Chimerism and central tolerance

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Chimerism and central tolerance Megan Sykes

Current Opinion in Immunology 1996, 8:694-703

the peripheral tissues. Peripheral tolerance has only been successfully induced in models involving relatively weak transplantation barriers such as those posed by MHC matched, minor histocompatibility antigen disparate grafts (reviewed in [2]) or by MHC mismatched cardiac and renal grafts in rodents. It is generally more difficult, however, to induce tolerance to allografts in large animals than in rodents. In contrast to central tolerance, peripheral mechanisms alone have not been sufficient to reliably overcome the most stringent transplantation barrier imposed by fully MHC mismatched primary skin allografts. Thus, central tolerance may prove to be critical to the development of clinical protocols that can ensure the induction of tolerance reliably enough to justify withholding chronic immunosuppressive therapy.

© Current Biology Ltd ISSN 0959-7915

Chimerism

Abbreviations APC antigen-presentingcell BMC bone marrow cell BMT bone marrow transplantation CTL cytotoxicT lymphocyte CTLp CTL precursor GVHD graft-versus-hostdisease mAb monoclonalantibody MLR mixed lymphocyte response NK natural killer PBL peripheralblood leukocyte TCR T cell receptor WBI whole body irradiation

The pioneering work of Owen, Medawar and others, beginning 50 years ago, led to the observation that hematopoietic chimerism (i.e. a condition in which hematopoietic cells from another animal exist in a recipient) can be associated with a state of donor-specific tolerance (reviewed in [3]). Tolerance can most readily be induced by allogeneic hematopoietic cells in animals that are developmentally immunodeficient, or in which immunodeficiency has been artificially induced. The capacity of hematopoietic cells to induce tolerance results largely from their ability to induce intrathymic clonal deletion of thymocytes with TCRs that recognize antigens expressed by the hematopoietic cells. This deletion, which probably occurs when an immature thymocyte binds with high affinity to a peptide-MHC complex presented in the thymus and hence undergoes apoptotic cell death, results in the generation of a T cell repertoire that is tolerant of antigens presented in the thymus. Several marrow-derived cell types, including dendritic cells, B cells, and thymocytes, as well as nonhematopoietic cells of the host thymus (reviewed in [4,5]), have the capacity to induce intrathymic tolerance by both deletional and nondeletional mechanisms. It seems that intrathymic deletion should result in a dependable form of tolerance, since the absence of lymphocytes with reactivity to the donor would ensure that a specific response to donor antigens could not be induced under any circumstances. Indeed, when pre-existing T cells are adequately eliminated and bone marrow engraftment is achieved, tolerance to the most immunogenic allografts, such as fully MHC mismatched skin grafts and small bowel grafts, is reliably attained [6,7].

With adequate depletion or inactivation of the pre-existing immune system and establishment of conditions permitting donor hematopoietic stem cell engraftment, a robust state of central deletional tolerance to allogeneic or xenogeneic donors can be induced. Advances have been made in the ability to achieve this permissive state without toxic, myeloablative conditioning, thus bringing this approach closer to clinical application.

Addresses Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital East, Building 149-5102, 13th Street, Boston, MA 02129, USA; e-mail: [email protected]

Introduction

While improvements in immunosuppressive drugs have dramatically increased the success of clinical organ transplantation, these drugs are associated with lifelong risks of infection, malignancy, and graft rejection. Chronic rejection still results in a constantly downsloping long-term survival curve for most organ allografts. Induction of donor-specific tolerance with preservation of otherwise normal immune responses is therefore a major goal of research in the field of transplantation. A second impetus for developing methods of inducing tolerance arises from the current shortage of allogeneic organs, a situation that has increased interest in the use of other species as xenograft donors. Immune barriers to xenografts may be even greater than those to allografts [1], however, and the induction of both B cell and T cell tolerance may be essential to the ultimate success of xenotransplantation. This review will focus on recent advances in the ability to induce T cell tolerance in the thymus, which is the central organ for T cell development. Therefore, this form of tolerance is referred to as central, as distinguished from the peripheral tolerance that may develop among already mature T cells when they encounter antigen in

and tolerance

In addition to this central deletional form of tolerance, the induction of hematopoietic chimerism may have the capacity to induce tolerance among pre-existing mature T cells in the peripheral lymphoid system. While deletion is a predictable outcome of high affinity antigen recognition

Chimerism and central tolerance Sykes

among immature thymocytes, it may not be unique to this stage of T cell development. Peripheral deletion, often following activation, has been described for mature T cells upon exposure to antigen in vivo [8,9]. In addition, veto cells (see below) can delete alloreactive cytotoxic T lymphocyte (CTL) precursors (CTLps). Certain types of donor-derived hematopoietic cells, such as B cells, which may be encountered in the periphery, can induce T cell anergy, which is an inability to respond upon recognition of antigen by a TCR. Methods of inducing T cell anergy (reviewed in [3,5,10]) include lack of costimulation upon antigen presentation, and presentation of antagonist peptides. Unlike deletional tolerance, however, the anergic state is reversible and can be overcome by infection [11] or by removal of the antigen 112,13].

Obstacles to the use of hematopoietic cell transplantation for the induction of central tolerance in man In view of its potent and reliable ability to induce intrathymic tolerance, it may seem surprising that hematopoietic cell transplantation has not yet been applied to the induction of tolerance in man. There are numerous immunological and physiological obstacles, however, to the engraftment and function of allogeneic and xenogeneic hematopoietic cells. Donor cells with long-term multilineage repopulating ability, known as pluripotent hematopoietic stem cells, must survive and function sufficiently to provide a lifelong source of donor antigens in the thymus. Furthermore, if the pre-existing peripheral alloreactivity is not adequately eliminated, eventual rejection may ensue even if initial intrathymic chimerism is achieved. T h e simplest and most widely used approach to overcome both the immunological and the physiological barriers to marrow engraftment in adult animals has been to pretreat recipients with lethal whole body irradiation (WBI) prior to bone marrow transplantation (BMT). While this procedure is well tolerated and reliably induces chimerism and tolerance in rodents, its use is unfortunately less feasible in humans. Lethal WBI is unduly toxic for use in patients who do not have malignant diseases and who have a high likelihood of survival in the absence of a bone marrow transplant. Furthermore, because of the potency of anti-MHC alloresponses, B M T across HLA barriers is associated with an inordinately high risk of graft-versus-host disease (GVHD) and engraftment failure (reviewed in [14]). Thus, studies in mice showing that sublethal irradiation, with or without the commonly used immunosuppressive drug cyclophosphamide, is sufficient to allow engraftment of allogeneic marrow given in conventional doses [15,161 are unlikely to be directly applicable to HLA-mismatched allogeneic or xenogeneic transplantation in humans. Studies reporting the existence

