Stable Chimerism Research Articles

Mini-Haploidentical Transplantation for Refractory Acute Myeloid Leukemia.

The quest for effective, yet less toxic means of treating advanced diseases has led the bone marrow transplantation (BMT) researchers and clinicians searching for the optimal balance between host immune suppression, stable chimerism, effective graft versus tumor response and overall survival. We asked the question “how low can you go?” and approached this from the minimalist view. Born out of murine studies, mini-HLA-identical bone marrow transplantation (BMT) using 100cGy as host conditioning followed by non-mobilized peripheral blood lymphocyte infusion of 1×108 CD3 cells/kg was successful in achieving a complete responses (CR) in 4 of 11 refractory hematologic malignancy patients (Blood100:442, 2002). This study has been extended using HLA-haploidentical donors. We have evaluated CD3+ cell dose escalation with HLA-haploidentical transplantation in patients with refractory malignancies. We have performed 42 HLA-haplo transplants with escalation of the CD3 dose from 1x106–2x108cells/kg using G-CSF primed peripheral blood stem cells, with a conditioning regimen of 100cGy TBI. Twenty patients were treated at the highest level. The CD34 dose infused was always >2x106cell/kg. Ages ranged from16–82 years. We have treated 15 patients with solid tumors and 27 patients with hematologic malignancies consisting of AML (13), NHL (5), Multiple Myeloma (5) CML (3), and HD (1). One treatment related death occurred from grade IV aGVHD. Most patients had a transient febrile engraftment syndrome believed to be immunologically based and represents neither hyperacute nor acute graft versus host disease. There were no objective responses in the patients with solid tumors. One of 5 patients with NHL had a partial response. We observed several encouraging responses in patients with refractory AML. We treated 13 patients with AML and have obtained a complete response in three patients and transient responses in 7 of 12 evaluable patients (survival >2wks after BMT). The median time of transient response was19 days± 5 days and was defined as a persistent loss of peripheral blood blasts and/or more than 50% reduction in marrow blasts as seen on biopsy. In these patients, improvement in blood counts was seen in 3 patients and an improved performance status in 6 of 12. All responses occurred at CD3 levels of 1–2x108 cells/kg. All complete and transient responses occurred in the absence of measurable donor chimerism (<5%). Donor cells labeled with indium111 analyzed 1,2,3 and six days following BMT in one patient showed persistent signal in the bone marrow and spleen. The kinetics of detectable donor cells in the peripheral blood and marrow show near complete homing to the marrow by 24 hours with a subsequent loss of detectable chimerism 6 days after BMT. Serial bone marrow biopsies performed in several patients show evidence for large tumor reduction and early resumption of normal hematopoiesis. In summary, TBI of 100cGy followed by HLA-haplo transplant is a biologically active therapy for refractory AML. The responses outside of chimersim are intriguing and warrant further investigation. This well tolerated treatment produced minimal toxicity for the majority of patients. Theories on biological effect include an initial graft vs. tumor cell kill, altered host immune response breaking host tumor tolerance, persistent non-detectable microchimerism or a combination of any of the three.

Keratinocyte Growth Factor Ameliorates Acute Graft-versus-Host Disease in a Novel Nonmyeloablative Haploidentical Transplantation Model.

Allogeneic stem cell transplantations (SCT) are currently being used as a therapy for hematological malignancies, some solid tumors and non-malignant bone marrow deficiencies. Nevertheless, clinical applicability is limited due to toxicity of conditioning regimens, graft-versus-host disease (GVHD) and the scarcity of HLA-identical family donors. New concepts are based on nonmyeloablative conditioning to reduce toxicity, prevention or amelioration of GVHD and the use of haploidentical donors to increase donor availability.To meet these requirements, we have developed a nonmyeloablative conditioning regimen in a haploidentical F1 → F1 mouse model. These mini-transplantations, consisting of low dose total body irradiation and cyclophosphamide-based chemotherapy, resulted in stable full donor chimerism, but also in the development of GVHD. Administration of keratinocyte growth factor (KGF) before and after SCT resulted in reduced GVHD, evident as reduced weight loss and a lesser degree of dermatitis than in saline-treated controls. KGF preserved plasma citrulline and TNF-a levels, both indicative for reduced radiation-induced injury to the gastrointestinal tract. Citruline has recently been described as a new marker for gastrointestinal toxicity. Six months after transplantation, survival rates were significantly higher in KGF-treated animals (57% as compared to PBS-treated controls (5.3%). [Display omitted] These data indicate that nonmyeloablative haploidentical transplantations might be feasible and that KGF might contribute substantially to reduction of lethal GVHD, also after nonmyeloablative procedures. For clinical transplantation such an effect would be significant if intensive T cell depletion might be prevented in haploidentical transplantation protocols and therefore reduced infection rates can be obtained.

