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Stem Cells 2003;21:21-32 www.StemCells.com
© 2003 AlphaMed Press

CONCISE REVIEW

Reprogramming Immune Responses: Enabling Cellular Therapies and Regenerative Medicine

Julian D. Down, Mary E. White-Scharf

BioTransplant Incorporated, Charlestown, Massachusetts, USA

Key Words. Immune system • Reprogramming • Cellular therapy • Stem cells • Immunosuppression • Tolerance

Mary E. White-Scharf, Ph.D., 19 Johnson Road, Winchester, Massachusetts 01890, USA. Telephone: 781-729-4079; e-mail:
maryewhite{at}attbi.com


   ABSTRACT
Top
 Abstract
Introduction
 Challenges for the Field
 Overview of Tolerance Induction
 Experience with Cellular...
 Induction of Central Tolerance:...
 Molecular Chimerism and...
 Conclusions and Future...
 References
 
Recent advances in cellular therapies have led to the emergence of a multidisciplinary scientific approach to developing therapeutics for a wide variety of diseases and genetic disorders. Although most cell-based therapies currently consist of heterogeneous cell populations, it is anticipated that the standard of care will eventually be well-characterized stem cell lines that can be modified to meet the individual needs of the patient. Many challenges have to be overcome, however, before such "designer cells" can become a clinical reality. One of the major hurdles will be to prevent immune rejection of the therapeutic cells. A patient’s immune system may react to genetically modified or allogeneic cells as foreign, leading to their destruction. We propose that specific reprogramming of the immune system to accept cellular therapies can be accomplished by establishing hematopoietic chimerism. Successful engraftment of hematopoietic stem cells (HSCs), which have the same origin as those cells intended for therapeutic use, should lead to a re-education of the immune system so that the donor cells are recognized as self and will not be rejected. Developing safe, nontoxic protocols for reprogramming the immune system is critical to the success of this approach. Two major requirements exist for achieving stable HSC engraftment: A) depletion or displacement of host stem cells, and B) adequate immune suppression. Available data indicate that an agent such as busulfan is effective in depleting stem cells and that immune suppression can be accomplished with monoclonal antibodies that specifically target immune-reactive cells in the periphery.


   INTRODUCTION
Top
 Abstract
 Introduction
Challenges for the Field
 Overview of Tolerance Induction
 Experience with Cellular...
 Induction of Central Tolerance:...
 Molecular Chimerism and...
 Conclusions and Future...
 References
 
Technical advances associated with the manipulation and growth of cells in vitro, especially the isolation and growth of human embryonic stem (ES) cells and nuclear transfer of genetic material from one cell type to another, have given rise to new areas of scientific investigation within the broad realm of transplantation. The widespread potential for eventual therapeutic application has encouraged multidisciplinary collaborations among scientists with expertise in areas including the basic biological sciences, engineering, and materials science, and has led to the emergence of a new field: regenerative medicine.

Cells are the basic building blocks of a living organism, the majority of which exist and function in a terminally differentiated state. Reservoirs of stem cells exist, however, that are defined by their capacity for self-renewal and differentiation. Stem cells in many adult mammalian tissues have now been identified and are thought to serve as a resource for replacing cells lost over time as a result of cell death or injury. Although these "adult" stem cells are associated with specific tissues of origin, some of them may have the capacity or plasticity to transdifferentiate into other types of tissue [1–7]. The "ultimate" stem cell, however, is the ES cell, which is found in the blastocyst stage of early mammalian embryos and has the capacity to differentiate into any cell type in the body [8]. It is intriguing to consider, then, that a therapeutic could eventually take the form of a well-characterized stem cell line that could be expanded and specifically modified and differentiated to meet the needs of an individual patient. Ideally, these cells could be cryopreserved after manipulation while quality control assays were performed. In the majority of cases, these cell lines would be, by definition, allogeneic with respect to prospective recipients and would be subject to immune surveillance and potential elimination by competent immune systems. Even if cells were collected from an individual and then modified to correct a defect, expression of foreign genes by a patient’s cells could lead to an immune response and subsequent elimination of the modified cells. Assuming such barriers can be overcome, the potential therapeutic applications for cellular therapies are extensive and include cancer; genetic disorders (such as hemophilia, thallassemia, and chronic granulomatous disease); neurological disorders (such as Parkinson’s and Huntington’s disease); tissue repair of damaged organs (such as cardiac, pancreatic, hepatic, and nervous), and autoimmune disease.