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of a novel CD3 +, TCRotl3-, TCRT~-, CD8 ÷ bone marrow cell population that facilitates peripheral hematopoietic stem cell engraftment [17] have recently received widespread attention. T h e identity of this unusual cell population and its mechanism of action remain to be determined. It will be necessary, therefore, to understand the immunological and physiological barriers to hematopoiesis in allogeneic and xenogeneic environments and to develop more specific methods of overcoming these barriers before the B M T approach to tolerance can be applied clinically. For the purpose of achieving normal immune function, it would be most desirable to achieve a state of mixed lymphohematopoietic chimerism rather than full donor reconstitution in completely M H C mismatched combinations. It has been observed that improved immunocompetence is achieved when host-type APCs are present in the peripheral tissues to allow optimal antigen presentation to T cells that have developed in the host thymus, and that are therefore skewed toward recognition of peptide antigens in the context of host MHC.

immunological barriers to allogeneic and xenogeneic hematopoietic cell engraftment A model that allows induction of mixed chimerism and donor-specific tolerance across M H C barriers in mice without myeloablative conditioning (nonmyeloblative conditioning allows sufficient host hematopoietic cells to survive so that a bone marrow transplant is not necessary to sustain life) has helped to define the immunological barriers to marrow engraftment [6]. This model involves the use of depleting CD4 and CD8 monoclonal antibodies (mAbs) followed by a low dose (3 Gy) of WBI and a higher dose (7Gy) of local irradiation to the thymus. Similar results have recently been achieved using anti-TCRcxl3 antibodies and 3 Gy WBI [18]. T h e s e and other studies have shown that either CD4÷ or CD8 ÷ T cells of the host can readily reject fully M H C mismatched marrow, and as expected, that CD4 ÷ cells reject M H C class II mismatched marrow while CD8 ÷ cells reject M H C class I disparate marrow [6,19,20]. Additionally, CD8 + cells also play a significant role in rejecting M H C class II mismatched marrow, and CD4 ÷ host cells pose a weak but detectable barrier to M H C class I mismatched marrow [19,20]. It is possible that this apparent violation of the usual CD4 ÷ and CD8 + associations with M H C class II and class I, respectively, may reflect recognition of M H C peptides presented by M H C molecules that are shared by the donor and recipient. Natural killer (NK) cells, which have long been assumed to resist allogcneic marrow engraftment on the basis of short-term studies of resistance to myeloid progenitor proliferation, actually present only a weak barrier to the engraftment of allogcncic peripheral hematopoietic stem cells [21]. NK cclls may play a more significant role, however, in resisting the cngr;iftmcnt of xenogeneic marrow [22]. Encouraging resuhs in a primate model using antibodies, sublethal WB1 and thynlic irradiation followed by allogeneic B M T

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and kidney transplantation [23 °°] provide hope that this approach may soon be applied in the clinical setting.

Physiological barriers to allogeneic and xenogeneic hematopoietic cell engraftment The concept that space must be created in the hematopoietic compartment in order to allow donor stem cells to engraft has long been widely accepted. In a syngeneic B M T system in which donors and hosts differed only by nonimmunogenic alleles of the leukocyte common antigen Ly-5, a low dose (1.5-3.0Gy) of WBI was required to permit engraftment of syngeneic marrow cells given in numbers similar to those that could be obtained from marrow of living human allogeneic marrow donors [24]. This requirement to make hematopoietic space, however, can be overcome by the administration of very high doses of syngeneic marrow [25]. The achievement in humans of successful engraftment of large numbers of T-cell-depleted HLA mismatched mobilized peripheral blood and marrow stem cells supports the concept that alloengraftment can also be enhanced by increasing the number of hematopoietic progenitors administered [26]. The dose of irradiation required to achieve allogeneic marrow engraftment in mice can be reduced by administering increasing numbers of donor marrow cells [27°]. We have recently demonstrated that engraftment of high doses of allogeneic marrow can be achieved without myelosuppressive treatment in mice that receive T-cell-depleting mAbs (M Sykes, G L Slot, K Swenson, DA Pearson, unpublished data). We have learned from these studies, however, that it is essential to create space in the thymus and to achieve high levels of early donor T cell repopulation in order to achieve permanent tolerance. Apparently, thymic space and peripheral hematopoietic space are regulated independently (M Sykes, G L Slot, K Swenson, DA Pearson, unpublished data). Using peripheral blood stem cells collected after cytokine-induced mobilization, or using cadaveric donors, it may be possible to transplant large numbers of allogeneic hematopoietic cells to humans. Advances in the ability to propagate primitive hematopoieric cells in vitro using cytokine combinations [28,29] and to reconstitute autologous hematopoiesis in patients with progenitor cells expanded in vitro [30] suggest that ex vivo expansion of allogeneic hematopoietic cells could have clinical utility. The ability to overcome the requirement for host myelosuppression by giving large numbers of hematopoietic cells, which might be available from inbred donors, also deserves exploration in xenotransplantation. The mechanism by which myelosuppression promotes marrow engraftment is not fully understood, and could include both the creation of physical niches due to the destruction of host hematopoietic cells, and the upregulation of cytokines that promote hematopoiesis. Homing to the marrow environment depends on interactions between adhesion molecules and their ligands [31°°], and active hematopoiesis may also depend on specific molecular interactions between the stroma and hematopoietic cells.

It is probably the species specificity of some of these interactions (cytokines, adhesion molecules and other cell surface signalling molecules) that accounts for the competitive advantage enjoyed by recipient marrow over xenogeneic donor marrow, as is discussed below. T h e mAb-based nonmyeloablative approach to host conditioning used in the mouse allogeneic BMT model described above has been extended to a xenogeneic combination, rat--~mouse. In the xenogeneic model, however, large numbers of donor bone marrow cells (BMCs) are needed to achieve engraftment, and the level of rat hematopoietic reconstitution gradually declines over time, despite persistent tolerance [22,32°]. This decline, which can be averted by the late administration of repeated marrow injections [32°], is due to a competitive advantage enjoyed by host hematopoietic cells over xenogeneic cells that becomes increasingly evident as recovery of the host from low dose WBI occurs [33°]. This competitive advantage has been demonstrated by comparing reconstitution in sublethally irradiated severe combined immunodeficient mice receiving rat BMCs alone or with added immunodeficient mouse marrow. The addition of unirradiated mouse marrow to the inoculum resulted in preferential early repopulation of myeloid lineages by the mouse instead of the rat donor [33°]. Achievement of xenogeneic hematopoietic repopulation has proved to be an even more formidable challenge in more disparate species combinations. Human and pig progenitor cells have clearly been shown to be capable of repopulating murine recipients at low levels ([34,35]; Pallavicini M, Flake AW, Bethel C, Langlois R, Madden D, Duncan BW, Haendel S, Montoya T, abstract, page 50, First International Congress on Xenotransplantation, Minneapolis, October 1991) but the species specificity of critical regulatory molecules may limit donor repopulation. Administration of exogenous donor species-specific cytokines can partially overcome this barrier [34,36].

