Induction Of Central Tolerance Research Articles

Vascularized thymic lobe transplantation in miniature swine: thymopoiesis and tolerance induction across fully MHC-mismatched barriers.

As the major site of self-nonself discrimination in the immune system, the thymus, if successfully transplanted, could potentially carry with it the induction of central tolerance to any other organ or tissue from the same donor. We have recently developed a technique for transplantation of an intact, vascularized thymic lobe (VTL) in miniature swine. In the present study, we have examined the ability of such VTL allografts to support thymopoiesis and induce transplantation tolerance across fully MHC-mismatched barriers. Six miniature swine recipients received fully MHC-mismatched VTL grafts with a 12-day course of tacrolimus. Three of these recipients were thymectomized before transplantation and accepted their VTL allografts long-term, with evidence of normal thymopoiesis. In contrast, three euthymic recipients rejected their VTL allografts. Donor renal allografts, matched to the donor VTL grafts, were transplanted without immunosuppression into two of the three thymectomized recipients, and one of the three euthymic recipients. These renal allografts were accepted by thymectomized recipients, but rejected by the euthymic recipient in an accelerated fashion. This study thus demonstrates that successful transplantation of a vascularized thymus across a fully MHC-mismatched barrier induces tolerance in this preclinical, large-animal model. This procedure should enable studies on the role of the thymus in transplantation immunology as well as offer a potential strategy for tolerance induction in clinical transplantation.

The Biology of CD45 and its Use as a Therapeutic Target

All mature hemopoietic lineage cells, with exclusion of platelets and mature erythrocytes, share the surface expression of a transmembrane phosphatase, the CD45 molecule. It is also present on hemopoietic stem cells and most leukemic clones and therefore presents as an appropriate target for immunotherapy with anti-CD45 antibodies. This short review details the biology of CD45 and its recent targeting for both treatment of malignant disorders and tolerance induction. In particular, the question of potential stem cell depletion for induction of central tolerance or depletion of malignant hemopoietic cells is addressed. Mechanisms underlying the effects downstream of CD45 binding to the cell surface are discussed.

Establishing insulin 1 and insulin 2 knockout congenic strains on NOD genetic background.

As insulin is a major autoantigen in autoimmune diabetes and because the insulin gene region locus in humans has been linked to diabetes risk, we have bred insulin gene knockouts onto the NOD mouse. Mice differ from humans in terms that they express two nonallelic genes of insulin. Insulin 2 is the murine homologue of the human insulin gene and is located on mouse chromosome 7. Insulin 1 is thought to have evolved by a gene duplication event, lacks the second intron of the insulin 2 gene, and is located on mouse chromosome 19. The differential thymic expression of the insulin gene may be important for central tolerance induction. Here, we present the initial establishment of congenic knockouts and characterization of the congenic intervals corresponding to insulin 1 and insulin 2 knockout genes on mouse chromosome 19 and 7, respectively.

Thymic medullary epithelial cell differentiation, thymocyte emigration, and the control of autoimmunity require lympho-epithelial cross talk via LTbetaR.

Thymocytes depend on the interaction with thymic epithelial cells for the generation of a diverse, nonautoreactive T cell repertoire. In turn, thymic epithelial cells acquire their three-dimensional cellular organization via instructive signals from developing thymocytes. The nature of these signals has been elusive so far. We show that thymocytes and medullary epithelial cells (MECs) communicate via the lymphotoxin β receptor (LTβR) signaling axis. Normal differentiation of thymic MECs requires LTβR ligand on thymocytes and LTβR together with nuclear factor–κB-inducing kinase (Nik) in thymic epithelial cells. Impaired lympho–epithelial cross talk in the absence of the LTβR causes aberrant differentiation and reduced numbers of thymic MECs, leads to the retention of mature T lymphocytes, and is associated with autoimmune phenomena, suggesting an unexpected role for LTβR signaling in central tolerance induction.

Autoreactive T cells can be protected from tolerance induction through competition by flanking determinants for access to class II MHC.

It is not clear why the N-terminal autoantigenic determinant of myelin basic protein (MBP), Ac1-9, is dominant in the B1O.PL (H-2(u)) mouse, given its weak I-A(u)-MHC binding affinity. Similarly, how do high-affinity T cells specific for this determinant avoid negative selection? Because the MBP:1-9 sequence is embryonically expressed uniquely in the context of Golli-MBP, determinants were sought within the contiguous N-terminal "Golli" region that could out-compete MBP:1-9 for MHC binding, and thereby prevent negative selection of the public response to Ac1-9, shown here to be comprised of a V beta 8.2J beta 2.7 and a V beta 8.2J beta 2.4 expansion. Specifically, we demonstrate that Ac1-9 itself can be an effective inducer of central tolerance induction; however, in the context of Golli-MBP, Ac1-9 is flanked by determinants which prevent its display to autoreactive T cells. Our data support competitive capture as a means of protecting high-affinity, autoreactive T cells from central tolerance induction.

