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Depletion of alloantigen-primed lymphocytes overcomes resistance to allogeneic bone marrow in mildly conditioned recipients
Blood Cells, Molecules, and Diseases 33 (2004) 238 – 247 www.elsevier.com/locate/ybcmd
Depletion of alloantigen-primed lymphocytes overcomes resistance to $ allogeneic bone marrow in mildly conditioned recipients Tatyana B. Prigozhina, Gregory Elkin, Olga Gurevitch, Shoshana Morecki, Elena Yakovlev, Sofia Khitrin, Shimon Slavin* Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah University Hospital, Jerusalem, Israel Submitted 28 July 2004 Available online 7 October 2004 (Communicated by M. Lichtman, M.D., 3 August 2004)
Abstract Objective: Successful implantation of allogeneic bone marrow (BM) cells after nonmyeloablative conditioning would allow to compensate for the inadequate supply of compatible grafts and to reduce mortality of graft-vs.-host disease (GVHD). Recently, we proposed to facilitate engraftment of mismatched BM by conditioning for alloantigen-primed lymphocyte depletion (APLD) with cyclophosphamide (CY). Here we summarize the experimental results obtained by this approach. Materials and methods: Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY (day 1) to deplete alloantigen-primed lymphocytes, and given a second C57BL/6 BM transplant (day 2) for engraftment. Recipients were repeatedly tested for chimerism in the blood and followed for GVHD and survival. The protocol was also tested for inducing tolerance to donor tissue and organ allografts, and for treatment of leukemia, breast cancer, and autoimmune diabetes in NOD mice. Results: APLD by 200 mg/kg CY provided engraftment of allogeneic BM from the same donor in 100% mildly irradiated recipients. Eighty percent chimeras remained GVHD-free more 200 days. All chimeras accepted permanently donor skin grafts and donor hematopoietic stromal progenitors. Allogeneic BM transplantation (BMT) after APLD had a strong therapeutic potential in BALB/c mice harboring malignant cells and in autoimmune NOD recipients. Tolerance-inducing CY dose could be reduced to 100 mg/kg. Conditioning for APLD resulted in engraftment of allogeneic BM after a significantly lower radiation dose than treatment with radiation and CY alone. Conclusion: Our results demonstrate that conditioning for APLD has a definite advantage over general immunosuppression with CY and radiation therapy. D 2004 Elsevier Inc. All rights reserved. Keywords: Transplantation tolerance; Bone marrow transplantation; Reduced-intensity conditioning; Cyclophosphamide; APLD (alloantigen-primed lymphocytes depletion)
Introduction Allogeneic bone marrow (BM) or mobilized stem cell transplantation is the treatment of choice for patients with
$ This paper is based upon a presentation at a Focused Workshop on Haploidentical Stem Cell Transplantation sponsored by The Leukemia and Lymphoma Society held in Naples, Italy from July 8 to 10, 2004. * Corresponding author. Department of Bone Marrow Transplantation, Hadassah University Hospital, PO Box 12000, 91120, Jerusalem, Israel. Fax: +972 2 642 2731. E-mail address: [email protected] (S. Slavin).
1079-9796/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2004.08.010
hematological malignancies and certain hereditary diseases [1,2]. BM chimeras can also permanently accept various organ transplants taken from the BM donor with no need for maintenance immunosuppressive therapy [3]. However, broad application of BM transplantation (BMT) in clinical practice is limited by graft failure and severe graft-vs.-host disease (GVHD) after transplantation. The data obtained in clinical practice and experiments in mice show, however, that reducing the intensity of immunosuppressive conditioning prior to BMT improves the safety of the procedure [4–10]. The progress in introducing reduced-intensity regimens into clinical prac-
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tice came with understanding that the generation of graftversus-leukemia (GVL) effects by transplanted BM does not require myeloablation of recipients, but does require persistent donor BM cell (BMC) engraftment. Substitution of myeloablative preconditioning by reduced-intensity regimens with fludarabine, busulfan, or cyclophosphamide (CY), with or without antibodies against T cells [6–10], diminished acute transplantation-related toxicity and mortality, while providing rapid and stable engraftment of HLA-compatible BM cells (BMCs). Unfortunately, rejection of haploidentically mismatched BM and organ transplants is difficult to overcome by reduced-intensity conditioning protocols available today. Lately, we proposed improving engraftment of incompatible BM grafts in reduced-intensity transplantation protocols by conditioning aimed to diminish the clone size of responding alloreactive T cells. This conditioning includes priming of host by inoculation of donor lymphocytes and subsequent depletion of activated donor-reactive host cells by CY, a drug that is predominantly toxic to proliferating cells [11]. The method was previously used by us and others to facilitate donor organ and skin transplant engraftment in the absence of BMT [12–15]. Application of this approach facilitated acceptance of donor organs and skin differing from recipient in minor histocompatibility antigens. Tolerance to fully allogeneic heart grafts could not be achieved unless recipients were thymectomized. Mice tolerant to allogeneic heart grafts nevertheless rejected the more immunogenic skin transplants derived from the same donor [12]. We concluded that the main failure of allogeneic organ and skin engraftment after CY-induced alloantigen-primed lymphocyte depletion (APLD) was due to minimal, if any, donor cell chimerism in recipients of organ grafts. Administration of CY after donor cell priming depleted not only activated donor-reactive host cells, but also donor lymphocytes used for priming because most of transplanted donor cells also proliferated in recipient after BMT [16]. Thus, to convert recipients to stable chimeras, we proposed performing a second BMT from the same donor following the disappearance of the cytotoxic CY metabolites. The results obtained by the application of our protocol for induction of donor-specific transplantation tolerance to allogeneic BMC and organ grafts are summarized in this report.
Materials and methods Animals BALB/c (H-2d), C57BL/6 (H-2b), and (C57BL/6 BALB/c) F1 (H-2b/d) hybrid mice (F1), CBA (H-2k), and NOD (H2-KdDb) were purchased from Harlan Hebrew University-Hadassah Medical School’s Animal Facility in Jerusalem, Israel. Two-month-old mice that were kept under
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standard conditions, with food and water ad libitum, were used in the study. Radiation Fractionated total lymphoid irradiation (TLI, 200 cGy/ fraction) was used for conditioning of mice in the initial experiments. Mice were anesthetized for proper positioning in an apparatus that exposes the major lymph nodes, thymus, and spleen to ionizing irradiation, while shielding most of the skull, ribs, lungs, hind limbs, and tail with lead, as previously described [4]. Total body irradiation (TBI) was given to mice as a single dose from a 6-MEV linear accelerator at a dose rate 1.9 Gy/min. The exact TBI doses given to recipients are presented in Results. Alloantigen-primed lymphocyte depletion (APLD) as conditioning to induce tolerance to allografts Naive BALB/c mice and mice exposed to a low-dose radiation were treated for APLD by an intravenous (IV) injection of 3 107 donor C57BL/6 BMC and by intraperitoneal (IP) injection of 200 mg/kg CY (Baxter, Frankfurt, Germany) on the next day. When specified, the CY dose was reduced to 100 or 60 mg/kg. A day after CYinduced APLD mice were transplanted intravenously with a second inoculum of donor BMC (3 107) for engraftment of donor hematopoietic cells. Assay for chimerism Mice were anesthetized and blood was taken from the retro-orbital sinus of the eye. WBC (2–8 105/sample) were separated, directly stained with anti-H-2Kb-FITC (IgG2a) or anti-H-2Kd-FITC (IgG2a) monoclonal antibodies (mAb) (Serotec, USA), and analyzed by FACS analysis (FACStar plus, Becton Dickinson, San Jose, CA, USA). Background binding of each H-2K-specific mAb was determined by staining with it the cells of nonrelevant haplotype. Skin grafting A full-thickness donor skin graft was transplanted to recipients on the day of priming with BMC or 20 days after completing the conditioning [16]. The graft was considered accepted when hair of donor color grew on the soft, flexible underlying skin, and rejected when donor hair and epithelium were lost. GVHD monitoring Clinical signs, for example, weight loss, diarrhea, and hunched back, were used to evaluate GVHD in chimeras. Animals were observed daily for survival. Postmortem
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analysis of spleens and lymph nodes showed that they were reduced in size. Treatment of malignant diseases BALB mice were inoculated intravenously with 106 spleen cells from mice with overt BCL1 leukemia [17] or transplanted ID into the right flank with 2 104 4T1 breast carcinoma cells [18] on day 0, exposed to 200 cGy TLI on day 1, treated for APLD by inoculation of 3 107 C57BL/6 BMC on day 2, and by injection of 200 mg/kg CY on day 3. One day after treatment with CY (day 4), recipients were given a second IV inoculum of donor BMC (3 107 cells/mouse). Treatment of autoimmune diabetes in NOD mice To induce consistent autoimmunity, diabetes-free female NOD mice were given 650 cGy TBI and 2 h later inoculated intravenously with 25 106 spleen cells obtained from overtly diabetic NOD [19]. Conditioning for APLD was started 3 days later. It lasted 2 days (see the previous section) and was followed by transplantation of BMC from C57BL/6 donors. Mixed lymphocyte reaction (MLR) T cells, enriched by passing spleen cells through a nylon wool column, were stimulated by equal numbers of stimulator cells (irradiated by 3000 cGy), cultured as described [16], pulsed on the third day with 1 mCi [3H]-thymidine, harvested on the fourth day, and thymidine incorporation was measured. Statistical analysis of the data The results were analyzed according to Student’s t test. Donor cell chimerism was presented as mean F SE.
