Jozef Goebels

Induction of specific transplantation tolerance across xenogeneic barriers in the T-independent immune compartment

After transplantation of primarily vascularized xenografts (Xgs), T-independent mechanisms may lead to Xg rejection before T-cell activation even takes place. The possibility of achieving T-independent xenotolerance was evaluated in nude rats that normally reject hamster cardiac Xgs within 4 days by non-T cell-mediated mechanisms. After donor antigen infusion, temporary NK-cell depletion and a 4-week administration of Leflunomide, hamster heart grafts survived even after withdrawal of immunosuppression. Tolerant rats accepted second hamster hearts, but promptly rejected mouse heart Xgs. In vivo immunization and in vitro cy-totoxicity assays indicated that this species-specific tolerance was based on B-lymphocyte and NK-cell tolerance respectively.

Transforming growth factor-beta inhibits lymphokine activated killer cytotoxicity of bone marrow cells: implications for the graft-versus-leukemia effect in irradiation allogeneic bone marrow chimeras.

BACKGROUND We have previously shown that allogeneic bone marrow (BM) chimeras preconditioned with total lymphoid irradiation and low-dose total body irradiation (TLI/TBI) develop a stronger graft-versus-leukemia (GVL) effect than chimeras preconditioned with high-dose total body irradiation only (TBI). Here, we report on the possible role of cytokines in the mechanism underlying this GVL effect. METHODS Splenic mRNA levels of the cytokines interleukin (IL)-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta (TGF-beta), and of inducible nitric oxide synthetase were determined by reverse transcription-polymerase chain reaction in TLI/TBI- or TBI-conditioned C3H/AKR BM chimeras challenged with AKR-type BW5147.3 leukemia cells. Ex vivo TGF-beta protein production by splenocytes was determined using ELISA. The possibility that cytokines influence the GVL effect by modulating the activity of IL-2-activated lymphocytes (LAK cells) was investigated by in vitro assays on donor-type BM cells. RESULTS Of all cytokine mRNA levels studied, those of TGF-beta and IL-7 were different between groups; both were significantly more elevated in TBI- than in TLI/ TBI-conditioned or normal mice. Differences were apparent after conditioning and were not influenced by additionally injected BM or leukemia cells. Cultured splenocytes of TBI-conditioned animals produced significantly more TGF-beta protein than those of TLI/TBI-conditioned ones or normal controls. r-TGF-beta but not r-IL-7 suppressed in vitro LAK activity of donor-type BM cells against BW5147.3 cells in a dose-dependent way. CONCLUSIONS High-dose TBI-induced, host-derived splenic TGF-beta may inhibit generation of LAK cells from subsequently transplanted donor BM cells, suppressing their capacity to generate cytotoxicity upon injection of leukemia cells. The cytokine profile, induced by irradiation in host hematopoietic organs, can significantly modify posttransplant immunological processes such as the GVL effect and graft-versus-host disease (GVHD).

CTLA-4 blockade in murine bone marrow chimeras induces a host-derived antileukemic effect without graft-versus-host disease

We studied the effect of CTLA-4 blockade on graft-versus-leukemia and graft-versus-host responses in a mouse model of minor histocompatibility-mismatched bone marrow transplantation. Early CTLA-4 blockade induced acute graft-versus-host disease. Delayed CTLA-4 blockade resulted in a lethal condition with lymphosplenomegaly, but with stable mixed T-cell chimerism, unchanged alloreactive T-cell frequencies and absent anti-host reactivity in vitro. In contrast, multiorgan lymphoproliferative disease with autoimmune hepatitis and circulating anti-DNA auto-antibodies were documented. Splenic lymphocytes exhibited ex vivo spontaneous proliferation and a marked proliferative response against host-type dendritic cells pulsed with syngeneic (host-type) tissue-peptides. Both phenomena were exclusively mediated by host and not donor T cells, supporting an autoimmune pathogenesis. Selectively host-derived T-cell immune reactivity was equally documented against leukemia-peptide-pulsed dendritic cells, and this was paralleled by a strong in vivo antileukemic effect in anti-CTLA-4-treated and subsequently leukemia-challenged chimeras. In conclusion, delayed CTLA-4 blockade induced a host-derived antileukemic effect, occurring in the context of an autoimmune syndrome and strictly separated from graft-versus-host disease. Both antileukemic and autoimmune responses depended on the allogeneic component, as neither effect was seen after syngeneic bone marrow transplantation. Our findings reveal the potential of using CTLA-4 blockade to establish antileukemic effects after allogeneic hematopoietic stem cell transplantation, provided autoimmunity can be controlled.

Transplantation Tolerance and Autoimmunity After Xenogeneic Thymus Transplantation

Successful grafting of vascularized xenografts (Xgs) depends on the ability to reliably induce both T cell-independent and -dependent immune tolerance. After temporary NK cell depletion, B cell suppression, and pretransplant infusion of donor Ags, athymic rats simultaneously transplanted with hamster heart and thymus Xgs developed immunocompetent rat-derived T cells that tolerated the hamster Xgs but provoked multiple-organ autoimmunity. The autoimmune syndrome was probably due to an insufficient development of tolerance for some rat organs; for example, it led to thyroiditis in the recipient rat thyroid, but not in simultaneously transplanted donor hamster thyroid. Moreover, grafting a mixed hamster/rat thymic epithelial cell graft could prevent the autoimmune syndrome. These experiments indicate that host-type thymic epithelial cells may be essential for the establishment of complete self-tolerance and that mixed host/donor thymus grafts may induce T cell xenotolerance while maintaining self-tolerance in the recipient.

