Constant Segers

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+CD45RClow 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.

Induction Of T-independent Xenotolerance In A Semi-discordant Hamster-to-presensitized, Nude Rat Model

BACKGROUND We have previously demonstrated that in a concordant hamster-to-nude rat cardiac transplant model, T-independent specific B-lymphocyte and natural killer (NK)-cell tolerance could be induced, leading to long-term xenograft (Xg) survival. Here, we investigated whether the same could be achieved in a clinically more relevant semi-discordant model involving hamster hearts transplanted into pre-sensitized, nude rats. METHODS Sensitized, nude rats with high titers of anti-hamster immunoglobulin (Ig)M xenoantibodies (XAbs) were prepared by transplanting a first hamster heart without treatment. One week after rejection, a complete tolerizing regimen was given, including the following: a) an i.v. injection of hamster heart antigens; b) a 4-week administration of malononitriloamide; and c) a single injection of an anti-NK antiserum. Two weeks later, a second hamster heart was grafted. The isotype and level of XAb were examined by fluorescence-activated cell sorting. NK cytotoxicity was evaluated by a standard 4-hr 51Cr release assay. Hamster heart Xgs were examined by conventional histologic and immunohistochemical analysis. RESULTS Untreated, presensitized, nude rats developing high titers of IgM XAb underwent hyperacute rejection within 4 hr (n=4) after transplantation of the second hamster heart. Immunohistochemical analysis showed intensive staining for IgM and C3 along the vascular endothelia in the rejected Xgs. In contrast, presensitized, nude rats receiving the complete tolerizing regimen had a rapid decrease in anti-hamster IgM XAb. The second hamster hearts were not rejected and showed long-term survival even after withdrawal of malononitriloamide (n=6). Moreover, tolerant rats showed specific B-lymphocyte tolerance and a specific continuous absence of anti-hamster NK-cell reactivity. CONCLUSION T-independent B-lymphocyte and NK-cell xenotolerance can also be achieved in recipients with pre-existing IgM XAb.