   CHALLENGES FOR THE FIELD
Top
 Abstract
 Introduction
 Challenges for the Field
Overview of Tolerance Induction
 Experience with Cellular...
 Induction of Central Tolerance:...
 Molecular Chimerism and...
 Conclusions and Future...
 References
 
In addition to the obvious immunological challenges for successful cellular therapies, other hurdles need to be overcome. Ethical concerns have been expressed in the use of embryonic stem cells, even for therapeutic applications. Initial reports suggested that adult stem cells could transdifferentiate into various cell types and potentially supplant the need for ES cells [5, 7]. Optimism in this area has become more guarded, however, as it has been demonstrated that adult stem cells migrate into other tissues and may adapt morphologically to their environment [9–11]. Such events occurring in tissues could be misinterpreted as transdifferentiation events. It may be that adult stem cells exhibit some degree of plasticity; however, it seems unlikely at this point that adult stem cells will be able to substitute for all potential applications of ES cells. These issues have been the subjects of a number of recent reviews.

Regardless of the origin of potential cell lines, standardized procedures that are commercially viable and Food and Drug Administration approvable for expansion, differentiation, and selection must be achieved. Several groups have been experimenting with means of expanding cord blood hematopoietic stem cells (HSCs) by carefully controlling growth conditions [12–16], and clinical trials have been initiated. Another approach for expanding cell lines is the use of telomerase, which appears to be able to promote expansion of dividing cells without transforming them [17–21]. When transdifferentiation is required, it may be more readily achievable by first expanding the cells and then inducing specific transcription factors for differentiation according to specifications for treatment. The assumption is that differentiated cells, or immediate precursors thereof, will home to the appropriate tissues when injected in vivo and maintain their differentiated state. However, this has yet to be confirmed in vivo.

Even though there are challenges associated with the scale-up and production of cell-based therapies, overcoming immune rejection remains a major hurdle. We know from the vast literature in transplantation that, although improvements in immunosuppressive agents have extended the lives of many transplant patients, chronic immunosuppression has a negative impact on quality-of-life issues. All too often, successfully transplanted patients succumb to opportunistic infections. In order to realize the full potential of this new and exciting field, it will be essential to find ways of reprogramming the immune system to accept modified autologous or transdifferentiated allogeneic cells. Progress toward this goal is the major focus of this review.


   OVERVIEW OF TOLERANCE INDUCTION
Top
 Abstract
 Introduction
 Challenges for the Field
 Overview of Tolerance Induction
Experience with Cellular...
 Induction of Central Tolerance:...
 Molecular Chimerism and...
 Conclusions and Future...
 References
 
Immunological tolerance refers to a state of nonresponsiveness to a specific set of antigens, the prototype being autologous or "self" antigens. The immune system of a healthy individual is able to distinguish between self and nonself antigens by reacting to and eliminating the latter as a means of protecting the body against infection. Mechanisms for inducing tolerance can be divided into two major categories: central and peripheral [22–26]. Central tolerance is induced by a thymic deletional mechanism. Basically, immature T cells migrate through the thymus where they come into contact with endogenous peptides bound to major histocompatibility complex (MHC) molecules expressed on antigen-presenting cells (APCs). Those T cells that exhibit high-affinity interactions with self peptides are eliminated (negative selection), whereas those that have low to intermediate affinities are positively selected and released into the periphery where they provide immune surveillance [27, 28]. Peripheral tolerance occurs when mature T cells in the periphery come into contact with antigen under conditions where the mature cells are activated in such a way that they are either deleted or rendered anergic [22]. Peripheral mechanisms are thought to be invoked for those antigens that are not presented in the thymus or that somehow escape central tolerance.

