Mixed Chimerism as an Approach for the Induction of Transplantation Tolerance1

Tolerance remains a much needed goal in organ transplantation. The reliable induction of donor-specific tolerance would be a solution to the high rates of late graft loss due to chronic rejection and would eliminate the toxic side effects associated with nonspecific immunosuppression (1-3). Furthermore, tolerance will probably be required to overcome the formidable immunological barriers posed by xenotransplantation (4, 5) that would otherwise require unacceptably high levels of chronic nonspecific immunosuppression (6, 7). There is no shortage of experimental strategies for the induction of tolerance. Numerous regimens leading to the indefinite acceptance of vascularized grafts have been reported in rodent models. The relevance of these models for developing a clinical protocol, however, is severely limited, as most of these strategies fail to achieve similar results when attempted in large animals. When developing a regimen in rodents, therefore, it is of critical importance to use the most stringent tests of tolerance induction [e.g., the permanent acceptance of fully major histocompatibility complex- (MHC*) mismatched skin grafts]. Furthermore, to be clinically useful a tolerance protocol needs to have a high degree of reliability. It is also of critical importance that a strategy is developed that leads to a robust state of tolerance that cannot be perturbed by intercurrent diseases or other events that may occur during the life of the patient. Although a nontoxic strategy has to be the eventual goal, any short-term risk of a tolerance-inducing regimen should be weighed against the morbidity and mortality associated with chronic immunosuppressive therapy used for conventional transplants. In view of recent progress in the field, strategies for tolerance induction that involve mixed hematopoietic chimerism have a strong possibility of fulfilling these requirements. These advances will be the main subject of this article. We will outline the current understanding of the concept of mixed chimerism and the underlying mechanisms of this strategy for inducing allo- and xenotolerance before reviewing recent advances that allow the induction of mixed chimerism through novel, considerably less toxic protocols in rodents. Finally, preclinical porcine and primate models and results from a clinical trial to induce mixed chimerism in patients with hematological malignancies will be discussed. THE CONCEPT OF TOLERANCE INDUCTION THROUGH MACROCHIMERISM More than 50 years ago, Owen (8) observed that in fraternal bovine twins that share a common placental circulation "a mixture of two distinct types of erythrocytes" can be found long after birth, describing a naturally occurring state of mixed chimerism. Shortly thereafter, Medawar (9, 10) showed that these chimeric twins were tolerant to skin grafts from their twin sibling. Medawar's group was also first to actively induce tolerance experimentally by injecting a tissue suspension containing hematopoietic cells into embryos or neonatal mice (11). These studies provided the basis for using bone marrow transplantation (BMT) to induce donor-specific tolerance in adult recipients. The clinical application of BMT for nonmalignant diseases has largely been prohibited, however, by the toxicity of the host conditioning necessary to allow bone marrow (BM) engraftment and the formidable problems of graft-versus-host disease (GVHD) and failure of engraftment. The latter complication occurs more frequently when GVHD-inducing T cells are depleted from donor marrow. These problems become prohibitive when major HLA barriers are transgressed (12, 13). Because of these factors, no BMT protocol is currently available that would be suitable for routine use in HLA-mismatched organ transplant recipients. We think that this situation emphasizes the importance of developing nontoxic, nonmyeloablative conditioning regimens that use more specific treatments to overcome host resistance to donor marrow engraftment, and that are not associated with GVHD. We will review advances that have been made in this direction, both experimentally and clinically. Two types of macrochimerism resulting from BMT into preconditioned hosts can be distinguished: full chimerism, wherein the entire recipient hematopoietic system is destroyed and replaced by donor cells, leading to complete or near-complete donor hematopoietic reconstitution (14); and mixed chimerism, which describes a state wherein hematopoietic populations of both the recipient and the donor coexist in the recipient (15, 16), and which can be achieved by milder forms of treatment which do not ablate the host hematopoietic system. In addition to requiring less toxic host conditioning, mixed chimerism offers additional advantages over full chimerism, including improved immunocompetence (17, 18) and reduced susceptibility to GVHD (19, 20). A third type of chimerism, termed