Induction of donor-specific tolerance to allogeneic hepatocytes by allogeneic bone marrow transplantation

Allogenic hepatocytes are rejected within a few days after transplantation without immunosuppression. We previously showed that the peripheral tolerance did not effectively prolong the survival of allogeneic hepatocytes transplanted in the spleen, because anergic T cells lysed allogeneic hepatocytes through Fas/Fas ligand system. The aim of this study was to address whether or not the central tolerance induced by allogeneic bone marrow transplantation (BMT) allows transplanted hepatocytes to survive in the spleen of the recipient. Bone marrow cells obtained from C57BL/6 (B6) donor mice were injected intravenously into lethally irradiated BALB/c recipients. Ninety percent of the recipient mice were able to survive more than 30 days, although all of the non-BMT controls died within 12 days. Full donor chimerism was observed in the recipient. Then hepatocytes from B6 mice were transplanted into the spleen of BALB/c mice at 30 days post-BMT. The transplanted donor-hepatocytes were able to survive for over 7 days, while those from third-party mice (C3H/He) were rejected within a few days. These findings demonstrate that donor-specific tolerance was induced by allogeneic BMT. Furthermore, the induction of central tolerance, but not peripheral tolerance, may be a useful strategy for prolonging the survival of allogeneic hepatocytes in the spleen.

Multiple sclerosis candidate autoantigens except myelin oligodendrocyte glycoprotein are transcribed in human thymus.

An important feature of central nervous system (CNS) immune privilege is that antigens expressed in CNS are sequestered and not available for central tolerance induction. Tissue distribution and, more specifically, thymic expression of many of the CNS putative autoantigens have not yet been clearly established in humans. We have addressed this question for the putative multiple sclerosis(MS) autoantigens alphaB-crystallin, S100beta, proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG)-alpha and MOG-beta isoforms, using quantitative RT-PCR on human thymus (total, cell fractions and microdissected specimen) and on a panel of peripheral tissues. alphaB-crystallin, S100beta and the DM20 isoform of PLP were clearly expressed in the thymus and also in selected peripheral tissues. In contrast, the expression of MOG out of the CNS was not observed. Within the human thymus, the level of CNS antigen expression was found higher in the stromal epithelial enriched cell fraction, and in microdissected samples of the medullary compartment. These results indicate that most of the antigens involved in MS are expressed in the thymus, suggesting a possible role in central tolerance. However, MOG and, to a lesser extent PLP, conform the classical concept of sequestered antigens, thus supporting the involvement of MOG in autoimmune demyelinating diseases.

Immunogenicity and tolerogenicity of hepatitis B virus structural and nonstructural proteins: implications for immunotherapy of persistent viral infections.

Persistent hepatitis B virus (HBV) infection is characterized by a weak and narrowly focused CD8+ T-cell response to HBV that is thought to reflect the induction of central and/or peripheral tolerance to HBV proteins in neonatal and adult onset infections, respectively. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may lead to viral clearance in chronically infected individuals. The present study was performed to compare the relative immunogenicities and tolerogenicities of HBV structural (envelope [ENV]) and nonstructural (polymerase [POL]) proteins at the CD8+ cytotoxic T lymphocyte (CTL) level in transgenic mice that replicate HBV in the liver and secrete infectious virus into the blood, thus representing an excellent model of persistent HBV infection. Interestingly, the mice were tolerant to the ENV but not to the POL proteins at the CTL level. Furthermore, the POL-specific CTLs had no impact on HBV replication or liver function in vivo, even though they were readily induced and reached the liver after DNA immunization, reflecting their relatively low avidity and the low level at which the POL protein is expressed by the hepatocyte. Collectively, these results suggest that the factors that make POL less tolerogenic also make POL-specific CTLs relatively inefficient effector cells when they reach the target organ. Immunotherapeutic strategies to control HBV infection by inducing virus-specific CTL responses in chronically infected subjects should be evaluated in light of this observation.

Cyclosporin A-Induced Autoimmunity in the Rat: Central versus Peripheral Tolerance

No abstract available

Evolution of adenoviruses as gene therapy vectors.

Evolution of adenoviruses as gene therapy vectors.