Results Scaling down the radiation dose and transplantation of non-T-cell-depleted donor BM to induce GVHD-free chimeras tolerant to donor skin grafts Our initial experiments revealed that conditioning of BALB/c mice with six fractions of TLI (200 cGy each) followed by selective depletion of alloantigen-primed lymphocytes (3 107 C57BL/6 BMC IV and on the next day 200 mg/kg CY IP) provided engraftment of allogeneic BMC from the same donor, but required transplantation with T-cell-depleted BM to prevent GVHD [16]. In order to decrease the incidence of GVHD after infusion of allogeneic non-T-cell-depleted BM (3 107 cells/mouse), conditioning for APLD was given to nonirradiated
recipients and to mice irradiated with one TLI fraction (200 cGy) instead of six TLI fractions [20]. Fig. 1 shows that BMT after reduced-intensity conditioning converted to chimeras all mice treated with a single TLI dose before APLD and 84% recipients treated for APLD alone. About 80% of these animals remained GVHD-free N200 days though they were transplanted with unmodified allogeneic BMC. In contrast, 90% of chimeras treated for APLD after conditioning with six TLI fractions died of GVHD after BMT. Specificity of transplantation tolerance to donor skin allografts A number of BALB mice tolerant to H-2b transplantation antigens were implanted with a skin graft obtained from a third party CBA (H-2k) donor 50–150 days after successful engrafting of C57BL/6 (H-2b) skin. All animals rejected the CBA skin grafts within 19 F 3 days while keeping the original C57BL/6 skin allografts intact (n = 10), indicating that recipients selectively tolerant to MHC antigens of the initial donor were capable of generating an immune response to unrelated transplantation antigens. Lymphocytes from full donor-type chimeras that maintained donor skin grafts did not respond to self-stimulators or to stimulators of donor or recipient type (Fig. 2). However, they retained significant in vitro reactivity to allogeneic stimulators obtained from third party CBA mice (Fig. 2). Engraftment of allogeneic hematopoietic stroma microenvironment (HSM) in recipients treated with fractionated TLI and conditioned for APLD Donor cells infused in the course of BMT seed in the host hematopoietic stromal microenvironment (HSM) and a direct interaction of host HSM with the donor stem cells is essential for differentiation and maturation of hematopoietic progenitor cells. In diseases associated with primary or secondary defects of HSM, namely, certain forms of aplastic anemia or myelofibrosis, and disorders resulting from intensive ionizing radiation or chemotherapy, graft failure, distinguishable from graft rejection, can occur. In such cases long-term hematopoietic engraftment of transplanted BM may ultimately require transplantation of donor HSM. Thus, we investigated the relationship between the intensity of the conditioning of recipients and the induction of tolerance to allogeneic HSM [21]. Fig. 3 shows that irradiating recipients with three instead of six TLI fractions (200 cGy each) and inoculation of donor BMC and CY prior to BM plug implantation provided consistent acceptance of stromal progenitors and formation of ectopic bone under the kidney capsule. Ectopic ossicles were maintained for the entire life of the animal. Transplantation of donor femoral plugs to recipients irradiated only once or not at all
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Fig. 1. Conditioning for alloantigen-primed lymphocyte depletion (APLD) by CY after low-dose TLI provides GVHD-free engraftment of non-T-cell-depleted donor BM and induces long-lasting tolerance to donor skin grafts. Nonirradiated BALB/c mice and BALB/c recipients given one or six daily TLI fractions (200 cGy each) were treated for alloantigen-primed lymphocyte depletion by intravenous priming with C57BL/6 BMC (3 107/mouse) and by IP inoculation of 200 mg/kg CY a day later. One day after CY administration, mice were transplanted intravenously with a second dose of C57BL/6 BMC (3 107/mouse). Percentage of mice with chimerism (N80% donor cells in the blood) was determined by FACS analysis 120 days after BMT. Skin grafting was performed 20 days after BMT. GVHDfree survival of recipients and maintenance of donor B6 skin allografts by survivors were followed for 200 days after BMT. Each group included 15 mice or more.
prior to APLD resulted in the development of ectopic bones in 45% of experimental animals. In mice conditioned only with TLI and CY, ectopic ossicles never developed. Acceptance of HSM in nonirradiated mice improved substantially when the BM plug was transplanted simultaneously with the intravenous infusion of donor BM cells. Intravenous priming of donor-reactive host cells by donor
Fig. 2. MLR response of T cells from BALB/c mice that either accepted or rejected B6 skin allografts. Spleen T cells of eight nonirradiated BALB/c chimeras bearing C57BL/6 skin transplant N100 days and of seven BALB/c recipients that rejected C57BL/6 BM skin transplants after conditioning with 200 mg/kg CY were tested in MLR.
BM could not be omitted or replaced by local plug transplantation.
Fig. 3. Engraftment of C57BL/6 hematopoietic stroma microenvironment (HSM) in BALB/c mice treated with fractionated TLI (200 cGy each fraction) and conditioned for alloantigen-primed lymphocyte depletion. Nonirradiated BALB/c mice and animals treated with TLI were conditioned for APLD (C57BL/6 BMC IV and 200 mg/kg IP on the next day). One day after injection of CY recipients were transplanted under the kidney capsule with two femoral BM plugs obtained from C57BL/6 donors irradiated with 400 cGy (empty columns) or received in addition to the femoral plug implantation an intravenous injection with 3 107 BMC from C57BL/6 donors (solid columns). One month later, recipients were anesthetized and checked for the presence of ectopic bone. Reproduced with permission from Gurevitch O et al., Transplantation 68 (1999) 1362. Copyright 1999, L.W.W.
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Application of APLD-based reduced-intensity conditioning for BMT aimed for treatment of murine leukemia and solid breast carcinoma To assess the efficacy and safety of our reduced-intensity BMT protocol for the treatment of leukemia and metastatic solid tumors, we performed allogeneic BMT to the animals inoculated with malignant cells before conditioning [22]. BALB/c mice were given intravenously 106 BCL1 cells from mice with overt leukemia or transplanted ID into the right flank with 2 104 4T1 breast carcinoma cells. Preparation for BMT consisted of TLI (200cGy) on day 1 and APLD (IV priming with 3 107 C57BL/6 BMC on day 2, and IP injection with 200 mg/kg CY on day 3), and was followed by BMT from the same donor (3 107 cells IV on day 4). The results presented in Fig. 4C show that BMT performed after reduced-intensity conditioning for APLD prevented development of leukemia in 90% of recipients. All control mice treated only with 200 cGy TLI and 200 mg/ kg CY died of the disease (Fig. 4A). GVL effects were evidently mediated by the engrafted second BM transplant, since most animals treated by TLI, donor BMC, and CY, but no second infusion of donor BMC, died of leukemia (Fig. 4B). Late GVHD-related mortality in mice treated for leukemia by reduced-intensity BMT was higher than in healthy recipients of BMC (compare GVHD-related mortality in Figs. 1 and 4C). However, 60% recipients remained leukemia- and GVHD-free N170 days after BMT (Fig. 4C). These results show that nonmyeloablative BMT has a significant curative effect in leukemia-bearing mice. BMT performed after TLI and APLD also delayed the appearance of palpable solid 4T1 tumors for 1–2 weeks.
Moreover, 40% (14 of 35) transplanted mice did not develop tumors at all (Fig. 5C). Here, as well as documented in mice treated by BMT for leukemia, the second BM transplant mediated eradication of malignant cells. All animals preconditioned by TLI, donor BMC, and CY, but without a second infusion with donor BM cells, developed tumors (Fig. 5B). Most of cured recipients (10 of 14) also did not develop GVHD and remained healthy for the entire observation period. Yet, the results obtained by application of the same BMT protocol for the treatment of leukemia and solid tumor show that eradication of solid tumor after allogeneic BMT is much less effective than eradication of leukemia blasts. Prevention of diabetes by induction of chimerism in NOD mice Allogeneic BMT can prevent and even reverse autoimmune insulitis and overt diabetes. At the same time, BMT can induce donor-specific tolerance to islet allografts [23]. However, before BMT can be considered in the clinical setting, transplantation-related morbidity and GVHD-related mortality must be better controlled. Therefore, we have investigated our new reduced-intensity BMT strategy for safer treatment of autoimmune diabetes [19,24]. Consistent diabetes was induced in NOD mice after irradiation (650 cGy TBI) and intravenous transplantation of 2.5 107 diabetogenic spleen cells from overtly diabetic NOD mice. Three days later, NOD recipients were primed intravenously with donor C57BL/6 BM (3 107 cells), a day later inoculated intraperitoneally with CY (200, 100, or 60 mg/kg) for APLD, and 1 day later transplanted with a
Fig. 4. Anti-leukemia effect induced by non-T-cell-depleted allogeneic BMC transplanted after mild TLI and antigen-primed lymphocyte depletion. BALB/c mice were inoculated intravenously on day 0 with 106 BCL1 cells and treated with (A) TLI (200cGy) on day +1 and Cy (200 mg/kg IP) on day +3 (n = 31); (B) TLI on day +1, C57BL/6 BM (3 107 cells IV) on day +2, and Cy on day +3 (n = 14); (C) as in group B and the second inoculation of C57BL/6 BM (3 107 cells IV) on day +4 (n = 43). Survival of recipients treated by BMT (group C) was significantly better than in control groups (PA–C and B–C = 0.0001). Reproduced with permission from T. B. Prigozhina et al., Exp. Hematol. 30 (2002) 89. Copyright 2002, Elsevier Inc.
