Tolerance In Allotransplantation Research Articles

PS-068 - Operational tolerance in allotransplantation by use of regulatory T cells with a MHC-specific chimeric antigen receptor

PS-068 - Operational tolerance in allotransplantation by use of regulatory T cells with a MHC-specific chimeric antigen receptor

A peptide tetramer Tk-tPN induces tolerance of cardiac allografting by conversion of type 1 to type 2 immune responses via the Toll-like receptor 2 signal-promoted activation of the MCP1 gene.

The plant protein trichosanthin (Tk) and its derived peptide tetramer Tk-tPN have been shown to stimulate the type 2 immune responses for treating autoimmune disease. This work explores the possibility of using Tk-tPN as a non-toxic immunosuppressant to induce transplantation tolerance using the mechanisms by which T-cell-mediated immune responses are transferred from type 1 to type 2 through innate immunity-related pathways. Immunocytes and cytokine secretions involved in the mouse cardiac allografting model with Tk-tPN treatment were characterized. Identification of critical genes and analysis of their functions through Toll-like receptor (TLR) -initiated signalling and the possible epigenetic changes were performed. Mean survival times of the cardiac allografts were delayed from 7.7 ± 0.3 days (control) to 22.7 ± 3.9 days (P < 0.01) or 79.1 ± 19.2 days (P < 0.0001) when Tk-tPN was introduced into the recipients alone or together with rapamycin, respectively. The grafting tolerance was donor-specific. The secretion pattern of the type 1 cytokine/transcription factor (IL-2(+) IFN-γ(+) T-bet(+)), which is responsible for the acute graft rejection, was shifted to the type 2 factor (IL-4(+) IL-10(+) Gata3+), together with a selective expansion of the IL-4/IL-10-producing CD8+ CD28- regulatory T-cell subset. A TLR2-initiated high expression of chemokine gene MCP1 was detectable simultaneously. Epigenetically Tk/Tk-tPN could also acetylate the histone H3K9 of MCP1 promoter to skew the immunity towards T helper type 2 responses. Tk/Tk-tPN is therefore capable of down-regulating the type 1 response-dominant rejection of cardiac allografts by evoking type 2 immunity through the activation of a TLR2-initiated signalling pathway and MCP1 gene to expand the IL-4/IL-10-secreting CD8+ CD28- regulatory T cells. Tk-tPN could be a promising novel immunosuppressant to induce tolerance in allotransplantation.

Syngeneic adipose-derived stem cells with short-term immunosuppression induce vascularized composite allotransplantation tolerance in rats

Background aimsA clinically applicable tolerance induction regimen that removes the requirement for lifelong immunosuppression would benefit recipients of vascularized composite allotransplantation (VCA). We characterized the immunomodulatory properties of syngeneic (derived from the recipient strain) adipocyte-derived stem cells (ADSCs) and investigated their potential to induce VCA tolerance in rats. MethodsADSCs were isolated from Lewis (LEW, RT1Al) rats; their immunomodulatory properties were evaluated by means of mixed lymphocyte reactions in vitro and VCAs in vivo across a full major histocompatibility complex mismatch with the use of Brown-Norway (BN, RT1An) donor rats. Two control and four experimental groups were designed to evaluate treatment effects of ADSCs and transient immunosuppressants (anti-lymphocyte globulin, cyclosporine) with or without low-dose (200 cGy) total body irradiation. Flow cytometry was performed to quantify levels of circulating CD4+CD25+FoxP3+ regulatory T cells (Tregs). ResultsCultured syngeneic ADSCs exhibited CD90.1+CD29+CD73+CD45−CD79a−CD11b/c− phenotype and the plasticity to differentiate to adipocytes and osteocytes. ADSCs dramatically suppressed proliferation of LEW splenocytes against BN antigen and mitogen, respectively, in a dose-dependent fashion, culminating in abrogation of allo- and mitogen-stimulated proliferation at the highest concentration tested. Accordingly, one infusion of syngeneic ADSCs markedly prolonged VCA survival in LEW recipients treated with transient immunosuppression; of these, 66% developed tolerance. Total body irradiation provided no additional VCA survival benefit. An important role for Tregs in tolerance induction/maintenance was suggested in vivo and in vitro. ConclusionsTreatment comprising syngeneic ADSCs and transient immunosuppression (i) increased levels of circulating Tregs and (ii) induced tolerance in 66% of recipients of major histocompatibility complex–mismatched VCAs.