SIROLIMUS CONTROLS GVH REACTIVITY IN ANTI-CD45 MAB CONDITIONED ALLOGENEIC BMT IN ORDER TO ACHIEVE TOLERANCE BY MIXED CHIMERISM

O271 Aims: We could recently introduce in a rat system less-toxic BMT conditioning using only an anti-CD45 mAb. As severe side effect in fully MHC mismatch setting risk of aplasia was observed and most likely provoked by an atypical GvH reaction on bone marrow level. In this study, unspecific immunosuppression (IS) was tested as addition to anti-CD45 mAb conditioning for establishment of a safe and tolerizing BMT protocol. Methods: The model consisted of LEW.1W (RT1u, RT7a) recipients, LEW.7B (RT1l,RT7b) BM donors, a single injection of anti-CD45 mAb (7.5 mg/kg BW) detecting RT7a as conditioning and orally applicated cyclosporine A (CsA 5 or 15 mg/kg BW) or sirolimus (1 or 2 mg/kg BW) as IS for 14 days. Animals were monitored for clinical status and haematological parameters. They were tested for multilineage chimerism at day 20 and day 100 by FACS. Long-term survivors received skin grafts of BM donor- and 3rd party-type on day 100. Results: Only animals of high-dosage sirolimus (2 mg/kg BW) treated group showed reproducible stable chimerism (9 of 12) compared to all other groups (no IS: 1 of 9, CsA 5: none of 9, CsA 15: 1 of 9, sirolimus 1: 1 of 9). Thereby, initial leukocyte depletion by anti-CD45 mAb as parameter for conditioning extent was highly critical for BMT success. Animals, which did not became stable chimeras, showed transient chimerism but suffered mainly from severe aplasia syndrome and died between days 29 to 70 (no IS: 7 of 9, CsA 5: 9 of 9, CsA 15: 6 of 9, sirolimus 1: 5 of 9, sirolimus 2: 3 of 12). GvHD signs were only seen in high-dosage CsA treated animals (2 of 7). The attempt to stabilize BM engraftment by prolongation of high-dosage CsA treatment (28 days) even aggravated GvHD symptoms (4 of 6) like thymic selection disturbance by thymic irradiation did in high-dosage sirolimus group (4 of 4). Moreover, analysis of persisting anti-CD45 mAb coated thymocytes suggested reduced thymocyte selection progress. Long-term lineage-typing of stable chimeras from high-dosage sirolimus group revealed mainly high-graded mixed chimerism in lymphoid and fully chimerism in myeloid lineages. Stable chimeras tolerated skin grafts only of donor-type (> 100 days), while acutely rejecting 3rd party control grafts like none chimeric survivors rejecting both graft-types. Conclusions: Addition of high-dosage sirolimus to isolated anti-CD45 mAb BMT conditioning reduced significantly risk of fatal aplasia in allogeneic BMT. This is in contrast to CsA, where the combination of intrathymically persisting anti-CD45 mAb and CsA seems to disturb TC selection processes leading to severe GvHD without stabilizing HSC engraftment. Without any clinical signs for GvHD the combination of anti-CD45 mAb and sirolimus in allogeneic BMT is promising for generation of stable mixed chimerism and tolerance induction.

