Intrathymic Tolerance Induction Research Articles

T cells specific for post-translational modifications escape intrathymic tolerance induction

Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism.

Efficient Intrathymic Gene Transfer Following In Situ Administration of a rAAV Serotype 8 Vector in Mice and Nonhuman Primates

The thymus is the primary site of T-cell development and plays a key role in the induction of self-tolerance. We previously showed that the intrathymic (i.t.) injection of a transgene-expressing lentiviral vector (LV) in mice can result in the correction of a T cell-specific genetic defect. Nevertheless, the efficiency of thymocyte transduction did not exceed 0.1-0.3% and we were unable to detect any thymus transduction in macaques. As such, we initiated studies to assess the capacity of recombinant adeno-associated virus (rAAV) vectors to transduce murine and primate thymic cells. In vivo administration of AAV serotype 2-derived single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors pseudotyped with capsid proteins of serotypes 1, 2, 4, 5, and 8 demonstrated that murine thymus transduction was significantly enhanced by scAAV2/8. Transgene expression was detected in 5% of thymocytes and, notably, transduced cells represented 1% of peripheral T lymphocytes. Moreover, i.t. administration of scAAV2/8 particles in macaques, by endoscopic-mediated guidance, resulted in significant gene transfer. Thus, in healthy animals, where thymic gene transfer does not provide a selective advantage, scAAV2/8 is a unique tool promoting the in situ transduction of thymocytes with the subsequent export of gene-modified lymphocytes to the periphery.

Efficiency of intrathymic tolerance induction in various inbred rat strains: relationship with T H1/T H2 status of the recipient?

Summary The simultaneous transplantation and intrathymic tolerance induction (STITTI) protocol induces a longlasting state of functional tolerance in over 90% of AO (RT1 u ) recipients transplanted with a fully MHC-incompatible PVG (RT1 c ) cardiac allograft. Similar results are obtained when using LEWIS (RT1 1 ) rats as recipients of either PVG or DA (RT1 avl ) grafts. However, when STITTI is performed on PVG and BN (RT1 n ) as recipient animals receiving spleen cells intrathymically and a cardiac allograft from respectively AO and PVG rats, this procedure results in significantly shorter graft survival (MST PVG → BN 25 ± 9 days; AO → PVG 31 ± 8 days) as compared to the combinations using AO (MST PVG → AO > 236 ± 28 days) and LEWIS (MST PVG → LEW > 366 ± 51 days; DA → LEW > 123 ± 33 days) rats as recipients. Since both PVG and BN rats are relatively deficient in their ability to produce IFNγ and intrathymic IFNγ responses are very dominant upon intrathymic injection of alloantigens, it is argued that the inability to effectively induce a longlasting state of functional tolerance in BN and PVG rats using the STITTI protocol may be related to their decreased IFNγ-production potential.

An alternative model to study intrathymic tolerance induction: the neonatal heart-in-ear transplantation model in the rat

An alternative model to study intrathymic tolerance induction: the neonatal heart-in-ear transplantation model in the rat

Direct allorecognition: a mechanism in intrathymic tolerance induction

Direct allorecognition: a mechanism in intrathymic tolerance induction

Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts.

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells.

Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells. (Hepatology 1997 Nov;26(5):1287-95)

Long-lasting adenovirus transgene expression in mice through neonatal intrathymic tolerance induction without the use of immunosuppression.

The major barrier to the clinical application of adenovirus gene therapy for diseases that require stable transgene expression is the immunogenicity of recombinant adenovirus, which ordinarily limits the duration of its effects to a period of about 2 weeks. We postulated that tolerance to adenovirus could be induced and transgene expression could be prolonged if T lymphocytes underwent thymic selection in the presence of adenovirus antigens. Mice were inoculated in the thymus with a recombinant adenovirus containing the lacZ marker gene during the neonatal period at a time before T-cell maturation had occurred. When the virus was administered intravenously to these mice in adulthood, they were found to have an impaired adenovirus-specific cytotoxic T-lymphocyte response which allowed prolonged hepatic lacZ expression, for up to 260 days. The ability to achieve unresponsiveness to a recombinant adenovirus after inoculation of the thymus in neonates extends the paradigm of intrathymic tolerance induction. Furthermore, this model will enable the study of stable adenovirus transgene expression in vivo without the use of immunosuppression and ultimately may have clinical utility.

A METHOD WITH CLINICAL POTENTIAL

It has been shown that donor-specific tolerance to cardiac allografts can be induced by pretreating the prospective recipient with injections of donor spleno-cytes (intrathymically) and antilymphocyte serum (in-traperitoneally) weeks or days before the actual transplantation. This procedure, however, lacks clinical relevance in the case of cadaver donors due to the obligatory interval between the start of the tolerance induction protocol and transplantation. We have tried to devise a protocol in which this interval is eliminated, thus allowing allotransplantation simultaneously with tolerance induction. Our results show that simultaneous cardiac allotransplantation and intrathymic tolerance induction by intrathymic injection of donor splenocytes and treatment with antilymphocyte serum is indeed possible in the PVG to AO high-responder rat strain combination, provided that low doses of cyclosporine are given intramuscularly on day 1, 2, and 3 after transplantation. As we now are able to combine the start of tolerance induction with the actual allotransplantation, this procedure may indeed have clinical potential.