microchimerism, occurs spontaneously in a fraction of organ transplant recipients as a result of persisting donor hematopoietic cells originally present in the transplanted organ (21-23). The level of chimerism in these cases is typically several orders of magnitude lower than that in full or mixed chimeras, and, more importantly, there is no clear evidence that microchimerism has any causal role in the induction of graft acceptance or tolerance (24-30). Before describing specific strategies for the creation of mixed chimerism in detail, the principal features of this approach will be discussed briefly. The fundamental concept underlying tolerance induction through mixed chimerism is to recapitulate nature's major strategy for "self" tolerance (Fig. 1). Several steps are necessary to achieve this goal. Because in an adult recipient mature T cells with anti-donor reactivity already exist in the periphery, they have to be eliminated or inactivated by the initial host conditioning (e.g., by means of exhaustive peripheral T cell depletion or by the use of costimulatory blocking reagents), to provide a "clean slate" for the newly developing T cell repertoire and to prevent the rejection of the infused donor BM. Hematopoietic stem cells contained in the donor BM inoculum then need to "home" to the BM compartment of the recipient. For sufficient levels of engraftment to occur, some form of recipient irradiation or other myelosuppressive therapy is usually necessary. Once stem cells have engrafted, they coexist with recipient stem cells and give rise to cells of all hematopoietic lineages throughout the lifetime of the recipient. In addition, hematopoietic progenitor cells need to seed the thymus through a separately regulated process (31), giving rise to T cells and to dendritic cells that mediate clonal deletion (32). Clonal deletion denotes the elimination of T cells with a certain antigen specificity. In the thymus, self-reactive T cells are clonally deleted during their maturation through the physiologic process of negative selection. Antigens expressed on cells of hematopoietic origin within the thymus are the most effective mediators of negative selection (33, 34). In the case of mixed chimeras, hematopoietic cells from both the recipient and the donor locate to the thymus and hence mediate the elimination of both host-reactive and donor-reactive T cells by negative selection (35, 36). Consequently, the newly developing T cell repertoire in mixed chimeras is tolerant towards the donor and the host and remains so as long as chimerism persists. When all of these steps are successfully accomplished, a robust state of central, deletional tolerance is achieved.Figure 1: Schematic illustration of the concept of tolerance induction through mixed chimerism. Donor hematopoietic stem cells contained in the hematopoietic cell inoculum (1) engraft in the recipient's BM compartment, where they permanently co-exist with hematopoietic cells from the host (2). Both host and donor stem cells give rise to cells of all hematopoietic lineages (3), including progenitor cells that engraft in the thymus (4), where they give rise to dendritic cells mediating clonal deletion (5) and to thymocytes. These newly maturing thymocytes (of both host and donor origin) emigrating from the thymus are tolerant toward both host antigens and donor antigens that are now regarded as self antigens (6).As stated above, protocols for inducing mixed chimerism have been developed that are substantially less toxic than the myeloablative conditioning regimens needed to achieve full allogeneic hematopoietic reconstitution. Nevertheless, the main obstacles precluding the introduction of the mixed chimerism approach into the clinical setting have been the remaining toxicity associated with the host conditioning used to achieve engraftment of allogeneic BM, and the difficulty in obtaining reagents for use in humans that have similar T cell-depleting or inactivating properties as those available for use in rodents. NONMYELOABLATIVE STRATEGIES FOR THE INDUCTION OF TOLERANCE THROUGH MIXED HEMATOPOIETIC CHIMERISM In 1986, Cobbold and Waldmann demonstrated that fully MHC-mismatched marrow engraftment and specific tolerance could be achieved by pretreating recipients with depleting doses of anti-CD4 and anti-CD8 monoclonal antibodies (mAb) along with a sublethal dose (6 Gy) of total body irradiation (TBI) (37). Subsequently, Sharabi and Sachs (16) showed that stable mixed chimerism and tolerance could be induced in mice conditioned with these mAb along with a lower dose of TBI [3 Gy, which is only minimally and transiently myelosuppressive in mice (38)] if additional selective irradiation was given to the thymic area (referred to as thymic irradiation, TI). Lasting multilineage mixed chimerism is achieved when such treatment is followed by the infusion of a conventional dose (15×106) of unseparated fully MHC-mismatched allogeneic BM (16, 39). Chimeras prepared