DIFFERENT ROUTES OF DONOR DERIVED HEMATOPOIETIC STEM CELLS TRANSPLANTATION FOR DONOR SPECIFIC CHIMERISM INDUCTION ACROSS MHC BARRIER

P940 Aims: Transplantation of donor–derived stem cells can improve organ allograft survival in animal models. This study was designed to investigate the effect of different routes of stem cell transplantation (SCT) on donor specific tolerance induction across MHC barrier under short-term CsA monotherapy and αβTCR/CsA treatment protocols. Methods: Forty-eight SCT were performed between BN (RT1n) donors and Lewis (RT1l) recipients. Intraosseous and intravenous SCT was studied in 6 groups of 8 animals each receiving 35x106 (n=4) and 70x106 (n=4) SCT. Groups I and II served as controls and received SCT but no treatment. Groups III and IV received CsA monotherapy for 7-days, Groups V and VI were under αβTCR/CsA protocol for 7-day. Flow cytometry analysis was used for evaluation of immunodepletion of T-lymphocytes and multilineage donor specific chimerism for MHC class-I (RT1n) for RT1n/CD4, RT1n/CD8 and RT1n/CD45RA antigens in peripheral blood of recipients at post-transplant days 7, 21, 35 and 63. Results: All animals survived without sign of graft versus host disease. At day 63 non-treatment groups receiving 70x106 SCT developed 4.3% of multilineage donor-specific chimerism following intraosseous transplantation and 1.0% following intravenous transplantation. At day 7 under CsA monotherapy and 70x106 SCT, chimerism level for 1.5% RT1n/CD4 was induced in intraosseous and 10.5% in intravenous transplanted groups. At day 63 following intraosseous transplantation chimerism for RT1n/CD4 was stable, but following intravenous transplantation significantly declined to 0.8%. Under αβTCR/CsA protocol T-lymphocyte subpopulation was significantly depleted at day 7 and repopulated to pre-transplant level at day 63. After intraosseous transplantation of 70x106 SCT at day 7 chimerism was 5.7% for RT1n/CD4, and increased up to 6.5% at day 63. In contrast in intravenously transplanted groups at day 7 chimerism was higher and showed 23.7% for RT1n/CD4 antigens, however declined to 0.9% at day 63. Multilineage chimerism was 75% higher in intraosseous (9.9%) when compared with intravenous (3.4%) transplant groups receiving 70x106 SCT at day 63. Following intraosseous SCT under αβTCR/CsA protocol, multilineage chimerism was 50% higher in group receiving 70x106 stem cells (9.9%) compared to 35x106 stem cells (4.9%). Conclusions: Stem cell transplantation under αβTCR/CsA protocol showed better engraftment of donor cells following intraosseous delivery leading to development of higher level (75%) of multilineage donor-specific chimerism when compared with intravenous transplantation. High level of chimerism in intravenously transplanted groups was transient, whereas intraosseous transplantation induced stable chimerism, which was maintained up to 63 days post-transplantation. This strategy of intraosseous stem cell transplantation under αβTCR/CsA protocol could be applied for chimerism and tolerance induction during transplantation of composite and solid organ allografts.

Modulating erythrocyte chimerism in a mouse model of pyruvate kinase deficiency

AbstractIn vivo selection may provide a means to increase the relative number of cells of donor origin in recipients with hemopoietic chimerism. We have tested whether in vivo selection using chemical inducers of dimerization (CIDs) can direct the expansion of transduced normal donor erythrocytes in recipients with chimerism using a mouse model of pyruvate kinase deficiency. Marrow cells from normal CBA/N mice were transduced with a vector (F36VmplGFP) that promotes cell growth in the presence of CIDs. Transduced cells were then transplanted into minimally conditioned, pyruvate kinase-deficient recipients (CBA-Pk-1slc/Pk-1slc) to establish stable chimerism. CID administration resulted in expansion of normal donor erythrocytes and improvement of the anemia. The preferential expansion of normal erythrocytes also resulted in a decrease in erythropoietin levels, reducing the drive for production of pyruvate kinase-deficient red blood cells. CID-mediated expansion of genetically modified erythrocytes could prove a useful adjunct to transplantation methods that achieve erythroid chimerism. (Blood. 2004;103:4432-4439)

Use of lentiviral vectors to induce long-term tolerance to gal(+) heart grafts.

