Prolonged allograft survival through conditional and specific ablation of alloreactive T cells expressing a suicide gene

BACKGROUND: Control of antidonor activated T cells involved in allograft rejection while preserving immunocompetence is a challenging goal in transplantation. Engineered T cells expressing a viral thymidine kinase (TK) suicide gene metabolize the nontoxic prodrug ganciclovir (GCV) into a metabolite toxic only to dividing cells. We evaluated this suicide gene strategy for inducing transplantation tolerance in mice. METHODS: Transgenic mice expressing TK in mature T cells were analyzed for (i) specific T-cell depletion under GCV treatment upon various stimulations; (ii) outcome of allogeneic nonvascularized skin or heart allografts under a short 14-day GCV treatment initiated at the time of transplantation; and (iii) the capacities of T cells from such allotransplanted mice to proliferate in mixed lymphocyte reactions and to induce graft-versus-host disease in irradiated recipients with the genetic background of the donor allograft. RESULTS: Upon in vitro or in vivo GCV treatment, only activated dividing TK T cells but not B cells were efficiently depleted. Acute rejection of allogeneic grafts was prevented and a significant prolongation of graft survival was obtained, although associated with signs of chronic rejection. Prolonged skin graft survival correlated with decreased in vitro and in vivo T-cell reactivities against donor alloantigens, whereas overall immunocompetence was preserved. CONCLUSIONS: Efficient and specific depletion of alloreactive TK T cells can be achieved by administrating GCV. These results open new perspectives for the control of allogeneic graft rejection using suicide gene therapy.     

Abnormal T cell selection on nod thymic epithelium is sufficient to induce autoimmune manifestations in C57BL/6 athymic nude mice

To investigate the role of primary T cell repertoire selection in the immunopathogenesis of autoimmune diseases, pure thymic epithelium (TE) from nonobese diabetic (NOD) embryos was grafted into non autoimmune prone newborn C57BL/6 athymic mice. The results show that NOD TE selects host T cell repertoires that establish autoimmunity in otherwise nondiabetic animals. Thus, such chimeras regularly show CD4 and CD8 T cell-mediated insulitis and sialitis, in contrast with syngeneic or allogeneic chimeras produced with TE from nonautoimmune strains. This is the first demonstration that autoimmunity to pancreatic β cells and salivary glands can be established by the sole alteration of the thymic environment involved in T cell selection, regardless of the nature and presentation of both major histocompatibility complex and tissue-specific antigens on the target organ. These data indicate that T cell repertoire selection by the NOD thymic epithelium is sufficient to induce specific autoimmune characteristics in the context of an otherwise normal host.     

Establishment of tissue-specific tolerance is driven by regulatory T cells selected by thymic epithelium

Grafts of thymic epithelium (TE) rudiments restore T cell development and function in allogeneic athymic mice. These TE chimeras are specifically tolerant to grafts of peripheral tissues (e.g. skin and heart) from the TE donor strain, although they harbor peripheral immunocompetent T cells capable of rejecting those grafts. Initial analysis has shown that TE chimeras also harbor TE-selected CD4 T lymphocytes that inhibit graft rejection by tissue-reactive T cells in immunocompetent recipients. Peripheral tolerance in TE chimeras is thus maintained by dominant mechanisms dependent on regulatory CD4 T lymphocytes. Here we show that TE-selected regulatory T cells recruit nontolerant tissue-reactive CD4 and CD8 T cells to express similar regulatory functions. Only recent thymic emigrants, but not peripheral resident mature T cells are susceptible to this process of functional education, which also requires exposure to specific antigens and occurs entirely in the periphery. We propose that these mechanisms play a major role in establishing and maintaining natural self tolerance to tissue-specific antigens.     

Transient control of a virus-induced immunopathology by genetic immunosuppression

The ability to control T cell reactivity using suicide genes opens new perspectives for the treatment of T cell-mediated diseases. The therapeutic effect is achieved by the selective killing of thymidine kinase gene-modified activated T cells by ganciclovir (GCV). This strategy has been shown to control T cell alloreactivity efficiently after bone marrow or solid organ transplantation. Here, we aimed to determine whether an immunopathological process induced by a viral infection could be controlled by GCV when T cells express a thymidine kinase transgene. When transgenic mice were infected with the lymphocytic choriomeningitis virus, administration of GCV resulted in an efficient, but only transient, control of the immunopathological immune response. Further analysis revealed the existence of a minute population of GCV-insensitive T cells. These cells expand in response to the virus despite the presence of GCV and cause immunopathology before viral elimination is finally obtained. Thus, when confronted with a replicative virus, the efficacy of this genetic immunosuppression strategy is highly dependent on the presence of even small numbers of GCV-insensitive cells. These results emphasize the need for sufficient preclinical investigations with regard to the pathology and the nature of the immune response if suicide gene transfer is envisioned for new therapeutic indications.     

Lymphocytes selected in allogeneic thymic epithelium mediate dominant tolerance toward tissue grafts of the thymic epithelium haplotype.