How much intrathymic chimerism is needed to achieve full deletional tolerance? In mice receiving allogeneic B M T after conditioning with CD4 and CD8 mAbs, 3 G y WBI and 7Gy thymic irradiation, the first wave of thymocyte recovery (day 10) included complete deletion of mature host thymocytes recognizing superantigens presented by donor I-E MHC molecules, despite the presence of only very small numbers of donor I-E-bearing cells in the thymus at this early time [37]. Thymic irradiation can be excluded from this conditioning regimen if a second pretransplant T cell mAb injection is given [38 °] to overcome residual intrathymic antidonor alloreactivity (B Nikolic, M Sykes, unpublished data). Thymocyte depletion is not increased by the second mAb injection, which reliably permits the intrathymic deletion of donor-reactive host thymocytes [39°°]. Some animals receiving this regimen (two mAb treatments without thymic irradiation), however, show a gradual

Chimerism and central tolerance Sykes 697

decline in chimerism among all lineages, and eventual loss of tolerance [38°]. Remarkably, this incomplete form of tolerance can be identified soon after BMT by observing the proportion of donor cells among the initial T cells that recover. A low fraction ( < 20%) of donor T cells reliably predicts an eventual loss of chimerism in all lineages, even if high levels of engraftment are initially seen in non-T-cell lineages [38"]. Since thymic dendritic cells and thymocytes are derived from a common progenitor [40], high levels of donor thymocyte repopulation might reflect high levels of dendritic cell chimerism. Very low levels of initial intrathymic chimerism, however, permit the induction of complete and lasting tolerance in mice receiving thymic irradiation [37], thus mitigating against an absolute requirement for high levels of dendritic cell chimerism. In animals not receiving thymic irradiation, the correlation of high levels of early donor T cell repopulation with permanent tolerance might reflect a role for donor thymocytes themselves in the induction of tolerance among residual host thymocytes. In a model involving lethal (i.e. myeloablative) irradiation and mixed BMT for the induction of tolerance to an allogeneic I-E antigen, >30% donor peripheral blood leukocyte (PBL) repopulation was required in order for complete deletional tolerance to ensue [41]. These studies were performed, however, with non-T-cell-depleted marrow that included alloreactive T cells in the host marrow population, and specific lineage analysis was not performed among PBLs, making the interpretation of this observation somewhat complex.

Thymic t r a n s p l a n t a t i o n as an approach to central t o l e r a n c e induction An alternative approach to inducing central tolerance obviates the requirement for engraftment of donor hematopoietic stem cells that can send progeny to the host thymus and does not depend on a functioning host thymus for the achievement of T cell recovery. Grafting of donor thymic tissue to recipients that have been thymectomized and depleted of peripheral T cells with mAbs allows host T cell recovery in the donor thymus. This approach would be most appropriately applied to discordant xenotransplantation, in which potentially unlimited amounts of donor thymic tissue could be obtained. Furthermore, the greatest difficulties in achieving donor hematopoietic cell migration to the host thymus have been observed in widely disparate species combinations such as pig---)mouse. We have recently shown that implantation of fetal pig thymus and liver fragments under the kidney capsule of C57BL/10 (B 10) thymectomized mice that have been treated with the same T-cell- and NK-cell-depleting mAb regimen described above for rat---)mouse chimeras can reconstitute immunocompetent CD4 ÷ T cells that are specifically tolerant of the discordant xenogeneic donor. Full maturation of mouse TCR(xl3÷ single positive thymocytes from progenitors is seen in these grafts, and CD4 ÷ cells repopulate the peripheral lymphoid tissues extensively [44]. A state of specific tolerance, that includes the absence of mixed lymphocyte responses (MLRs) to the donor [44] and the acceptance of donor swine leukocyte antigen-matched pig skin (Y Zhao, JJ Sergio, JS Am, DH Sachs, M Sykes, unpublished data), exists in these animals. To our knowledge, these results provide the first demonstration that donor-specific skin graft tolerance can be induced across widely disparate (discordant) species barriers.

Thymic reconstitution potential in adults Depletion or inactivation of pre-existing T cells, along with the minimal amount of host conditioning required to allow induction of mixed chimerism, could provide a potent and practical approach to central tolerance induction only if the host thymus is capable of reconstituting the immune system with new T cells. This is likely to be the case in children. T h e human thymus involutes at puberty, however, and little is known about its capacity to function when peripheral T cell depletion provides a physiologic stimulus for thymopoiesis. Heavily treated cancer patients demonstrate an age related delay in the recovery of thymically derived CD45RAhig h naive-type CD4 ÷ cells [42,43"']. Nevertheless, in B M T patients of all ages who do not develop GVHD, thymic function appears to eventually allow naive CD4 ÷ recovery [43"']. T h e potential of the thymus to generate naive T cells after peripheral T cell depletion in older patients who have not received chemotherapy or radiotherapy remains to be determined. Thus, the age groups for whom peripheral T cell depletion followed by induction of mixed chimerism and intrathymic tolerance will be practical is currently unclear.

Other studies have demonstrated the capacity of allogeneic or closely related xenogeneic thymic stromal grafts to induce tolerance to antigens expressed by the graft (reviewed in [45,46]). The grafted thymic stroma in these studies was devoid of hematopoietic cells. Donor-specific skin graft tolerance was observed, but in vitro responsiveness to the donor persisted [45,46]. Recently, tolerance in this system has been shown to involve an immunoregulatory CD4 ÷ cell population [47°], and donor-reactive T cells clearly persist [48"]. An important difference between those studies and the above pig---)mouse model is that the fetal pig thymus grafts transplanted into mice were already seeded with pig hematopoietic cells, including some that expressed MHC class II, at the time of harvest. Since bone marrow derived cells may be more efficient than thymic epithelial cells at inducing clonal deletion of developing thymocytes, these pig hematopoietic cells may be responsible for the MLR tolerance to donor pig antigens displayed by mouse T cells that have developed in pig thymi. Analyses of deletion

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of T cell families using certain VI3 suggest that tolerance to both donor and host developed by an intrathymic deletional mechanism in these animals. Consistent with this possibility, both murine and porcine MHC class Ilhigh cells are detectable in swine thymus grafts (Y Zhao, JJ Sergio, JS Arn, DH Sachs, M Sykes, unpublished data). A surprisingly high level of immunocompetence and host-restricted antigen reactivity has been observed for T cells that develop in pig thymic grafts in these mice. Since published studies suggest that thymic epithelial cells determine MHC restriction of CD4 ÷ T cells (reviewed in [4]), the basis for this immunocompetence and host restriction in thymic xenografts is currently the subject of active investigation. T h e excellent immunocompetence that has been observed in children with congenital thymic aplasia (i.e. the DiGeorge anomaly) after grafting with HLA mismatched allogeneic thymi [49] suggests that MHC restriction may not be imposed to a major extent by thymic epithelium in man.

