Intrathymic Inoculation Research Articles

Immune Suppression Produced by Intrathymic Inoculation With Xenogeneic Antigen and Whole-Body γ-Irradiation in a Pig-to-Monkey Heart Transplantation Model

Objective To validate the use of pig-to-monkey transplantation as an experimental animal model for study of the induction of immune suppression of cardiac xenografts via intrathymic inoculation with xenogeneic spleen cells and whole-body γ-irradiation. Materials and Methods Donors (Meishan pigs) and recipients (Rhesus monkeys) were randomized into 3 groups. Group 2 was the control group; group 2 received whole-body irradiation, and group 3 received whole-body irradiation and intrathymic inoculation. In each group, survival time of heterotopic heart xenografts in monkeys were observed. A mixed lymphocyte reaction assay with 3H thymidine was used to examine changes in lymphocyte function in all groups on the day of transplantation. In each group, recipient serum was harvested for analysis of IL-2 and IL-10 concentrations. Results Mean (SD) survival time of donor hearts in group 3 was significantly longer (91.1 [22.8] hours) than in group 1 (36.6 [5.8] hours) ( P < .01). The results of mixed lymphocyte reaction assay demonstrated a significant reduction in the stimulation index in group 3 compared with groups 1 and 2 ( P < .01) when recipient splenocytes responded to the donor stimulator. The IL-2 concentration in recipients in all groups was much higher during rejection of xenografts than before transplantation ( P < .01). Conclusion These results suggest that pretreatment with intrathymic inoculation or whole-body irradiation induces T-cell immunosuppression. The IL-2 concentration is closely correlated with xenograft rejection.

Activation of Complement in the Discordant Heart Xenotransplantation of Pig-to-Monkey Model and the Impact of Intrathymic Inoculation of Xenogeneic Antigen Combined With Whole-Body γ-Radiation

Objective This study investigated changes in complement in discordant heart xenotransplantation using a pig-to-monkey model as well as the impact of intrathymic inoculation (IT) with xenogeneic antigen combined with whole-body γ-radiation (WBI). Methods In this experiment, pigs and monkeys selected as donors and recipients, respectively, were randomly divided into three groups: a blank group (group A), a whole-body irradiation group (group B) and an irradiation plus intrathymic injection group (group C). In every group, monkeys underwent heterotopic heart xenotransplantation. Results The results showed that the survival of donor hearts in group C was significantly longer than that of group A ( P < .01). In mixed lymphocyte reactions, there was a significant reduction of the stimulation index in group C compared with group A. After xenotransplantation, the level of xenoreactive antibody in group C rose slower than that in group A or group B ( P < .01). After rejection, the levels of CD46 and C3 declined greatly. Conclusions These results suggested that pretreatment with IT and WBI induced T-cell immunosuppression, restraining elicited xenoreactive antibody production of both immunoglobulin M and immunoglobulin G classes. However, it did not hinder complement activation via the classical pathway during hyperacute rejection and consequent xenograft rejection.

Intrathymic inoculation of donor HLA class I–derived peptide generates donor-specific CD4 +CD25 + regulatory T cells

CD4 +CD25 + regulatory T cells are selected in the thymus to control autoreactive thymic escapees preventing autoimmunity that cannot be achieved by negative selection or deletion alone, thus playing an important role in the maintenance of immunological homeostasis. Not only significant in preventing autoimmunity, CD4 +CD25 + regulatory T cells have also been shown to be involved in allograft tolerance in organ transplantation. We have formerly introduced two lines of HLA class I transgenic mice to elucidate the role of HLA class I molecules in transplantation biology. Using a heterotopic cardiac transplantation model, we show herein that intrathymic inoculation of donor HLA class I–derived synthetic peptide results in the generation of CD4 +CD25 + regulatory T cells, which induce graft specific tolerance without any preconditioning of the recipient or use of immunosuppressive drugs. This study provides evidence of the novel therapeutic potential of CD4 +CD25 + regulatory T cells for clinical transplantation.

