Aminoacyl-tRNA synthetase inhibitors as potent and synergistic immunosuppressants.

The aminoacyl-tRNA synthetase family of enzymes is the target of many antibacterials and inhibitors of eukaryotic hyperproliferation. In screening analogues of 5'-O-(N-L-aminoacyl)-sulfamoyladenosine containing all 20 proteinogenic amino acids, we found these compounds to have potent immunosuppressive activity. Also, we found that combinations of these compounds inhibited the immune response synergistically. Based on these data, analogues with modifications at the aminoacyl and ribose moieties were designed and evaluated, and several of these showed high immunosuppressive potency, with one compound having an IC50 of 80 nM, when tested in a cellular mixed lymphocyte reaction assay. Apart from showing the potential of aminoacyl-tRNA synthetase inhibitors as immunosuppressants, the current study also provides arguments for careful evaluation of the immunosuppressive activity of developmental antibacterials that target these enzymes.

Occurrence of autoimmunity after xenothymus transplantation in T-cell-deficient mice depends on the thymus transplant technique.

Xenothymus transplantation under the kidney capsule in athymic rodents frequently leads to multiorgan autoimmunity. Herein, we explore whether this is an intrinsic risk of xenothymus grafting or whether it depends on the transplant technique. We developed a new technique of "venous pouch" thymus grafting (heart-xenothymus) and compared this with the conventional kidney subcapsular technique (kidney-xenothymus) in a rat-into-nude-mouse model. Whereas lethal autoimmunity developed in 90% of kidney-xenothymus recipients, all heart-xenothymus grafted mice remained completely healthy. Autoimmunity in heart-xenothymus recipients was absent despite a significantly improved T-cell generation and was associated with significantly higher CD4+CD25+ T-cell frequencies and CD4+CD25+ cell Foxp3 mRNA levels than those observed in kidney-xenothymus recipients. In conclusion, we describe a novel vascular pouch technique of xenothymus transplantation that prevents the development of autoimmunity in nude mice. Our data further suggest that prevention of autoimmunity is related to a superior development of regulatory T-cells.

Rapidly induced, T-cell independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells.

Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell-independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic recipient mice, that rapidly induced, T-cell-independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell-mediated IFN-gamma production, nor NK-cell-mediated cytolytic killing of xenogeneic target cells. The T-cell-independent IgM xenoantibody response could be partially suppressed by CD40L blockade.

Induction and maintenance of T-dependent or T-independent xenotolerance by nonprimarily-vascularized skin or thymus grafts.

BACKGROUND: The success of clinical xenotransplantation will depend on induction of xenotolerance. We have previously shown that combined xenothymus and vascularized xenoheart transplantation under the coverage of a tolerizing regimen (TR) can induce and maintain full xenotolerance. Here, induction/maintenance of xenotolerance using nonprimarily-vascularized thymus and/or skin grafts was investigated. MATERIALS AND METHODS: Hamster skin or thymus or combined skin and thymus transplantation was performed in nude rat recipients with or without administering a TR (NK cell depletion, day -14; xenoantigen infusion, day -14; Leflunomide, day -14 through +14). Xenotolerance was confirmed by subsequent transplantation of a vascularized hamster heart, measurement of xenoantibody formation, or mixed lymphocyte reaction (MLR). RESULTS: Skin grafts were as effective as vascularized heart grafts to induce/maintain T-independent xenotolerance. Even without TR and despite being rejected themselves, xenoskin grafts lead to progressively developing xenononreactivity. Xenothymus transplantation induced xenotolerance in the T-dependent but not in the T-independent immune compartment, leading to rejection of subsequently transplanted hamster hearts by T-independent mechanisms (production of IgM but not IgG xenoantibodies (Xabs), presence of antihamster MLR nonresponsiveness). Combined skin and thymus xenotransplantation sensitized the T-cell compartment, leading to hyperacute rejection of subsequently transplanted hamster hearts. This was not the case when the skin grafts were transplanted late (2 months) after the thymus grafts. CONCLUSIONS: Xenogeneic skin and xenogeneic thymus grafts have opposite xenotolerance inducing capacities in the T-independent as compared to the T-dependent immune compartment. Thymus grafts induce and maintain T-dependent but not T-independent xenotolerance. Skin grafts alone induce T-independent xenotolerance but sensitize the T-cell compartment when transplanted concomitantly with thymus grafts.

