Mixed chimerism: Preclinical studies and clinical applications

Biology of Blood and Marrow Transplantation 5:192–203 (1999) © 1999 American Society for Blood and Marrow Transplantation

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#99-26 McSweeney

Mixed chimerism: preclinical studies and clinical applications Peter A. McSweeney, Rainer Storb Clinical Research Division, Fred Hutchinson Cancer Research Center, and the Department of Medicine, University of Washington, Seattle, WA Offprint requests: Peter A. McSweeney, MD, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, D1100, P.O. Box 19024, Seattle, WA 98109-1024; e-mail: [email protected]. (Received 20 May 1999; accepted 9 June 1999)

ABSTRACT Traditional approaches to allogeneic stem cell transplantation have relied on the use of toxic high-dose conditioning therapy to achieve allogeneic engraftment and control of underlying disease. Preclinical studies and clinical observations have shown that, for engraftment purposes, conditioning regimens can be reduced in intensity, resulting in reduced treatment toxicities. In preclinical canine studies, the use of potent pre- and postgrafting immunosuppression allowed for reduction in conditioning regimens and development of stable mixed chimerism. If these newer approaches using attenuated conditioning regimens can be successfully applied to human transplantation, an improved safety profile will allow potentially curative treatment of patients not currently offered such therapy. Mixed chimerism per se could prove curative of disease manifestation for various nonmalignant disturbances of the hematopoietic and immune systems. For patients with malignancy, infusion of additional donor lymphocytes may be needed to effectively treat underlying disease.

KEY WORDS: Adoptive immunotherapy

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Mixed chimerism

INTRODUCTION Successful transplantation of allogeneic hematopoietic stem cells (HSC) requires that two immunologic barriers be overcome. The first, a host-vs.-graft (HVG) reaction, leads to graft rejection. The second, a graft-vs.-host (GVH) reaction, leads to an immunologic attack on the graft recipient. Traditional approaches to controlling these two reactions have followed distinctly different principles. Control of HVG reactions has been achieved through the use of regimens delivered before transplant, with the goal of completely eradicating the host’s immune responses. To accomplish this, high-dose conditioning therapy has been given, which also has the purpose of eliminating the patient’s underlying disease (e.g., leukemia). Attempts to reduce the risk of graft rejection have included intensification of conditioning regi-

Supported in part by grants HL03701, HL36444, DK42716, CA78902, and CA15704 from the National Institutes of Health,Bethesda, MD. P.A.M. was supported by a grant from the Gabriella Rich Leukemia Foundation. R.S. received support through a prize from the Josef Steiner Krebsstiftung, Bern, Switzerland, and the Laura Landro Salomon Endowment Fund.

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mens and addition of antibodies targeting T cells. The intensity and relative lack of specificity of conditioning regimens results in almost complete destruction of any vestiges of the patient’s normal hematopoietic system. Acute and profound pancytopenia ensues, and the immediate role of the subsequent stem cell graft is to promptly restore hematopoiesis. Control of the GVH reactions involves targeting donor cells through agents delivered after transplant or through removal of T cells from the graft. Given the fragility of the transplanted HSC graft and the need for rapid engraftment, postgrafting immunosuppression must be minimally myelosuppressive and, ideally, would affect only those donor cells that mediate the GVH reaction. In attempts to achieve therapeutic goals (eradication of malignancy and prevention of graft rejection), conditioning regimens have been intensified such that dose-limiting toxicities are frequently incurred. Profound pancytopenia predisposes to the development of serious and potentially fatal infections. Therapy-related organ toxicities, particularly those affecting liver, kidneys, and gastrointestinal tract, can impair adequate delivery of postgrafting agents such as cyclosporine and methotrexate given to control GVHD.