INTRATHYMIC INOCULATION OF DONOR HLA CLASS I DERIVED PEPTIDE GENERATES DONOR SPECIFIC CD4+CD25+ REGULATORY T CELLS.

Abstract CD4 + CD25 + regulatory T cells are selected in the thymus to control autoreactive thymic escapees preventing autoimmunity that cannot be achieved by negative selection or deletion alone, thus playing an important role in the maintenance of immunological homeostasis. Not only significant in preventing autoimmunity, CD4 + CD25 + regulatory T cells have also been shown to be involved in allograft tolerance in organ transplantation. We have formerly introduced two lines of HLA class I transgenic mice to elucidate the role of HLA class I molecules in transplantation biology. Using a heterotopic cardiac transplantation model, we show herein that intrathymic inoculation of donor HLA class I–derived synthetic peptide results in the generation of CD4 + CD25 + regulatory T cells, which induce graft specific tolerance without any preconditioning of the recipient or use of immunosuppressive drugs. This study provides evidence of the novel therapeutic potential of CD4 + CD25 + regulatory T cells for clinical transplantation.

Fonction thymique et auto-immunité

Purpose. – Thymus is the site of T-cell development and is essential for the induction of self-tolerance, by deletion of autoreactive T lymphocytes (negative selection) and by generation of regulatory T cells. Defect of the selection mechanism of both types of lymphocytes lead to autoimmune diseases. Current knowledge and key points. – Elimination of potentially self-reactive T cells in the thymus requires the intrathymic expression of ubiquitous and “tissue-specific” antigens. Some thymic antigen expressions are dependant on AIRE expression. Mutations in the AIRE gene that are associated with the absence of autoantigen expression in the thymus, defects in the peptide presentation or in apoptosis can allow autoreactive T cells to escape negative selection, and are associated with autoimmune diseases. Recent data are now available concerning the thymic selection of autoreactive regulatory T cells. The Foxp3 gene was recently shown to be predominantly expressed in regulatory T cells and could be a more specific marker of regulatory T cells than phenotypic markers. Future prospects and projects. – Animal models show that regulatory T cells injection or intrathymic inoculation of antigen lead to immunological tolerance in autoimmunity and transplantation. These novel strategies could be used in human.

Induction of thymic tolerance as possibility in prevention of recurrent spontaneous abortion

A major process through which the immune system becomes tolerant to self-proteins involves the deletion of self-reactive cells in the thymus and/or inhibition of specific Th 1 cells clones. Deletion process includes two selection mechanisms in which the thymus eliminates unwanted thymocytes are known as positive selection and negative selection. The thymus is an antigenically privileged site, mainly for it is discrete by blood-thymus barrier. Many researches were shown that intrathymic inoculation of any antigen resulted in specific tolerance induction. The embryo/fetus and placenta are an allograft to which the mother must remain immunologically tolerant in order for the fetus to survive. Today, there is much interest focused on the immunology of recurrent spontaneous abortion (RSA). Up to 50% of RSA may be mediated by the immune system via inadequate maternal anti-paternal response. Nature of this maternal–fetal disturbance represents disbalance in Th 1/Th 2 activity. Contra-shift in Th 1/Th 2 activity is the basis for immunotherapy with paternal leukocyte immunization (PLI). PLI induce some kind of peripheral tolerance on embryonic/fetal/trophoblast antigens, but problems of central tolerance are still open. Intrathymic inoculation of fetal or paternal cells (like leukocyte, thymic dendritic cells, trophoblast cells) or paternal set of MHC molecules may cause central specific tolerance and may be a new possibility for immunotherapy in RSA patients.

CD25+ immunoregulatory CD4 T cells mediate acquired central transplantation tolerance.