Effects of a short course of leflunomide on T-independent B-lymphocyte xenoreactivity and on susceptibility of xenografts to acute or chronic rejection.

BACKGROUND: Leflunomide is a novel immunosuppressive agent with promising activity for xenotransplantation. It is not clear yet which mechanisms of action of leflunomide are responsible for that. METHODS: In a hamster-to-C57BL/6 nude mouse heart transplantation model, a 2-week course of leflunomide was used after transplantation or for pretreating donors. Nontolerant B lymphocytes were transferred to recipients after transplantation of first or second xenogeneic heart grafts that were transplanted with or without leflunomide treatment. RESULTS: Hamster xenogeneic hearts transplanted into athymic C57BL/6 nude mice receiving leflunomide did not induce immunoglobulin (Ig) M xenoantibodies (XAb) and survived without signs of chronic rejection. Second xenogeneic hearts transplanted 4 weeks after withdrawal of leflunomide survived without induction of XAb but developed chronic vascular lesions. After injection of naive B lymphocytes at 6 weeks after grafting a first or second hamster heart, only in the latter case were XAb induced. These were deposited in, and provoked acute rejection of, only the second grafts. Pretreatment of donors with leflunomide decreased the ex vivo xenoantibody deposition on the xenogeneic heart endothelia. CONCLUSIONS: A short posttransplant course of leflunomide induces T-independent B-lymphocyte xenotolerance. Leflunomide treatment also influences xenoantigen expression, as nontolerant B lymphocytes provoke IgM XAb formation and rejection of only second xenografts (transplanted without leflunomide) and not of first xenografts (transplanted with leflunomide treatment). The ex vivo experiments that show that XAb deposition is decreased in leflunomide-pretreated xenografts further confirm this. The latter may also explain the resistance of first and not second xenografts against chronic rejection.

Pathogenesis of autoimmunity after xenogeneic thymus transplantation.

Thymus transplantation is a promising strategy to induce xenotolerance, but may also induce an autoimmune syndrome (AIS). The pathogenesis of this AIS was explored using nude rats as recipients. Thymus grafts consisted of fetal hamster thymic tissue with or without mixing with fetal rat tissue such as thymus, thyroid, salivary gland, and heart. All hamster thymus recipients died of AIS within 2-3 mo. In most recipients of xenothymus mixed with rat tissues such as thymus, thyroid, and salivary gland, but not heart, AIS was prevented, indicating an insufficient presence of rat epithelial cell Ags within the xenothymus. AIS could be transferred to control nude rats by whole splenocytes or by splenocyte subpopulations such as CD3(+), CD3(-), and B lymphocytes, but not by non-T, non-B cells from AIS animals. This transfer could be suppressed by cotransferring either CD4(+) or CD8(+) lymphocytes from euthymic rats, but not by splenocytes from recipients of syngeneic or xenogeneic thymus mixed with rat tissue, indicating a defective generation of regulatory lymphocytes. As for CD4(+) regulatory cells this defect was probably qualitative, because the percentages of CD4(+)CD25(+) or CD4(+)CD45RC(low) populations were normal after xenothymus transplantation. As for the CD8(+) regulatory cells, the defect was quantitative, as CD8(+) cell levels always remained low. The latter was related to the nonvascularized nature of thymus grafts. In conclusion, AIS after xenothymus transplantation in nude rats is due to a combination of insufficient intrathymic presence of host-type epithelial cell Ags and a defective generation of regulatory T lymphocytes.