A METHOD WITH CLINICAL POTENTIAL

It has been shown that donor-specific tolerance to cardiac allografts can be induced by pretreating the prospective recipient with injections of donor splenocytes (intrathymically) and antilymphocyte serum (intraperitoneally) weeks or days before the actual transplantation. This procedure, however, lacks clinical relevance in the case of cadaver donors due to the obligatory interval between the start of the tolerance induction protocol and transplantation. We have tried to devise a protocol in which this interval is eliminated, thus allowing allotransplantation simultaneously with tolerance induction. Our results show that simultaneous cardiac allotransplantation and intrathymic tolerance induction by intrathymic injection of donor splenocytes and treatment with antilymphocyte serum is indeed possible in the PVG to AO high-responder rat strain combination, provided that low doses of cyclosporine are given intramuscularly on day 1, 2, and 3 after transplantation. As we now are able to combine the start of tolerance induction with the actual allotransplantation, this procedure may indeed have clinical potential.

INDUCTION OF TOLERANCE TO SKIN ALLOGRAFTS BY INTRATHYMIC INJECTION OF DONOR SPLENOCYTES

The effect of donor-recipient strain combination and supplemental rapamycin (Rapa) on tolerance induction by intrathymic (IT) injection of donor splenocytes was examined in a mouse skin allograft model. In an MHC class I-mismatched C3H/He skin to (C57BL/6 x A)F1 (B6AF1) mouse combination, IT injection of 50 x 10(6) donor splenocytes with transient immunosuppression by rabbit anti-mouse lymphocyte serum (ALS) induced significant prolongation of skin allograft survival with a median survival time (MST) of 115 days versus an MST of 24.5 days in controls given ALS alone. With an additional short course of supplemental Rapa treatment at a dose of 1.5 mg/kg i.p. every other day from day 0 to 12, all C3H/He skin allografts survived indefinitely (> 350 days) in ALS-treated, donor splenocyte intrathymically injected B6AF1 recipient mice. Tolerance was antigen-specific, since the second donor-type skin allografts were accepted while third-party skin allografts were acutely rejected in these mice bearing long-term C3H/He skin allografts. In MHC class I- and II-disparate (DBA/2 to B6AF1) and fully MHC-incompatible (AKR to B6) strain combinations, IT injection of donor splenocytes and ALS treatment failed to prolong skin allograft survival over ALS controls. When supplemental Rapa was used, long-term skin allograft acceptance was observed with an MST of 127 days for the DBA/2 to B6AF1 combination and 70 days for the AKR to B6 combination. In contrast, supplemental treatment with cyclosporine was not effective in these combinations, which suggests that supplemental Rapa may have a unique effect in augmenting IT tolerance induction. Thymectomy within 7 days after IT injection significantly shortened the allograft survival, which suggests that interaction of the host thymus and the injected donor splenocytes, which takes place early after IT injection, plays an important role in the induction of allograft tolerance in this model.

Reentry of T cells to the adult thymus is restricted to activated T cells.

To seek information on the capacity of mature T cells to migrate to the thymus, mice were injected with Thy-1-marked populations enriched for resting T cells or T blast cells; localization of the donor cells in the host thymus was assessed by staining cryostat sections of thymus and by FACS analysis of cell suspensions. With injection of purified resting T cells, thymic homing was extremely limited, even with injection of large doses of cells. By contrast, in vivo generated T blast cells migrated to the thymus in substantial numbers. Thymic homing by T blasts was greater than 50-fold more efficient than with resting T cells. Blast cells localized largely in the medulla and remained in the thymus for at least 1 mo post-transfer. Interestingly, localization of T blasts in the thymus was 10-fold higher in irradiated hosts than normal hosts. Thymic homing was especially prominent in mice injected with T blasts incubated in vitro with the DNA precursor, 125I-5-iodo-2'deoxyuridine (125IDUR); with transfer of 125IDUR-labeled blasts to irradiated hosts, up to 5% of the injected counts localized in the host thymus. These data suggest that thymic homing by T blasts might be largely restricted to cells in S phase. The physiological significance of blast cell entry to the thymus is unclear. The possibility that these cells participate in intrathymic tolerance induction is discussed.

Intrathymic tolerance induction: determination of tolerance to class II major histocompatibility complex antigens in maturing T lymphocytes by a bone marrow-derived non-lymphoid thymus cell.