Development of chimerism and tolerance without T-cell-depleting or myelosuppressive treatment In view of the potency of ailoresponses and xenoresponses, it would seem unlikely that donor hematopoietic cells could engraft and subsequently induce central tolerance without potent host immunosuppression. Based on the recent observation that microchimerism (i.e. chimerism below the level of detection by flow cytometry) can exist for many years in the tissues of human solid organ ailograft recipients [50], however, it has been hypothesized that microchimerism leads to a state of donor-specific tolerance [51]. Since it would be unethical to test this hypothesis by intentionally depleting donor hematopoietic cells from patients, it is unlikely that the question of whether chimerism induces tolerance or is an epiphenomenon that reflects either tolerance or adequate immunosuppressive pharmacotherapy will be resolved in the near future. There are a number of mechanisms by which microchimerism could, in theory, induce peripheral T cell tolerance. These include nonprofessional APC function of donor-derived T and B cells, that can anergize responding T cells. In addition, veto activity of T cells, NK cells and other cell types eliminates C T L s reactive against antigens expressed on the veto cells (reviewed in [3]). Putative veto activity was detected among donor cells isolated from a long-term renal allograft recipient [52°°], providing the first evidence for a specific mechanism by which incidental chimerism may lead to tolerance in human solid organ allograft recipients. Confirmation in additional patients and further analyses of the relationship between tolerance and chimerism, however, are needed before a basis for discontinuing immunosuppressive therapy in humans can be comfortably discerned.

In addition to the above mechanisms by which chimerism might induce peripheral tolerance, donor leukocytes migrating to recipient thymi might induce central tolerance among T cells that develop subsequent to the time of donor engraftment. Recent evidence suggests that liver grafts may contain self-renewing hematopoietic stem cells [53°,54°]. These could potentially ensure the lifelong presence of tolerance-inducing donor cells within the host thymus. Indeed, dendritic cell progenitors have been detected in the marrow of recipients of spontaneously accepted mouse liver allografts [55°]. T h e observation of lasting microchimerism in long-term human allograft recipients has prompted examination of the role of chimerism in a variety of animal models in which peripheral tolerance is induced. In a mouse model, in which donor-specific transfusions are given with depleting CD4 mAbs prior to cardiac allografting, tolerance has been shown to require the presence of donor antigens in the induction phase, but its maintenance is independent of the lasting presence of donor hematopoietic cells. Antigens presented by the heart allograft, on the other hand, appear to be critical for maintenance of the tolerant state in these mice [56"]. The presence of microchimerism failed to predict tolerance in a neonatal tolerance model [57"] and was not always observed in tolerant rat recipients of cardiac allografts [58]. Thus, it has become clear that microchimerism neither predicts tolerance nor is required to maintain tolerance under all circumstances. Attempts :~ correlate in vitro measures of donor-specific hyporcsponsiveness (MLR, cell-mediated lympllaolysis [CML]) with chimerism and in vivo tolerance [59] have not all shown a correlation [60"] and it is not clear that these tests could predict which patients would tolerate withdrawal of immunosuppressive therapy without rejection. Since a fraction of noncompliant long-term allograft recipients who discontinue immunosuppressive therapy do not reject their grafts, and most patients can be maintained on relatively low levels of long-term immunosuppression, it is possible that a state of partial or complete donor-specific tolerance exists in some patients. In view of the high rate of chronic graft rejection, however, it seems unlikely that the level of tolerance in most patients is adequate to allow safe removal of immunosuppressive drugs. Of course it is possible that immunosuppressive therapy interferes with the induction of tolerance. Attempts to increase the level of chimerism have been made in human and monkey solid organ transplant recipients by administering donor bone marrow around the time of organ transplantation, without giving specific myelosuppressive therapy [60",61,62]. T h e clinical studies have not, however, demonstrated a reduction in the number of rejection episodes [60",62], even when

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chimerism has been enhanced [60°]. These studies did not include intentional peripheral T cell depletion. In the primate model, which includes recipient pretreatment with antilymphocyte serum for T cell depletion and, for optimal results, total lymphoid irradiation, veto cells in donor bone marrow that inactivate recipient C T L p s may promote graft acceptance [63]. Only a fraction of recipients show long-term graft acceptance, however, with the best results obtained when the donor and recipient share a DR class II allele [61]. Chimerism might contribute to tolerance without the use of myelosuppressive or immunosuppressive conditioning in recipients that are developmentally immunodeficient. In neonatally tolerized animals, several mechanisms have been invoked to explain the observed tolerance. Specific suppressor T cells have been detected [64], and recent studies suggest that a Th2-type response to the donor may be responsible for the inability to reject donor-specific skin grafts in such animals [65°,66°°]. In addition, lasting microchimerism has been detected in some neonatally tolerized mice and evidence to support an intrathymic deletional mechanism has been obtained in some, although not all, strain combinations [64]. Furthermore, the presence of microchimerism does not always predict skin graft tolerance in recipients of allogeneic lymphocytes perinatally, and nontolerant animals can still maintain microchimerism following rejection of donor skin grafts [57°',67]. Similarly, induction of low levels of chimerism by intrauterine BMT in sheep was not associated with tolerance to donor organ allografts [68]. Perhaps the persisting donor cells in these nontolerant animals are T cells that are able to avoid their own destruction through a veto mechanism, but that are insufficient in number to ensure a systemic state of tolerance in the recipient. Recent studies have challenged the view that neonates are immunoincompetent and hence uniquely permissive for tolerance induction, or, in fact, for induction of a Th2 response. T h e tendency of allogeneic cell infusions to result in tolerance and of antigen exposure to result in Th2 responses in this period may be due to the high ratio of noncostimulatory APCs (T and B cells) in donor inocula to recipient T cells in the neonate, rather than to any unique susceptibility to tolerance induction. Neonatal mice can clearly mount T h l responses and can be immunized to ailoantigens and pathogens [69°°-71°°]. In fact, the ability of neonates to mount host-antigraft responses may play an important role in the induction of cell populations that suppress antidonor responses. In contrast, when intrathymic deletional tolerance is induced in animals in which the pre-existing peripheral T cell response has been fully ablated, suppressive responses to the donor are notably absent, and tolerance is readily broken by the infusion of nontolerant host-type lymphocytes [72,73°°]. In contrast, tolerance cannot be easily broken by the infusion of nontolerant host-type lymphocytes in neonatally tolerized animals [64,74,75].