Facial Subunit Composite Tissue Allografts in Nonhuman Primates: I. Technical and Immunosuppressive Requirements for Prolonged Graft Survival

Widespread application of composite tissue allotransplantation has been impeded by risks of rejection and conventional immunosuppression. The authors have developed a nonhuman primate composite tissue allotransplantation model that demonstrated reliable and long-term success necessary to progress to preclinical studies. Composite facial subunits (e.g., skin, muscle, and bone) were transplanted between mismatched cynomolgus monkeys. Vascular supply was based on the common carotid artery and external and internal jugular veins. Facial allografts were heterotopically transplanted to the recipient's lower abdomen with end-to-side anastomoses of the common carotid artery to the common femoral artery and of both the internal and external jugular veins to the common femoral vein. Animals received tacrolimus monotherapy. Grafts were inspected daily, submitted to biopsy regularly, and studied with magnetic resonance imaging. Thirteen transplants were performed. Two grafts based on the common carotid artery and only the internal jugular vein failed within 3 to 5 days because of venous thrombosis not related to rejection. Subsequent transplants included anastomoses of both the internal and external jugular veins to the common femoral vein without thromboses. Immunosuppression consisted of tacrolimus monotherapy and was tolerated without complications. Long-term success was achieved with rejection-free graft survival (60 to 177 days). The authors have developed the first successful model of facial composite tissue allotransplantation in a nonhuman primate. Technical requirements include preservation of both internal and external jugular venous outflow. Tacrolimus monotherapy permitted prolonged rejection-free graft survival without early complications. This model provides a platform for further investigation of composite tissue allotransplantation tolerance and requirements for indefinite survival.

Involvement of Dendritic Cells in Allograft Rejection New implications of Dendritic Cell-Endothelial Cell Interactions

For almost half a century immunologists have tried to tear down the MHC barrier, which separates two unrelated individuals during transplantation. Latest experimental data suggest that a breakthrough in vitro is imminent. Dendritic cells (DCs), which activate naïve allo-reactive T-cells (TCs), play a central role in the establishment of allo-antigen-specific immunity. Allograft solid organ rejection is initiated at the foreign endothelial cell (EC) layer, which forms an immunogenic barrier for migrating DCs. Thus, DC/EC interactions might play a crucial role in antigen-specific allograft rejection. Organ rejection is mediated by host allo-reactive TCs, which are activated by donor DCs (direct activation) or host DCs (indirect activation). Direct allo-antigen presentation by regulatory dendritic cells (DCreg) can play an instructive role towards tolerance induction. Several groups established that, DCregs, if transplanted beforehand, enter host thymus, spleen, or bone marrow where they might eventually establish allo-antigen-specific tolerance. A fundamental aspect of DC function is migration throughout the entire organism. After solid organ transplantation, host DCs bind to ECs, invade allograft tissues, and finally transmigrate into lymphoid vessels and secondary lymphoid organs, where they present allo-antigens to naïve host TCs. Recent data suggest that in vitro manipulated DCregs may mediate allo-transplantation tolerance induction. However, the fundamental mechanisms on how such DCregs cause host TCs in the periphery towards tolerance remain unclear. One very promising experimental concept is the simultaneous manipulation of DC direct and indirect TC activation/suppression, towards donor antigen-specific allo-transplantation tolerance. The allo-antigen-specific long-term tolerance induction mediated by DCreg pre-transplantation (with simultaneous short-term immunosuppression) has become reproducible in the laboratory animal setting. Despite the shortcomings of laboratory animal studies, strong promises are deriving from these studies for clinical kidney, heart, and liver transplantation.

Detection of chimerism following vascularized bone allotransplantation by polymerase chain reaction using a Y-chromosome specific primer

Chimerism following allogeneic organ transplantation is a phenomenon known to occur and be associated with development of immunologic tolerance in allotransplantation. However, little is known about graft cell migration following vascularized bone allografting. In this study, chimerism was assessed following vascularized tibia transplantation from male DA or PVG donors to female PVG rat recipients using a semi-quantitative polymerase chain reaction for the Y-chromosome. FK-506 (Tacrolimus) was administered after transplantation for immunosuppression. All immunosuppresssed PVG rat recipients of PVG bone grafts showed a high level of chimerism (1%) in the thymus, spleen, liver and cervical lymph nodes at 18 weeks post-transplant. Donor cells were also detected in the contralateral tibia and humerus. In non-immunosuppressed PVG rat recipients of DA bone grafts, donor cells were detected in the spleen in three of five rats within 2 weeks post-transplant. In these animals the bone grafts were severely rejected. In immunosuppressed PVG rat recipients of DA bone grafts, two of five, four of eight and eight of 10 rats showed low level chimerism (0.1%) in peripheral blood at 1, 12, and 18 weeks post-transplant. Six rats showed a high level of chimerism in the spleen and thymus. Histological studies revealed no rejection findings through 18 weeks post-transplant. Our results indicate that chimerism, or the presence of graft cells in host tissue, may occur in the face of acute rejection and be demonstrable following vascularized isograft and allograft living bone transplantation when chronic immunosuppression is maintained. Graft vascular patency during the short-term likely allows cellular migration, even in the face of acute rejection. Long-term survival and proliferation of graft marrow elements in host tissue may be possible with adequate immunosuppression.