with this regimen demonstrate donor-specific tolerance, as they permanently accept donor skin grafted any time after BMT. At the same time, they demonstrate immunocompetence, as shown by the rejection of third party grafts in the usual time frame, and by other parameters (40, 41). This normal immunocompetence distinguishes them from full allogeneic chimeras, which suffer some degree of immunoincompetence (17, 18). In full chimeras, antigen is presented in the context of donor-type MHC on donor antigen-presenting cells (APC), because no host hematopoietic elements, and thus no host APCs, persist in appreciable quantities after myeloablation. Donor T cells, however, develop in the host thymus and are therefore positively selected by host MHC, which could make them less than fully effective when they encounter antigen presented in the context of donor MHC. This problem does not occur in mixed chimeras, because the host contribution to hematopoiesis ensures a life-long supply of host-type APC in the periphery. Thymic irradiation can be replaced in the above nonmyeloablative model for the induction of chimerism by the repeated administration of anti-CD4 and anti-CD8 mAbs in addition to a first dose on the toxicity of the conditioning a of T cell-depleting mAbs leads to near-complete depletion of the peripheral T cell donor-reactive thymocytes persist in the thymus and rejection of donor cells the thymus, which leads to failure to the T cell repertoire and is not to after if high levels of peripheral chimerism are achieved A cell this thymic by mechanisms that are not fully In addition to recipient T cells, recipient cells also a to allogeneic BM engraftment have their role in engraftment of multilineage hematopoietic stem cells, and have observed that they present only resistance to this cell This resistance is overcome by giving marrow doses to allogeneic recipients cells a much more to marrow than to allogeneic engraftment Furthermore, recent studies that cells of the two a mixed may not be of MHC A failure of cell tolerance the possibility that donor and host cells hematopoietic cells of the other if such occurs in the it does not to have any clinical and does not prevent stable mixed chimerism from persisting for the life of the Numerous other protocols using forms of and T cell elimination for the induction of mixed chimerism in have been reported is the that a the of the chimerism and tolerance induction in and MHC mismatched has been reported for the in BMT models remains to be In nonmyeloablative model it was shown that chimerism and tolerance could be achieved with a conditioning regimen that on the of BMT the of this approach for organ transplantation. chimerism and tolerance could also be induced with this protocol when stem cells or BM cells were transplanted of unseparated adult BM deletion as the main of tolerance induction in mixed Several of evidence demonstrate that deletion is the only major for the of tolerance in mixed to MHC in and are by T cells certain of the in their T cell of the of the using recipient that the the MHC required to present in with donor that provide the required MHC for of the the deletion of donor-reactive T cells can be In the above model and other models mixed chimerism newly developing host and donor thymocytes that presented by donor MHC are and permanently deleted in the thymus, and are from the and where mature T cells have been eliminated by the T cell depleting antibodies in the This specific deletion is throughout the system for the life of the Because from in some however, a more to T cells is to use recipients with specific for a transplantation In hosts with a specific for a donor deletion of donor-reactive cells could be shown by the of thymocytes the Furthermore, donor cells with dendritic cells, which are mediators of negative can be found in mixed chimeras throughout their as as a after BMT The role of deletion as the only by which tolerance is in this nonmyeloablative model is also by the of evidence for or was by studies in which chimeras were before donor antigen was by administration of a mAb specific for donor MHC Tolerance in these mice the of donor which would be required for of tolerance through does not a major role as chimerism and tolerance were in chimeras by the infusion of host-type cells or by the of antigen when the host thymus was so that T cells could be in the thymus clonal deletion leads to the complete of donor-reactive cells, it is a robust form of tolerance major mechanisms for inducing T cell tolerance, such as of T and to a much less stable state of tolerance. Because in these donor-reactive cells are from their persist in large tolerance be overcome such as or of antigen from the where it is needed to tolerance of GVHD in mixed GVHD is of the in clinical allogeneic and has therefore been a in tolerance protocols using BMT. however, is not in the above rodent models for the induction of mixed chimerism, or in a primate model the use