The relationship b e t w e e n peripheral T cell tolerance and central tolerance T h e topic of peripheral tolerance is covered in detail elsewhere in this issue. It should be pointed out here, however, that the distinction between central and peripheral tolerance may not always be clear. In a pig peripheral tolerance model, in which a kidney is grafted with a short course of immunosuppressive therapy [76], the thymus appears to play a role in the induction of tolerance (K Yamada, P Gianello, DH Sachs, personal communication). Although passenger leukocytes from the graft that enter the thymus might help to tolerize subsequently developing thymocytes, the role of the thymus in tolerizing pre-existing T cells in the periphery requires further explanation. It is possible that T cells that are activated in the periphery by the organ allograft recirculate to the thymus as has been described [77], and encounter donor antigen there, perhaps on passenger leukocytes, which then inactivate the T cells (this could be a fail-safe mechanism for ensuring that T cells activated in the periphery of an animal are switched off if the same antigens are present on intrathymic leukocytes). Or it is possible that the migration of donor antigen to the thymus may result in the development of T cells that specifically recognize the donor antigen and downregulate the activity of destructive alloreactive T ceils, perhaps by secreting Th2-associated cytokines [78,79]. A second situation in which the boundary between central and peripheral tolerance is uncertain results from the intrathymic injection of donor antigens. While initial uses of this approach included antibody treatment to deplete peripheral T cells, more recent studies have shown that tolerance to soluble alloantigens can be induced by intrathymic injection without peripheral T cell depletion [80]. Since removal of the thymus before or within the first few days of allografting results in rejection of the allograft [81], the thymus must play an active role in tolerizing pre-existing peripheral T cells. This inactivation could be due to recirculation of activated T cells through the thymus, or to emigration from the thymus of regulatory T cells. Active regulatory cell populations have been reported in rats receiving intrathymic injections of allogeneic BMCs [82]. T h e role of the allograft in inducing tolerance in animals receiving intrathymic injections should not be ignored. Transferable tolerance is not induced by intrathymic marrow injection alone without an organ allograft in rats [82], suggc~,:ing that the graft itself helps to tolerize the pre-existing T cell repertoire, perhaps by presenting antigen in the absence of costimulation, or by activating recipient T cells that then recirculate to the thymus to induce tolerance by the mechanisms described above. In contrast to intrathymic injection, pure intrathymic deletional tolerance induced by bone marrow engraftment is not dependent on the continual presence of antigen in the periphery [73°°]. Donor tissue can be grafted at any

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time, and tolerance is assured. T h e intrathymic injection approach has not been successful in high responder rat strain combinations and may even induce allosensitization [83°,84•]. In contrast to bone marrow transplantation, intrathymic injection has not been successfully used to facilitate xenotolerance induction.

Conclusions Intrathymic tolerance can be induced by a variety of mechanisms, including deletion, anergy and suppression, when allogeneic or xenogeneic hematopoietic cells or thymic tissue are successfully engrafted. In order to rely purely on intrathymic deletion for tolerance induction, it is essential to adequately eliminate or inactivate the pre-existing peripheral immune system, and to ensure a continual supply of tolerance-inducing antigens to the thymus. Advances in the ability to achieve engraftment of hematopoietic and thymic tissue without ablative treatment of the host, and the demonstration of efficacy in primate models, have brought these approaches closer to clinical application. A deeper understanding of the physiological mechanisms regulating hematopoietic stem cell engraftment, hematopoietic function, and homing to the thymus should allow the development of minimal host conditioning regimens that will make reliable allogeneic and xenogeneic tolerance feasible. T h e observation that long-term human allograft recipients frequently contain donor hematopoietic cells in their peripheral lymphoid tissues has stimulated intensive examination of the relationship between chimerism and tolerance in animal models. These studies have shown that a state of chimerism is not always necessary for tolerance to exist, and that the presence of microchimerism does not necessarily imply a state of tolerance. T h e mechanisms and conditions under which peripheral chimerism can induce T cell tolerance are likely to be further clarified in future studies.

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Nomoto K, Yung-Yun K, Omoto K, Umesue M, Murakami Y, Matsuzaki G: Tolerance induction in a fully allogeneic combination using anti-T cell receptor-monoclonal antibody, low dose irradiation, and donor bone marrow transfusion. Transplantation 1995, 59:395-401.

19.

Sharabi Y, Sachs DH, Sykes M: T cell subsets resisting induction of mixed chimerism across various histocompetibility barriers. In Progress in Immuno/ogy. V/I/. Edited by Gergely J, Benczur M, Falus A, F(Jst GY, Medgyesi G, Petr~nyi GY, RajnavSIgyi E. Heidelberg: Springer-Verlag; 1992:801-805.

20.

Vallera DA, Taylor PA, Sprent J, Blazar BR: The role of host T cell subsets in bone marrow rejection directed to isolated major histocompatibility complex class I versus class II differences of bml and bm12 mutant mice. Transplantation 1994, 57:249-256.

21.

Lee I_A, Sergio JJ, Sykes M: Natural killer cells weakly resist engraftment of allogeneic Iong-tarm multilineage-repopulatin9 hematopoietic stem cells. Transplantation 1996, 61:125-132.

22.

Sharabi Y, Aksentiievich I, Sundt TM III, Sachs DH, Sykes M: Specific tolerance induction across a xenogeneic barrier: production of mixed rat/mouse lymphohematopoietic chimeras using a nonlethal preparative regimen. J Exp Mad 1990, 172:195-202.

Acknowledgements I thank Boris Nikolic and Shiv Pillai for helpful comments and review of the manuscript and Diane Plemenos for expert secretarial assistance.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • ••

of special interest of outstanding interest

1.

Auchincloss HA: Why is cell-mediated xenograft rejection so strong? Xenotransplantation 1995, 3:19-22.

2.

Cobbold SP, Adams E, Marshall SE, Davies JD, Waldmann H: Mechanisms of peripheral tolerance and suppression induced by monoclonal antibodies to CD4 and CD8. Immunol Rev 1996, 149:5-34.

3.

Charlton B, Auchincloss H Jr, Fathman CG: Mechanisms of transplantation tolerance. Annu Rev Immunol 1994, 12:707-734.

4.

Matzinger P: Why positive selection? Immunol Rev 1993, 135:81-117.

23. ,,=

Kawai T, Cosimi AB, Colvin RB, Powelson J, Eason J, Kozlowski T, Sykes M, Monroy R, Tanaka M, Sachs DH: Mixed allogeneic chimerism and renal allograft tolerance in cynomologous monkeys. Transplantation 1995, 69:256-262. Reliable tolerance induction is demonstrated in a class I and class II MHC mismatched primates using low-dose WBI, thymic irradiation, and antilymphocyte serum, followed by donor marrow infusion and renal allografting, with one month of cyclosporine therapy. This study demonstrates that BMT can be used after nonmyeloablative conditioning to consistently induce donor-specific tolerance in primates. 24.

TomitaY, Sachs DH, Sykes M: Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow. Blood 1994, 83:939-948.

Chimerism and central tolerance Sykes

25.

Ramshaw HS, Crittenden RB, Dooner M, Peters SO, Rao SS, Quesenberry PJ : High levels of engraftment with a single infusion of bone marrow cells into normal unprepared mice. Biol Blood Marrow Transplant 1995, 1:74-80.

26.

Averse F, Tabilio A, Terenzi A, Velardi A, Falzetti F, Gionnoni C, lacucci R, Zei T, Martelli MP, Gambelunghe C e t el.: Successful engraftment of T-cell-depleted haploidentical three-loci incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factormobilized peripheral blood progenitor cells to bone marrow inoculum. Blood 1994, 84:3948-3955.