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Fig. 5. Treatment of breast cancer by transplantation of non-T-cell-depleted allogeneic BM following mild TLI and antigen-primed lymphocyte depletion. BALB/c mice, transplanted ID with 2 104 4T1 breast carcinoma cells on day 0, received (A) no treatment (n = 12); (B) TLI on day +1, 3 107 C57BL/6 BM cells on day +2, 200 mg/kg CY on day +3 (n = 32); C - as in group B and the second inoculation of C57BL/6 BM (3 107 cells IV) on day +4 (n = 35). Reproduced with permission from T. B. Prigozhina et al., Exp. Hematol. 30 (2002) 89. Copyright 2002, Elsevier Inc.
second inoculum of C57BL/6 BM (3 107 cells). All mice treated for APLD before BMT converted to chimeras with a high percentage of donor cells (Fig. 6A, 1–4). None of them developed diabetes for N3 months, demonstrating that BMC from a diabetes-resistant murine strain prevents immune destruction of the pancreas by diabetogenic effector cells. Unfortunately, all mice converted to chimeras by protocols
utilizing 200 mg/kg CY died later of chronic GVHD (Fig. 6B). When the second BM inoculum was omitted, donor chimerism was not achieved and all NOD mice died of diabetes (Fig. 6A, Gr. 5). Reducing the CY dose to 100 or 60 mg/kg caused significant improvement of the chimera’s survival. Most animals survived N300 days, remaining chimeras free of GVHD and diabetes (Fig. 6B).
Fig. 6. Allogeneic BMT prevents development of diabetes in NOD mice. Consistent diabetes was induced in NOD mice after irradiation (650 cGy TBI) and intravenous inoculation of 2.5 107 diabetogenic spleen cells from overtly diabetic NOD mice. Three days after transfer of diabetogenic cells, NOD recipients were primed intravenously with donor C57BL/6 BMC (3 107/mouse), a day later inoculated intraperitoneally with CY (200, 100, or 60 mg/kg), and 1 day after that transplanted with a second inoculum of donor BMC (3 107 cells). Omission or use of the first or the second BMC inoculation is shown in Fig. 5A by symbols or +. After transplantation, recipients were tested every 7 days for glucosuria and every 100 days for donor cell chimerism.
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Promotion of allogeneic BM engraftment after APLD by a reduced CY dose (100 mg/kg) The therapeutic effects of large CY doses are often associated with systemic toxicity. Therefore, with a view to safer clinical application in the future, we reduced the CY dose in APLD protocol to 100 mg/kg (in press). Fig. 7A shows that modified treatment converted to chimeras 46% of recipients (12 of 26) irradiated with 100 cGy TBI, 90% (9 of 10) recipients irradiated with 200 cGy TBI, and all recipients treated with 300 cGy TBI (n = 12). When priming with donor BMC was excluded from the protocol and all the C57BL/6 BMCs (6 107) were given as a bmegadoseQ [25] of BMC after CY to mice that had been exposed to 300 cGy TBI, only 33% (3 of 10) became chimeras. These results definitely show that conditioning for APLD facilitates engraftment of mismatched BMC significantly better than amplification of donor BMC numbers after general immunosuppression. Eighty percent chimeras obtained by BMT after low-dose TBI and APLD remained GVHD-free for N150 days (data not shown). Although donor T cells significantly facilitate engraftment of allogeneic BMC, most clinically approved protocols recommend depleting donor T cells from haplotype-mis-
Fig. 7. Evaluation of the minimal conditioning required for induction of transplantation tolerance with a reduced CY dose (100 mg/kg). (A) Transplantation of allogeneic BMC. BALB/c mice were treated with a range of TBI doses, inoculated with 100 mg/kg CY IP (day 1), and transplanted with 6 107 C57BL/6 BM cells intravenously (day 2, white bars). Alternatively, TBI-treated mice received intravenous inoculation of 3 107 C57BL/6 BMC (day 1), IP injection of 100 mg/kg CY (day 2), and 3 107 C57BL/6 BM (day 3, black bars). Data from several experiments were pooled (9 or more mice/group) and presented as a frequency of recipients with N25% donor cells in the blood as measured by FACS analysis 50 days after BMT. (B) Transplantation of semiallogeneic (C57BL/6 BALB/c) F1 BMC. Comparison of the efficacy of F1 BM cell engraftment in BALB/c recipients prepared for BMT by general immunosuppression with irradiation and 100 mg/kg CY (dense pattern columns) and in BALB/c mice prepared for BMT by irradiation and antigen-primed lymphocyte depletion (gray columns). Each group included nine mice or more.
matched BM transplants to avoid GVHD-related complications. Therefore, we examined the efficacy of allogeneic BMT under stringent conditions: low dose TBI, reduced CY dose (100 mg/kg), and transplantation of semiallogenic (C57BL/6 BALB/c) F1 BMCs that express transplantation antigens of both parents (H-2b and H-2d in our case) and lack anti-parental T cell clones (in press). Fig. 7B shows that the minimal radiation dose providing acceptance of semiallogenic BMC is significantly higher than the dose required for the engraftment of allogeneic BMC. However, the advantage of the selective APLD protocol over the bmegadoseQ protocol is also evident after transplantation of F1 BMC.
Discussion Two immunological barriers must be overcome for a successful mismatched BMT. One is rejection and the other is GVHD. Engraftment of mismatched BMC requires pretransplant depletion of most donor-reactive lymphocytes. All clinically approved anti-rejection therapies available today accomplish this task by providing general depletion of the recipients’ immune system. Myeloablative conditioning generally leads to engraftment of allogeneic BM but is associated with a high incidence of GVHDrelated mortality of recipients. Low-intensity preparative regimens allow inducing sufficient immunosuppression to permit engraftment of compatible hematopoietic stem cells and reduce GVHD-related mortality after BMT. Several factors may contribute to the low risk of GVHD after reduced-intensity BMT. Mild conditioning preserves the integrity of the gastrointestinal system and keeps the level of pro-inflammatory cytokines low [26,27]. Reducedintensity conditioning also preserves low numbers of T and bvetoQ cells of the host that cannot reject donor BMC but can resist or suppress their unlimited expansion [20,28]. However, though reduced-intensity regimens for BMT gained significant popularity, myeloablative immunosuppression is still required for providing acceptance of strongly immunogenic haploidentical hematopoietic stem cells. The results of our investigation show that selective depletion of alloantigen-activated donor-reactive cells by CY allows significant reduction of radiation dose essential for BM engraftment across strong MHC barriers, providing the advantages of reduced-intensity conditioning to recipients of allogeneic BM transplants. Engraftment of allogeneic BMC after reduced-intensity conditioning for APLD guarantees conversion of most recipients to GVHD-free chimeras and, in contrast to the old reduced-intensity experimental protocols recommending organ transplantation directly after APLD, provides acceptance of various donor tissue and organ allografts, including donor hematopoietic stromal progenitors and, most immunogenic, donor skin transplants.