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) from day 10 embryos before hematopoietic cell colonization reconstitute normal numbers of T cells and exhibit full life-long tolerance to skin grafts of the TE haplotype. Intravenous transfers of splenic cells, from these animals to adult syngeneic athymic recipients, reconstitute T-cell compartments and the ability to reject third-party skin grafts. The transfer of specific tolerance to skin grafts of the TE donor strain, however, is not observed in all reconstituted recipients, and the fraction of nontolerant recipients increases with decreasing numbers of cells transferred. Furthermore, transfers of high numbers of total or CD4+ T cells from TE chimeras to T-cell receptor-anti-H-Y antigen transgenic immunocompetent syngeneic hosts specifically hinder the rejection of skin grafts of the TE haplotype that normally occurs in such recipients. These observations demonstrate (i) that mice tolerized by allogeneic TE and bearing healthy skin grafts harbor peripheral immunocompetent T cells capable of rejecting this very same graft; and (ii) that TE selects for regulatory T cells that can inhibit effector activities of graft-reactive cells.     

Suicide gene-mediated modulation of graft-versus-host disease.

The development of suicide genes and progress in retroviral gene transfer to T-cells open new perspectives for the treatment of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) for leukemia and lymphoma. Indeed, suicide genes that metabolize inactive prodrugs into compounds toxic for dividing cells provide a powerful means for the pharmacogenetic control of T-cell reactivity. Here, we demonstrate the selective destruction of activated TK-transgenic T-cells in vivo and develop two new transgenic lines which should be useful for preclinical studies of suicide gene therapy strategies for GVHD.     

Tolerance induced without immunosuppression in a T-lymphocyte suicide-gene therapy cardiac allograft model in mice

BACKGROUND: Life-long immunosuppression is a major cause of mortality and morbidity in transplant recipients. Gene therapy could provide new ways to obtain tolerance and avoid indefinite immunosuppression. EpTK mice are derived from the FVB/N strain (H2q) and express the thymidine kinase gene of herpesvirus in all mature T cells. Thus any mature dividing T cell can be killed in the presence of ganciclovir. We investigated the survival of alloincompatible C57B1/6 (H2b) hearts heterotopically transplanted into EpTK mice given only ganciclovir from day 0 to day 7 or 14. METHODS: Abdominal cardiac transplantations were performed in 22 control mice (untreated FVB [n = 15], ganciclovir-treated FVB [n = 5], and untreated EpTK mice [n = 2]) and in 28 EpTK mice given ganciclovir from day 0 to day 7 (n = 15) or day 14 (n = 13). Rejection was defined as complete cessation of cardiac beat. Histologic examination of the grafts was performed at rejection, at day 7, or at day 100. Lymphocyte proliferation assays (concanavalin A stimulation or mixed lymphocyte reaction) were performed at day 7 and at day 100. RESULTS: All control animals rejected transplants in 7 days (range, 5-9 days), whereas indefinite survival (>100 days) was observed in 89% of the ganciclovir-treated EpTK group, irrespective of the duration of ganciclovir treatment. Graft histology showed extensive cellular infiltrates with myocyte necrosis and arteritis in the control animals but only a mild infiltrate without necrosis or arteritis in the ganciclovir-treated EpTK group. The proliferative responses of the tolerant mice at day 100 were identical to those of naive mice, including a preserved proliferation against the donor's lymphocytes in mixed lymphocyte reaction. CONCLUSION: Functional transplantation tolerance of a fully incompatible heart can be achieved without immunosuppressive drugs in this model of suicide gene therapy.     

Lymphocyte population kinetics during the development of the immune system. B cell persistence and life-span can be determined by the host environment

We have investigated the kinetic behaviour of LPS reactive Bcells during the development of the immune system. By studyingthe persistence of LPS reactive spleen cells transferred fromadult C57BL/6 donor mice into histocompatible C57BL/10 Sc.CrLPS non-responder mice we have confirmed that B cell populationsobtained from adult donor mice decay rapidly after transferinto adult recipients. In contrast, the same cell populationsafter transfer into neonatal reclpients are able to divide andmaintain their numbers for {small tilde}2-5 weeks in the host'sspleen. In fact, comparison of hosts at different ages showthat with the increasing age of the host the fate of donor Bcells evolves to mimic the behaviour observed upon transferinto adult recipients. Kinetic studies of LPS reactive spleencells obtained from newborn (2 weeks old) C57BL/6 donors aftertransfer into adult C57BL/10 Sc.Cr mice have shown that youngdonor cells were able to keep at constant numbers in the adultenvironment for the first {small tilde}10–15 days aftertransfer and to decay afterwards in the host's spleen at thesame rate as observed upon transfer of spleen cells from adultdonors into adult hosts. These studies provide the first evidencefor the different kinetic behaviour of lymphoid B cell populationsin developing and mature immune systems, confirm that cell persistencecorrelates with cell activation and division, and show thatB cell life-span is also dependent on the host environment.     

Thymic epithelium induces full tolerance to skin and heart but not to B lymphocyte grafts

Athymic nude mice reconstituted at birth with allogeneic thymic epithelia (TE) from day 10 embryos (E10), show life-long specific tolerance to skin and heart grafts, but eliminate B lymphocytes of the TE donor haplotype, nearly as well as those from a third strain. Previous immunizations with B cells do not alter the state of tolerance to skin grafts, but specifically accelerate elimination of lymphocytes. In contrast, transplantation of E15 allogeneic thymuses already seeded by hematopoietic cells resulted in chimeras tolerant to both skin and B lymphocytes. In vitro reactivities towards stimulator spleen cells of the haplotype of the thymus were observed in both E10 TE and E15 thymus chimeras. We conclude that induction of full in vivo tolerance to B cells requires hematopoietic cells, while this is not the case for induction of tolerance to skin and heart tissues; furthermore, in vitro reactivity to stimulator spleen cells of the tolerized haplotype is independent of in vivo tolerance.