Tolerance to organ grafts has been achieved by establishing a state of stable mixed-cell chimerism after bone marrow transplantation. Gene therapy has been applied to establish chimerism for cells expressing galactose alpha 1,3 galactose in alpha 1,3 galactosyltransferase deficient (gal knockout) mice using retroviral vectors. Limitations to the success of this methodology include short-term expression of the introduced gene and rejection of gal hearts transplanted into these animals within a month. Autologous bone marrow from gal knockout mice was transduced with a lentiviral vector expressing porcine alpha 1,3 galactosyltransferase and transplanted into lethally irradiated gal knockout mice. Chimerism was monitored by flow cytometry. Hearts from wild type mice (gal/) were transplanted into these animals and palpated daily. Xenoantibodies directed at the gal carbohydrate or porcine xenoantigens were detected by enzyme-linked immunosorbent assay. Hearts from wild-type gal/ donors were permanently accepted in all mice receiving autologous, transduced bone marrow before heart transplantation. Control mice rejected gal hearts within 12 to 14 days. Histologic analysis demonstrated classical signs of rejection in controls and normal myocardium with no evidence of rejection in mice chimeric for the gal carbohydrate. Anti-gal xenoantibodies were not produced in gal chimeras, but normal antibody responses to other xenoantigens were detected. Specific tolerance for the gal carbohydrate was achieved by this procedure. These experiments report the first demonstration of permanent survival of gal hearts after transplantation with autologous, transduced bone marrow. Transduction with lentiviral vectors results in long-term, stable chimerism at levels sufficient to induce long-term tolerance to heart grafts in mice.

Stem cell–mediated tolerance inducing strategies in organ transplantation

The scope of possible tools to modulate the recipients immune response towards tolerance induction basically includes deletional and non deletional mechanisms, which are currently targeted by various strategies including monoclonal antibodies, cytokine deviation, chimerism induction and the support of regulating T-cells. Here we summarize the main findings in the field derived from experimental animal studies and currently performed clinical studies. This review focuses to give a clinically relevant overview over relevant tolerance inducing concepts, taking into consideration risk profiles and clinical efficacy associated with specific immunosuppressive regiments currently applied in the clinical setting of transplantation.

Nonmyeloablative bone marrow transplantation of BXSB lupus mice using fully matched allogeneic donor cells from green fluorescent protein transgenic mice.

Male BXSB mice, a mouse model of systemic lupus erythematosus, were given bone marrow transplants (BMT) at 20 wk of age using MHC-matched donor cells and nonmyeloablative conditioning (550 cGy irradiation). Transplanted mice and irradiation controls were followed for a period of 20 wk. Mice transgenic for green fluorescent protein were used as donors to allow tracking of donor cells and a determination of chimerism. Radiation controls had reduced renal pathology at 10 wk posttransplant, but not at 20 wk compared with untreated mice, while nonmyeloablative BMT mice had significantly reduced pathology at both time intervals. The monocytosis characteristic of older BXSB mice was also reduced by BMT, but the treatment did not prevent production of Ab to dsDNA. A stable chimerism of 24-40% donor CD45-positive cells was achieved in spleen and bone marrow, and there was no evidence of clinical graft vs host disease. Donor cells were detected in most recipient organs, notably the thymus and renal glomeruli. The results suggest that complete depletion of mature lymphocytes or of progenitor stem cells is not required to control lupus nephritis in BXSB mice.

Tubular chimerism occurs regularly in renal allografts and is not correlated to outcome.

Recent studies have demonstrated an integration of recipient-derived progenitor cells into solid allografts with differentiation into parenchymal cells. Whether or to what extent this phenomenon influences allograft outcome has still to be elucidated. To detect epithelial chimerism tubular cells were harvested from sequential renal allograft biopsy samples by laser microdissection in 36 patients. Recipient-derived cells were detected by short-tandem repeat-based genotyping. In cases with gender-mismatched transplantation, chimerism was semiquantitatively evaluated by in situ hybridization for the Y-chromosome. Findings were correlated to different pathomechanisms of epithelial injury as well as to morphologic and clinical outcome. Epithelial chimerism was detectable as early as 8 d after transplantation and lasted for 8 yr. A total of 88% of the patients showed an epithelial chimerism; 72% had a stable chimerism in sequential biopsy samples. Evaluation of Y-chromosome by in situ hybridization revealed low percentages of chimerical tubular epithelial cells (2.4% to 6.6%). No correlation to morphology was found. Chimerism was detectable in inconspicuous protocol biopsy samples, cases of drug toxicity, and rejected allografts with and without chronic changes. No correlation was found to allograft function. Epithelial microchimerism is an early and persistent phenomenon after renal transplantation. There is no correlation to morphologic or functional outcome. Probably recipient-derived stem cells contribute in a minor fashion to tissue homeostasis, and cell turnover in renal allografts is predominantly enabled by donor cell renewal.