Transplantation tolerance is induced reliably in experimental animals following intrathymic inoculation with the relevant donor strain Ags; however, the immunological mechanisms responsible for the induction and maintenance of the tolerant state remain unknown. We investigated these mechanisms using TCR transgenic mice (TS1) that carry T cells specific for an immunodominant, MHC class II-restricted peptide (S1) of the influenza PR8 hemagglutinin (HA) molecule. We demonstrated that TS1 mice reject skin grafts that have transgene-encoded HA molecules (HA104) as their sole antigenic disparity and that intrathymic but not i.v. inoculation of TS1 mice with S1 peptide induces tolerance to HA-expressing skin grafts. Intrathymic peptide inoculation was associated with a dose-dependent reduction in T cells bearing high levels of TCR specific for HA. However, this reduction was both incomplete and transient, with a full recovery of S1-specific thymocytes by 4 wk. Peptide inoculation into the thymus also resulted in the generation of immunoregulatory T cells (CD4+CD25+) that migrated to the peripheral lymphoid organs. Adoptive transfer experiments using FACS sorted CD4+CD25- and CD4+CD25+ T cells from tolerant mice revealed that the former but not the latter maintain the capacity to induce rejection of HA bearing skin allografts in syngeneic hosts. Our results suggest that both clonal frequency reduction in the thymus and immunoregulatory T cells exported from the thymus are critical to transplantation tolerance induced by intrathymic Ag inoculation.

Intrathymic inoculation of donor bone marrow induces long-term acceptance of lung allografts

BackgroundWe investigated whether intrathymic inoculation of donor bone marrow at the time of transplantation induced long-term acceptance of lung allografts. MethodsFour- to-six-week-old August Copenhagen Irish (ACI) and Wistar Furth (WF) rats were used as donors and recipients, respectively. After being inoculated intrathymically with either donor-specific (ACI) or third-party (F344) bone marrow (2.0 × 107 cells/lobe), the recipient (WF) animal received a left lung transplant from an ACI donor. A short course of tacrolimus (1 mg/kg per day for 5 days) was administered. Animals were sacrificed at timed intervals after transplantation, and rejection was graded on a scale of 0 (none) to 4 (severe). ResultsAt 28 days, animals receiving donor-specific bone marrow have lower (p < 0.01) median rejection grade (MRG = 0.25; n = 6) than those receiving third-party bone marrow (MRG = 3; n = 6) and controls (no bone marrow; MRG = 2.5; n = 6). Animals receiving intrathymic donor bone marrow accepted lung allografts up to 380 days with minimal rejection (MRG = 2; n = 6). Long-term lung recipients also accepted a challenging donor-specific heart graft (n = 4) for more than 150 days. In mixed lymphocyte reaction assays, T lymphocytes of WF recipients that had received intrathymic bone marrow (from ACI donor) exhibited low response (similar to self antigens) to donor (ACI) cells, but reacted strongly (five times higher) to third-party (F344) cells. ConclusionsIntrathymic inoculation of donor bone marrow at the time of transplantation along with a short course of tacrolimus induces long-term acceptance of lung allografts in rats. This simple approach of tolerance induction may have clinical application.

Immunotolerance of liver allotransplantation induced by intrathymic inoculation of donor soluble liver specific antigen.

To study the effects of liver specific antigen (LSA) on the immunoreaction of liver allotransplantation and its significance. Orthotopic liver transplantation was used in this study. Group I: syngeneic control (Wistar-to-Wistar); Group II: acute rejection (SD-to-Wistar). Group III: acute rejection treated by intramuscular injection of cyclosporine A (CsA) (SD-to-Wistar+CsA). Group IV: Intrathymic inoculation of SD rat LSA one week before transplantation (LSA+SD-to-Wistar). The common situation and survival time, rejection grades, NF-kappaB activity of splenocytes and intragraft cytokine gene expression were observed to analyze the acute rejection severity and immune state of animals. The common situation of Wistar-to-Wistar group was very good after the transplantation and no signs of rejection were found. Recipients of SD-to-Wistar group lost body weight progressively. All died within 9 to 13 days after transplantation with the median survival time of 10.7+/-0.51 days. It was an optimal control for acute rejection. The common situation of SD-to-Wistar+CsA group was bad during CsA medication but only with mild rejection. As for LSA+SD-to-Wistar group, 5 of 6 recipients survived for a long time and common situation was remarkably better than that of SD-to-Wistar group and SD-to-Wistar+CsA group. Its rejection grades were significantly lower than that of SD-to-Wistar group (P=0.026). Furthermore, no significant discrepancies of rejection were found between SD-to-Wistar group and LSA+SD-to-Wistar group at day7 and day12 (P=0.067). NF-kappaB activity, IFN-gamma and IL-2mRNA expression were significantly inhibited in LSA+SD-to-Wistar group compared with that of SD-to-Wistar group (P<0.05). LSA is an important transplantation antigen which involves in the immunorejection of liver transplantation directly. We reported for the first time that intrathymic inoculation of LSA can induce immnotolerance of liver allotransplantation and grafts can survive for a long time thereby, thus leading to a novel way to liver transplantation immunotolerance.