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The inability of older patients to tolerate these combined insults has restricted the use of HSC allografting to relatively young patients who can better tolerate intensive therapy. To manage the side effects of HSCT, it has traditionally been delivered in highly specialized hospital wards. An important goal is to develop safer approaches to allografting that can also be extended to older patients or patients with preexisting organ toxicities that preclude the use of high-dose conditioning regimens. Implicit in these aims is that treatment of the underlying diseases would not depend on the use of nonspecific high-dose myeloablative chemoradiotherapy, but rather on disease-specific targeting mediated by alloreactive immune cells, possibly in combination with other cell-directed targeting of the malignancy, such as monoclonal antibodies recognizing cell-surface antigens. Several important observations from HSC allografting relevant to this goal have emerged over the years. First, advanced hematologic malignancies are rarely cured using even the most intensive conditioning regimens, echoing observations in the 1960s by Burchenal et al. [1] that murine leukemia could be eradicated only through the use of totalbody irradiation (TBI) doses of several thousand centigray, which would be incompatible with human survival after transplant. Second, beyond rescuing patients from hematopoietic toxicity, allografts provide a graft-vs.-leukemia (GVL) effect, which has been responsible for many observed cures, particularly in patients with advanced leukemias. This was predicted from murine studies reported by Barnes et al. [2] and for which Mathé et al. [3] later coined the term “adoptive immunotherapy.” GVL effects in humans were first described by Weiden and colleagues in 1979 and 1981 [4,5]. Their observations were subsequently confirmed by others [6–10] and led to the testing of donor buffy coat infusions to augment GVL activity of marrow allografts [11] and subsequently to the use of donor lymphocyte infusions (DLI) as a treatment for patients whose malignancy had relapsed after allografting [12–16]. Complete clearing of malignant and nonmalignant host hematopoiesis through alloimmune reactions supports the concept that grafts can create their own marrow space without need for myelosuppressive conditioning therapy. Third, in a small number of patients transplanted for either thalassemia major or sickle cell disease, stable mixed chimerism has been observed and found to result in the resolution of clinical manifestations of disease [17,18]. Fourth, in transplants from major histocompatibility complex (MHC)-identical donors, both HVG and GVH reactions are mediated by T cells, a fact that stimulated studies on whether postgrafting immunosuppression designed to prevent GVHD could also be used to suppress host immunity and thereby enhance allogeneic engraftment. These observations have provided underlying principles for the development of novel, safer transplant protocols that can be carried out in an ambulatory care setting and extended to older patients or those with organ toxicities that would otherwise preclude allogeneic transplantation.

MIXED CHIMERISM IN HUMANS Prior observations Mixed chimerism was first described in leukemia patients who received cyclophosphamide-based regimens

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before allogeneic HSC transplantation [19]. In patients with aplastic anemia conditioned only with high doses of cyclophosphamide before marrow allografting from HLAidentical sibling donors, mixed donor-host chimerism was found in a substantial proportion of patients and was associated with less risk of GVHD than in patients who were complete donor chimeras [20,21]. Branch et al. [22] and Petz et al. [23] reported finding mixed chimerism in patients transplanted for hematologic malignancies using unmodified bone marrow grafts after myeloablative therapy. This finding did not predict subsequent risk of disease relapse, although others have made an association between mixed chimerism and relapse risk. Mixed chimerism has been found most frequently in patients who either received T cell–depleted marrow grafts or underwent transplants for genetic diseases and received unmodified grafts. Depletion of T cells from the graft has also been associated with an increased risk of graft rejection and risk of disease relapse with these outcomes [24,25]. A period of unstable mixed chimerism most likely precedes the adverse event. In contrast, transplantation of CD6-depleted marrow after high-dose conditioning regimens for hematologic malignancy resulted in stable mixed chimerism in ~51% of patients and was associated with protection from GVHD and relapse [26]. In other reports, stable mixed chimerism after marrow allografting has been associated with clinical benefit in patients with b-thalassemia, sickle cell disease, mucopolysaccharidosis, and immunodeficiency diseases. Observations of so-called microchimerism have been made in solid organ transplant recipients, raising the question of whether microchimerism contributes to survival of the organ graft [27–29]. Although it might be anticipated that a patient with mixed chimerism induced by marrow allografting would accept a donor solid organ without rejection, it is not yet clear whether the microchimerism observed after solid organ transplants represents an epiphenomenon as a result of patient immunosuppression or is indeed a factor in long-term graft survival. A number of studies are ongoing to determine whether the administration of donor bone marrow in conjunction with organ grafts can enhance graft survival. These objectives are supported by findings in mice and later in dogs that irradiation chimeras tolerate organ transplants from their HSC donors without the need for prolonged immunosuppression. Potential clinical applications Stable mixed hematopoietic chimerism implies a state of mutual donor–host tolerance and, thus, control of GVH and HVG reactions. Ideally, this state would be associated with normal immune responses against other antigens. Development of successful strategies for establishing stable mixed chimerism without need for continued use of immunosuppressive agents would have important therapeutic implications for a number of diseases. To be clinically useful for some diseases, such as thalassemia, a substantial donor component of hematopoiesis may be required to alleviate clinical symptoms. In some settings, such as autoimmune disease or organ transplants, it has been speculated that low levels of stable donor chimerism may be adequate to achieve the clinical goals of controlling autoreactivity and preventing graft rejection, respectively. In patients with malignancy, it is like-

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Table 1. Potential clinical applications of mixed chimerism Disease Hemoglobinopathies Genetically based immunodeficiency and enzyme deficiency Malignancy Organ transplantation HIV infection

Autoimmune diseases

Goal Correction of anemia, iron overload Provide normal lymphocyte function

Eradicate disease using adoptive immunotherapy Tolerance of organ graft without prolonged immunosuppressive therapy Restore lymphocyte numbers and function; boosts from donor as needed; eradicate viruscontaining host cells Control autoreactivity using donor immunity or subsequent adoptive immunotherapy to eradicate autoreactive host lymphocytes

ly that conversion of mixed chimerism to full donor chimerism would be necessary to treat malignancy and prevent disease recurrence. Table 1 summarizes some potential applications of mixed chimerism for the treatment of disease in humans. To be clinically useful and acceptable to patients in many of the clinical situations suggested, mixed chimerism would need to be established with minimal toxicity to the patient. Thus developing nontoxic, nonmyelosuppressive regimens that can be readily applied in an outpatient setting should be the goal of new strategies to establish mixed chimerism in humans. An alternative, the establishment of full donor chimerism with minimally toxic immunosuppressive regimens, would also represent a major advance in allografting for some diseases.