Disorganization of Thymic Medulla Precedes Evolution Towards Diabetes in Female NOD Mice

The thymic medulla is a complex microenvironment which plays a crucial role in central tolerance induction. Using a quantitative histological analysis of non-obese diabetic (NOD) mice, we show that the medulla undergoes several structural modifications during the course of the disease in NOD mice. Indeed, the majority of 70-day-old NOD mice show a scattering of medullary epithelial cells in the cortex which is associated with a reduction in the size of the medulla in heavily disorganized thymuses. The severity of this phenotype is shown to correlate with the subsequent appearance of diabetes in older female NOD mice. This trait is mainly controlled by non-major histocompatibility complex NOD genes since C57BL/6 H-2g7 congenic mice have a normal medulla. It persists in conditions where effector lymphocytes that lead to diabetes are inhibited in periphery. These results suggest that primary alterations of the thymic stroma might play a role in the progression towards diabetes in NOD mice.

Transfer of porcine MHC DRalpha into IEalpha-deficient murine bone marrow results in reduced IE-restricted Vbeta usage.

Allogeneic bone marrow transplantation has proven effective for inducing specific tolerance to subsequent solid organ allografts, although the clinical applicability of this approach is limited by the morbidity and mortality associated with this procedure. As an alternative, we are investigating the transfer of allogeneic MHC class II genes into recipient bone marrow cells (BMC), using the miniature swine as a model. To understand the mechanism of tolerance induction achieved through class II gene transfer, BMC from C57BL/10 mice, which lack expression of the MHC class II DRalpha equivalent (H-2 IEalpha), were transduced with a retrovirus vector for swine DRalpha. Expression of the DRA-vector in bone marrow-derived cells was demonstrated by Northern analysis of colonies grown in vitro from transduced myeloid progenitors. Taking advantage of the fact that the introduced DRalpha chain was able to form heterodimers with endogenous IEbeta, surface expression of the transgene was demonstrated on splenocytes harvested 1, 17, and 28 weeks after bone marrow transplantation. Transgene expression was confirmed by reverse transcriptase-polymerase chain reaction in the thymus of those animals killed at weeks 17 and 28. Finally, the effects of bone marrow transduction on central tolerance induction was demonstrated by the progressive decrease of IE-reactive T-cell clones bearing Vbeta5 and Vbeta11 T cell receptors in the peripheral blood cells of engineered recipients. Our results support the notion that transplantation tolerance, induced by class II gene transfer into syngeneic BMC, results in part from durable deletional unresponsiveness of graft-specific alloreactive T cells.

T Lymphocyte Tolerance: From Thymic Deletion to Peripheral Control Mechanisms

This chapter discusses the central and peripheral tolerance in the T-cell population and self-tolerance in the B-cell population. The understanding of tolerance induction in lymphocyte subpopulations is exponentially advanced in the generation of T- and B-cell receptor transgenic mice. Initial contradictions that seem to appear as different transgenic models are published and have given way to overall consensus; at least as far as central tolerance induction in the thymus is concerned. The first direct demonstration for the elimination of potentially autoreactive cells in the thymus came from studies of the T-cell repertoire to endogenous superantigens. T cells bearing Vβ segments that conferred binding to endogenous mouse mammary tumor viruses (Mtv) were absent from mice carrying these superantigens in their genome. One mechanism of deletion is terminal differentiation to short-lived effector cells that may die by apoptosis because of lymphokine depletion. The latter mechanism could be prominent for CD8 T cells. The activation of T cells requires at least two signals: a signal through the T-cell receptor and a second signal presumed to involve a co-stimulatory molecule(s) on an antigen-presenting cell.

Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning.

Donor-specific tolerance induced by bone marrow transplantation (BMT) would allow organ allografting without chronic immunosuppressive therapy. However, the toxicity of conditioning regimens used to achieve marrow engraftment has precluded the clinical use of BMT for tolerance induction. We have developed a BMT strategy that achieves alloengraftment without toxic or myelosuppressive host conditioning. B6 mice received depleting anti-CD4 and anti-CD8 monoclonal antibodies, local thymic irradiation, and a high-dose (174 x 10(6)) of major histocompatibility (MHC)-mismatched B10.A bone marrow cells (BMCs) divided over days 0 through 4. High levels of donor cells were observed among white blood cells (WBCs) of all lineages. Permanent, multilineage mixed chimerism; donor-specific skin-graft tolerance; and in vitro tolerance were observed in most animals. Large numbers of donor class II(high) cells were detected in thymuses of long-term chimeras, and their presence was associated with intrathymic deletion of donor-reactive host thymocytes. The treatment was not associated with significant myelosuppression, toxicity, or graft-versus-host disease (GVHD). Thus, high levels of allogeneic stem-cell engraftment can be achieved without myelosuppressive host conditioning. As stem-cell mobilization and in vitro culture techniques have increased the feasibility of administering high doses of hematopoietic cells to humans, this approach brings hematopoietic cell transplantation closer to clinical use for the induction of central deletional T-cell tolerance.