Vascular endothelial cell apoptosis induced by anti-donor non-MHC antibodies: a possible injury pathway contributing to chronic allograft rejection

Backgound Non–major histocompatibility complex (non-MHC) alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized. Methods The kinetics of alloantibody production and the mechanism by which non-MHC alloantibodies cause graft injury were investigated in a Lewis-to-Fischer 344 (LEW-to-F344) rat model of cardiac transplantation. Results Flow cytometry detected that all the F344 recipients of LEW allografts produced anti-donor immunoglobulin G (IgG) antibodies reactive with LEW lymphocytes and endothelial cells. A sub-group of recipients that rejected their grafts in 30 to 60 days exhibited markedly increased levels of anti-donor IgG antibodies ( n = 6, mean fluorescence intensity [MFI]:23.85 ± 2.7) than recipients with long-surviving allografts ( n = 4, MFI:11.23 ± 0.81; p = 0.00058). Passive transfer of anti-donor sera induced chronic rejection of LEW heart allografts in an immune non-responsiveness model of F344 rats induced by intrathymic inoculation of donor-specific lymphocytes. Immunoglobulin G antibodies purified from the anti-LEW sera exhibited complement-dependent cytotoxicity against LEW vascular endothelial cells in flow-cytometric cytotoxicity assay. The targeted endothelial cells displayed early (annexin V+) and late (TUNEL+) evidence for programmed cell death. Western blot analysis of poly (ADP-ribose) polymerase (PARP) demonstrated that the 25-kD PARP-cleavage fragment was present at the lysates of the vascular endothelial cells treated with anti-donor IgG antibodies, indicating apoptosis-associated caspase activity in these cells. In situ teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining demonstrated that vascular endothelial cell apoptosis was consistently present in all LEW heart allografts with chronic rejection. Conclusions Non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury through an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.

Evolution of adenoviruses as gene therapy vectors.

Evolution of adenoviruses as gene therapy vectors.

Vascular endothelial cell apoptosis induced by non-MHC alloantibodies: an important injury mechanism in the development of chronic graft rejection

Non-MHC alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized. This study investigated the kinetics of antidonor antibody production and the injury mechanism in a LEW-to-F344 rat model of cardiac transplantation, in which donor and recipient strains differ for non-MHC alloantigens. All the F344 recipients of LEW allografts produced antidonor IgG antibodies reactive with LEW endothelial cells (EC). A subgroup of recipients that rejected their grafts in 30-60 days exhibited markedly higher levels of anti-donor IgG antibodies (n=6, MFI:23.85±2.7) prior to graft rejection when compared to those with long-surviving allografts (n=4, MFI:11.23±0.81; P=0.00058). F344-anti-LEW sera were capable of inducing lesions of chronic rejection when passively transferred in an antibody-free heart graft model in which immune non-responsiveness of F344 rats to LEW alloantigens was induced by intrathymic inoculation of LEW lymphocytes. Five of six cardiac allografts exposed to the antisera were rejected in 2-5 weeks after passive transfer and developed severe chronic lesions while control allografts (N=3) treated with normal rat sera survived for > 90 days (p=0.015). IgG antibodies purified from anti-LEW sera exhibited complement-dependent cytotoxicity against LEW EC in culture. The EC displayed early (Annexin V+) and late (TUNEL+) evidence for programmed cell death examined with flowcytometry. Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage showed that an 116 kDa intact form of PARP was degraded into 85 kDa and 25 kDa fragments in lysates of the ECs treated with the IgG, indicating an apoptosis-associated caspase activity. In addition, in situ TUNEL experiment demonstrated that vascular EC apoptosis was consistently present inall the heart allografts examined. We conclude that non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury via an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.