Central tolerance is considered to be more stable since potentially reactive T cells are systematically deleted prior to maturation and release. In contrast, peripheral tolerance requires that mature T cells actually come into contact with antigen so that there is a concern for potential escape of T cells or the potential for overcoming anergy. Central tolerance is induced to those antigens present in the thymus during the normal development of the immune system. It is much more difficult to achieve central tolerance to newly introduced antigens once the immune system has developed. Since mature T cells are long lived, peripheral T cells reactive with the intended tolerogen must be deleted or inactivated when the new antigen is presented in the thymus in order for the process of thymic re-education to be translated into modified immune surveillance in the periphery.

Although, historically, central and peripheral mechanisms of tolerance induction have been described as being distinct processes, with deletion associated with central tolerance and anergy associated with peripheral tolerance, recent studies suggest that this division may not be quite so clear cut. Investigations of a regulatory T-cell subset, characterized phenotypically as CD4+CD25+, suggest that these cells may be common to both pathways [29–31]. These regulatory, or suppressor, T cells are present in the thymus [32–34], and in the periphery [31, 35]. Apparently, CD4+CD25+ cells are derived intrathymically from thymocytes manifesting high-affinity interactions with self peptides presented by MHC. At least some of these high-affinity thymocytes are selected as regulatory cells rather than for deletion [29]. There may be peripheral mechanisms for their development as well [30, 31]. Regulatory T cells have been associated with the prevention of autoimmune disease for some time [36–39]. More recently, however, they have also been shown to be required for the induction of tolerance to alloantigens via peripheral mechanisms [40, 41]. Mechanisms leading to T regulatory cell development and function are still poorly understood. T regulatory cells have been implicated in Notch signaling pathways in the thymus and in the periphery [42–44]. Overexpression of the Notch receptor, Serrate 1, on APCs leads to the development or activation of T regulatory cells in response to antigen being presented by the APCs. T regulatory cells have also been implicated in mechanisms of "infectious tolerance" or linked immunosuppression [35]. A better understanding of these mechanisms may eventually lead to additional modalities for modulating immune responses [41, 45].


   EXPERIENCE WITH CELLULAR THERAPIES: LESSONS FROM HSCS
Top
 Abstract
 Introduction
 Challenges for the Field
 Overview of Tolerance Induction
 Experience with Cellular...
Induction of Central Tolerance:...
 Molecular Chimerism and...
 Conclusions and Future...
 References
 
It is probably not coincidental that among the adult stem cells identified, HSCs have been the most extensively investigated, and these are the cells that are essential in establishing central tolerance to associated antigens. HSCs are pluripotent, giving rise to erythroid, myeloid, and lymphoid lineages. Dendritic cells are derived from hematopoietic precursors of either the myeloid or lymphoid lineage. Some of these dendritic cells home to the thymus where they serve as APCs and participate in thymic education. Others enter the circulation where they are distributed among tissues and organs. They are known to have an important role in the induction and regulation of immune responses [46–48]. Subclasses of dendritic cells have been identified and are being actively investigated, but their functions are not yet fully understood [49]. Other cells from the lymphoid compartment develop into lymphocytes, a subset of which migrate through the thymus where they become "educated" T cells before joining their B-cell counterparts in the periphery to provide immune surveillance.

Using rodent models, it has been clearly and reliably demonstrated that donor-specific tolerance can be induced by establishing hematopoietic chimerism, as evidenced by long-term graft acceptance [24, 50–55]. Tolerance is induced only to those antigens introduced to the recipient by hematopoietic cell precursors that engraft or seed hematopoietic reservoirs such as the bone marrow. For example, skin-specific antigens present on skin but not on the corresponding bone marrow cells used to establish chimerism may elicit an immune response resulting in skin graft rejection [25, 56–58]. In the translation of these studies to large animal models, however, two major problems have arisen that have not been major issues in the rodent studies: achieving stable engraftment of hematopoietic cell precursors in the bone marrow and preventing graft-versus-host disease (GVHD) once engraftment is accomplished. In fact, achieving donor-specific tolerance in clinical transplantation continues to be referred to as the "elusive Holy Grail" [22].