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The potential importance of the transplantation of donor BM within its own microenvironment cannot be overestimated. It may provide an optimal opportunity for BM engraftment, especially for patients suffering from diseases caused by primary HSM abnormalities as well as from secondary HSM defects induced by intensive irradiation or chemotherapy. The reduced-intensity BMT protocol that we present here also provides significant direct graft-vs.-malignancy and graft-vs.-autoimmunity effects. In both cases therapeutic potential of BMT depended on the robust engraftment of donor BMC. One of the ways to improve engraftment of mismatched BM allografts in reduced-intensity protocols is to escalate the dose of transplanted cells (the bmegadoseQ approach, ref. [25]). Facilitation of engraftment by infusion of larger numbers of BM cells is based on the fact that donor T cells, NK cells, and veto cells within the CD34+hematopoietic progenitor cell population play an active role in engraftment and can remove recipients’ residual donor-reactive cells. Our results show, however, that conditioning for APLD provided significantly better engraftment of allogeneic and semiallogenic BMC in reduced-intensity protocols than transplantation of the same total cell dose as a bmegadoseQ after nonselective immunosuppression with identical radiation and CY doses. Thus, we consider that treatment of recipients with CY soon after priming with lymphoid cells of the future donor results in better eradication of donor-reactive host clones than relatively weak bvetoQ activity mediated after transplantation by hematopoietic stem cells. All donor hematopoietic cells that we tested (BMC, spleen cells, and blood cells) could be effectively used for priming of donor-reactive host cells [20]. We believe that unmanipulated BMC or even spleen cells or blood mononuclear cells can be safely used for priming in APLD protocols because donor cells used for priming are eradicated after optimally timed administration of CY. Thus, application of APLD conditioning in future BMT protocols may possibly allow for a reduction in both the radiation dose essential for engraftment of T-cell-depleted allogeneic BMC, and the numbers of donor stem cells that must be selected for transplantation by immunomagnetic separation methods. Systemic (IV) inoculation of a large dose of donor lymphocytes was found to be essential for the priming. Transplantation of the intravenously effective numbers of donor BM cells under the kidney capsule as a plug or local transplantation of a donor skin graft (data not shown) could not provide significant APLD after CY administration. We suppose that only circulating donor lymphocytes induce systemic activation of donor-reactive host cells essential for profound APLD by CY. Recent generation by Quezada et al. of a T-cell receptor transgenic model of CD4+T-cellmediated rejection allowed visualization of the fate and function of CD4+T cells, specific to MHC donor antigens. The results obtained in their study directly demonstrate that
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robust systemic proliferation of donor-reactive CD4+T cells is seen only after IV donor cell transfusion [29]. Although our reduced-intensity protocol proved effective for induction of transplantation tolerance to allografts, its wider clinical application was suspended because of the relatively high CY dose considered essential for APLD. Mild irradiation of recipients prior to priming with donor BMC allowed the tolerance-inducing dose of CY to be reduced to a clinically acceptable level (100 mg/kg). Binding of a specific antibody to the CD154 molecule (CD40 ligand) expressed on recently activated CD4+T cells, but absent on naive lymphocytes, and subsequent eradication of activated donor-reactive cells by antibody and complement [30], may present another example of selective depletion of alloantigen-primed cells for tolerance induction. CD154-specific antibodies are especially potent in animals primed by donor-specific transfusion (DST) and subsequently transplanted with donor BMC [31,32]. DST causes a robust systemic proliferation of donor-reactive CD4+T cells that is followed by a state of temporary hyporesponsiveness of CD4+[29] and CD8+T cells [31]. Application of CD154-specific antibodies after DST additionally reduces expansion of allograft-responding CD4+T cells, causes significant depletion of responding CD4+clones [29], and facilitates acceptance of donor BMC in mildly irradiated recipients. As in our experimental model, induction of robust donor cell chimerism after formation of donor-specific transplantation tolerance with DST- and CD154-specific antibody is essential for permanent acceptance of donor organ and skin transplants. Both chimeras obtained by BMT after APLD and chimeras induced by BMT after conditioning with CD154-specific antibodies are specifically unresponsive to host and donor transplantation antigens in vitro [20,32,33] and are capable of rejecting unrelated skin grafts. Kurtz et al. [33] were unable to find evidence of regulatory/suppressor mechanisms for maintenance of transplantation tolerance in chimeras generated by donor BMT into mice treated with CD154-specific mAbs and therefore came to conclusion that donor-reactive host cells are eliminated or become irreversibly anergic during formation of the transplantation tolerance. Our present data (in press) revealed that donor spleen cells of full chimeras obtained by BMT after APLD conditioning can neither generate GVHD after transplantation to irradiated F1 recipients nor suppress GVHD generated by spleen cells of naive C57BL/6 mice. These experimental data support our assumption that the two methods are based on the same principles: selective elimination of most activated donorreactive lymphocytes during the conditioning phase of the protocol and facilitation of organ engraftment by elimination of residual donor-reactive lymphocytes after donor BMT and chimerism formation. We revealed that CD154-specific antibodies synergize with CY [34]. A short course of CD154-specific antibodies helps to reduce the APLD-effective CY dose to 100 mg/kg without additional radiation of recipients. CD154-specific
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antibodies can also downregulate the ability of transplanted BMC to induce GVHD [35]. However, our experimental data and results of others have documented that persisting CD154-specific mAb impair generation of GVL effects after BMT [35,36]. Furthermore, CD154-specific mAb may cause thromboembolic complications in some patients [36], and therefore this mAb was not approved for clinical application. In conclusion, our data and results obtained by other investigators indicate that the future of the reduced-intensity BMT regimens lies in the development of selective immunosuppression methods that would allow targeted eradication of donor-reactive host cells. We feel that today, in view of the nonavailability of safe antibodies against the CD154 molecule or other clinical grade antibodies to the markers of activated T cells, conditioning for alloantigenprimed lymphocyte depletion (APLD) by a low CY dose may represent the first clinically acceptable step in this direction. This conditioning method is significantly more effective in the formation of transplantation tolerance to BM allografts than bmegadoseQ BMT after nonselective immunosuppression. The treatment is inexpensive, relatively safe, and potentially effective for BMT carried out for the therapy of malignant and nonmalignant diseases and for organ transplantation. The introduction of APLD into clinical practice may improve current reduced-intensity conditioning protocols for transplantation of BMC from haploidentically mismatched and unrelated donors. References [1] P.L. Weiden, K.M. Sullivan, N. Flournoy, R. Storb, E.D. Thomas, Antileukemic effect of chronic graft-versus-host disease: contribution to improved survival after allogeneic marrow transplantation, N. Engl. J. Med. 304 (1981) 1529 – 1533. [2] R. Hirschhorn, Treatment of genetic diseases by allotransplantation, Birth Defects, Orig. Artic. Ser. 16 (1980) 429 – 444. [3] P.B. Medawar, Two notes on immunological tolerance, Transplant. Bull. 4 (2) (1957) 72 – 74. [4] S. Slavin, S. Strober, Z. Fuks, H.S. Kaplan, Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts, J. Exp. Med. 146 (1977) 34 – 48. [5] Y. Sharabi, D.H. Sachs, Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen, J. Exp. Med. 169 (2) (1989) 493 – 502. [6] S. Giralt, E. Estey, M. Albitar, et al., Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative therapy, Blood 89 (1997) 4531 – 4536. [7] S. Slavin, A. Nagler, E. Naparstek, et al., Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases, Blood 91 (1998) 756 – 763. [8] I.F. Khouri, M. Keating, M. Korbling, et al., Transplant-lite: induction of graft-versus-malignancy using fludarabine-based nonablative chemotherapy and allogeneic blood progenitor-cell transplantation as treatment for lymphoid malignancies, J. Clin. Oncol. 16 (1998) 2817 – 2824.
[9] P. McSweeney, D. Niederwieser, J. Shizuru, et al., Outpatient allografting with minimally myelosuppressive, immunosuppressive conditioning of low-dose TBI and postgrafting cyclosporine (CSP) and mycophenolate mofetil (MMF), Blood 94 (10 Pt. 1) (1999) 347a, Abstract 1742. [10] A.M. Carella, R. Champlin, S. Slavin, P. McSweeney, R. Storb, Miniallografts: ongoing trials in humans, Bone Marrow Transplant. 25 (2000) 345 – 350. [11] J.F. Bach, The mode of action of immunosuppressive agents, Front. Biol. 41 (1975) 1 – 374. [12] I.Y. Chernyakhovskaya, E.V. Nagurskaya, G.B. Shaposhnikova, T.B. Prigozhina, L.N. Fontalin, Tolerance to allogeneic and to xenogeneic heart grafts provided by thymectomy of adult mice combined with donor cell and cyclophosphamide inoculation, Transplantation 29 (1980) 409 – 412. [13] I.Y. Chernyakhovskaya, T.B. Prigozhina, E.V. Nagurskaya, L.N. Fontalin, Immunological status of mice with long-standing allogeneic or xenogeneic neonatal heart grafts, Transplantation 38 (3) (1984) 267 – 272. [14] H. Mayumi, K. Himeno, T. Shin, K. Nomoto, Drug-induced tolerance to allografts in mice: VI. Tolerance induction in H-2-haplotype-identical strain combinations in mice, Transplantation 40 (1985) 188 – 194. [15] H. Mayumi, M. Umesue, K. Nomoto, Cyclophosphamide-induced immunological tolerance: an overview, Immunobiology 195 (1996) 129 – 139. [16] T.B. Prigozhina, O. Gurevitch, J. Zhu, S. Slavin, Permanent and specific transplantation tolerance induced by a non-myeloablative treatment to a wide variety of allogeneic tissues: I. Induction of tolerance by a short course of total lymphoid irradiation and selective elimination of the donor-specific host lymphocytes, Transplantation 63 (1997) 1394 – 1399. [17] S. Slavin, S. Strober, Spontaneous murine B-cell leukaemia, Nature 272 (1978) 624 – 626. [18] D.L. Dexter, H.M. Kowalski, B.A. Blazar, Z. Fligiel, R. Vogel, G.H. Heppner, Heterogeneity of tumor cells from a single mouse mammary tumor, Cancer Res. 38 (1978) 3174. [19] G. Elkin, T.B. Prigozhina, O. Gurevitch, S. Slavin, Nonmyeloablative bone marrow transplantation based on deletion of host-anti-donor alloreactive cells prevents autoimmune insulitis and diabetes in nonobese diabetic mice, Transplant. Proc. 34 (4) (2002) 1303 – 1306. [20] T.B. Prigozhina, O. Gurevitch, S. Slavin, Nonmyeloablative conditioning to induce bilateral tolerance after allogeneic bone marrow transplantation in mice, Exp. Hematol. 27 (1999) 1503 – 1510. [21] O. Gurevitch, T.B. Prigozhina, T. Pugatsch, S. Slavin, Transplantation of allogeneic or xenogeneic bone marrow within the donor stromal microenvironment, Transplantation 68 (1999) 1362 – 1368. [22] T.B. Prigozhina, O. Gurevitch, S. Morecki, E. Yakovlev, G. Elkin, S. Slavin, Nonmyeloablative allogeneic bone marrow transplantation as immunotherapy for hematologic malignancies and metastatic solid tumors in preclinical models, Exp. Hematol. 30 (1) (2002) 89 – 96. [23] R. Yasumizu, K. Sugiura, H. Iwai, et al., Treatment of type 1 diabetes mellitus in non-obese diabetic mice by transplantation of allogeneic bone marrow and pancreatic tissue, Proc. Natl. Acad. Sci. U. S. A. 84 (18) (1987) 6555 – 6557. [24] G. Elkin, T.B. Prigozhina, S. Slavin, Prevention of diabetes in nonobese diabetic mice by nonmyeloablative allogeneic bone marrow transplantation, Exp. Hematol. 32 (6) (2004) 579 – 584. [25] E. Bachar-Lustig, N. Rachamim, H.W. Li, F. Lan, Y. Reisner, Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice, Nat. Med. 1 (12) (1995) 1268 – 1273. [26] P. Reddy, J.L. Ferrara, Immunobiology of acute graft-versus-host disease, Blood 17 (4) (2003) 187 – 194. [27] C.Q. Xun, J.S. Thompson, C.D. Jennings, S.A. Brown, M.B. Widmer, Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute and chronic graft-versus-host disease in H-2-incompatible transplanted SCID mice, Blood 83 (8) (1994) 2360 – 2367.