The role of dendritic cells in T cell activation.

Dendritic cells (DC) are distinguishable from other antigen-presenting cells by their potent antigen-presenting capacity. They are not only efficient at presenting peptide antigen but can also process and present soluble protein antigen sto antigen-specific T cells and cloned T cell lines. They are very strong stimulators of both allogeneic and syngeneic mixed lymphocyte reactions and have a unique capacity to stimulate naive T cells. The potent functional capacity of DC is related to a high-level expression of major histocompatibility complex class I/II molecules and constitutive expression of costimulatory molecules, such as CD80/CD86, as well as heat stable antigen, CD40 and the leucocyte function antigen (LFA) family of adhesion molecules. Recent studies have shown that DC are also involved in regulation of the immune response via induction of both central and peripheral tolerance.

Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long-term gene therapy in Gunn rats.

Recombinant adenoviruses are highly efficient at transferring foreign genes in vivo. However, duration of gene expression is limited by the host antiviral immune response which precludes expression upon viral readministration. We tested the feasibility of prolonging gene expression by induction of central tolerance to adenoviral antigens in bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats. Tolerance was induced by intraperitoneal injection of antilymphocyte serum, followed by intrathymic inoculation of one of the following: a recombinant adenovirus (Ad), adenovirus human UDP-glucuronosyltransferase (Ad-hBUGT1) carrying the hBUGT1 gene; a protein extract of the same virus; or viral infected hepatocytes. Controls received intrathymic injections of normal saline. After 12 d all groups were injected intravenously with 5 x 10(9) pfu of either Ad-hBUGT1 or adenovirus beta-galactosidase (Ad-LacZ) (expressing the Escherichia coli beta-galactosidase [LacZ] gene). In all three groups of tolerized rats, hBUGT1 was expressed in the liver after administration of Ad-hBUGT1, with glucuronidation of biliary bilirubin of above 95%. Serum bilirubin levels decreased from 7.2 to 1.8 mg/dl within 1 wk and remained low for 7 wk. Similar findings were observed following repeat injections given on days 45 and 112. In control rats serum bilirubin levels were reduced for only 4 wk, and viral readministration was ineffective. In all tolerized groups, but not in controls, there was a marked inhibition of appearance of neutralizing antibodies and cytotoxic lymphocytes against the recombinant adenovirus. Injection of wild type adenovirus-5 (Ad5) into the tolerized rats elicited a wild type-specific cytotoxic lymphocyte response. This is the first demonstration of Ad-directed long-term correction of an inherited metabolic disease following central tolerization with thymic antigen.

Mechanism by which additional monoclonal antibody (mAB) injections overcome the requirement for thymic irradiation to achieve mixed chimerism in mice receiving bone marrow transplantation after conditioning with anti-T cell mABs and 3-Gy whole body irradiation.

A relatively nontoxic method of conditioning mice has been developed recently that allows allogeneic bone marrow engraftment and specific skin allograft tolerance induction. This regiment included anti-CD4 and anti-CD8 mAbs administered on day -5, followed by 3-Gy whole body irradiation (WBI) and 7-Gy thymic irradiation (TI) on day 0. We have recently shown that the potential toxicity of this regimen can be further reduced by replacing TI with additional anti-T cell mAb injections before and after bone marrow transplantation. Mixed chimerism and prolonged donor-specific skin graft acceptance are induced in 90% of B10 mice conditioned with anti-CD4 and anti-CD8 mAbs on days -6 and -1 and 3-Gy WBI on day 0 without TI, but only in a small fraction of mice receiving a similar regimen, except that mAbs are given on day -5 only. To determine the mechanism of tolerance induction in the former group, we compared the two groups for the extent of thymocyte depletion, for the timing of development of intrathymic and extrathymic chimerism, and for clonal deletion of host-type thymocytes with TCR recognizing superantigens presented by donor class II molecules. The results suggest that administration of a second mAb injection depletes or inactivates residual host thymocytes that are capable of causing intrathymic rejection of donor hematopoietic cells even when peripheral engraftment is achieved. The presence of donor class II+ hematopoietic cells in the thymus on day 14 correlated with marked deletion of mature host-type V beta 11+ thymocytes that recognize donor I-E plus endogenous superantigen. This suggests that tolerance is achieved primarily through a central deletional mechanism when peripheral and intrathymic host T cells are adequately inactivated or depleted by mAbs and 3-Gy WBI. In addition, the higher incidence of early failure of peripheral chimerism in mice conditioned with a single injection rather than than two mAb injections prior to bone marrow transplantation suggests that nontolerant residual host thymocytes can also induce early peripheral rejection after mAbs have cleared from the circulation. This early rejection is prevented by the longer persistence of anti-T cell mAbs observed in mice receiving two pretransplant mAb injections. Thus, administration of sufficient depleting anti-T cells mAbs followed by 3-Gy WBI allows the induction of central deletional tolerance while minimizing the toxicity of the conditioning regimen.