Targeted bone marrow radioablation with 153Samarium-lexidronam promotes allogeneic hematopoietic chimerism and donor-specific immunologic hyporesponsiveness

Transplantation tolerance, defined as acceptance of a graft by an otherwise fully immunocompetent host, has been an elusive goal. Although robust tolerance has been achieved by the induction of stable hematopoietic chimerism after bone marrow transplantation, lethal or sublethal radiation conditioning used to induce long-term chimerism precludes its clinical use. We studied whether targeted delivery of radiation to bone marrow could allow for bone marrow cell (BMC) engraftment, chimerism, and donor-specific tolerance in the absence of the side effects associated with external irradiation. We administered a radioactive bone-seeking compound (Samarium-Lexidronam, Quadramet, Berlex Laboratories, Wayne, NJ) together with transient T-cell costimulatory blockade to recipient mice. Allogeneic BMCs were given 7 or 14 days after preconditioning. Costimulatory blockade was obtained by the use of an anti-CD154 antibody for 4 weeks. Chimerism was assessed by flow cytometry. Mice then received donor-specific and third-party skin grafts. Graft survival was analyzed with mechanisms of donor-specific hyporesponsiveness. High levels of stable chimerism across an allogeneic barrier were achieved in mice by a single administration of Samarium-Lexidronam, transient T-cell costimulatory blockade, and BMC transplantation. A large percentage of chimeric animals retained donor-derived skin grafts for more than 120 days without requiring additional immunosuppression, suggesting that harsh cytotoxic preconditioning is not necessary to achieve stable chimerism and donor specific hyporesponsiveness. Analysis of the T-cell repertoire in chimeras indicates T-cell deletional mechanisms. These data broaden the potential use of BMC transplantation for tolerance induction and argue for its potential in treating autoimmune diseases.

No evidence for significant transdifferentiation of bone marrow into pancreatic beta-cells in vivo.

Several recent studies have suggested that the adult bone marrow harbors cells that can differentiate into tissues from all three germ layers. Other reports have contradicted these findings or attributed them to cell fusion. In this study, we investigated whether bone marrow-derived cells contribute to the renewal of adult pancreatic endocrine cells, in particular insulin-producing beta-cells, in vivo. To address this issue, we studied mice transplanted with green fluorescent protein (GFP)-positive, sex-mismatched bone marrow. We also extended our studies to pancreatic injury models (partial pancreatectomy and streptozotocin administration). All animals showed stable full donor chimerism in the peripheral blood and microscopic analysis at 4-6 weeks and 3 months after transplantation, indicating that the GFP(+) and Y chromosome-positive donor bone marrow contributed substantially to blood, lymphatic, and interstitial cells in the pancreas. However, after examining >100,000 beta-cells, we found only 2 beta-cells positive for GFP, both of which were in control animals without pancreatic injury. Thus our study results did not support the concept that bone marrow contributes significantly to adult pancreatic beta-cell renewal.

Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine.