T.B. Prigozhina et al. / Blood Cells, Molecules, and Diseases 33 (2004) 238–247 [28] B.R. Blazar, C.J. Lees, P.J. Martin, et al., Host T cells resist graft-versus-host disease mediated by donor leukocyte infusions, J. Immunol. 165 (2000) 4901 – 4909. [29] S.A. Quezada, B. Fuller, L.Z. Jarvinen, M. Gonzalez, B.R. Blazar, A.Y. Rudensky, T.B. Strom, R.J. Noelle, Mechanisms of donor-specific transfusion tolerance: preemptive induction of clonal T-cell exhaustion via indirect presentation, Blood 102 (5) (2003) 1920 – 1926. [30] N.J. Monk, R.E. Hargreaves, J.E. Marsh, et al., Fc-dependent depletion of activated T cells occurs through CD40L-specific antibody rather than costimulation blockade, Nat. Med. 9 (2003) 1275 – 1280. [31] E. Seung, J.P. Mordes, A.A. Rossini, D.L. Greiner, Hematopoietic chimerism and central tolerance created by peripheral-tolerance induction without myeloablative conditioning, J. Clin. Invest. 112 (5) (2003) 795 – 808. [32] Y. Takeuchi, H. Ito, J. Kurtz, T. Wekerle, L. Ho, M. Sykes, Earlier low-dose TBI or DST overcomes CD8+T-cell-mediated alloresistance to allogeneic marrow in recipients of anti-CD40L, Am. J. Transplant. 4 (1) (2004) 31 – 40.
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[33] J. Kurtz, J. Shaffer, A. Lie, N. Anosova, G. Benichou, M. Sykes, Mechanisms of early peripheral CD4 T cell tolerance induction by anti-CD154 monoclonal antibody and allogeneic bone marrow transplantation: evidence for anergy and deletion, but not regulatory cells, Blood 103 (11) (2004) 4336 – 4343. [34] T.B. Prigozhina, O. Gurevitch, G. Elkin, S. Morecki, E. Yakovlev, S. Slavin, CD40 ligand-specific antibodies synergize with cyclophosphamide to promote long-term transplantation tolerance across MHC barriers but inhibit graft-vs-leukemia effects of transplanted cells, Exp. Hematol 31 (1) (2003) 81 – 88. [35] M. Abedi, D. Greer, J.F. Lambert, G.A. Colvin, M.S. Dooner, C.I. McAuliffe, D. Demers, B.E. Moore, P.J. Quesenberry, Tolerance induction by costimulator blockade in 100 cGy treated hosts with varying degrees of genetic disparity, Leukemia 17 (9) (2003) 1871 – 1879. [36] T. Kawai, D. Andrews, R.B. Colvin, D.H. Sachs, A.B. Cosimi, Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand, Nat. Med. 6 (2) (2000) 114.
Depletion of alloantigen-primed lymphocytes overcomes resistance to $ allogeneic bone marrow in mildly conditioned recipients Tatyana B. Prigozhina, Gregory Elkin, Olga Gurevitch, Shoshana Morecki, Elena Yakovlev, Sofia Khitrin, Shimon Slavin* Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah University Hospital, Jerusalem, Israel Submitted 28 July 2004 Available online 7 October 2004 (Communicated by M. Lichtman, M.D., 3 August 2004)
Abstract Objective: Successful implantation of allogeneic bone marrow (BM) cells after nonmyeloablative conditioning would allow to compensate for the inadequate supply of compatible grafts and to reduce mortality of graft-vs.-host disease (GVHD). Recently, we proposed to facilitate engraftment of mismatched BM by conditioning for alloantigen-primed lymphocyte depletion (APLD) with cyclophosphamide (CY). Here we summarize the experimental results obtained by this approach. Materials and methods: Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY (day 1) to deplete alloantigen-primed lymphocytes, and given a second C57BL/6 BM transplant (day 2) for engraftment. Recipients were repeatedly tested for chimerism in the blood and followed for GVHD and survival. The protocol was also tested for inducing tolerance to donor tissue and organ allografts, and for treatment of leukemia, breast cancer, and autoimmune diabetes in NOD mice. Results: APLD by 200 mg/kg CY provided engraftment of allogeneic BM from the same donor in 100% mildly irradiated recipients. Eighty percent chimeras remained GVHD-free more 200 days. All chimeras accepted permanently donor skin grafts and donor hematopoietic stromal progenitors. Allogeneic BM transplantation (BMT) after APLD had a strong therapeutic potential in BALB/c mice harboring malignant cells and in autoimmune NOD recipients. Tolerance-inducing CY dose could be reduced to 100 mg/kg. Conditioning for APLD resulted in engraftment of allogeneic BM after a significantly lower radiation dose than treatment with radiation and CY alone. Conclusion: Our results demonstrate that conditioning for APLD has a definite advantage over general immunosuppression with CY and radiation therapy. D 2004 Elsevier Inc. All rights reserved. Keywords: Transplantation tolerance; Bone marrow transplantation; Reduced-intensity conditioning; Cyclophosphamide; APLD (alloantigen-primed lymphocytes depletion)
Introduction Allogeneic bone marrow (BM) or mobilized stem cell transplantation is the treatment of choice for patients with
$ This paper is based upon a presentation at a Focused Workshop on Haploidentical Stem Cell Transplantation sponsored by The Leukemia and Lymphoma Society held in Naples, Italy from July 8 to 10, 2004. * Corresponding author. Department of Bone Marrow Transplantation, Hadassah University Hospital, PO Box 12000, 91120, Jerusalem, Israel. Fax: +972 2 642 2731. E-mail address: [email protected] (S. Slavin).
1079-9796/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2004.08.010
hematological malignancies and certain hereditary diseases [1,2]. BM chimeras can also permanently accept various organ transplants taken from the BM donor with no need for maintenance immunosuppressive therapy [3]. However, broad application of BM transplantation (BMT) in clinical practice is limited by graft failure and severe graft-vs.-host disease (GVHD) after transplantation. The data obtained in clinical practice and experiments in mice show, however, that reducing the intensity of immunosuppressive conditioning prior to BMT improves the safety of the procedure [4–10]. The progress in introducing reduced-intensity regimens into clinical prac-
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tice came with understanding that the generation of graftversus-leukemia (GVL) effects by transplanted BM does not require myeloablation of recipients, but does require persistent donor BM cell (BMC) engraftment. Substitution of myeloablative preconditioning by reduced-intensity regimens with fludarabine, busulfan, or cyclophosphamide (CY), with or without antibodies against T cells [6–10], diminished acute transplantation-related toxicity and mortality, while providing rapid and stable engraftment of HLA-compatible BM cells (BMCs). Unfortunately, rejection of haploidentically mismatched BM and organ transplants is difficult to overcome by reduced-intensity conditioning protocols available today. Lately, we proposed improving engraftment of incompatible BM grafts in reduced-intensity transplantation protocols by conditioning aimed to diminish the clone size of responding alloreactive T cells. This conditioning includes priming of host by inoculation of donor lymphocytes and subsequent depletion of activated donor-reactive host cells by CY, a drug that is predominantly toxic to proliferating cells [11]. The method was previously used by us and others to facilitate donor organ and skin transplant engraftment in the absence of BMT [12–15]. Application of this approach facilitated acceptance of donor organs and skin differing from recipient in minor histocompatibility antigens. Tolerance to fully allogeneic heart grafts could not be achieved unless recipients were thymectomized. Mice tolerant to allogeneic heart grafts nevertheless rejected the more immunogenic skin transplants derived from the same donor [12]. We concluded that the main failure of allogeneic organ and skin engraftment after CY-induced alloantigen-primed lymphocyte depletion (APLD) was due to minimal, if any, donor cell chimerism in recipients of organ grafts. Administration of CY after donor cell priming depleted not only activated donor-reactive host cells, but also donor lymphocytes used for priming because most of transplanted donor cells also proliferated in recipient after BMT [16]. Thus, to convert recipients to stable chimeras, we proposed performing a second BMT from the same donor following the disappearance of the cytotoxic CY metabolites. The results obtained by the application of our protocol for induction of donor-specific transplantation tolerance to allogeneic BMC and organ grafts are summarized in this report.