of unseparated donor bone marrow cells This is most by the of the T cell-depleting antibodies in the of the hosts the time of BMT may also the susceptibility of mixed chimeras to GVHD (19, of mixed chimerism with a minimally conditioning Although the risk to a a nonmyeloablative dose of TBI is it may be associated with It would therefore be to eliminate TBI from a clinical tolerance in an hematopoietic stem cells do not engraft when in a conventional a dose of TBI to in an system (38)] is required for the induction of stable chimerism in models This for conditioning can be overcome by injecting large quantities of BM these to the allogeneic mixed chimerism it has been to TBI with the of a high dose of BM are conditioned with T cell-depleting mAbs and and a total of unseparated than the conventional levels of chimerism and donor-specific tolerance are in of TBI could be successfully eliminated from the conditioning, thymic irradiation was a of this deletion has been as the major for tolerance induction in this protocol, and GVHD was not of BM would not be available from a or a donor in peripheral stem cells could be used from a donor and in hematopoietic cell could make this approach clinically for in the INDUCTION OF MIXED CHIMERISM The use of costimulatory blocking reagents as of the host conditioning has to much less toxic protocols for the induction of mixed chimerism. In the nonmyeloablative model above, the use of or an mAb the for or repeated of T cell-depleting antibodies and to chimerism and donor-specific tolerance The need for exhaustive recipient T cell depletion in a clinical protocol would additional because the of the adult thymus to T cells after such treatment and to a of reduced immunoincompetence Furthermore, T cell-depleting antibodies currently available for clinical use also do not the near-complete degree of depletion in rodent models. are by recent results that the addition of a costimulatory can the of T cell-depleting needed for induction of high levels of chimerism and tolerance. depletion of the recipient's peripheral T cell repertoire is needed for this to be achieved when a of is also and T cell depletion is achieved in Because studies in to a toxicity for both and these reagents could be of use in clinical chimerism protocols that involve peripheral depletion with T cell-depleting A common to all the mixed chimerism protocols above is the nonspecific of mature recipient T cells in the conditioning by mAbs or myeloablation. it has been to achieve high levels of allogeneic chimerism in the of a mature host T cell T cell by using T cell costimulatory blocking reagents this protocol, mice a nonmyeloablative dose of a conventional dose of fully MHC-mismatched BM, and of an These mice demonstrated high levels of stable multilineage chimerism, and permanently donor-type skin third party skin in the usual time clonal deletion was present during A in this model was that specific deletion of donor-reactive T cells was in the in the after BMT. The mechanisms that mediate this of costimulatory after BMT to be in and their in large has to be that mature donor-reactive host T cells are clonally deleted by an within the first after BMT with costimulatory donor-reactive cells that this peripheral deletion be or In this a "clean slate" in the peripheral T cell repertoire is by only donor-reactive T cells, T cell BM engraftment is thus and accomplished, mixed chimerism the of donor-specific tolerance in the usual through It is also of that the conditioning in this model on the of which thus it to for the induction of mixed chimerism and tolerance with BMT and costimulatory T cell use of costimulatory blocking reagents on their has for the induction of tolerance of vascularized grafts or grafts in can be observed with results when both the and are Although of skin graft has been shown skin graft tolerance has not been achieved by costimulatory in recipients mice accept skin grafts permanently after treatment with and donor In a primate transplantation model treatment with was effective in rejection and graft in reactivity against donor antigen and it is indefinite graft could be achieved chronic treatment with costimulatory blocking reagents used therefore, immunosuppressive effects and a toxicity have not been shown to be to induce transplantation tolerance in it is not costimulatory can be used as a tolerance-inducing on studies of BMT with costimulatory a protocol has now been developed that achieved mixed chimerism irradiation, treatment with or T cell depletion in The of mice with this regimen developed multilineage chimerism with donor-specific tolerance. BMT with costimulatory to the goal of and donor-reactive T cells nonspecific or of the remaining T cell and the approach developed to for inducing mixed chimerism. MIXED CHIMERISM has so been considerably more to induce in large than in rodents. The recent in from models to large and humans that this goal can be of the in results from to