Bone marrow transplantation (BMT) has been in practice for many years for the purpose of reconstituting the hematopoietic system in treating blood cancers. In autologous transplants, bone marrow is collected prior to treatment with myeloablative or lethal chemotherapeutic agents, which are administered in an effort to destroy remaining cancer cells. The bone marrow is then enriched for hematopoietic precursors and purged of cancer cells before being returned to the patient to reconstitute hematopoiesis. Allogeneic transplants are potentially more efficacious for two reasons. The first is that the donor cells are free of cancer cells and the second is that, because the cells are allogeneic, they provide an additional antitumor effect. The risks associated with allogeneic transplantation are substantial, however, and include the potential failure to engraft and the development of GVHD when engraftment does occur. These risks become greater with increasing disparity between donor and recipient.

Initially, myeloablative conditioning protocols were used for allogeneic BMT [59–62], but, in recent years, there has been a shift to nonmyeloablative protocols [63–68], as can be seen in Figure 1Go. Nonmyeloablative protocols have the advantage of being less toxic, and these protocols often result in mixed chimerism (the presence of both donor and recipient cells), which may provide advantages as well [24, 69].



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  		Figure 1. Increasing use of nonmyeloablative compared to myeloablative conditioning in clinical allogeneic stem cell transplantation. Data were compiled from The European Group for Blood and Marrow Transplantation (EBMT) registry.

*For the year 2001, the results are extrapolated from incomplete data presented in oral form at the Annual EBMT Meeting, March 24-27, 2002, Montreux, Switzerland. These less-intensive conditioning regimens for 2001 from the International Bone Marrow Transplant Registry (IBMTR) are reported to be comparable (about 25% of allotransplants) (IBMTR/ABMTR Newsletter 9:Issue 1, February 2002).

 
Although hematopoietic chimerism is associated with tolerance induction, achieving chimerism does not insure the induction of tolerance. Examples have been cited where organ graft rejection occurred in the presence of lymphoid chimerism; however, since T lymphocytes are long lived, lymphoid chimerism is not necessarily indicative of stem cell engraftment [70]. Homing of donor bone-marrow-derived APCs to the thymus is important as well for the induction of central tolerance [58, 71]. With insufficient conditioning, donor APCs may not be able to home to and reside in the thymus, and appropriate education of developing cells will fail to occur. In contrast, organ graft acceptance has been documented in the absence of stable chimerism [72–74]. These data suggest that transient chimerism is sufficient to initiate tolerance induction but that peripheral mechanisms may participate once a graft is in place. Organ grafts such as liver, kidneys, and heart may be susceptible to such mechanisms since they tend to contain passenger cells of lymphoid origin, which may play a role in peripheral mechanisms [75]. Such apparent discrepancies may be resolved eventually with a better understanding of the precise mechanisms that lead to tolerance induction.

While hematologists have been actively pursuing the use of HSC transplantation for the treatment of hematological cancers, immunologists have focused on achieving successful organ transplantation by establishing hematopoietic chimerism to induce donor-specific tolerance. Perhaps it has been the recognition of common goals and associated challenges that has brought about the convergence of these two fields and led to an integrated effort to resolve the hematological and immunological complications that are associated with achieving allogeneic hematopoietic cell transplantation.


   INDUCTION OF CENTRAL TOLERANCE: DISSECTING THE REQUIREMENTS
Top
 Abstract
 Introduction
 Challenges for the Field
 Overview of Tolerance Induction
 Experience with Cellular...
 Induction of Central Tolerance:...
Molecular Chimerism and...
 Conclusions and Future...
 References
 
Although exceptions have been noted, stable hematopoietic chimerism is associated with the induction of central tolerance. Two major requirements must be met in order for engraftment to occur: some of the recipient stem cells must be depleted or displaced and immune resistance from the host must be overcome. Increasing the genetic disparity between donor and recipient increases the difficulty of achieving engraftment.

Stem Cell Depletion
In order to look at the requirements for engraftment alone (in the absence of immune resistance), syngeneic animal models bearing nonimmunogenic markers to distinguish donor and host cells have been used [76, 77]. Engraftment of HSCs requires depletion or displacement of at least a portion of recipient stem cells. Strategies for accomplishing this include irradiation, treatment with chemotherapeutic agents such as busulfan, infusion of very high doses of autologous HSCs or of allogeneic T cells, administration of radioactively tagged stem-cell-reactive antibodies, and cytokine mobilization of stem cells into the periphery. It is important to note that acute myelosuppression (or in its severest form, myeloablation) does not necessarily include stem cell depletion. Agents such as thiotepa, melphalan, and cyclophosphamide, which are myelosuppressive when given at high doses, actually have little effect on quiescent long-term repopulating stem cells [78, 79]. Some of the conditioning agents commonly used in bone marrow transplantation and their biological properties are listed in Table 1Go.