Materials and methods Animals BALB/c (H-2d), C57BL/6 (H-2b), and (C57BL/6 BALB/c) F1 (H-2b/d) hybrid mice (F1), CBA (H-2k), and NOD (H2-KdDb) were purchased from Harlan Hebrew University-Hadassah Medical School’s Animal Facility in Jerusalem, Israel. Two-month-old mice that were kept under
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standard conditions, with food and water ad libitum, were used in the study. Radiation Fractionated total lymphoid irradiation (TLI, 200 cGy/ fraction) was used for conditioning of mice in the initial experiments. Mice were anesthetized for proper positioning in an apparatus that exposes the major lymph nodes, thymus, and spleen to ionizing irradiation, while shielding most of the skull, ribs, lungs, hind limbs, and tail with lead, as previously described [4]. Total body irradiation (TBI) was given to mice as a single dose from a 6-MEV linear accelerator at a dose rate 1.9 Gy/min. The exact TBI doses given to recipients are presented in Results. Alloantigen-primed lymphocyte depletion (APLD) as conditioning to induce tolerance to allografts Naive BALB/c mice and mice exposed to a low-dose radiation were treated for APLD by an intravenous (IV) injection of 3 107 donor C57BL/6 BMC and by intraperitoneal (IP) injection of 200 mg/kg CY (Baxter, Frankfurt, Germany) on the next day. When specified, the CY dose was reduced to 100 or 60 mg/kg. A day after CYinduced APLD mice were transplanted intravenously with a second inoculum of donor BMC (3 107) for engraftment of donor hematopoietic cells. Assay for chimerism Mice were anesthetized and blood was taken from the retro-orbital sinus of the eye. WBC (2–8 105/sample) were separated, directly stained with anti-H-2Kb-FITC (IgG2a) or anti-H-2Kd-FITC (IgG2a) monoclonal antibodies (mAb) (Serotec, USA), and analyzed by FACS analysis (FACStar plus, Becton Dickinson, San Jose, CA, USA). Background binding of each H-2K-specific mAb was determined by staining with it the cells of nonrelevant haplotype. Skin grafting A full-thickness donor skin graft was transplanted to recipients on the day of priming with BMC or 20 days after completing the conditioning [16]. The graft was considered accepted when hair of donor color grew on the soft, flexible underlying skin, and rejected when donor hair and epithelium were lost. GVHD monitoring Clinical signs, for example, weight loss, diarrhea, and hunched back, were used to evaluate GVHD in chimeras. Animals were observed daily for survival. Postmortem
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analysis of spleens and lymph nodes showed that they were reduced in size. Treatment of malignant diseases BALB mice were inoculated intravenously with 106 spleen cells from mice with overt BCL1 leukemia [17] or transplanted ID into the right flank with 2 104 4T1 breast carcinoma cells [18] on day 0, exposed to 200 cGy TLI on day 1, treated for APLD by inoculation of 3 107 C57BL/6 BMC on day 2, and by injection of 200 mg/kg CY on day 3. One day after treatment with CY (day 4), recipients were given a second IV inoculum of donor BMC (3 107 cells/mouse). Treatment of autoimmune diabetes in NOD mice To induce consistent autoimmunity, diabetes-free female NOD mice were given 650 cGy TBI and 2 h later inoculated intravenously with 25 106 spleen cells obtained from overtly diabetic NOD [19]. Conditioning for APLD was started 3 days later. It lasted 2 days (see the previous section) and was followed by transplantation of BMC from C57BL/6 donors. Mixed lymphocyte reaction (MLR) T cells, enriched by passing spleen cells through a nylon wool column, were stimulated by equal numbers of stimulator cells (irradiated by 3000 cGy), cultured as described [16], pulsed on the third day with 1 mCi [3H]-thymidine, harvested on the fourth day, and thymidine incorporation was measured. Statistical analysis of the data The results were analyzed according to Student’s t test. Donor cell chimerism was presented as mean F SE.
Results Scaling down the radiation dose and transplantation of non-T-cell-depleted donor BM to induce GVHD-free chimeras tolerant to donor skin grafts Our initial experiments revealed that conditioning of BALB/c mice with six fractions of TLI (200 cGy each) followed by selective depletion of alloantigen-primed lymphocytes (3 107 C57BL/6 BMC IV and on the next day 200 mg/kg CY IP) provided engraftment of allogeneic BMC from the same donor, but required transplantation with T-cell-depleted BM to prevent GVHD [16]. In order to decrease the incidence of GVHD after infusion of allogeneic non-T-cell-depleted BM (3 107 cells/mouse), conditioning for APLD was given to nonirradiated
recipients and to mice irradiated with one TLI fraction (200 cGy) instead of six TLI fractions [20]. Fig. 1 shows that BMT after reduced-intensity conditioning converted to chimeras all mice treated with a single TLI dose before APLD and 84% recipients treated for APLD alone. About 80% of these animals remained GVHD-free N200 days though they were transplanted with unmodified allogeneic BMC. In contrast, 90% of chimeras treated for APLD after conditioning with six TLI fractions died of GVHD after BMT. Specificity of transplantation tolerance to donor skin allografts A number of BALB mice tolerant to H-2b transplantation antigens were implanted with a skin graft obtained from a third party CBA (H-2k) donor 50–150 days after successful engrafting of C57BL/6 (H-2b) skin. All animals rejected the CBA skin grafts within 19 F 3 days while keeping the original C57BL/6 skin allografts intact (n = 10), indicating that recipients selectively tolerant to MHC antigens of the initial donor were capable of generating an immune response to unrelated transplantation antigens. Lymphocytes from full donor-type chimeras that maintained donor skin grafts did not respond to self-stimulators or to stimulators of donor or recipient type (Fig. 2). However, they retained significant in vitro reactivity to allogeneic stimulators obtained from third party CBA mice (Fig. 2). Engraftment of allogeneic hematopoietic stroma microenvironment (HSM) in recipients treated with fractionated TLI and conditioned for APLD Donor cells infused in the course of BMT seed in the host hematopoietic stromal microenvironment (HSM) and a direct interaction of host HSM with the donor stem cells is essential for differentiation and maturation of hematopoietic progenitor cells. In diseases associated with primary or secondary defects of HSM, namely, certain forms of aplastic anemia or myelofibrosis, and disorders resulting from intensive ionizing radiation or chemotherapy, graft failure, distinguishable from graft rejection, can occur. In such cases long-term hematopoietic engraftment of transplanted BM may ultimately require transplantation of donor HSM. Thus, we investigated the relationship between the intensity of the conditioning of recipients and the induction of tolerance to allogeneic HSM [21]. Fig. 3 shows that irradiating recipients with three instead of six TLI fractions (200 cGy each) and inoculation of donor BMC and CY prior to BM plug implantation provided consistent acceptance of stromal progenitors and formation of ectopic bone under the kidney capsule. Ectopic ossicles were maintained for the entire life of the animal. Transplantation of donor femoral plugs to recipients irradiated only once or not at all
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Fig. 1. Conditioning for alloantigen-primed lymphocyte depletion (APLD) by CY after low-dose TLI provides GVHD-free engraftment of non-T-cell-depleted donor BM and induces long-lasting tolerance to donor skin grafts. Nonirradiated BALB/c mice and BALB/c recipients given one or six daily TLI fractions (200 cGy each) were treated for alloantigen-primed lymphocyte depletion by intravenous priming with C57BL/6 BMC (3 107/mouse) and by IP inoculation of 200 mg/kg CY a day later. One day after CY administration, mice were transplanted intravenously with a second dose of C57BL/6 BMC (3 107/mouse). Percentage of mice with chimerism (N80% donor cells in the blood) was determined by FACS analysis 120 days after BMT. Skin grafting was performed 20 days after BMT. GVHDfree survival of recipients and maintenance of donor B6 skin allografts by survivors were followed for 200 days after BMT. Each group included 15 mice or more.
prior to APLD resulted in the development of ectopic bones in 45% of experimental animals. In mice conditioned only with TLI and CY, ectopic ossicles never developed. Acceptance of HSM in nonirradiated mice improved substantially when the BM plug was transplanted simultaneously with the intravenous infusion of donor BM cells. Intravenous priming of donor-reactive host cells by donor
Fig. 2. MLR response of T cells from BALB/c mice that either accepted or rejected B6 skin allografts. Spleen T cells of eight nonirradiated BALB/c chimeras bearing C57BL/6 skin transplant N100 days and of seven BALB/c recipients that rejected C57BL/6 BM skin transplants after conditioning with 200 mg/kg CY were tested in MLR.
BM could not be omitted or replaced by local plug transplantation.
Fig. 3. Engraftment of C57BL/6 hematopoietic stroma microenvironment (HSM) in BALB/c mice treated with fractionated TLI (200 cGy each fraction) and conditioned for alloantigen-primed lymphocyte depletion. Nonirradiated BALB/c mice and animals treated with TLI were conditioned for APLD (C57BL/6 BMC IV and 200 mg/kg IP on the next day). One day after injection of CY recipients were transplanted under the kidney capsule with two femoral BM plugs obtained from C57BL/6 donors irradiated with 400 cGy (empty columns) or received in addition to the femoral plug implantation an intravenous injection with 3 107 BMC from C57BL/6 donors (solid columns). One month later, recipients were anesthetized and checked for the presence of ectopic bone. Reproduced with permission from Gurevitch O et al., Transplantation 68 (1999) 1362. Copyright 1999, L.W.W.