large have been due to in the types of reagents available and in the that are given in the For T cell-depleting antibodies have not been given to in that achieve the level of T cell depletion that has been achieved in models for mixed chimerism this approach to the as as it will be critical to develop models that use treatments in and that those that can be and clinically. mixed chimerism has been achieved in using a nonmyeloablative protocol a of immunosuppression after BMT with this approach has to be demonstrated MHC results in a porcine model that that successfully induce mixed chimerism and tolerance in mice can be successfully to large when suitable reagents are This has been achieved in using a conditioning regimen on a depleting the first available that T cell depletion in the TBI and thymic irradiation with conventional stem cell doses and in Furthermore, using the stem cell transplantation approach that the need for TBI in the this group has demonstrated that mixed chimerism and tolerance to full MHC can be achieved using the and high stem cell along with thymic irradiation and in Tolerance induction through mixed chimerism has also been to a primate were conditioned with TBI and before transplantation of unseparated bone marrow and a from the same was given for after BMT with no The of was necessary to donor-reactive host T cells not depleted by during the critical The of TBI the toxicity of the regimen and in chimerism was in for to after BMT and stable graft more than was that the conditioning BM infusion their grafts that BMT was necessary for tolerance to occur in this tolerance long after chimerism that the graft probably in the of tolerance. Although these results are and life-long on deletion for tolerance induction through stable chimerism probably has to be achieved in are in progress to the to achieve improved chimerism and to the need for in this model by a of costimulatory to the and results have been MIXED CHIMERISM MHC BMT NONMYELOABLATIVE mixed chimeras that of donor T cells to full chimerism developing GVHD a of effects of from to GVHD. In a of nonmyeloablative BMT regimen was given of and infusion was shown to mixed chimeras to full chimeras GVHD This model was to the of mixed chimerism induction followed by to mediate effects GVHD in patients with hematological treatment and and this protocol, mixed chimerism was induced in a of donor BMT and also in a of HLA-mismatched BMT recipients to HLA antigen in the and have been achieved in some These results have demonstrated the that mixed chimerism can be achieved HLA barriers in humans myeloablative host conditioning, and that an protocol for use in organ transplant recipients may be The results in this trial also the view that induction of mixed chimerism with nonmyeloablative conditioning is associated with reduced to GVHD to that observed in recipients of HLA-mismatched BMT. INDUCTION OF MIXED CHIMERISM The mixed chimerism approach has been successfully to including several rodent (15, and a primate model In a to model a nonmyeloablative regimen for the induction of mixed chimerism that was similar to the allogeneic above, skin graft acceptance is chimerism is not stable have been found to be for this in chimerism and these are an additional to the of stable mixed chimerism in cells persist in the host thymus for a long after chimerism has in the periphery, and the of donor cells is of deletional tolerance in these are to be the most suitable donor for transplantation to humans antibodies may an additional to BM engraftment in both and In humans and the major by on porcine is a chimerism was shown in the above to BMT model to be to induce both T cell tolerance and tolerance of cells that The to induce cell tolerance for the most antigen by on porcine has been demonstrated after the induction of mixed chimerism in hosts BM from This approach has been shown to be in in which cells are not depleted by conditioning in approach to tolerance of cells against used of BM hosts were with BM that been with a for the of by This protocol to of for the time of using BM this model any engraftment barriers due to It remains to be which of the mechanisms of cell tolerance are in these models. of mixed chimerism and leads to a level of T and cell tolerance that the experimental The and of tolerance induced by the most of tolerance, make this approach a for clinical It has been demonstrated that hematopoietic chimerism can induce tolerance in allogeneic BMT for hematological and a transplant from the same donor the graft major MHC barriers In acceptance of donor skin was also demonstrated chimerism is thus a to achieve transplantation tolerance. progress in experimental models and in clinical BMT for the treatment of malignancies has reduced the toxicity of the conditioning required for allogeneic BM engraftment and offers that the of this approach to tolerance induction will be made from to The and for critical review of the Sachs for and and for