Induction of donor-specific unresponsiveness to rat cardiac allografts by pretreatment with intrathymic donor MHC class I antigens.

Since intrathymic injection of UV-B-irradiated spleen cells induces donor-specific unresponsiveness in the sublethally irradiated (200 rads TBI) recipients, while intrathymic injection of naive SC leads to acute graft rejection, we hypothesized that presentation of MHC class I rather than MHC class II antigens to immature T cells in the thymus may convey a tolerogenic signal to the recipient. The present study was designed to examine if intrathymic injection of naive MHC class I-positive resting T lymphocytes can induce antigen-specific unresponsiveness to cardiac allografts in the Lewis-to-ACI rat combination. The results showed that intrathymic injection of resting Lewis T cells consistently induced indefinite graft survival (> 300 days) in sublethally irradiated (200 rads TBI) ACI recipients while similar treatment failed to prevent the rejection of third-party (Wistar-Furth) cardiac allografts, thus demonstrating the specificity of the immunologic unresponsiveness to donor alloantigens. Examination of the timing of intrathymic antigen presentation relative to cardiac transplantation that would achieve 100% permanent graft survival in the Lewis-to-ACI rat combination showed that the optimal time was 7 days before allografting, while peritransplant and immediate post-transplant intrathymic inoculation of donor T cells was relatively ineffective in the induction of unresponsiveness to donor grafts. We also showed that removal of the antigen-containing thymus in the sublethally irradiated recipients with functioning cardiac allografts consistently caused graft rejection if performed earlier than 21 days after heart transplantation; thymectomy after 21 days of organ transplantation did not affect indefinite survival of the grafts. Thus, it appears that the maintenance of peripheral tolerance to the grafts after 21 days of transplantation may be dependent on the presence of a new clone of antigen-specific tolerant host T cells. These results confirm the immunologic privileged position of the thymus in the induction of central and peripheral tolerance, and suggest that pretreatment with intrathymic MHC class I alloantigens is potentially useful in the induction of unresponsiveness to donor vascularized allografts in adult animals and in man.

Ber-ACT8: new monoclonal antibody to the mucosa lymphocyte antigen.

Using a newly established HTLV-1 positive T cell line as an immunogen, a new monoclonal antibody, Ber-ACT8, was produced. It reacts with in vitro activated T cells and a small subset of normal resting T cells, but not with resting B cells or any of the 29 established human permanent cell lines tested. Immunohistological analysis of a wide spectrum of human tissues showed that Ber-ACT8 reactivity is restricted to a few T cells in the peripheral blood, the extrafollicular areas of lymph nodes and tonsils, and splenic red pulp. In the gut Ber-ACT8 labelled most intraepithelial T cells and up to 50% of lamina propria T cells. The antibody also immunostained T cells present in the oral and bronchial mucosa. Double labelling on splenic cells, fresh blood lymphocytes, and in vitro activated T cells showed that most Ber-ACT8 positive cells coexpressed CD8. Ber-ACT8 did not react with any of the 14 Hodgkin's lymphomas nor any of the 172 non-Hodgkin's lymphomas tested, with the exception of 10 cases of T cell lymphomas, five of which were located in the jejunum and associated with coeliac disease, and one B cell lymphoma, and most cases of hairy cell leukaemia tested. Parallel immunostainings with Ber-ACT8, anti-TCR-beta (beta F1), and anti-TCR-delta showed that most Ber-ACT8 positive T cells carry the TCR of alpha beta type. Comparison of Ber-ACT8 with HML-1, B-ly7, and LF61 showed essentially the same reactivity and an identical molecular target. The molecular structure recognised seems to be a trimeric molecule with components of 150, 125 and 105 kilodaltons, with the Ber-ACT8 epitope localised on the 150 kilodalton chain. The 150 kilodalton molecule contains an 0-linked carbohydrate moiety of about 10 kilodaltons. Because of its very selective distribution, the trimeric antigen is a powerful reagent for the diagnosis of gut T cell-derived T cell lymphomas and other extranodal T cell lymphomas, as well as hairy cell leukaemia.