27. •

Bachar-Lustig E, Rachamin N, Li H-W, Lan F, Reisner Y: Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice, Nat Mad 1995, 1:1268-1273. It is shown that the dose of WBI required to achieve allogeneic marrow engraftment and tolerance induction can be reduced by increasing the number of allogeneic hematopoietic cells administered. 28.

29.

30.

39. ••

TomitaY, Khan A, Sykes M: Mechanism by which additional monoclonal antibody 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. Transplantation 1996, 61:477-485. In this paper, along with [38"], it is shown that the potential toxicity of a mAb-based nonmyeloablative regimen allowing induction of mixed alloganeic chimerism and donor-specific tolerance can be further reduced by removing thymic irradiation from the conditioning regimen. Successful tolerance is induced by administration of two (instead of the standard single) CD4 and CD8 mAb injections prior to low dose WBI (3 Gy) and allogeneic BMT. Intrathymic deletion is the major mechanism of tolerance in these animals, and the second mAb injection appears to inactivate residual alloreactive host thymocytes that could otherwise reject donor cells intrathymically. Such intrathymic rejection can result in the eventual loss of chimerism even if initial engraftment is achieved in the periphery. 40.

Petzer AL, Hagge DE, Lansdorp PM, Reid DS, Eaves C J: Selfrenewal of primitive human hematopoietic cells (long-termculture-initiating cells) in vitro and their expansion in defined medium. Proc Natl Acad Sci USA 1996, 93:1470-1474.

Ardavin C, Wu L, Li C-L, Shortman K: Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature 1993, 362:761-763.

41.

EmersonSG: Ex vivo expansion of hematopoietic precursors, progenitors, and stem cells: the next generation of cellular therapeutics. Blood 1996, 87:3082-3088.

Taniguchi HAbe M, Shirai T, Fukao K, Nakauchi H: Reconstitution ratio is critical for alloreactive T cell deletion and skin graft survival in mixed bone marrow chimeras. J Immunol 1995, 12:5631-5636.

42.

Mackall CL, Fleisher TA, Brown MR, Andrich MR Chen CC, Feuerstein IM, Horowitz ME, Magrath IT, Shad AT, Steinberg SM et el.: Age, thymopoiesis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy. N Engl J Mad 1995, 332:143-149.

Brugger W, Heimfeld S, Berenson RJ, Mertelsmann R, Kanz L: Reconstitution of hematopoiesis after high-dose chemotherapy by autologous progenitor cells generated ex vivo. N Engl J Mad 1995, 333:283-287.

31. ••

Papayannopoulou T, Craddock C, Nakamoto B, Priestley GV, Wolf NS: The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc Nat/Acad Sci USA 1995, 92:9647-9651. Inhibition of the very late antigen 4/vascular cell adhesion molecule-1 (VLA4/VCAM-1) adhesion pathway is shown to mobilize and prevent the homing of hematopoietic progenitor cells to the marrow and to increase their numbers in peripheral blood and spleen. While previous in vitro studies have implicated this adhesion pathway in hematopoiesis, this is the first demonstration of its importance in vivo. Lee LA, Sergio JJ, Sykes M: Evidence for non-immune mechanisms in the loss of hematopoietic chimerism in rat-->mouse mixed xenogeneic chimeras. Xenotransplantation 1995, 2:57-66. Despite the gradual loss of chimerism in mixed xenoganeic chimeras prepared with a nonmyeloablative mAb-based conditioning regimen, donor-specific tolerance persists. Chimerism can be boosted long after the initial BMT with a low dose of WBI and repeat donor marrow infusion. Loss of chimerism is therefore most likely due to a nonimmune mechanism such as hematopoietic competition as the recipient recovers from irradiation. The study in [33"] confirms that recipient hematopoietic cells have a competitive advantage over the xenogeneic donor in this species combination.

43. ••

Weinberg K, Annett G, Kashyap A, Lenarsky C, Forman S J, Parkman R: The effect of thymic function on immunocompetence following bone marrow transplantation. Biol Blood Marrow Transplant 1995, 1:18-23. An age-associated reduction in the number of naive type CD45RAhigh CD4 + cells, presumably from the thymus, is demonstrated following recovery from BMT in humans. Patients of all ages, however, are shown to recover CD45RAhigh cells and immune function within one year if chronic GVHD does not develop. This paper provides evidence that the adult thymus is functional if it is not damaged by GVHD following allogeneic BMT. 44.

Lee LA, Gritsch HA, Sergio JJ, Arn .IS, Glaser RM, Sablinski T, Sachs DH, Sykes M: Specific tolerance across a discordant xenogeneic transplantation barrier. Proc Nat/Acad Sci USA 1994, 91:10864-10867.

45.

Bonomo A, Matzinger P: Thymus epithelium induces tissuespecific tolerance. J Exp Mad 1993, 177:1153-1164.

46.

MartinC, Ohki-Hamazaki H, Corbel C, Coltey M, Le Douarin NM: Successful xenogeneic transplantation in embryos: induction of tolerance by extrathymic chick tissue grafted into quail. Dev Immuno11991, 1:265-277.

32. •

33. •

Gritsch HA, Sykes M: Hematopoietic competition limits xenogeneic myeloid reconstitution in SCID mice. Transplant Proc 1996, 28:708. See annotation [32°]. 34.

Lapidot T, Pfiumia F, Doesdens M, Murdoch B, Williams DE, Dick JE: Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 1992, 255:1137-1143.

35.

Gritsch HA, Glaser RM, Emery DW, Lee LA, Smith CV, Sablinski T, Arn JS, Sachs DH, Sykes M: The importance of nonimmune factors in reconstitution by discordant xenogeneic hematopoietic cells. Transplantation 1994, 57:906-917.

36.

Yang Y-G, Sergio JJ, Swanson K, Glaser RM, Monroy R, Sykes M: Donor-specific growth factors promote swine hematopoiesis in SCID mice. Xenotransplantation 1996, 3:92-101.

37.

38. •

701

Tomita Y, Khan A, Sykes M: Role of intrathymic clonal deletion and peripheral anergy in transplantation tolerance induced by bone marrow transplantation in mice conditioned with a non-myeloablative regimen. J Immunol 1994, 153:1087-1098.

Tomita Y, Sachs DH, Khan A, Sykes M: Additional mAb injections can replace thymic irradiation to allow induction of mixed chimerism and tolerance in mice receiving bone marrow transplantation after conditioning with anti-T cell mAbs and 3 Gy whole body irradiation. Transplantation 1996, 61:469-477. See annotation [39"'].

47.