ASSESSMENT OF HEMATOPOIETIC CHIMERISM For practical purposes, mixed hematopoietic chimerism can be defined as the detection of 2.5–97.5% cells of donor origin in hematopoietic tissues, which approximately defines the sensitivity of routinely used assays for quantitating chimerism. Quantitation depends on identifying and measuring genetic markers that differ between the donor and the graft recipient. Conventional cytogenetic analysis has limited sensitivity due to the analysis of only a small number of dividing cells. Fluorescent in situ hybridization (FISH) with X and Y chromosome–specific probes evaluates all nucleated cells whether arresting or dividing and, thus, allows the study of a larger number of cells in the setting of a sex-mismatched transplant. Polymerase chain reaction (PCR)-based assays of polymorphic mini- or microsatellite markers provide a quantitation of chimerism in sex-matched and -mismatched transplant settings. The detection of microchimerism (<2.5% of donor cells) requires the use of more sensitive assays than those routinely employed to detect Y chromosome sequences in male cells transplanted into the female recipient. In transplants for malignancy, tumor-specific markers may be evaluated by sensitive techniques such as PCR to detect persistence of host cells derived from the underlying malignancy.

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Additional information may be obtained through the use of fluorescence-activated cell sorting (FACS) to isolate specific cell populations that can then be evaluated using the above techniques. In transplants to establish mixed chimerism, assays performed on T cells and other phenotypically defined cell populations may prove informative in evaluating clinical outcomes. Because T cells are the critical cell population mediating HVG and GVH reactions, monitoring T cell chimerism is an important aspect of evaluating the immunologic status of the individual. This may be especially important when admixtures of malignant cells coexist with normal cell populations as demonstrated in Figure 1.

ESTABLISHING MIXED CHIMERISM IN EXPERIMENTAL MODELS Rodent studies A number of investigators have reported establishing mixed chimerism in mice using regimens of less intensity than those conventionally used to establish allografts. In a number of these studies, in vivo T cell depletion using monoclonal antibodies has been combined with TBI at doses that, at least in mice, were sublethal. In other reports, sublethal TBI was given along with thymic irradiation and postgrafting cyclosporine or high-dose cyclophosphamide. In a study reported by Cobbold et al. [30], pretransplant treatment with monoclonal antibodies against CD4 and CD8 was combined with 600–850 cGy TBI given at 35 cGy/min. In another report, nonobese diabetic (NOD) mice were conditioned for transplant with ≥750 cGy TBI followed by infusion of large numbers of allogeneic bone marrow cells (>1.53109/kg) [31]. In another report, mice were successfully conditioned for transplant with 500 cGy TBI and 200 mg/kg cyclophosphamide given after transplantation. Other investigators combined monoclonal antibodies against CD4 and CD8 with 300 cGy TBI and 700 cGy thymic irradiation to achieve engraftment [32]. They further reported that thymic irradiation could be omitted by using injections of monoclonal antibodies against CD4 and CD8 after transplant [33,34]. Subsequently, mixed chimerism was achieved through pretreatment with the same monoclonal antibodies, thymic irradiation, and the injection of large doses of marrow cells (8.73109/kg) [35]. In another study, sustained mixed chimerism for up to 16 weeks posttransplant was achieved after recipients were given monoclonal antibodies to CD4 and CD8 combined with 300 cGy TBI administered at 100 cGy/min before transplant, followed by monoclonal antibodies against natural killer cells for 16 weeks posttransplant [36]. It has been reported that a novel CD3+, CD8+, T cell receptor (TCR) ab–, TCR gd–, CD8+ lymphocyte population can enhance allogeneic engraftment after less than optimal immunosuppression [37]. Seledtsov et al. [38] used 200 mg/kg cyclophosphamide to condition mice with p815 leukemia to achieve mixed chimerism and prolonged recipient survival compared with syngeneic controls. Koch and Korngold [39] established sustained chimerism in MHC-mismatched murine allograft recipients after pretreatment with a synthetic-based CD4CDR3 peptide analog combined with TBI. Mixed chimerism has also been reported in mice given allogeneic marrow,

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Figure 1. Fractionation of peripheral blood for chimerism analysis by cell sorting in a patient with chronic lymphocytic leukemia A. Flow cytometry profiles of peripheral blood cell populations isolated for chimerism analysis by FISH to detect X and Y chromosomes. R1 is the mononuclear cell gate from which cells expressing CD3 (R2 ) or CD19 (R3 ) were isolated by cell sorting. B. Percent of donor cells in peripheral blood. Whereas unfractionated PBMC are 15% donor, T cells and granulocytes are 98 and 99% donor, respectively. Although mixed chimerism resulted from a large admixture of host B cells, the development of GVHD at this time was consistent with high-level donor engraftment in the T cell and granulocyte compartments.