Indirect allorecognition in acquired thymic tolerance: induction of donor-specific permanent acceptance of rat islets by adoptive transfer of allopeptide-pulsed host myeloid and thymic dendritic cells.

Pancreatic islet transplantation remains a promising approach to the treatment of type 1 diabetes. Unfortunately, graft failure continues to occur because of immunologic rejection, despite the use of potent immunosuppressive agents. It is therefore reasoned that induction of peripheral tolerance by the use of self-dendritic cells (DCs) as a vehicle to deliver specific target antigens to self-T-cells without ex vivo manipulation of the recipient is an attractive strategy in the treatment of type 1 diabetes. The finding that intrathymic inoculation of an immunodominant WF major histocompatibility complex (MHC) Class I (RT1.A(u)) peptide five (P5) or P5-pulsed host myeloid DCs induces acquired thymic tolerance raises the possibility that adoptive transfer of allopeptide-primed host myeloid or lymphoid DCs might induce transplant tolerance. To address this hypothesis, we studied the effects of intravenous transfer of in vitro P5-pulsed syngeneic myeloid DCs or in vivo P5-primed syngeneic lymphoid (thymic) DCs on islet survival in the WF-to-ACI rat combination. In vivo primed thymic DCs isolated from ACI rats given intrathymic inoculation of P5 for 2 days were capable of in vitro restimulation of in vivo P5-primed T-cells (memory cells). In the first series of studies, we showed that intravenous-like intrathymic-inoculation of in vitro P5-pulsed host myeloid DCs induced donor-specific permanent acceptance of islets in recipients transiently immunosuppressed with antilymphocyte serum (ALS). We next examined whether thymic DCs isolated from animals that had been previously intrathymically inoculated with P5 could induce T-cell tolerance. The results showed that intravenous adoptive transfer of in vivo P5-primed thymic DCs led to donor-specific permanent acceptance of islets in recipients transiently immunosuppressed with ALS. This finding suggested that the thymic DCs take up and present P5 to developing T-cells to induce T-cell tolerance, thus providing evidence of a direct link between indirect allorecognition and acquired thymic tolerance. The second series of studies examined the mechanisms involved in this model by exploring whether in vivo generation of peptide-specific alloreactive peripheral T-cells by intravenous inoculation of P5-pulsed self-DCs was responsible for the induction of T-cell tolerance. Intrathymic inoculation of splenic T-cells obtained from syngeneic ACI rats primed with intravenous injection of P5-pulsed DCs with a high in vitro proliferative response to P5 in the context of self-MHC induced donor-specific permanent acceptance of islets from WF donors. In addition, the clinically relevant model of intravenous injection of P5-activated T-cells combined with transient ALS immunosuppression similarly induced transplant tolerance, which was then abrogated by thymectomy of the recipient before intravenous injection of the activated T-cells. These data raise the possibility that circulation of peptide-activated T-cells to the host thymus plays a role in the induction and possibly the maintenance of T-cell tolerance in this model. Our findings suggest that intravenous administration of genetically engineered host DCs expressing alloMHC peptides might have therapeutic potential in clinical islet transplantation for the treatment of autoimmune diabetes.

Induction of Transplant Tolerance with Immunodominant Allopeptide-pulsed Host Lymphoid and Myeloid Dendritic Cells