Tolerance to composite tissue allografts might allow the widespread clinical use of reconstructive allotransplantation if protocols to achieve this could be rendered sufficiently nontoxic. The authors investigated whether tolerance could be generated in miniature swine to composite tissue allografts across a major histocompatibility (MHC) barrier. A clinically relevant tolerance protocol involving hematopoietic cell transplantation without the need for irradiation or myelosuppressive drugs was tested. Seven recipient animals were transiently T-cell depleted and a short course of cyclosporine was initiated. Twenty-four hours later, a donor hematopoietic cell transplant consisting of cytokine-mobilized peripheral blood mononuclear cells or bone marrow cells and a heterotopic limb transplant were performed. In vitro anti-donor responsiveness was assessed by mixed-lymphocyte reaction and cell-mediated lympholysis assays. Acceptance of the limb allografts was determined by gross and histologic appearance. Chimerism in the peripheral blood and lymphohematopoietic organs was assessed by flow cytometry. All seven experimental animals accepted the musculoskeletal elements but rejected the skin of the allografts. All but one of the animals displayed donor-specific unresponsiveness in vitro. The animals that received cytokine mobilized-peripheral blood mononuclear cells showed chimerism but had clinical evidence of graft-versus-host disease (GVHD). None of the animals that received bone marrow cells showed stable chimerism and none developed GVHD. This protocol can achieve tolerance to the musculoskeletal elements of composite tissue allografts across an MHC barrier in miniature swine. Stable chimerism does not appear to be necessary for tolerance and may not be desirable because of the risk of GVHD.

Development of donor‐specific chimerism and tolerance in composite tissue allografts under αβ‐T‐cell receptor monoclonal antibody and cyclosporine a treatment protocols

Recently, we induced donor-specific tolerance to rat hindlimb allografts under a 35-day course of alphabeta-T-cell receptor monoclonal antibody (alphabeta-TCR mAb) and cyclosporine A (CsA). In this report, we investigated the role of shorter alphabeta-TCR/CsA protocols on tolerance induction. We performed 52 hindlimb transplantations, between Lewis-Brown-Norway (LBN, F1) donors and Lewis recipients to test the impact of 21-, 7-, and 5-day protocols of combined alphabeta-TCR/CsA treatment on tolerance induction. Donor-specific tolerance and immunocompetence were tested by mixed lymphocyte reaction (MLR) in vitro and by standard skin grafting in vivo. The efficacy of immunosuppressive protocol and donor-specific chimerism was assessed by flow cytometry. All transplants under 5, 7, and 21 days of combined alphabeta-TCR/CsA therapy survived over 350 days. Clinical tolerance and immunocompetence were confirmed by skin grafting in vivo and MLR in vitro. Flow cytometry revealed a high level of donor chimerism in the peripheral blood of long-term survivors. The extension of survival of limb allografts and allo-unresponsiveness were directly associated with the development of stable chimerism in the tolerant recipients.

Correction of a large animal model of type I Glanzmann's thrombasthenia by nonmyeloablative bone marrow transplantation

Objective The purpose of this study was to determine if nonmyeloablative bone marrow transplantation would induce stable hematopoietic chimerism that would correct the bleeding diathesis associated with type I Glanzmann's thrombasthenia (GT). Methods Three young dogs (less than 12 weeks of age) with GT were transplanted with DLA-matched bone marrow from littermates. Recipients received a sublethal dose (200 cGy) of total-body irradiation (TBI) prior to infusion with bone marrow (1–4×10 8 cells/kg). Recipient dogs were immunosuppressed with cyclosporine (15 mg/kg) and mycophenolate mofetil (10 mg/kg). Chimerism was determined by quantitation of donor microsatellite repeat polymorphisms in peripheral blood DNA and by flow cytometry to detect the presence of glycoproteins IIb and IIIa on platelets. Platelet function was assessed by a clot retraction test. Results One dog died one week posttransplant due to hemorrhage. Another dog died four weeks posttransplant from an unrecognized congenital heart defect and complications due to canine distemper virus infection. At the time of death, microsatellite analysis indicated 35 to 50% chimerism. Flow cytometry showed 20% of circulating platelets positive for glycoproteins IIb and IIIa. The third dog is alive and doing well approximately two years posttransplant. Hematopoietic chimerism has been sustained at 35 to 60% with approximately 30% of the platelets positive for glycoproteins IIb and IIIa. Platelet function is normal based on clot retraction. The animal does not have clinical signs of bleeding. Conclusions These observations suggest that GT and perhaps other severe inherited platelet disorders can be corrected using nonmyeloablative bone marrow transplantation to establish partial chimerism with normal platelets in the platelet compartment.