FK506, and treatment with granulocyte-macrophage colonystimulating factor (GM-CSF) or flt3-ligand [40]. In general, it is difficult to translate the rodent findings directly to humans. Engraftment across MHC barriers in mice has not been predictive for humans, appears to be more readily achieved, and is associated with less GVHD than observed in the human setting. Most of the regimens used in the rodents are too intensive for the goal of establishing mixed chimerism in humans with minimally toxic therapy.

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Primate studies There are several reports of attempts to establish mixed chimerism in conjunction with organ grafting across MHC barriers in primates. In one study, cynomolgus monkeys were pretreated with antithymocyte globulin (ATG), 300 cGy TBI, and 700 cGy thymic irradiation with postgrafting cyclosporine for 4 weeks [41]. Marrow chimerism persisted for 18–51 days posttransplant with maximum levels ranging from 1.5 to 57.8%. Of note, kidney grafts persisted for extended periods of time without immunosuppression

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despite the fact that the marrow grafts had been rejected. In subsequent studies, a TBI dose of 250 cGy was less successful and the addition of deoxyspergualin to TBI was not effective [42]. In a study by Thomas et al. [43], the use of peritransplant anti-CD3 immunotoxin combined with bone marrow depleted of mature antigen-presenting cells was associated with persistence of mixed chimerism in two of six rhesus monkeys studied and with microchimerism in two of three additional animals for periods ranging between 6 and 16 weeks posttransplant. This was associated with survival of kidney grafts for periods between 120 days and >1.5 years. Canine studies A significant proportion of this review focuses on canine preclinical studies because of important recent observations that are relevant to current human clinical studies. Distinct advantages of the dog model over other large animal models are the availability of MHC typing for determination of MHC relationships between potential littermate donors and the ready availability of dog leukocyte antigen (DLA)-identical littermates as a model for HLA-identical transplants in humans. The following section describes transplants between healthy, randomly bred dogs using DLA-identical marrow grafts. The focus was on establishing engraftment across minor histocompatibility barriers through immunosuppression specifically targeting host and donor T cells. TBI has been an integral part of these studies, and it has an important role in the development of nonmyeloablative transplant approaches because of important immunosuppressive properties. Marrow toxicity of TBI [44]. TBI delivered in a single fraction at 7 cGy/min in dogs subsequently given intensive supportive care but no HSC grafts causes lethal marrow failure at ≥400 cGy. After 300 cGy, approximately two-thirds of dogs die; after 200 cGy, endogenous marrow recovery usually occurs (only one of 19 animals died); and after 100 cGy, all 12 dogs studied survived. Thus a dose of 200 cGy should pose little risk of irreversible marrow failure should graft rejection occur. Immunosuppressive properties of TBI. Results of unmodified marrow grafting given without postgrafting immunosuppression after conditioning therapy with various doses of TBI are shown in Table 2 [45,46]. A dose of 920 cGy was sufficiently immunosuppressive to allow stable engraftment in 95% of dogs transplanted. With reduced doses of TBI, the frequency of sustained engraftment dropped. At 450 cGy, only 41% of dogs had stable engraftment as mixed or full donor chimeras, whereas most dogs rejected their grafts and either died with marrow aplasia (23%) or survived with eventual autologous recovery (36%). Transient hematopoietic support provided by the allograft accounts for autologous recovery seen in some dogs that received otherwise lethal TBI. Engraftment is facilitated by postgrafting immunosuppressive drug therapy [47]. In these experiments, immunosuppressive agents were discontinued by 35 days after transplant. At 450 cGy TBI, cyclosporine and prednisone, two drugs commonly used in GVHD prevention, were tested for enhancing engraftment. Cyclosporine was given orally at 15 mg/kg b.i.d. starting on day –1 and continued through day 35. Prednisone was given at 12.5 mg/kg orally b.i.d. on days –5

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Table 2. Effect of TBI dose and postgrafting immunosuppression on engraftment of DLA-identical marrow grafts TBI dose (cGy)

Postgrafting immunosuppression

Dogs with stable engraftment*/Total transplants

920 450 450 450 200 200 200 100

None None Cyclosporine* Prednisone† Cyclosporine* Methotrexate‡/cyclosporine* MMF§/cyclosporine* MMF§/cyclosporine*