Fate of donor cells in vascularized bone grafts: identification of systemic chimerism by the polymerase chain reaction.

Systemic chimerism, or the movement of cells from a transplanted tissue into host organs, is a phenomenon known to occur in association with development of immunological tolerance in allotransplantation. However, little is known about the fate and movement of cells into or out of autogenous free tissue transfers, including vascularized bone grafts. The purpose of this study was to identify systemic chimerism in vascularized bone grafts by transplantation of a vascularized tibiofibular graft from isogenous (inbred) male Lewis rats to female recipients. Donor (male) cells could be identified in the recipient (female) tissues by semiquantitative polymerase chain reaction analysis for a Y chromosome-specific DNA sequence. Chimerism was assessed at 1, 12, 18, and 24 weeks after transplantation. Competitive polymerase chain reaction study using the specific primers for a Y-chromosome marker ( gene) and an autosomal gene (GAPDH) allowed detection of small amounts of male cells in a large pool of female cells and measurement of their relative proportions as a function of time. Of 19 nonimmunosuppressed recipients, nine animals (47 percent) showed low-level chimerism (<0.1 percent) in the peripheral blood. Nine (47 percent), three (16 percent), and two (11 percent) recipients showed high-level chimerism (>1 percent) in the spleen, liver, and thymus, respectively, at final assessment. Donor cells were detected in all bone grafts and in six contralateral tibial bones (i.e., 67 percent of sampled contralateral tibial bones) at 18 and 24 weeks after transplantation. Twenty-four recipients were immunosuppressed with FK506 (tacrolimus) to suppress reaction to a minor histocompatibility barrier present on the Y chromosome. In this group, 14 animals (58 percent) showed low-level chimerism in peripheral blood and 12 (50 percent), eight (33 percent), and one (4 percent) recipients showed high-level chimerism in the spleen, thymus, and liver, respectively. Transplanted cells were detected in nine contralateral tibial bones (i.e., 60 percent of sampled contralateral tibial bones) at 12 and 18 weeks after surgery. The results indicate that polymerase chain reaction for the Y chromosome is a useful tool for differentiating between donor and recipient cell populations experimentally using sex-mismatched tissues in a rat model. This study demonstrated that systemic chimerism occurs after successful vascularized bone transplantation. Transplanted cells not only survive in the graft but also gradually migrate into the recipient's body.

Abolition of anti-CD4-induced allotransplantation tolerance by exogenous IL-2

Abolition of anti-CD4-induced allotransplantation tolerance by exogenous IL-2

Long-term survival of hamster islet xenografts in mice under short-course treatment with nondepleting versus depleting anti-CD4 monoclonal antibodies.

Xenogeneic grafts provide a potential alternative to the current shortage of human organs for transplantation. However, the prevention of rejection and tolerance induction of xenografts still remain to be further explored. Islet xenografts appear more promising than vascularized whole organ xenografts and additionally also more resistant to the recurrence of autoimmune disease than allografts. Recently, the nondepleting monoclonal antibody (mAb), which blocks the CD4 molecule on lymphocytes, was reported to be able to induce tolerance in allotransplantation and CD4 positive cells were further confirmed to be a major factor responsible for cellular xenograft rejection. Therefore, we hypothesize that anti-CD4 nondepleting mAb could also be effective in protecting cellular xenografts and inducing unresponsiveness of recipients. We studied the effect of the nondepleting anti-CD4 mAb YTS177.9 on islet xenograft survival by using the hamster-to-mouse islet transplantation model. Results were compared with that of the depleting anti-CD4 mAb GK1.5 that was shown to have similar binding sites on the CD4 molecule to mAb YTS177.9. Our data show that mAb YTS177.9 did effectively prolong the survival of islet xenografts and, in addition, also successfully did induce long-term acceptance of 40% grafts after only three perioperative injections of 0.5 mg mAb per mouse. The average survival of the graft was markedly prolonged to >66.8+/-37.1 days compared with controls (8.3+/-1.4 days) or with the depleting anti-CD4 mAb GK1.5 (25.7+/-5.5 days). However, the latter displayed a more profound inhibition in in vitro and ex vivo mixed lymphocyte xenoreaction than mAb YTS177.9. Moreover, the activity of this nondepleting mAb was found to be dose-dependent and 80% of grafts survived permanently when the dose was increased to six injections of 0.5 mg mAb. Like mAb GK1.5, mAb YTS177.9 also prevented rejection when given after a delay of two days posttransplant. In addition, we found that neither depleting nor nondepleting anti-CD8 mAb was effective in this model. Our results strongly suggest that an anti-CD4 nondepleting or blocking mAb alone is able to induce long-term acceptance of islet xenografts and that blocking the CD4 molecule is significantly superior to depleting CD4 positive cells for the protection of islet xenografts. This may indicate that CD4 cells play a major role in xenograft tolerance induction.