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Application of APLD-based reduced-intensity conditioning for BMT aimed for treatment of murine leukemia and solid breast carcinoma To assess the efficacy and safety of our reduced-intensity BMT protocol for the treatment of leukemia and metastatic solid tumors, we performed allogeneic BMT to the animals inoculated with malignant cells before conditioning [22]. BALB/c mice were given intravenously 106 BCL1 cells from mice with overt leukemia or transplanted ID into the right flank with 2 104 4T1 breast carcinoma cells. Preparation for BMT consisted of TLI (200cGy) on day 1 and APLD (IV priming with 3 107 C57BL/6 BMC on day 2, and IP injection with 200 mg/kg CY on day 3), and was followed by BMT from the same donor (3 107 cells IV on day 4). The results presented in Fig. 4C show that BMT performed after reduced-intensity conditioning for APLD prevented development of leukemia in 90% of recipients. All control mice treated only with 200 cGy TLI and 200 mg/ kg CY died of the disease (Fig. 4A). GVL effects were evidently mediated by the engrafted second BM transplant, since most animals treated by TLI, donor BMC, and CY, but no second infusion of donor BMC, died of leukemia (Fig. 4B). Late GVHD-related mortality in mice treated for leukemia by reduced-intensity BMT was higher than in healthy recipients of BMC (compare GVHD-related mortality in Figs. 1 and 4C). However, 60% recipients remained leukemia- and GVHD-free N170 days after BMT (Fig. 4C). These results show that nonmyeloablative BMT has a significant curative effect in leukemia-bearing mice. BMT performed after TLI and APLD also delayed the appearance of palpable solid 4T1 tumors for 1–2 weeks.
Moreover, 40% (14 of 35) transplanted mice did not develop tumors at all (Fig. 5C). Here, as well as documented in mice treated by BMT for leukemia, the second BM transplant mediated eradication of malignant cells. All animals preconditioned by TLI, donor BMC, and CY, but without a second infusion with donor BM cells, developed tumors (Fig. 5B). Most of cured recipients (10 of 14) also did not develop GVHD and remained healthy for the entire observation period. Yet, the results obtained by application of the same BMT protocol for the treatment of leukemia and solid tumor show that eradication of solid tumor after allogeneic BMT is much less effective than eradication of leukemia blasts. Prevention of diabetes by induction of chimerism in NOD mice Allogeneic BMT can prevent and even reverse autoimmune insulitis and overt diabetes. At the same time, BMT can induce donor-specific tolerance to islet allografts [23]. However, before BMT can be considered in the clinical setting, transplantation-related morbidity and GVHD-related mortality must be better controlled. Therefore, we have investigated our new reduced-intensity BMT strategy for safer treatment of autoimmune diabetes [19,24]. Consistent diabetes was induced in NOD mice after irradiation (650 cGy TBI) and intravenous transplantation of 2.5 107 diabetogenic spleen cells from overtly diabetic NOD mice. Three days later, NOD recipients were primed intravenously with donor C57BL/6 BM (3 107 cells), a day later inoculated intraperitoneally with CY (200, 100, or 60 mg/kg) for APLD, and 1 day later transplanted with a
Fig. 4. Anti-leukemia effect induced by non-T-cell-depleted allogeneic BMC transplanted after mild TLI and antigen-primed lymphocyte depletion. BALB/c mice were inoculated intravenously on day 0 with 106 BCL1 cells and treated with (A) TLI (200cGy) on day +1 and Cy (200 mg/kg IP) on day +3 (n = 31); (B) TLI on day +1, C57BL/6 BM (3 107 cells IV) on day +2, and Cy on day +3 (n = 14); (C) as in group B and the second inoculation of C57BL/6 BM (3 107 cells IV) on day +4 (n = 43). Survival of recipients treated by BMT (group C) was significantly better than in control groups (PA–C and B–C = 0.0001). Reproduced with permission from T. B. Prigozhina et al., Exp. Hematol. 30 (2002) 89. Copyright 2002, Elsevier Inc.
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Fig. 5. Treatment of breast cancer by transplantation of non-T-cell-depleted allogeneic BM following mild TLI and antigen-primed lymphocyte depletion. BALB/c mice, transplanted ID with 2 104 4T1 breast carcinoma cells on day 0, received (A) no treatment (n = 12); (B) TLI on day +1, 3 107 C57BL/6 BM cells on day +2, 200 mg/kg CY on day +3 (n = 32); C - as in group B and the second inoculation of C57BL/6 BM (3 107 cells IV) on day +4 (n = 35). Reproduced with permission from T. B. Prigozhina et al., Exp. Hematol. 30 (2002) 89. Copyright 2002, Elsevier Inc.
second inoculum of C57BL/6 BM (3 107 cells). All mice treated for APLD before BMT converted to chimeras with a high percentage of donor cells (Fig. 6A, 1–4). None of them developed diabetes for N3 months, demonstrating that BMC from a diabetes-resistant murine strain prevents immune destruction of the pancreas by diabetogenic effector cells. Unfortunately, all mice converted to chimeras by protocols
utilizing 200 mg/kg CY died later of chronic GVHD (Fig. 6B). When the second BM inoculum was omitted, donor chimerism was not achieved and all NOD mice died of diabetes (Fig. 6A, Gr. 5). Reducing the CY dose to 100 or 60 mg/kg caused significant improvement of the chimera’s survival. Most animals survived N300 days, remaining chimeras free of GVHD and diabetes (Fig. 6B).
Fig. 6. Allogeneic BMT prevents development of diabetes in NOD mice. Consistent diabetes was induced in NOD mice after irradiation (650 cGy TBI) and intravenous inoculation of 2.5 107 diabetogenic spleen cells from overtly diabetic NOD mice. Three days after transfer of diabetogenic cells, NOD recipients were primed intravenously with donor C57BL/6 BMC (3 107/mouse), a day later inoculated intraperitoneally with CY (200, 100, or 60 mg/kg), and 1 day after that transplanted with a second inoculum of donor BMC (3 107 cells). Omission or use of the first or the second BMC inoculation is shown in Fig. 5A by symbols or +. After transplantation, recipients were tested every 7 days for glucosuria and every 100 days for donor cell chimerism.
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Promotion of allogeneic BM engraftment after APLD by a reduced CY dose (100 mg/kg) The therapeutic effects of large CY doses are often associated with systemic toxicity. Therefore, with a view to safer clinical application in the future, we reduced the CY dose in APLD protocol to 100 mg/kg (in press). Fig. 7A shows that modified treatment converted to chimeras 46% of recipients (12 of 26) irradiated with 100 cGy TBI, 90% (9 of 10) recipients irradiated with 200 cGy TBI, and all recipients treated with 300 cGy TBI (n = 12). When priming with donor BMC was excluded from the protocol and all the C57BL/6 BMCs (6 107) were given as a bmegadoseQ [25] of BMC after CY to mice that had been exposed to 300 cGy TBI, only 33% (3 of 10) became chimeras. These results definitely show that conditioning for APLD facilitates engraftment of mismatched BMC significantly better than amplification of donor BMC numbers after general immunosuppression. Eighty percent chimeras obtained by BMT after low-dose TBI and APLD remained GVHD-free for N150 days (data not shown). Although donor T cells significantly facilitate engraftment of allogeneic BMC, most clinically approved protocols recommend depleting donor T cells from haplotype-mis-
Fig. 7. Evaluation of the minimal conditioning required for induction of transplantation tolerance with a reduced CY dose (100 mg/kg). (A) Transplantation of allogeneic BMC. BALB/c mice were treated with a range of TBI doses, inoculated with 100 mg/kg CY IP (day 1), and transplanted with 6 107 C57BL/6 BM cells intravenously (day 2, white bars). Alternatively, TBI-treated mice received intravenous inoculation of 3 107 C57BL/6 BMC (day 1), IP injection of 100 mg/kg CY (day 2), and 3 107 C57BL/6 BM (day 3, black bars). Data from several experiments were pooled (9 or more mice/group) and presented as a frequency of recipients with N25% donor cells in the blood as measured by FACS analysis 50 days after BMT. (B) Transplantation of semiallogeneic (C57BL/6 BALB/c) F1 BMC. Comparison of the efficacy of F1 BM cell engraftment in BALB/c recipients prepared for BMT by general immunosuppression with irradiation and 100 mg/kg CY (dense pattern columns) and in BALB/c mice prepared for BMT by irradiation and antigen-primed lymphocyte depletion (gray columns). Each group included nine mice or more.
matched BM transplants to avoid GVHD-related complications. Therefore, we examined the efficacy of allogeneic BMT under stringent conditions: low dose TBI, reduced CY dose (100 mg/kg), and transplantation of semiallogenic (C57BL/6 BALB/c) F1 BMCs that express transplantation antigens of both parents (H-2b and H-2d in our case) and lack anti-parental T cell clones (in press). Fig. 7B shows that the minimal radiation dose providing acceptance of semiallogenic BMC is significantly higher than the dose required for the engraftment of allogeneic BMC. However, the advantage of the selective APLD protocol over the bmegadoseQ protocol is also evident after transplantation of F1 BMC.