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  		Table 1. Conditioning agents and their biological properties
 
Stewart and coworkers demonstrated that creating "niches" or "space" may not be required for engraftment, in that transplanting very large numbers of HSCs can overcome the need for traditional methods of making space in the bone marrow [76]; this has since been confirmed by other groups as well [58, 80, 81]. However, such large numbers of cells are often not practical, if not impossible, to attain in a clinical setting. The high doses used in the animal studies probably facilitate engraftment by displacing some of the host stem cells, and perhaps by an active mechanism, such as a veto effect [82, 83].

Results from rodent studies have clarified that some depletion or displacement of recipient HSCs is required for engraftment of donor HSCs in the bone marrow [76, 78, 84, 85]. Many of the current nonmyeloablative protocols being used in the clinic rely on the presence of donor T cells in the graft, but this effect is difficult to manage without evoking GVHD [65, 67, 69]. This point is well illustrated in our own modeling of engraftment in which different doses of T-depleting monoclonal antibodies (mAbs) were administered as part of a cyclophosphamide/thymic-irradiation regimen (Fig. 2Go) [86]. When donor and recipient T cells were depleted (administration of high-dose anti-CD4/CD8 mAbs), very little chimerism was observed, whereas mice were almost 100% chimeric when no T-depleting antibodies were administered. These mice suffered severe weight loss, however, indicative of GVHD. Note that an intermediate dose of antibodies resulted in highly variable responses in individual animals (low-dose anti-CD4/CD8 mAbs), suggesting that the effect of donor T cells is very difficult to regulate. Comparison with a much lower level of chimerism (about 5%) following the same conditioning regimen but with syngeneic BMT strongly suggests that allogeneic donor T cells procure engraftment via depletion of recipient marrow stem cells. The difficulty in managing donor T-cell activity has been borne out in the clinic in that the presence of GVHD in successfully engrafted patients undergoing nonmyeloablative protocols has been a common observation [66, 67, 69]. These data serve to emphasize the need to identify agents that can deplete host stem cells without the assistance of T cells in the donor graft, especially for indications other than cancer where a graft-versus-tumor effect is not needed.



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  		Figure 2. Schematic and results of protocol. A) Lymphoid (CD3+) and myeloid (CD11b+) donor-type blood leukocyte chimerism (mean ± 1 standard error) in groups of five C57BL/6 (H2b) mice pretreated with 200 mg/kg cyclophosphamide (CY), 7 Gy thymic irradiation (TI), with or without anti-CD4 and anti-CD8 mAbs, followed by mixed bone marrow ([BM] 15 x 106 cells) and splenocyte ([SPL] 10 x 106 cells) transplantation from MHC-mismatched B10.A (H2a) donor mice. The T-depleting antibodies were administered at either a low dose (0.3 mg for anti-CD4 and 0.14 mg for anti-CD8 at 14 days) or a high dose (2 mg for anti-CD4 and 1.4 mg for anti-CD8 at 5 days), giving average blood antibody levels of 3 and 500 µg/ml, respectively, at the time of BMT (B).

 
In syngeneic mouse models, busulfan is one of the few drugs that enables engraftment of bone marrow cells [78, 84, 87]. It has been demonstrated that busulfan depletes cobblestone-area forming cells in long-term cultures derived from host bone marrow [87] and that there is a direct correlation between the dose of busulfan and the amount of HSC depletion. In an extension of this study, we have observed in an MHC-mismatched model with T-cell depletion and thymic irradiation, that the levels of chimerism achieved are also dependent on the dose of busulfan administered and that the dose range lies below half the maximal tolerated dose (Fig. 3AGo). These data confirm that, when immune resistance is overcome, the amount of chimerism established is related to the extent of recipient stem cell depletion. Interestingly, when these mice were examined for donor-specific tolerance induction by challenging with skin grafts from the donor or a third party strain, tolerance induction was observed with very low doses of busulfan (Fig. 3BGo), indicating that tolerance can be achieved with minimal levels of chimerism. Similar results have been observed by Adams and coworkers when titrations of busulfan were administered together with costimulatory blockade [84].