20/21 6/17 7/7 0/5 0/4 2/5 11/12 0/6

*Mixed or full donor chimerism. *Cyclosporine 15 mg/kg p.o. b.i.d. days –1 to 35. † Prednisone 12.5 mg/kg p.o. days –5 to –3 with tapering to day 32. ‡ Methotrexate 0.4 mg/kg i.v. days 1,3, 6, and 11. § Mycophenolate mofetil 10 mg/kg b.i.d. s.c. days 0–27.

to 3 with tapering through day 32. The prednisone regimen was designed based on studies that used high-dose chemotherapy as part of conditioning for second marrow grafts [48,49]. All seven cyclosporine recipients had stable engraftment without GVHD, significantly better than six of 17 dogs that did not receive cyclosporine (p = 0.01) and consistent with a hypothesis that postgrafting immunosuppression could control HVG reactions. High-dose prednisone did not enhance engraftment: none of the five transplanted dogs had sustained allogeneic engraftment. After 200 cGy TBI, cyclosporine alone failed to establish stable grafts (Table 2), and four dogs had endogenous hematopoietic recovery after early graft rejection. Cyclosporine was then combined with methotrexate, a drug shown to be synergistic with cyclosporine in preventing GVHD in dogs and humans. Two of five evaluable dogs became stable mixed chimeras, whereas three rejected their grafts. Mycophenolate mofetil (MMF), an antimetabolite that blocks de novo purine synthesis, was then tested in combination with cyclosporine. In studies of GVHD prevention, a combination of MMF and cyclosporine appeared synergistic and was superior to methotrexate/cyclosporine [50]. With the use of MMF/cyclosporine after 200 cGy TBI, 10 of 11 dogs became stable mixed chimeras for up to 130 weeks posttransplant (Table 2). GVHD was not observed in these dogs. Thus, dual-agent postgrafting immunosuppression with MMF/cyclosporine allowed a reduction of 720 cGy in the TBI dose needed to achieve sustained allogeneic engraftment. The contribution of donor cells to hematopoiesis ranged from 45 to 85%, and donor chimerism was usually higher in myeloid cells than in lymphocytes or lymph nodes. Based on peripheral blood CD4/CD8 ratios, the humoral responses to sheep red blood cells, and in vitro studies of lymphocyte responsiveness to concanavalin A and to alloantigens, immune function of the stable mixed chimeras appeared to be in the normal range. Skin grafts from the marrow donors were accepted but third-party skin grafts were rejected, indicating specific tol-

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Figure 2. Establishment of stable mixed chimerism after nonmyeloablative conditioning A. Platelet and neutrophil counts after 200 cGy TBI, a marrow graft from a DLA-identical sibling on day 0, and postgrafting MMF/cyclosporine to 5 weeks posttransplant. B. Microsatellite marker analysis by PCR showing pretransplant samples (left) and recipient cells after transplantation (right). DSB, donor specific band; RSB, recipient specific band; PBWC, unfractionated peripheral blood white cells.

erance to donor cells. Figure 2 shows the peripheral blood counts and microsatellite marker studies from one of these dogs to illustrate the transplant procedure. Persistence of donor- and host-specific bands in marrow, peripheral blood, and lymph node cells was evidence of stable mixed chimerism after transplant. After 100 cGy TBI, MMF/cyclosporine did not allow for the establishment of stable mixed chimerism [47]. However, when granulocyte colony-stimulating factor (G-CSF)mobilized peripheral blood stem cells (PBSC) were used, stable mixed chimerism was observed in two of five dogs, and in the others that rejected, longer graft survival was

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observed than in dogs that rejected bone marrow transplants [51]. Studies in other transplant settings have demonstrated an engraftment-enhancing property of unmodified PBSC [52], and along with these results, suggest that PBSC facilitate engraftment with less immunosuppression than bone marrow grafts. Mechanism of low-dose TBI: Host immunosuppression or creating marrow space? An established concept has been that regimens administered before transplant are necessary to create marrow space in which the incoming graft can settle. To address the role of TBI in establishing mixed chimerism, six dogs were given marrow grafts and postgrafting