Modigliani Y, Tomas-Vaslin V, Bandeira A, Coltey M, Le Douarin NM, Coutinho A, Salaun J: Lymphocytes selected in allogeneic thymic epithelium mediate dominant tolerance toward tissue grafts of the thymic epithelium haplotype. Proc Nat/Acad Sci USA 1995, 92:7555-7559. See annotation [48"]. •

48. •

Modigliani Y, Persira P, Thomas-Vaslin V, Salaun J, BurlenDefranoux O, Coutinho A, Le Douarin N, Bandeira A: Regulatory T cells in thymic epithelium-induced tolerance. I. Suppression of mature peripheral non-tolerant T ceils. Eur J Immuno11995, 25:2563-2571. This paper, along with [47"], provides evidence that the mechanism by which pure thymic epithelial grafts, devoid of hematopoietic cells, induce tolerance to antigens of the allogeneic thymic graft involves a mechanism other than deletion or energy. These papers add to an increasing body of evidence that at least one mechanism by which the thymus induces tolerance is the production of an active regulatory cell population. 49.

Hong R, Moore AL: Organ culture for thymus transplantation. Transplantation 1996, 61:444-448.

50.

Starzl TE, Demetris AJ, Trucco M, Zeevi A, Ramos H, Terasaki P, Rudert WA, Kocova M, Ricordi C, Ildstad S e t aL: Chimerism and donor-specific nonreactivity 27 to 29 years after kidney allotransplantation. Transplantation 1993, 55:1272-1277.

51.

Starzt TE, Murase N, Thomson A, Demetris AJ: Liver transplants contribute to their own success. Nature Mad 1996, 2:163-165.

52. ,,•

Burlingham WJ, Grailer AP, Fechner JH Jr, Kusaka S, Trucco M, Kocova M, Belzer FO, Sollinger HW: Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal

702

Transplantation

energy) in a tolerant renal transplant recipient. Transplantation 1995, 59:1147-1155. Using repeated stimulation in the presence of interleukin-2 after depleting donor (chimeric) cells, the authors demonstrate the presence of antidonor CTLs in PBLs of a long-term renal allograft recipient who had discontinued immunosuppressivs therapy. Addition of fresh recipient PBLs, however, inhibited the development of these CTLs. Removal of donor cells from patient PBLs removed the suppressive activity. This study demonstrates that donor microchimerism can actively suppress antidonor responses in a tolerant human.

53. •

Murase N, Starzl TE, Ye Q, Tsamandas A, Thomson AW, Rao AS, Demetris AJ: Multilineage hematopoietic reconstitution of supralethally irradiated rats by syngeneic whole organ transplantation with particular reference to the liver. Transplantation 1996, 61:1-4. See annotation [54"]. 54. •

Taniguchi H, Toyoshima T, Fukao K, Nakauchi H: Presence of hematopoietic stem cells in the adult liver. Nat Med 1996, 2:198-203. This paper, along with [53"], demonstrates that adult rat and mouse livers, respectively, contain sufficient hematopoietic cells to protect animals from death due to irradiation-induced aplasia. The latter paper also demonstrates the presence of cells with the phenotype of pluripotent stem cells in the adult mouse liver. These studies provide a basis for the possibility that long-term chimerism in liver graft recipients is due to ongoing differentiation from selfrenewing hematopoietic progenitors or stem cells derived from the organ graft. 55. •

Lu L, Rudert WA, Qian SG, McCaslin D, Fu RM, Ran AS, Trucco M, Fung JJ, Starzl TE, Thomson AW: Growth of donor-derived dendritic cells from the bone marrow of murine liver allograft recipients in response to granulocyte/macrophage colonystimulating factor. J Exp Med 1995, 182:379-387. This study demonstrates the presence of cells in recipient marrow of donorderived progenitors that could potentially be involved in tolerance induction. Bushell A, Pearson TC, Morris PJ, Wood KJ: Donor-recipient microchimerism is not required for tolerance induction following recipient pretreatment with donor-specific transfusion and anti-CD4 antibody. Transplantation 1995, 59:1367-1371. The role of chimerism is examined when routine cardiac allograft tolerance is facilitated by prior donor-specific transfusion and CD4 mAbs. It is demonstrated that repeated injection, for a limited time period, of irradiated donorspecific transfusions is as effective as a single untreated donor-specific transfusion at facilitating tolerance induction. Thus, the authors conclude that lasting chimerism from the donor-specific transfusion is not required for the maintenance of tolerance in this model.

tolerance induced by donor bone marrow in rhesus monkeys. Transplantation 1995, 59:245-255. 62.

Rolles K, Burroughs AK, Davidson BR, Karatapanis S, Prentice HG, Hamon MD: Donor-specific bone marrow infusion after orthotopic liver transplantation. Lancet 1994, 343:263-265.

63.

Verbanac KM, Carver FM, Halsch CE, Thomas JM: A role for transforming growth factor-beta in the veto mechanism in transplant tolerance. Transplantation 1994, 57:893-900.

64.

Streilein JW: Neonatal tolerance of H-2 alloantigens. Transplantation 1991, 52:1-10.

65. •

Chen N, Field EH: Enhanced type 2 and diminished type 1 cytokines in neonatal tolerance. Transplantation 1995, 59:933-941. It is shown that acceptance of skin grafts from the strain to which animals had been neonatally tolerized is associated with increased intsrleukin-4 and decreased interferon-y production, whereas rejection of a third party graft leads to predominant interferon-y production. This result suggests that neonatal tolerance may be due to immune deviation toward a Th2-1ike response that may prevent graft rejection by inhibiting Thl responses. 66. oo

Donckier V, Wissing M, Bruyns C, Abramowicz D, Lybin M, Vanderhaeghen M-L, Goldman M: Critical role of interleukin 4 in the induction of neonatal transplantation tolerance. Transplantation 1995, 59:1571-1576. It is shown that neutralization of interleukin-4 prevents neonatal tolerance induction, and permits development of Thl cytokine responses. This result implicates interleukin-4 and Th2 responses in the induction of neonatal tolerance. 6'7.

Smith JP, Kasten-Jolly J, Field I1, Thomas JM: Assessment of donor bone marrow cell-derived chimerism in transplantation tolerance using transgenic mice. Transplantation 1994, 58:324-329.

68.

Hsdrick MH, Rice HE, MacGillivray TE, Bealer JF, Zanjani ED, Flake AW: Hematopoietic chimerism achieved by in utero hematopoietic stem cell injection does not induce donorspecific tolerance for renal allografts in sheep. Transplantation 1994, 58:110-111.

56. *•

57, ••

Alard P, Matriano JA, Socarras S, Ortega M-A, Streilein JW: Detection of donor-derived cells by polymerase chain reaction in neonatally tolerant mice. Microchimerism fails to predict tolerance. Transplantation 1995, 60:1125-1130. Microchimerism is shown to be present at the time of skin grafting in mice that received neonatal hematopoietic cell injections that failed to induce skin graft tolerance. This is an important demonstration that the presence of microchimerism is insufficient in itself to maintain tolerance in animals receiving hematopoietic cell injections without suppression of the pre-existing immune system. Chimerism persists even after skin graft rejection, suggesting that surviving donor hematopoietic cells may be resistant to destruction by host T-ce)ls, but are insufficient to maintain systemic tolerance. 58.

