocytes and 3.54% CD45RA/RT1n B-lymphocytes; and in ACI recipients 10.55% CD4/RT1a and 4.59% CD8/RT1a T-lymphocytes. MLR assay at day 160 post-transplant revealed suppressed responses against LBN donor antigens in Group 4, but moderate reactivity to the ACI donor antigens in Group 5. Grade II rejection pattern was established in skin biopsies of rejecting LBN and ACI recipients at days 140 and 180 post-transplant. Conclusion: Functional tolerance was induced in hemifacial allograft transplants across MHC barrier under CsA monotherapy protocol. This was associated with the presence of donor specific chimerism in T- and B-cell subpopulations.

MHC CLASS I ALLOCHIMERIC MOLECULE MODULATES ALLOANTIBODY RESPONSE AND UPREGULATES EXPRESSION OF PROTECTIVE GENES: INFLUENCE ON DEVELOPMENT OF GRAFT ACCOMMODATION

P1028 Aims: Specific transplantation tolerance can be induced in rat recipients treated during peri-transplant period with MHC class I antigens in which 4 donor a.a. are substituted onto host MHC class I sequences. The tolerance characterized with upregulation of cyto-protective and anti-apoptotic genes and accumulation of IgG type alloantibodies in the vessel endothelium. This study was designated to investigate possible influence of this therapy on development of graft accommodation through passive transfer of alloantibodies to a new cohort of recipients. Methods: 1 ml of serum from tolerant ACI rats after allochimeric protein therapy was injected through the portal vein to lightly irradiated (130 Rad) ACI recipients of WF hearts. Grafts were evaluated at 100 days. Results: Passive transfer of sera from rats conditioned with allochimeric therapy, but not from naive rats, to lightly irradiated syngeneic ACI recipients of WF hearts resulted in indefinite survival of cardiac allografts (MST 135+21.9, n=13). Histopathology examination revealed intact myocardial architecture, absent or minimal intramiocardial inflammation, and no evidence of neointimal hyperplasia. The degree of neointimal proliferation and TVS within each vessel was quantitated by the neointimal index (NI). Serum conditioned hosts demonstrated NI of 6.5 with 32% of vessels effected. In contrast, control group receiving low dose radiation in conjunction with naive ACI serum had NI of 39 with 70% of vessels affected. Upregulation of genes for Th2-type cytokines (IL-10 and IL-4) was found in tolerated grafts in comparison to control. FACScan analysis of serum from passive transferred tolerated ACI rats revealed low level of IgM alloantibodies. IgG production was markedly abrogated but noticeably higher than in naive control, IgG isotype analysis showed upregulation for IgG1 and IgG2a alloantibodies. Immunohistology staining of tolerant grafts with anti-IgM and IgG antibodies showed deposits of IgG granules around of internal intima when in control hearts after high doses of CsA such patterns were not revealed. To detect level of apoptotic cells within myocardium of long term survived tolerant grafts in compare to chronic rejected tissue in situ hybridization was performed (TUNEL). Analysis showed decreased number of apoptotic cells in tolerant hosts as compared to controls (7.5+1.2; 13.4+ 2.5, per 5-6 fields, respectively). Conclusions: Allochimeric MHC class I therapy orchestrates the development of donor-specific alloantibodies than can be transferred to new recipient and might cause allograft vascular accommodation, characterized by inhibition of apoptotic cell death, upregulation of cyto-protective genes and IgG antibody binding to vascular endothelium.