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MMF/cyclosporine after 450 cGy irradiation (at 200 cGy/min) given to cervical, thoracic, and upper abdominal lymph nodes while at the same time shielding marrow-containing bones with lead blocks [53]. Each dog showed evidence of initial mixed chimerism. Whereas myelosuppression was minimal, with granulocyte nadirs of 3500/µL and platelet nadirs >100,000/µL, profound lymphocytopenia ensued, with nadirs ~200/µL and slow recovery. Two dogs rejected their grafts by weeks 8 and 18, respectively; one dog died with allogeneic engraftment from GVHD; and three remained stable mixed chimeras with follow-up of 38–90 weeks. Marrow samples from unirradiated bones showed mixed chimerism from as early as 4 weeks posttransplant and persisting for the period of observation. These results support the hypothesis that marrow grafts can create their own marrow space, most likely through a mechanism of subclinical GVH reactions, and suggest that engraftment can be achieved across minor histocompatibility barriers, specifically through suppression of host T cells. Thus, TBI may be replaced by the use of other immunosuppressive agents such as monoclonal antibodies to CD3 or TCR, and using molecules to target costimulation reactions,such as CTLA4, CD154 (CD40 ligand). Mixed chimerism in a model of severe hereditary hemolytic anemia. Severe canine hereditary hemolytic anemia due to pyruvate kinase (PK) deficiency can be considered a model for genetically determined hemolytic anemia in humans. HSC transplants from healthy littermates after high-dose conditioning corrected hemolytic anemia and led to resolution of preexisting severe hepatic iron overload [54], findings that encouraged HSC transplantation as a treatment for b-thalassemia. Using the canine PK deficiency model, studies were initiated to test the hypothesis that establishing mixed chimerism can correct the phenotypic manifestations of a genetic disease. Support for the hypothesis has come from an initial transplant of a 5-year-old Basenji dog with PK deficiency. Immediately pretransplant, the dog had a hematocrit of 24% with a reticulocyte count of 30%. Pretransplant biopsies of liver and marrow showed extensive hemosiderosis and fibrosis along with extramedullary hepatic hematopoiesis. After 200 cGy TBI before and MMF/cyclosporine after marrow transplant from a healthy DLA-identical littermate, mixed chimerism was established without GVHD and resulted in improvement of the hematocrit to 44% and the reticulocyte count to 1.2%. Engraftment was evident at 3 weeks posttransplant, and mixed chimerism stabilized, with blood and marrow showing 80% donor cells. Longer follow-up will determine whether preexisting hemosiderosis and extramedullary hematopoiesis resolve in the setting of mixed chimerism.

THERAPEUTIC APPLICATION OF MIXED CHIMERISM Conceptual basis To date, only a few reports document a specific goal of establishing stable mixed hematopoietic chimerism in patients with hematologic diseases. The safety and efficacy of the canine preclinical studies helped develop a hypothetical conceptual schema for studies pertaining to the clinical application of mixed chimerism (Fig. 3). Because the use of cytotoxic agents does not appear to be necessary to create marrow

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space, in patients with profound T cell deficiencies a conditioning regimen may not be required before HSC transplant, and postgrafting immunosuppression may suffice. Immunocompetent patients would require treatment with an immunosuppressive, but not myelosuppressive, regimen before transplant. After transplantation all patients would receive immunosuppression, for instance MMF/cyclosporine, to prevent both rejection and GVHD. In the absence of myeloablation, host hematopoietic cells would survive in various proportions along with the engrafted donor cells, resulting in stable mixed chimerism. An adequate percentage of donor cells in the mixture, such as 20–30%, would be expected to correct phenotypic manifestations of most genetic hematologic diseases as suggested by observations of mixed chimerism in allograft recipients with thalassemia major [17] and sickle cell disease [18]. If the clinical response of the nonmalignant disease to mixed chimerism was inadequate, complete chimerism could be achieved through DLI (Fig. 3). The successful use of DLI has already been described in a patient with b-thalassemia [55]. For the treatment of malignant diseases, mixed chimerism per se would not be expected to be effective in preventing relapse. Thus, DLI would be required after initial engraftment to convert mixed to complete donor chimerism and to treat any persisting host malignancy. In some cases, the establishment of all-donor hematopoiesis may occur with the initial transplant, thus obviating the need for DLI. Some degree of effectiveness of DLI in eradicating recurrent malignancy after conventional transplantation has been demonstrated for most hematologic malignancies, but DLI is most potent against chronic myeloid leukemia. For application to treatment of solid tumors, innovative approaches to targeting the malignancy will be required. Strategies to develop more effective, safer approaches to DLI are being tested. These include the insertion of a suicide gene into the lymphocytes before infusion and the use of lymphocytes that target either tumorspecific antigens or minor histocompatibility antigens expressed on tissues from which the tumor is derived. Initial clinical experiences Postgrafting MMF/cyclosporine as conditioning for patients with severe T cell immunodeficiency. The hypothesis that patients with severe T cell immunodeficiencies would not require pretransplant conditioning is supported by recent observations [56]. A 30-year-old male with a common variable immunodeficiency, a history of recurrent serious infections, and active Mycobacterium avium infection received an HSC transplant from his HLA-identical sibling without pretransplant TBI, but with postgrafting MMF/cyclosporine. At 3 months posttransplant, donor cells comprised 50% of bone marrow cells, 90% of peripheral blood granulocytes, and ~95% of monocytes. The T cell count was >1000/µL, with 30% donor cells. Acute GVHD grade II developed and was effectively treated with steroids and cyclosporine. Despite this, infectious complications resolved, including bone marrow granulomas secondary to mycobacterial infection. Similar observations were made in a second patient with a T cell immunodeficiency syndrome conditioned in the same way. Low-dose TBI and MMF/cyclosporine as conditioning for patients with hematologic malignancies. Based on results of the