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.

Increased mortality and impaired clonal deletion after staphylococcal enterotoxin B injection in old mice: Relation to cytokines and nitric oxide production

In the present study peripheral T cell tolerance and the occurrence of shock were evaluated in young and old mice after injection of Staphylococcal enterotoxin B (SEB). In young mice SEB immunization leads to tolerance based on deletion and anergy of SEB‐reactive Vβ8+ T cells. With aging, mice developed resistance to SEB‐induced deletion of Vβ8+ T cells as well as a high sensitivity to toxic shock. Compared to young mice, older mice injected with SEB showed increased serum levels of interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2) and IL‐4. These results were confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR), as splenic mRNA levels taken 2 h after SEB injection showed higher values of IL‐2, IL‐4, and IFN‐γ in old mice. In contrast, mRNA levels for FasL and tumour necrosis factor‐α (TNF‐α) were lower. No difference in IL‐10 mRNA was found. Compared to young mice, old mice showed a high, but statistically not significantly different (P = 0.20), production of nitric oxide (NO). Blocking of IFN‐γ with antibodies or reducing IFN‐γ by depletion of natural killer (NK) cells resulted, respectively, in a complete or partial protection against mortality in aged mice. Suppressing the NO production by the NO synthase inhibitor N‐nitro‐L‐arginine methylester (L‐NAME) increased the mortality in both young and old mice, and abrogated clonal deletion in the surviving young mice. In conclusion, in young mice NO production is a key factor in the protection against mortality and the development of clonal deletion after SEB injection. The higher mortality seen in older mice is mainly related to the elevated production of IFN‐γ and occurs despite a sufficient production of NO. The decreased clonal deletion of old mice may be related to their decreased expression of Fas ligand or TNF‐α after SEB injection.

Effect of treatments with cyclosporin A and anti-interferon-gamma antibodies on the mechanisms of immune tolerance in staphylococcal enterotoxin B primed mice

The authors were interested to investigate the effect of Cyclosporin A (CsA), known to block interleukin‐2 (IL‐2) production, or of anti‐interferon‐γ antibodies (anti‐IFN‐γ Abs) in a model of T cell tolerance induced by the injection of the superantigen Staphylococcal Enterotoxin B (SEB) in BALB/c mice. After SEB immunization, tolerance was mainly achieved through deletion and anergy of SEB‐reactive Vβ8+ T cells. Association of CsA treatment with SEB led to a greater decrease of the percentage of Vβ8+ CD4+ lymphocytes in the spleen and an abolition of clonal anergy. In contrast, treatment of SEB primed mice with anti‐IFN‐γ Abs resulted in an increased percentage of Vβ8+ CD4+ cells without affecting the induction of clonal anergy. The authors found that 1–2 h after SEB priming, splenic mRNA levels of IFN‐γ and IL‐4 were decreased by either CsA and anti‐IFN‐γ Abs, whereas FasL, Bcl‐2, p. 53, and c‐myc levels were not influenced by either treatment. However, SEB‐induced IL‐2 and IL‐10 mRNA expression was suppressed only by CsA, whereas tumour necrosis factor‐α (TNF‐α) was decreased only by anti‐IFN‐γ Abs. To investigate whether the effect of CsA on the tolerance mechanisms was related to suppression of IL‐2, CsA was administered together with recombinant IL‐2. Whereas anergy was not influenced, the decreased percentage of Vβ8+ CD4+ cells seen in CsA‐treated animals in the second week after SEB injection was partially corrected by the administration of IL‐2. Experiments involving bromodeoxiuridine incorporation revealed that the latter effect of IL‐2 was mainly due to a correction of the defective proliferation of Vβ8+ T cells after SEB injection in CsA‐treated mice. These results suggest that the effect of CsA and anti‐IFN‐γ Abs on tolerance mechanisms are in part explained by their action on cytokines.

Rejection of cardiac xenografts by NK cells and macrophages

Abstract DUE TO the nature of hyperacute rejection of xenografts transplanted between phylogenetically disparate species, cellular immune responses in xenotransplantation are less well understood. Recent advances in controlling hyperacute rejection have allowed the survival of vascularized xenografts to the point where these may elicit cellular immune responses. There is increasing evidence showing that, if hyperacute rejection is circumvented, discordant xenografts may undergo rejection within a few days. This delayed xenograft rejection is immunopathologically characterized by a prominent cellular infiltration consisting of mainly natural killer (NK) cells, macrophages and T cells.1,2 In the present study, the role played by NK cells and macrophages in xenograft rejection was further studied in athymic nude rats receiving concordant xenografts. As these nude rats were previously shown to have strong NK cell activity, we were interested to see how this would influence the capacity of these T-deficient rats to reject hamster heart xenografts.

Stable mixed chimerism induced by total lymphoid irradiation or by total body irradiation is maintained by different mechanisms and leads to different graft versus leukemia or graft rejection reactions

Abstract ALLOGENEIC bone marrow (BM) transplantation is an effective procedure for the treatment of various malignant disorders of hematologic origin. 1,2 Several reports indicate that the fatality rate in leukemic patients can be significantly decreased after allogeneic bone marrow transplantation. 1,2 Previously, we demonstrated that mice conditioned with TLI or TBI were capable of developing stable bone marrow chimerism without signs of graft-versus-host-disease (GVHD) or rejection. 3 In the present work, we examined the mechanisms that maintained the state of the chimerism and which determined graft-versus-leukemia (GVL) or breaking of the tolerance in these stable chimeras.