We have studied the effects of adoptive transfer of host thymic dendritic cells pulsed with immunodominant WF Class I peptide 5 (residues 93–109) on cardiac allograft survival in the WF-to-ACI rat combination. Our results showed that, whereas intrathymic inoculation of WF peptide 5-pulsed ACI thymic dendritic cells alone on day −7 did not prolong graft survival, similar treatment combined with 0.5 mL antilymphocyte serum (ALS) led to 100% permanent acceptance (> 200 d) of donor-specific cardiac allografts. Extension of our study to systemic administration of peptide 5-pulsed host thymic dendritic cells confirmed that intravenous injection of peptide 5-pulsed self thymic dendritic cells combined with ALS transient immunosuppression resulted in 100% permanent donor-specific graft survival (> 200 d). These results were reproducible in a clinically relevant model using intravenous injection of peptide-pulsed host myeloid dendritic cells. In contrast, thymectomy prior to adoptive transfer of peptide-pulsed host dendritic cells resulted in acute graft rejection at times equivalent to rejection in thymectomized controls. The long-term unresponsive recipients challenged with second-set grafts accepted permanently (> 100 d) donor-type (WF) but not third party (Lewis) cardiac allografts. This study suggests that intravenous administration of genetically engineered dendritic cells expressing donor MHC molecules has the potential of inducing transplant tolerance.

Induction of Transplant Tolerance with Immunodominant Allopeptide-pulsed Host Lymphoid and Myeloid Dendritic Cells

We have studied the effects of adoptive transfer of host thymic dendritic cells pulsed with immunodominant WF Class I peptide 5 (residues 93–109) on cardiac allograft survival in the WF-to-ACI rat combination. Our results showed that, whereas intrathymic inoculation of WF peptide 5-pulsed ACI thymic dendritic cells alone on day −7 did not prolong graft survival, similar treatment combined with 0.5 mL antilymphocyte serum (ALS) led to 100% permanent acceptance (> 200 d) of donor-specific cardiac allografts. Extension of our study to systemic administration of peptide 5-pulsed host thymic dendritic cells confirmed that intravenous injection of peptide 5-pulsed self thymic dendritic cells combined with ALS transient immunosuppression resulted in 100% permanent donor-specific graft survival (> 200 d). These results were reproducible in a clinically relevant model using intravenous injection of peptide-pulsed host myeloid dendritic cells. In contrast, thymectomy prior to adoptive transfer of peptide-pulsed host dendritic cells resulted in acute graft rejection at times equivalent to rejection in thymectomized controls. The long-term unresponsive recipients challenged with second-set grafts accepted permanently (> 100 d) donor-type (WF) but not third party (Lewis) cardiac allografts. This study suggests that intravenous administration of genetically engineered dendritic cells expressing donor MHC molecules has the potential of inducing transplant tolerance.

Intrathymic inoculation of donor B cells induced indefinite prolongation of fully allogeneic cardiac grafts but not islet or skin grafts

Intrathymic inoculation of donor B cells induced indefinite prolongation of fully allogeneic cardiac grafts but not islet or skin grafts

Induction of acquired thymic tolerance by intrathymic inoculation of allomhc class 1 peptide-specific T cell line with immunoregulatory functions

Induction of acquired thymic tolerance by intrathymic inoculation of allomhc class 1 peptide-specific T cell line with immunoregulatory functions

Evidence that non-deletional mechanisms are responsible for inducing and maintaining unresponsiveness after intrathymic injection of non-professional antigen presenting cells

Introduction: Intrathymic inoculation of donor alloantigen and concomitant immunosuppressive treatment can induce immune unresponsiveness to alloantigen. To examine the role of non-deletional mechanisms in the development of unresponsiveness, fractionated splenocytes were injected into only 1 lobe of the thymus. Methods and resultsUntreated CBA (H2k) mice or controls pre-treated with anti-CD4 monoclonal antibody alone (on Day −28 and −27 relative to transplantation) acutely rejected C57BL/10 (H2b) cardiac allografts. Intrathymic inoculation of unfractionated splenocytes, resting B (rB) cells, or dendritic cells into both thymic lobes with the antibody resulted in indefinite survival of cardiac allografts. In contrast, when donor rB cells or dendritic cells were delivered into a single lobe of the thymus with the antibody, only rB cells induced indefinite prolongation of graft survival; unfractionated splenocytes or dendritic cells were markedly less effective. Mice that had 1 of the 2 thymic lobes removed were able to reject grafts even when treated with the antibody 27 days before transplantation. Therefore, T-cell export from 1 thymic lobe was sufficient to induce graft rejection. Finally, adoptive transfer of splenocytes from mice with long-term surviving primary grafts resulting from the intrathymic injection of rB cells significantly prolonged a graft from the same donor strain in a naive syngeneic recipient. ConclusionTaken together, these data suggest that regulatory mechanisms generated by intrathymic injection of a non-professional antigen presenting cell, in this study donor rB cells, suppressed the rejection response mediated by T cells exported from the uninjected lobe.