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Figure 3. Conceptual approach to clinical application of mixed chimerism Microsatellite markers represent chimerism analysis of hematopoietic cell DNA using PCR and gel electrophoresis to identify donor and recipient specific bands.

canine preclinical studies reviewed earlier, a clinical trial was initiated to evaluate whether mixed chimerism can be established using the approach developed in the dogs and used as an immunologic platform for subsequent DLI to treat underlying hematologic malignancy [57]. Patients were conditioned with 200 cGy TBI followed by PBSC allografting from HLA-identical siblings and then postgrafting immunosuppression with MMF/cyclosporine for 28 and 35 days, respectively. The study targeted patients aged 50–75 years old, a population for which allografting is used sparingly due to the increased toxicities seen with increasing age. The goal was to develop a nontoxic protocol that would allow allografting to be performed as an outpatient procedure. Initial experience has confirmed the feasibility and safety of this approach. Patients have experienced minimal nausea, vomiting, diarrhea, and no alopecia, side effects almost invariably associated with high-dose conditioning regimens, and patients up to the age of 72 years have been transplanted in an outpatient setting. Myelosuppression was mild, particularly in patients with relatively normal counts pretransplant, and in several cases no transfusion support was required. Of the initial 26 patients studied, each has shown evidence of donor engraftment with mixed chimerism sustained up to at least 2 months posttransplant. Three patients rejected their grafts between 60 and 90 days posttransplant and recovered

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endogenous hematopoiesis. The first patient treated, who had chronic lymphocytic leukemia, showed complete clearing of leukemia and became PCR-negative for the tumorspecific immunoglobulin gene rearrangement as determined by assays at 7 and 10 months posttransplant. Although fol-

Table 3. Reduced-intensity nonmyeloablative conditioning regimens used in treatment of malignancy Regimen

Reference

1. Fludarabine 30 mg/m 3 4, cytosine arabinoside 2 g/m 3 4, idarubicin 12 mg/m2 3 3 2. Fludarabine 30 mg/m2 3 4, melphalan 140 mg/m2 3 1 3. 2-Chlorodeoxyadenosine 12 mg/m2/d 3 5, cytosine arabinoside 1 g/m2 3 5 4. Fludarabine 30 mg/m2 3 3–5, cyclophosphamide 300 mg/m2 to 1000 mg/m2 3 1–2 5. Fludarabine 30 mg/m2 3 2, cytosine arabinoside 500 mg/m2 3 2, cisplatin 25 mg/m2 3 4 6. Fludarabine 30 mg/m2 3 6, busulfan 4 mg/kg/d 3 2, antithymocyte globulin 10 mg/kg/d 3 4 7. Cyclophosphamide 50 mg/kg 3 4, ATG 3 3 or 4 pre- and posttransplant, ( thymic irradiation 7 Gy 3 1 8. Fludarabine 25 mg/m2 3 4, cyclophosphamide 60 mg/kg 3 2 2

2

[58] [58] [58] [59] [59] [60] [62] [61]

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low-up is relatively short and conclusions cannot be drawn as to overall therapeutic effectiveness, the ability to establish allografts and GVL reactions has been documented. Reduced dose conditioning regimens for allografting in patients with malignancy. Several groups of investigators have reported a more conventional approach to reducing regimen-related toxicities. The approach involves the use of regimens of lower intensity than typical high-dose conditioning regimens aimed at suppressing both host malignancy and HVG reactions. The regimens have been of varying intensity (Table 3) and have involved either dose reduction of conventional transplant agents or the use of modified conventional chemotherapy regimens for specific diseases. PBSC grafts were used in most of these transplants. Investigators speculated that because of the reduced intensity of conditioning regimens, mixed chimerism might result, and the regimen could be followed as necessary by DLI. Many of the patients treated with these approaches have been heavily pretreated, a factor that may contribute to successful engraftment but may also reduce the likelihood of stable mixed chimerism developing through suppressing recipient immunity. Investigators from the M.D. Anderson Cancer Center in Houston have based their conditioning programs on the use of purine analogs, fludarabine or 2-chlorodeoxyadenosine [58,59]. A multiplicity of regimens was used to provide effective chemotherapy of underlying malignancy and to facilitate donor cell engraftment. For example, fludarabine/idarubicin/cytosine arabinoside was used in patients with myeloid malignancies, whereas fludarabine/ cytosine arabinoside/platinum or fludarabine/cyclophosphamide was used in patients with lymphoid malignancies. Of 15 patients with myeloid malignancies (median age 59 years), eight had donor cell engraftment documented in the bone marrow within 30 days of transplant. Although one patient achieved a sustained complete response that appeared to be attributable to a GVH reaction, overall survival in this group was poor, no doubt in part because of the advanced state of the patients’ underlying malignancies. More encouraging results were reported in patients with lymphoid malignancies, suggesting the responsiveness of low-grade non-Hodgkins lymphoma and chronic lymphocytic leukemia to this approach. Slavin et al. [60] performed PBSC allografts in 26 patients using a regimen of fludarabine 30 mg/m 2 3 6, busulfan 8 mg/kg, and ATG 40 mg/kg. This study included not only patients with malignancies, but also three patients with b-thalassemia, Blackfan-Diamond anemia, and Gaucher disease. Compared with the Houston studies, patients had less advanced malignancy and were younger (median age 31 years), and overall survival in the study was very good (event-free survival 77.5% at 9 months). Nine of 26 patients developed transient mixed chimerism and went on to become full donor chimeras. Four patients died of GVHD, however, which appeared due to the use of inadequate GVHD prophylaxis in patients who had otherwise good-risk hematologic disease, and clinical evidence of veno-occlusive disease of the liver developed in 50% of the patients. Others have reported using modifications of a fludarabine/cyclophosphamide combination [61] or high-dose cyclophosphamide plus ATG with or without thymic irradiation [62]. In those studies, the regimens were designed to