Discussion Two immunological barriers must be overcome for a successful mismatched BMT. One is rejection and the other is GVHD. Engraftment of mismatched BMC requires pretransplant depletion of most donor-reactive lymphocytes. All clinically approved anti-rejection therapies available today accomplish this task by providing general depletion of the recipients’ immune system. Myeloablative conditioning generally leads to engraftment of allogeneic BM but is associated with a high incidence of GVHDrelated mortality of recipients. Low-intensity preparative regimens allow inducing sufficient immunosuppression to permit engraftment of compatible hematopoietic stem cells and reduce GVHD-related mortality after BMT. Several factors may contribute to the low risk of GVHD after reduced-intensity BMT. Mild conditioning preserves the integrity of the gastrointestinal system and keeps the level of pro-inflammatory cytokines low [26,27]. Reducedintensity conditioning also preserves low numbers of T and bvetoQ cells of the host that cannot reject donor BMC but can resist or suppress their unlimited expansion [20,28]. However, though reduced-intensity regimens for BMT gained significant popularity, myeloablative immunosuppression is still required for providing acceptance of strongly immunogenic haploidentical hematopoietic stem cells. The results of our investigation show that selective depletion of alloantigen-activated donor-reactive cells by CY allows significant reduction of radiation dose essential for BM engraftment across strong MHC barriers, providing the advantages of reduced-intensity conditioning to recipients of allogeneic BM transplants. Engraftment of allogeneic BMC after reduced-intensity conditioning for APLD guarantees conversion of most recipients to GVHD-free chimeras and, in contrast to the old reduced-intensity experimental protocols recommending organ transplantation directly after APLD, provides acceptance of various donor tissue and organ allografts, including donor hematopoietic stromal progenitors and, most immunogenic, donor skin transplants.
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The potential importance of the transplantation of donor BM within its own microenvironment cannot be overestimated. It may provide an optimal opportunity for BM engraftment, especially for patients suffering from diseases caused by primary HSM abnormalities as well as from secondary HSM defects induced by intensive irradiation or chemotherapy. The reduced-intensity BMT protocol that we present here also provides significant direct graft-vs.-malignancy and graft-vs.-autoimmunity effects. In both cases therapeutic potential of BMT depended on the robust engraftment of donor BMC. One of the ways to improve engraftment of mismatched BM allografts in reduced-intensity protocols is to escalate the dose of transplanted cells (the bmegadoseQ approach, ref. [25]). Facilitation of engraftment by infusion of larger numbers of BM cells is based on the fact that donor T cells, NK cells, and veto cells within the CD34+hematopoietic progenitor cell population play an active role in engraftment and can remove recipients’ residual donor-reactive cells. Our results show, however, that conditioning for APLD provided significantly better engraftment of allogeneic and semiallogenic BMC in reduced-intensity protocols than transplantation of the same total cell dose as a bmegadoseQ after nonselective immunosuppression with identical radiation and CY doses. Thus, we consider that treatment of recipients with CY soon after priming with lymphoid cells of the future donor results in better eradication of donor-reactive host clones than relatively weak bvetoQ activity mediated after transplantation by hematopoietic stem cells. All donor hematopoietic cells that we tested (BMC, spleen cells, and blood cells) could be effectively used for priming of donor-reactive host cells [20]. We believe that unmanipulated BMC or even spleen cells or blood mononuclear cells can be safely used for priming in APLD protocols because donor cells used for priming are eradicated after optimally timed administration of CY. Thus, application of APLD conditioning in future BMT protocols may possibly allow for a reduction in both the radiation dose essential for engraftment of T-cell-depleted allogeneic BMC, and the numbers of donor stem cells that must be selected for transplantation by immunomagnetic separation methods. Systemic (IV) inoculation of a large dose of donor lymphocytes was found to be essential for the priming. Transplantation of the intravenously effective numbers of donor BM cells under the kidney capsule as a plug or local transplantation of a donor skin graft (data not shown) could not provide significant APLD after CY administration. We suppose that only circulating donor lymphocytes induce systemic activation of donor-reactive host cells essential for profound APLD by CY. Recent generation by Quezada et al. of a T-cell receptor transgenic model of CD4+T-cellmediated rejection allowed visualization of the fate and function of CD4+T cells, specific to MHC donor antigens. The results obtained in their study directly demonstrate that
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robust systemic proliferation of donor-reactive CD4+T cells is seen only after IV donor cell transfusion [29]. Although our reduced-intensity protocol proved effective for induction of transplantation tolerance to allografts, its wider clinical application was suspended because of the relatively high CY dose considered essential for APLD. Mild irradiation of recipients prior to priming with donor BMC allowed the tolerance-inducing dose of CY to be reduced to a clinically acceptable level (100 mg/kg). Binding of a specific antibody to the CD154 molecule (CD40 ligand) expressed on recently activated CD4+T cells, but absent on naive lymphocytes, and subsequent eradication of activated donor-reactive cells by antibody and complement [30], may present another example of selective depletion of alloantigen-primed cells for tolerance induction. CD154-specific antibodies are especially potent in animals primed by donor-specific transfusion (DST) and subsequently transplanted with donor BMC [31,32]. DST causes a robust systemic proliferation of donor-reactive CD4+T cells that is followed by a state of temporary hyporesponsiveness of CD4+[29] and CD8+T cells [31]. Application of CD154-specific antibodies after DST additionally reduces expansion of allograft-responding CD4+T cells, causes significant depletion of responding CD4+clones [29], and facilitates acceptance of donor BMC in mildly irradiated recipients. As in our experimental model, induction of robust donor cell chimerism after formation of donor-specific transplantation tolerance with DST- and CD154-specific antibody is essential for permanent acceptance of donor organ and skin transplants. Both chimeras obtained by BMT after APLD and chimeras induced by BMT after conditioning with CD154-specific antibodies are specifically unresponsive to host and donor transplantation antigens in vitro [20,32,33] and are capable of rejecting unrelated skin grafts. Kurtz et al. [33] were unable to find evidence of regulatory/suppressor mechanisms for maintenance of transplantation tolerance in chimeras generated by donor BMT into mice treated with CD154-specific mAbs and therefore came to conclusion that donor-reactive host cells are eliminated or become irreversibly anergic during formation of the transplantation tolerance. Our present data (in press) revealed that donor spleen cells of full chimeras obtained by BMT after APLD conditioning can neither generate GVHD after transplantation to irradiated F1 recipients nor suppress GVHD generated by spleen cells of naive C57BL/6 mice. These experimental data support our assumption that the two methods are based on the same principles: selective elimination of most activated donorreactive lymphocytes during the conditioning phase of the protocol and facilitation of organ engraftment by elimination of residual donor-reactive lymphocytes after donor BMT and chimerism formation. We revealed that CD154-specific antibodies synergize with CY [34]. A short course of CD154-specific antibodies helps to reduce the APLD-effective CY dose to 100 mg/kg without additional radiation of recipients. CD154-specific
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antibodies can also downregulate the ability of transplanted BMC to induce GVHD [35]. However, our experimental data and results of others have documented that persisting CD154-specific mAb impair generation of GVL effects after BMT [35,36]. Furthermore, CD154-specific mAb may cause thromboembolic complications in some patients [36], and therefore this mAb was not approved for clinical application. In conclusion, our data and results obtained by other investigators indicate that the future of the reduced-intensity BMT regimens lies in the development of selective immunosuppression methods that would allow targeted eradication of donor-reactive host cells. We feel that today, in view of the nonavailability of safe antibodies against the CD154 molecule or other clinical grade antibodies to the markers of activated T cells, conditioning for alloantigenprimed lymphocyte depletion (APLD) by a low CY dose may represent the first clinically acceptable step in this direction. This conditioning method is significantly more effective in the formation of transplantation tolerance to BM allografts than bmegadoseQ BMT after nonselective immunosuppression. The treatment is inexpensive, relatively safe, and potentially effective for BMT carried out for the therapy of malignant and nonmalignant diseases and for organ transplantation. The introduction of APLD into clinical practice may improve current reduced-intensity conditioning protocols for transplantation of BMC from haploidentically mismatched and unrelated donors. References [1] P.L. Weiden, K.M. Sullivan, N. Flournoy, R. Storb, E.D. Thomas, Antileukemic effect of chronic graft-versus-host disease: contribution to improved survival after allogeneic marrow transplantation, N. Engl. J. Med. 304 (1981) 1529 – 1533. [2] R. Hirschhorn, Treatment of genetic diseases by allotransplantation, Birth Defects, Orig. Artic. Ser. 16 (1980) 429 – 444. [3] P.B. Medawar, Two notes on immunological tolerance, Transplant. Bull. 4 (2) (1957) 72 – 74. [4] S. Slavin, S. Strober, Z. Fuks, H.S. Kaplan, Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts, J. Exp. Med. 146 (1977) 34 – 48. [5] Y. Sharabi, D.H. Sachs, Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen, J. Exp. Med. 169 (2) (1989) 493 – 502. [6] S. Giralt, E. Estey, M. Albitar, et al., Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative therapy, Blood 89 (1997) 4531 – 4536. [7] S. Slavin, A. Nagler, E. Naparstek, et al., Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases, Blood 91 (1998) 756 – 763. [8] I.F. Khouri, M. Keating, M. Korbling, et al., Transplant-lite: induction of graft-versus-malignancy using fludarabine-based nonablative chemotherapy and allogeneic blood progenitor-cell transplantation as treatment for lymphoid malignancies, J. Clin. Oncol. 16 (1998) 2817 – 2824.
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