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  		Figure 3. The level of MHC-mismatched (B10.A, H2a) hematopoietic chimerism at 4 months post-BMT (15 x 106 cells) is dependent on the total dose of busulfan administered intraperitoneally as 10 mg/kg daily doses (-5 to -2 days) to C57BL/6 recipient mice (H2b) in combination with anti-CD4 and anti-CD8 antibodies (-5 days) and 7 Gy thymic irradiation (-5 hours). The maximal tolerated dose of four daily fractions of busulfan was determined to be 100 mg/kg. Each dose group consists of 10 to 4 mice pooled from one to three separate experiments (A). The incidence of donor-type skin graft survival out to 200 days post-skin transplant (performed 2 months after BMT) was also dependent on busufan dose, while all dose groups rejected third-party skin grafts from SJL donor mice (H2s) within 20 days (B).

 
Busulfan has been used clinically for a number of years as a conditioning agent for BMT. Considerable toxicity has also been reported with busulfan regimens, but these consisted of high doses of an oral formulation, so actual doses attained were variable. More consistent dosing has been achieved since the introduction of a soluble formulation, and this has led to a decrease in drug-related toxicities [88, 89]. Our data predict that, with adequate immune suppression, engraftment of HSCs in the bone marrow can be achieved with very low doses of a stem cell-depleting agent. Clinical studies confirm that engraftment of hematopoietic cells from HLA-matched donors can be accomplished with a reduced busulfan regimen in combination with immunosuppressive agents such as fludarabine [63, 90, 91]. Recently, the use of a low-dose regimen of busulfan alone has been attributed to the success of gene-corrected autologous stem cell transplantation in patients with adenosine deaminase-deficient severe combined immunodeficiency [92].

Immunosuppression
Central tolerance related to donor HSC chimerism can only be achieved if immunocompetent lymphocytes in the periphery are first eliminated or inactivated to circumvent initial immunological rejection of the stem cell graft. Although numerous chemotherapeutic agents have immunosuppressive properties, most of these are insufficient as single agents. Fludarabine, a potent drug capable of depleting lymphocytes, is now being used increasingly in nonmyeloablative conditioning regimens [64]; however, questions have arisen as to whether this agent may be harmful to target tissues, which could lead to transplant-related complications and perhaps even potentiation of immune reactivity [93]. A synergistic effect has been described for the combined administration of fludarabine with cyclophosphamide in suppressing immune resistance [94, 95], and consistent engraftment has been observed in protocols using this combined regimen [66]. Nevertheless, this regimen appears to be associated with a high incidence of GVHD [96, 97], and our own murine studies have indicated enhanced pulmonary toxicity after combining these two drugs [98, unpublished observations].

In animal models, the use of CD40/CD40L-mediated costimulatory blockade has also been effective in overcoming immune resistance [24, 84, 99]; however, adverse events (thromboembolic) were reported when mAbs to CD40L were clinically evaluated. Recent functional studies of the transmembrane protein CD40L indicate that it is involved not only in modulating immune responsiveness but also in platelet activation and thrombosis [100]. Blocking the CD40L/CD40 interaction in vivo could potentially render platelet plugs unstable and increase the likelihood of embolism development.

The use of T-cell-depleting mAbs has at least two potential advantages: the inherent specificity associated with carefully selected mAbs contributes to a strong safety profile, and the dual function provided of immune suppression of the recipient and depletion of T cells present in the donor graft reduces the likelihood of GVHD. In animal models, high-dose combinations of T-cell-depleting antibodies have been very effective in accomplishing both functions [54]. T-cell-directed immunotoxins also have been effective in providing immune suppression [58, 71], but their short half-life limits their usefulness in depleting donor T cells in the incoming graft. Many clinical protocols have included polyclonal T-cell-depleting sera [69, 83, 101, 102] such as antithymocyte globulin (ATG), since effective monoclonal agents previously have not been available for clinical use. Currently, however, several mAbs that are capable of selectively targeting lymphocyte subsets are being evaluated clinically in transplantation protocols.