Indirect allorecognition in acquired thymic tolerance: induction of donor-specific tolerance to rat cardiac allografts by allopeptide-pulsed host dendritic cells.

Presentation of peptides either by recipient or donor MHC molecules displayed on the surface of antigen-presenting cells is an essential element in the induction of T cell responses to transplant antigens. The finding that intrathymic (IT) injection of an immunodominant peptide induces acquired thymic tolerance suggests an indirect pathway of allorecognition in the thymus. To address this theory, we studied the effects of IT injection of host bone marrow (BM)-derived dendritic cells (DC)-pulsed with the immunodominant Wistar Furth (WF) MHC class I (RT1.Au) peptide 5 (93-109) on cardiac allograft survival in the WF-to-ACI rat combination. DC were propagated from cultures of ACI (recipient) bone marrow (BM) maintained in a medium supplemented with granulocyte-macrophage colony-stimulating factor and IL-4. The BM-derived DC after 8 days of culture were pulsed in vitro with a single WF MHC class I peptide (Residue 93-109) with the dominant epitope, washed, and injected into the thymus of ACI rats. The ACI recipients received donor-type (WF) or 3rd party (Lewis) cardiac allografts 7 days after IT immunization with peptide-pulsed DC. BM-derived DC cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 for 8 days have a strong allostimulatory ability and present peptide 5 to naive syngeneic T cells in mixed lymphocyte reaction. IT inoculation of 300 microg RT1.Au peptide 5 combined with transient antilymphocyte serum immunosuppressive therapy induced donor-specific tolerance to cardiac allografts. Extension of this finding to peptide-pulsed self DC showed that IT injection of peptide 5-pulsed host DC consistently led to permanent acceptance (>150 days) of donor-type (WF) cardiac allografts, whereas third-party (Lewis) grafts were acutely rejected. The long-term unresponsive recipients challenged with second-set grafts accepted permanently (>100 days) donor-type(WF) grafts while rejecting third-party (Lewis) grafts without the rejection of the primary WF grafts. This novel finding that allopeptide-pulsed host DC induces tolerance to cardiac allografts suggests that the induction of acquired tolerance is dependent on the indirect allorecognition pathway. The results further suggest that genetically engineered DC expressing donor MHC class I or II molecules or a peptide analogue might have therapeutic potential in the induction of transplant tolerance and in the treatment of autoimmune diseases.

Toward tolerance: lessons learned from liver transplantation.

*Define the various concepts of transplantation tolerance: Immunologically: unresponsiveness to donor antigens Clinically: ability to discontinue nonspecific immunosuppression Outcome-based: ability to prevent long-term immunologically mediated graft loss (i.e., chronic rejection). *Understand the various possible mechanism(s) involved in developing transplantation tolerance: Central tolerance: clonal deletion Peripheral tolerance: Blocking antibodies Cytokine imbalance Clonal T-cell anergy Active regulation of T- and B-cell proliferation. *Methods to achieve transplantation tolerance: Macrochimerism: recipient cytoablation and donor reconstitution Microchimerism: bone marrow augmentation, growth factors Intrathymic inoculation: central tolerance? T-cell costimulatory blockade: induction of T-cell anergy. *Limitations of achieving transplantation tolerance: No markers to define tolerance Poor understanding of acute and chronic rejection mechanisms (e.g., direct v indirect antigen presentation, high- v low-affinity T cells for alloantigen) What cells are involved in the development of tolerance? How stable is clinical tolerance: are the dynamics influenced by nontransplant factors (e.g., antigenic stimulation by viral factors)? Need for a two-pronged approach: nonspecific phase followed by specific phase?