An anti-CD2 mAb induces immunosuppression and hyporesponsiveness of CD2+ human T cells in vitro.

We describe here the potent specific immunosuppression obtained in vitro by LO-CD2a, a rat mAb directed against the human CD2 molecule. Addition of low dose LO-CD2a (40 ng/ml) at the time of mixed lymphocyte culture (MLC) initiation inhibits 80% of the proliferation and, more impressive, addition of the mAb 4 days after culture initiation at a similar concentration still suppresses 50% of the MLC. When responder T cells previously treated with LO-CD2a are challenged a second time by the same donor or third party allogeneic cells, hyporesponsiveness occurs in both cases, although reactivity to T cell mitogenic stimulation persists. Finally, the low production of cytokines such as tumor necrosis factor-alpha and IFN-gamma after incubation of human T cells with LO-CD2a suggests the absence of T cell activation. These results demonstrate that LO-CD2a mAb has a significant immunosuppressive effect and induces hyporesponsiveness in vitro, thereby suggesting potential efficacy in vivo for the treatment of acute rejection and for the induction of tolerance in allotransplantation.

Inhibition of rat mixed lymphocyte pancreatic islet cultures with anti-Ia immunotoxin.

Studies reported here indicate that an anti-Ia immunotoxin can eliminate the allostimulatory subpopulation of cells present within the islets of Langerhans without damaging the hormone-secreting cells. Such studies made use of an in vitro correlate of transplantation rejection, the mixed lymphocyte islet cell (MLIC) reaction. Using the MLIC, it was demonstrated that an anti-Ia immunotoxin removed cells capable of stimulating the MLIC in a dose-dependent fashion without altering the hormone-secreting functions of the remaining cells when challenged with glucose and theophylline. These studies suggest the feasibility of using such anti-Ia immunotoxins in islet allograft transplantation models to circumvent problems inherent in complement-mediated cytotoxicity, a previously documented effective form of inducing islet allotransplantation tolerance.

Modification of suppressor/cytotoxic and helper subsets in lentil lectin-pretreated mice.

The effect of lentil lectin (LCA) on various lymphocyte subpopulations in the spleen, lymph nodes and thymus of CBA mice was studied. The most marked effect after five 1-mg LCA doses was observed in the spleen. LCA caused a drop in both the total and relative number of cells carrying all the markers examined, i.e., Thy 1, L3/T4a, Lyt 2, and sIg, so that large numbers of lymphocytes did not express any of them. A dramatic decrease in the Thy 1-positive cell number is mainly due to depletion of the L3/T4a (helper) T cell subpopulation, whereas the Lyt 2+ (suppressor/cytotoxic) cells are less affected. The decrease in the Thy 1+, Lyt 1+ and L3/T4a+ cell numbers in the thymus and lymph nodes is smaller, the percentage of Lyt 2+ cells is even increased after LCA treatment. Our results indicate that two mechanisms play a role in the induction of allotransplantation tolerance by LCA: 'depletion' of helper T cells and a relative increase in the regulatory (suppressor) cell ratio, which may support allograft survival.

The effect of H-2IJ disparity on induction of allotransplantation tolerance by lentil lectin.

The effect of an H-2IJ disparity on skin graft survival was studied in 18 mouse donor-recipient strain combinations, in which the recipients were treated with an efficient immunosuppressant, lentil lectin (LCA). The simultaneous I-J disparity essentially had no (or a slightly adverse) effect on the graft survival times in strain combinations differing at the K and I-A loci or in the entire H-2 complex. In two strain combinations incompatible at the D locus, the simultaneous I-J disparity promoted graft survival. The disparity at the I-J locus therefore seems to have only a marginal effect on the survival of allografts in most of the LCA-treated recipients, but it may promote graft survival in some animals. A similar tolerance-promoting effect was also observed with D disparity.