Donor Graft Composition
Optimization of the donor graft composition may vary depending on genetic differences between donor and recipient, as well as on the specific indication for therapy. For example, in closely matched transplants where GVHD is less of an issue, T cells have not been rigorously depleted, and limited numbers are beneficial for providing graft-versus-tumor effects and for maintaining immune competency of the recipient. Stringent T-cell depletion is required in mismatched transplants, however, in order to prevent GVHD [83]. It is important to note that although the presence of limited numbers of T cells may be beneficial in some cases, purified populations of HSCs can engraft across MHC barriers when recipients are appropriately conditioned [103]. Furthermore, they can also reconstitute hematopoiesis and mediate positive and negative T-cell selection in the induction of tolerance to alloantigens [104]. Thus, although the inclusion of donor T cells may be beneficial in some situations, they are not required for engraftment.


   MOLECULAR CHIMERISM AND TOLERANCE INDUCTION
Top
 Abstract
 Introduction
 Challenges for the Field
 Overview of Tolerance Induction
 Experience with Cellular...
 Induction of Central Tolerance:...
 Molecular Chimerism and...
Conclusions and Future...
 References
 
Gene therapy approaches involve the use of autologous cells that have been modified to express foreign genes. Efficacy is often reduced, however, by the fact that gene products inserted into autologous cells are frequently immunogenic and result in clearance of the transduced cells [105–107]. A technical problem in gene transfer has been the difficulty of transducing primitive HSCs; however, new vectors and methods have resulted in improved transduction efficiencies. Several groups have now demonstrated, by gene transfer and engraftment of modified cells under myeloablative conditions, that molecular chimerism is sufficient to induce tolerance [108]. Since a general observation of both preclinical and clinical studies has been that the requirements for achieving engraftment become less stringent with increasing genetic identity between donor and recipient, we tested the assumption that the requirements for engrafting donor cells with a single antigen difference should be minimal. Using a C57BL/6J (B6) transplant model, HSCs expressing the enhanced green fluorescent protein (EGFP) were transplanted into B6 mice under nonmyeloablative conditions where engraftment was established following conditioning with low-dose busulfan alone. Tolerance for EGFP was established by the acceptance of B6-EGFP-transgenic skin grafts on chimeric mice and by their rejection on control mice that were transplanted with non-EGFP-expressing bone marrow cells. Third-party grafts were rejected by both experimental and control groups [109]. These studies provide encouragement for cellular therapies in that if genetic differences can be minimized, mild conditioning regimens should be effective for seeding the bone marrow with HSCs from the donor or therapeutic cell line. Furthermore, they confirm that molecular chimerism within the hematopoietic system can be used to induce tolerance to foreign antigens expressed elsewhere in the body.


   CONCLUSIONS AND FUTURE DIRECTIONS
Top
 Abstract
 Introduction
 Challenges for the Field
 Overview of Tolerance Induction
 Experience with Cellular...
 Induction of Central Tolerance:...
 Molecular Chimerism and...
 Conclusions and Future...
References
 
Cellular Therapies of the Future
Appreciation of the broad therapeutic potential together with advances in cell biology and methodologies for ex vivo growth and manipulation of cells have contributed substantially to the expanding interest in the rapidly growing fields of cellular therapies and regenerative medicine. Although cellular therapies currently under clinical evaluation have all been derived from heterogeneous populations of cells, one can envision that those of the future will consist of a well-characterized, homogeneous population of cells that has been clonally derived from stem cell banks. Depending on a patient’s needs, cells could be genetically altered, expanded, and differentiated into the appropriate cell type before transplant. Many challenges need to be overcome before such "designer cells" can become a reality, not the least of these being to gain immunological acceptance by the recipient.

Approaches to Overcoming Immunological Rejection
Several approaches to the problem of immunological rejection have been suggested by various investigators [8]. One possibility is to knock out MHC genes creating a so-called "null cell," which could then have MHC genes inserted to match those of the recipient. Although less stringent conditioning regimens are required to transplant MHC-matched cells, rejection can still occur as a result of non-MHC antigenic differences.