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achieve both antitumor effects and engraftment leading to a GVL effect. Severe GVHD was encountered in a significant number of patients. Of note, engraftment was achieved in HLA-mismatched transplants, resulting in mixed chimerism and GVL effects [62]. Despite the use of nonmyeloablative conditioning, many of these regimens are still quite toxic and some involve using high-dose chemotherapy (e.g., 200 mg/kg cyclophosphamide). The busulfan regimen caused significant toxicities, including veno-occlusive disease in 13 of 26 patients treated [60]. Although many of the patients were pretreated, the busulfan-based regimen did allow engraftment in seven patients with CML and three patients with nonmalignant disorders who presumably had not received prior therapy that would have significantly diminished HVG reactions. This, in turn, suggests that substantial dose reductions in conventional conditioning regimens are possible without exposing patients to a major risk of graft rejection when using HLA-identical donors and PBSC as a source of stem cells. With the exception of the fludarabine and low-dose cytoxan regimen, these regimens appear too toxic to be used routinely in an outpatient setting.

CONCLUSIONS AND FUTURE DIRECTIONS Preclinical studies and results of initial preclinical studies indicate that for many clinical circumstances pertaining to the use of stem cell transplantation, currently used intensive and toxic conditioning regimens may ultimately be replaced by nonmyeloablative immunosuppression because allogeneic stem cell grafts create their own space in the host’s bone marrow. Immunosuppression delivered before transplant may affect exclusively host cells, while immunosuppression delivered after transplant can be used with the intent of establishing mutual graft–host tolerance that would be manifest as stable mixed chimerism. Observations that mixed chimerism can be protective against GVHD suggest that the establishment of mixed chimerism with low-dose conditioning should be a priority in the use of allografting in nonmalignant diseases compared with the induction of full donor chimerism. In contrast, for patients with malignancy, the goal is to establish full allogeneic donor chimerism through either the initial PBSC graft or subsequent DLI. The latter is attractive for the treatment of less aggressive diseases, such as chronic myeloid and chronic lymphocytic leukemias, in which establishing allografts without GVHD in an outpatient setting would be a desirable initial step. This would ultimately allow for development of DLI strategies that are less toxic and more tumor specific. An improved toxicity profile and lower cost of allografting would be critical to developing new strategies that target solid tumors. The application of this approach to aggressive, rapidly growing malignancies is somewhat problematic. Because GVL responses may occur slowly, strategies to augment rapidly developing and effective antitumor responses that do not result in overwhelming GVHD are needed. While intensive chemotherapy has been used for this purpose to date, disease targeting with monoclonal antibodies may have an important future role in such circumstances. Preclinical canine studies demonstrated the utility of using low-dose TBI for establishing mixed chimerism and,

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in a disease model of PK deficiency, that the correction of severe hemolytic anemia can be achieved through the establishment of mixed chimerism. This may have major implications for the treatment of diseases such as sickle cell anemia and b-thalassemia. Because even the use of low doses of irradiation may predispose to malignancy, the development of newer, less toxic approaches to establishing donor-recipient tolerance are of continued importance. These may include the use of T cell–specific monoclonal antibodies and blockade of T cell costimulatory pathways such as a CD40CD154 and B7-CTLA4/CD28, which may further lead to specific donor-host tolerance without impairing broader T cell reactivity. Finally, initial promising results with attenuated conditioning regimens have been achieved, primarily in transplants with HLA-identical siblings. Achieving engraftment with nontoxic regimens using related MHC-mismatched or unrelated MHC-matched donors may require considerably more potent immunosuppression, both to prevent rejection and to control GVHD.

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