Two new experimental approaches were established to analyse the influence of the thymus on tolerance induction to major histocompatibility complex (MHC) antigens: The aim of the first experiment was to perform successful transplantation of adult allogeneic thymus tissue into nude mice, an attempt that has been unsuccessful in the past. Tolerance for the MHC genotype of a prospective thymus graft recipient (A) was induced in mice of strain B by injection of (A X B) splenocytes during the neonatal period. Adult thymic tissue obtained from these allogeneic donors (B) were grafted into the nude mice of strain A. The allogeneic thymus was accepted by the nude mice and immunoreconstitution was achieved. Subsequently the recipients developed tolerance to the MHC antigens of the allogeneic thymus donor as proved by mixed lymphocyte cultures and the acceptance of skin grafts. The second experiment was designed to determine which Ia-positive thymic compartment participates in conferring tolerance to MHC antigens in maturing T lymphocytes. Chimaeric thymus grafts were created by transplantation of neonatal thymus (A) into allogeneic nude mice (B) for a period of 8 weeks. The graft was populated with host bone marrow-derived Ia antigen-positive cells. The chimaeric thymuses consisting of type A epithelium but populated with both type A and B lymphocytes and non-lymphoid cells (i.e. Ia-positive macrophages and dendritic cells), were newly transplanted into nude mice of strain A. The engraftment lead to immunological reconstitution and the nude mice acquired tolerance to the MHC antigens expressed by the allogeneic Ia-positive cells populating the chimaeric graft. Irradiation of the chimaeric thymus prior to transplantation allowed transplantation of chimaeric thymus devoid of living thymocytes but still populated with functionally intact Ia-positive non-lymphoid cells. Transplantation of irradiated chimaeric thymuses resulted in immunoreconstitution and induced exactly the same allotolerance pattern as described above. The results demonstrate that not thymus epithelial cells but a bone-marrow-derived non-lymphoid thymus cell, most likely the Ia-antigen-positive thymic macrophage of dendritic cell, is responsible for the induction of tolerance to MHC antigens in developing T lymphocytes.

PROMOTION OF INTRATHYMIC T-CELL DEVELOPMENT BY MHC-Ia-POSITIVE THYMIC MACROPHAGES (Ia + Mø)

The generation of the peripheral T-cell pool is attributed to maturation and differentiation events occurring within the thymus. Three thymic compartments, thymic epithelial cells, thymic hormons and thymic Mo are considered to contribute to the intrathymic T-cell development. While, as we have shown earlier, murine as well as human MHC-Ia-negative-Mo (Ia−Mo) regulate thymocyte (TH) differentiation via suppressive effects (Thymus,6:295,1984) and MHC-Ia+-Mo are involved in intrathymic tolerance induction (Pediatr Res,15:800, 1984), now the contribution of Ia+Mo to intrathymic lymphopoiesis was investigated. An isolation method yielding cell suspensions highly enriched for Ia+ thymic Mo was performed (BSA-gradient,antibody treatment,irradiation). Cocultivation of these Ia+Mo with TH showed that Ia+Mo strongly augmented the mitogen-induced proliferation of TH by about 200%. The effect was dependent on the number of Ia+Mo added and on the degree of TH maturity: stronger augmentation occurred at higher Mo concentrations immature TH showed highest susceptibility to the Ia+Mo mediated effect. Cell-cell-contact was an important prerequisite for the proliferation amplifying effect as demonstrated by use of Cytochalasin B. In addition, humoral factors produced by Ia+Mo also improved the proliferative capacity of TH, similar to results obtained using interleukin II. The results described support the view that thymic Mo populations act as a main regulatory principle in T-cell development. Ia+Mo augment intrathymic lymphopoiesis and participate in tolerance induction,while Ia−Mo control the number of TH finally leaving the thymus gland.

RELEVANCE OF MESENCHYMAL THYMUS CELLS (MC) FOR ACCEPTANCE OF ALLOGENEIC MATURE THYMUS GRAFTS AND INDUCTION OF ALLOTOLERANCE IN NUDE MICE

Allogeneic thymus (aTh) from an adult immunocompetent donor cannot be transplanted successfully in thymusless nude mice even if the graft is irradiated. The graft is destroyed for unknown reasons and no immunocompetence is achieved in the host. Two approaches were established to achieve acceptance of aTh. In experiment 1, mature aTh was transplanted in nude mice, after specific tolerance to the recipient was induced in the donor during the neonatal period. This resulted in acceptance of the aTh, immunological reconstitution and tolerance to the donor's and recipient's MHC-haplotype as proved by skin-grafts and MLC. In experiment 2, neonatal thymus was grafted into allogeneic nude mice and retransplanted 6 weeks later into nude mice syngeneic to the thymus donor.Before retransplantation, investigation of the thymus tissue by monoclonal antibodies showed predominant MHC-antigen expression of the allogeneic host's haplotype on the MCs. Again,acceptance and immunological reconstitution was obtained; tolerance to the MHC-haplotype present on the allogeneic MCs was observed. These results demonstrate that mature aTh can be successfully transplanted if intrathymic tolerance to the recipient has been induced during the immature status of the thymus. After acceptance of the thymus, tolerance to the donor and recipient MHC-haplotype is achieved, apparently transmitted by the MCs. Intrathymic tolerance induction, therefore, may provide a new approach in the treatment of thymus-deficient patients.