FisherRA, Cohen DS, Ben-Ezra JM, Sallade RE, Tawes JW, Tarry WC: Induction of long-term graft tolerance and donor/recipient chimerism. J Surg Res 1996, 60:181-185.

59.

ReinsmoenNL, Jackson A, McSherry C, Ninova D, Wiesner RH, Kondo M, Krom RAF, Hertz MI, Bolman RM III, Matas AJ: Organspecific patterns of donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism in lung, kidney, and liver transplant recipients. Transplantation 1995, 60:1546-1554.

Shapiro R, Rao AS, Fontes P, Zeevi A, Jordan M, Scantlebury VP, Vivas C, Gritsch HA, Corry RJ, Egidi F et al.: Combined simultaneous kidney/bone marrow transplantation. Transplantation 1995, 60:1421-1425. Despite increased levels of early chimerism no difference has been seen thus far in the outcome of allografting when donor bone marrow is administered with standard immunosuppression, without specific myelosuppression. These data suggest that in humans, as in rodents, the mere presence of chimerism is insufficient to ensure a state of tolerance; specific conditions must be established under which chimerism can lead to tolerance.

69. .•

Ridge JP, Fuchs El, Matzinger P: Neonatal tolerance revisited: turning on newborn T cells with dendritic cells. Science 1996, 271:1723-1726. This study demonstrates that the immune system of the neonate does not have any special propensity toward tolerance induction upon alloantigen exposure. Instead, the animals are shown to be capable of mounting antiH-Y CTL responses if exposed in the neonatal period to an appropriately immunogsnic type of APC expressing male-specific H-Y minor antigens. The authors conclude that the relative ease with which neonatal mice are rendered tolerant to antigens compared to adult mice is due to the high ratio of tolerogenic APCs (T and B cells) to recipient T cells to which the neonates are exposed. Exposure of adults to a similar APC population in similar ratio to total recipient T cell numbers has the same outcome as in neonates. 70. -•

Sarzotti M, Robbins DS, Hoffman PM: Induction of protective CTL responses in newborn mice by a murine retrovirus. Science 1996, 271:1726-1728. It is shown that the apparent inability of neonatal mice to mount CTL responses to a virus is due to the relatively high dose of virus used, which leads to a Th2 response. When lower viral doses are administered, the neonate mounts a Thl response, which results in CTL development. 71. •is

Forsthuber T, Yip HC, Lehmann PV: Induction of Thl and Th2 immunity in neonatal mice. Science 1996, 271:1728-1730. shown that neonates do not have a special propensity to develop Th2 responses upon exposure to antigen. If exposed to protein antigen in an appropriately immunogenic fashion, neonates are quite capable of mounting Thl responses.

~t

72.

60. •

61.

Thomas JM, Verbanac KM, Smith JP, Kasten-Jolly J, Gross U, Rebellato LM, Haisch CE, Carver FM, Thomas FT: The facilitating effect of one-DR antigen sharing in renal allograft

73. •o

Sykes M, Sheard MA, Sachs DH: Effects of T cell depletion in radiation bone marrow chimeras. II. Requirement for allogeneic T cells in the reconstituting bone marrow inoculum for subsequent resistance to breaking of tolerance. J Exp IVied 1988, 168:661-673.

KhanA, Tomita Y, Sykes M: Thymic dependence of loss of tolerance in mixed allogeneic bone marrow chimeras after depletion of donor antigen. Peripheral mechanisms do not contribute to maintenance of tolerance. Transplantation 1996, in press. The ability to break tolerance in mixed allogeneic chimeras by eliminating chimerism with mAbs administered in vivo is shown to require a host thymus. Tolerance persists in the absence of antigen if the thymus is removed. Tolerance is readily broken by infusion of nontolerant host-type lymphocytes. These results indicate that in mixed allogeneic chimeras produced by a nonmyeloablative regimen that adequately eliminates the pre-existing T cell

Chimerism and central tolerance Sykes

repertoire, the maintenance of tolerance is due to ongoing intrathymic deletion, with no significant contribution form the mechanisms of anergy or suppression. 74.

Roser BJ: Cellular mechanisms in neonatal and adult tolerance. Immunol Rev 1989, 107:179-902.

75.

Lubaroff DM, Silvers WK: The importance of chimerism in maintaining tolerance of skin allografts in mice. J Immunol 1973, 111:65-71.

76.

Gianello PR, Yamada K, Fishbein JM, Lorf T, Nickeleit V, Colvin RB, Am JS, Sachs DH: Long-term acceptance of primarily vascularized renal allografts in miniature swine. Transplantation 1996, 61:503-522.

77.

Agus DB, Surh CD, Sprent J: Reentry of T cells to the adult thymus is restricted to activated T cells. J Exp Med 1991, 173:1039-1046.

78.

Blancho G, Gianello P, Germana S, Baetscher M, Sachs DH, LeGuern C: Molecular identification of porcine interleukin 10: regulation of expression in a kidney allograft model. Proc Nat/ Acad Sci USA 1995, 92:2800-2804.

79.

Pearce NW, Spinelli A, Gudey KE, Hall BM: Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. V. Dependence of CD4 + suppressor cells on the presence of alloantigen and cytokines, including interleukin-2. Transplantation 1993, 55:374-379.

80.

Oluwole SF, Jin M-X, Chowdhury NC, Ohajewkwe OA: Effectiveness of intrathymic inoculation of soluble antigens

703

in the induction of specific unresponsiveness to rat islet allografts without transient recipient immunosuppression. Transplantation 1994, 58:1077-1081. 81.

Sayegh MH, Perico N, Gallon L, Irnberti O, Hancock WW, Remuzzi G, Carpenter CB: Mechanisms of acquired thymic unresponsiveness to renal allografts. Thymi¢ recognition of immunodominant alIo-MHC peptides induces peripheral T cell anergy. Transplantation 1994, 58:125-132.

82.

Odorico JS, OConnor T, Campos L, Barker CF, Posselt AM, Naji A: Examination of the mechanisms responsible for tolerance induction after intrathymic inoculation of allogeneic bone marrow. Ann Surg 1993, 218:525-531.

83.

Debruin RWF, Vanrossum TJ, Scheringa M, Bonthuis F, Ijzermans JNM, Marquet RL: Intrethymic injection of alloantigen may lead to hyperacute rejection and prolonged graft survival of heart allografts in the rat. Transplantation 1996, 60:1061-1063. See annotation [84"]. •

84. •

Alfrey EJ, Wang X, Lee L, Holm B, Kim J, Adams G, DaFoe DC: Tolerance induced by direct inoculation of donor antigen into the thymus in low and high responder rodents. Transplantation 1 9 9 5 , 59:11 71-1176. This reference, along with [83"], shows that the induction of tolerance versus sensitization by the intrathymic injection of alloantigen is dependent on the strength of the alloresponse between the strain combination examined. These results suggest that a better understanding of the mechanism by which this approach can induce tolerance is needed in order to find reliable predictors of its potential efficacy in humans.