A second approach is to use nuclear transfer technology to insert a nucleus from the recipient into the donor cell line. While this is certainly an attractive approach for reconstitution of tissues or organs that have been damaged or destroyed, the technology is not yet sufficiently reliable or efficient to make this a practical alternative. There is also the possibility that such cells could still be rejected as a result of cytoplasmic influences on antigen expression (differences in glycosylation, etc.).

While both of these approaches would alter the donor cells in an attempt to make them acceptable to the recipient, a third approach is to reprogram the immune system of the recipient to accommodate the donor cells or tissue. This would seem to be the most reliable approach, given the caveats of the others discussed, and thus would be preferable, assuming that it can be accomplished with minimal, if any, adverse effects on the patient.

Reprogramming the immune system to accept foreign antigens as "self" can be accomplished by establishing stable hematopoietic chimerism between donor and recipient. The difficulty of transitioning preclinical models into the clinic underscores the fact that the underlying mechanisms involved are not yet completely understood. A number of investigators have used rodent models in an effort to investigate the requirements for thymic re-education or central tolerance induction after the immune system is fully developed. Shizuru and coworkers have shown that purified HSCs alone are capable of reconstituting hematopoiesis and inducing tolerance to alloantigens [103, 104]. Furthermore, recent reports suggest that HSCs can be generated in vitro from ES cells [110, 111]. Thus, even if the therapeutic application were for tissue repair of a peripheral organ such as the heart or liver, donor-specific tolerance could be induced by using HSCs derived from the same source as the therapeutic cells to establish hematopoietic chimerism in the recipient.

Hematopoietic stem cells will engraft if host stem cells are depleted and immune resistance is overcome. This can be accomplished with incomplete depletion of host stem cells. In fact, there seems to be a correlation between the extent of depletion required and the genetic disparity between donor and recipient (unpublished results). Many of the nonmyeloablative protocols being used clinically inadvertently rely on donor T cells to provide this function; however, GVHD frequently develops and requires immunosuppression for clinical management. Incorporating a stem cell-depleting agent such as busulfan into the conditioning regimen alleviates the need for donor T cells. As discussed earlier, stable hematopoietic chimerism has been achieved in both preclinical and clinical studies using protocols with low-dose busulfan.

In order to overcome immune resistance, recipient-derived mature lymphocytes must be eliminated or inactivated. Otherwise, even if donor HSCs were allowed to engraft and newly developing T cells underwent thymic education, they would be rapidly eliminated upon entry into the periphery. Current methods used clinically to overcome immune resistance include the use of immunosuppressive drugs or mAbs directed at lymphoid cells. Effective mAbs are preferable to drugs because of their specificity and lower toxicity. In cases with limited genetic disparity between donor and recipient, such as in a gene therapy approach using modified autologous cells, an even milder approach would be to selectively deplete antigen-specific T cells. Adams et al. [84] have demonstrated that costimulatory blockade (anti-CD40L + CTLA4-Ig) can provide immune suppression in rodents. Clinical trials with antibodies to CD40L have been terminated, however, as a result of serious adverse events. These adverse events may be explained by recently published functional studies suggesting that disrupting platelet-associated CD40/CD40L interactions could have serious ramifications [100]. The involvement of dendritic cells, or APCs, in immune regulation and that of CD4+CD25+ regulatory T cells in suppressing immune responses have been recognized for a number of years. Their broad presence in both thymus and periphery and their links to pathways involving central tolerance induction and costimulatory blockade have recently generated increased interest in these cells [29, 31, 33, 38, 39, 42–44, 49]. Gaining more insight into their involvement in suppression may provide additional avenues for overcoming immune resistance.

Potential Combination Approach for Optimal Success
The observation that molecular chimerism is sufficient to induce tolerance and can be achieved with minimal conditioning is very encouraging for the future of cellular therapies. Ultimately then, the goal should be to match donor cell lines with recipients as closely as possible, and then with minimal conditioning, to reprogram the recipient’s immune system to accept as self the remaining unshared antigens on the donor cells.


   REFERENCES
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 Abstract
 Introduction
 Challenges for the Field
 Overview of Tolerance Induction
 Experience with Cellular...
 Induction of Central Tolerance:...
 Molecular Chimerism and...
 Conclusions and Future...
 References
 

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