Allogeneic Parenchymal and Hematopoietic Tissues Differ in Their Ability to Induce Deletion of Donor-Reactive T Cells

The establishment of immune tolerance to self antigen expressed exclusively in the periphery is a crucial yet incompletely understood feature of the immune system. A dominant concept of peripheral tolerance has been that exposure of T cells to signal one, the TCR–MHC interaction, in the absence of signal two, or costimulation, is a major mechanism of peripheral tolerance. This model suggests that any cell type that expresses MHC-peptide complexes, be they of self or foreign origin, should have the capacity to tolerize antigen-specific T cells when critical costimulatory interactions are interrupted. However, a spectrum of responses, from permanent engraftment to rapid rejection, has been observed in various transplantation models utilizing costimulatory blockade. Therefore we undertook a series experiments to directly assess the tolerogenic potential of donor hematopoietic and parenchymal cells. We find that allogeneic tissues differ profoundly in their ability to promote peripheral tolerance concurrent with combined blockade of B7-CD28 and CD40-CD40L pathways. Non-vascularized and vascularized parenchymal grafts as well as donor-specific transfusions promote varying degrees of donor-specific hyporesponsiveness, but fail to induce donor-reactive T-cell deletion; whereas establishment of stable hematopoietic chimerism promotes specific tolerance mediated by deletion of donor-reactive cells in the periphery.

Adoptive Immunotherapy in Chimeras with Donor Lymphocytes

Allogeneic stem cell transplantation has a well-defined indication in the treatment of hematological malignancies. The beneficial immune effect of allogeneic marrow transplantation has long been known, but only recently have methods been developed to separate the graft-versus-leukemia (GVL) effect from graft-versus-host disease (GVHD). Animal experiments have shown that lymphocytes from the marrow donor can be transfused without causing severe GVHD if stable chimerism and tolerance is established. First clinical studies have been preformed in patients with recurrent chronic myelogenous leukemia. In these patients complete molecular remissions were induced that persist without further maintenance treatment. These results have been confirmed in larger multicenter studies in Europe and the USA. The best results were obtained in chronic myelogenous leukemia (CML); repeated successes have been reported in relapsing acute myeloid leukemia (AML), myelodysplastic syndromes and multiple myeloma (MMY), and rare responses were reported for acute lymphoid leukemia. Contrary to animal experiments GVHD has been observed in human patients although to a lesser extent than expected in transplants not given immunosuppression. Secondly myelosuppression has been observed in patients treated with relapsing CML. In CML the incidence of GVHD could be reduced by depleting CD8+ T cells from the donor lymphocyte concentrate. Alternatively only small numbers of T lymphocytes can be transfused and in the case of failing responses, the numbers of donor lymphocytes may be increased. Results in recurrent AML have been improved by the use of low-dose cytosine arabinoside, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor mobilized blood cells as compared to lymphocytes only. In MMY the response rate is higher than in AML, but the remissions are of limited duration in most patients. Several protocols have been designed to include preemptive donor lymphocyte transfusion in patients with a high relapse risk after transplantation. Problems remain to avoid chronic GVHD and to circumvent the immune escape mechanisms of leukemia.

Engraftment of quiescent progenitors and conversion to full chimerism after nonmyelosuppressive conditioning and hematopoietic cell transplantation in miniature swine

Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoietic cell transplantation that leads to mixed chimerism and allograft tolerance in miniature swine across minor and major histocompatibility disparities. Stable chimerism persisted in most of these animals but was restricted to T cells and confined to peripheral blood. Because of the importance of myeloid and erythroid progenitors for the treatment of hematologic disorders, the objective of this study was to assess whether such cells existed in the bone marrow of these lymphoid chimeras as an indication of functional engraftment. Colony-formation assays were performed on donor inocula before infusion and on bone marrow cells harvested from the transplant recipients. Donor-origin myeloid/erythroid progenitor colonies were detected in bone marrow from 6 of 7 lymphoid chimeric recipients. A delayed donor leukocyte infusion successfully converted a stable lymphoid chimera to full multilineage chimerism within 2 weeks. Donor-origin myeloid/erythroid progenitors could be detected in the bone marrow of a host-matched recipient after myeloablation and adoptive transfer of mobilized cells from one of the engrafted lymphoid chimeras. These data suggest that even when only lymphoid chimerism is readily detected by flow cytometry, dormant myeloid/erythroid progenitors can exist and subsequent conversion to full donor chimerism can be achieved. The ability to establish multilineage engraftment and chimerism without significant toxicity may have important clinical implications for the management of nonmalignant hematopoietic disorders and hematologic malignancies.

Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versus-host disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation

In an attempt to capture graft-versus-tumor effects without graft-versus-host disease (GVHD), the authors initiated a trial of nonmyeloablative allogeneic bone marrow transplantation (BMT) in patients with advanced hematologic malignancies, with the majority of patients having chemotherapy-refractory disease. Forty-two patients received an HLA-matched related donor BMT after a cyclophosphamide and antithymocyte globulin-based conditioning that also included thymic irradiation for patients who had not received prior mediastinal radiotherapy. Prophylactic donor leukocyte infusion (pDLI) at a dose of 1 × 10 7 CD3 + cells per kilogram were given beginning 5 weeks post-BMT to 16 patients with mixed chimerism (MC) but without GVHD, whereas 26 patients did not receive pDLI, either because of GVHD or early relapse. Twelve of 16 patients (75%) receiving pDLI had T cell chimerism at the time of pDLI ≥40%. These patients, by day 100 post-BMT, either converted to full donor chimerism (FDC) (n = 10) or had an increase in or stable donor chimerism (n = 2) after pDLI. Four of 4 patients whose T cell chimerism was ≤20% at the time of pDLI, lost the graft. In contrast, only 5 of 18 evaluable patients (28%) not receiving a pDLI converted to FDC by day 100 post-BMT, 7 maintained MC, and 10 of an evaluable 22 lost the graft. Patients who had undergone a previous autologous stem cell transplant had a higher rate of conversion to FDC (69% v 31%) and higher incidence of GVHD (69% v 34%) compared with those who did not have a previous autologous SCT. Eleven of 16 patients (69%) who received a pDLI achieved a remission with 50% 1-year progression-free survival rate and 44% 3-year overall survival rate. Nineteen of 42 patients (45%) had ≥grade II acute GVHD, including 12 after BMT and 7 after pDLI. Approximately one third of patients, after having initial MC, eventually lost their donor graft. The authors conclude that (1) pDLI has the potential to convert MC to FDC; (2) sustained remissions can be achieved in patients with chemorefractory hematologic malignancies who receive a pDLI, albeit with a significant risk of acute GVHD; and (3) the degree of donor T cell chimerism at the time of pDLI is predictive of the fate of MC, ie, donor T cell chimerism ≥40% or ≤20% at the time of pDLI correlates with conversion of MC or loss of the graft, respectively.

Nonlethal conditioning for the induction of allogeneic chimerism and tolerance to islet allografts.

We reported that tolerance to skin grafts can be achieved by chimerism induction by way of nonlethal conditioning. In the present study, we evaluated the outcome of islet allografts implanted either simultaneously or after donor bone marrow cell (BMC) infusion when nonlethal conditioning was used. B10 (H-2b) mice were conditioned with antilymphocyte serum (ALS), 100 cGy total body irradiation (TBI), and given 30 x 10(6) allogeneic (B10.BR, H-2k) BMC on day 0. On day 2, cyclophosphamide was given intraperitoneally (IP), followed by a second BMC infusion on day 3. After chimeras were typed for allogeneic BMC engraftment on day 28, animals were rendered diabetic chemically and transplanted under the kidney capsule with islet allografts genetically matched or disparate to the BM. Donor-specific islet grafts were accepted (median survival time [MST] > 180 days, n=6), whereas all major histocompatibility complex (MHC)-disparate third-party BALB/c (H-2d) islet grafts were rejected (MST=13.8 days, n=4). When B10.BR BMC and islets were given simultaneously, graft acceptance (MST >140 days, n=4) was observed. Surprisingly, when MHC-disparate third-party islets (BALB/c) were given together with B10.BR BMC, long-term survival was also observed (MST >100 days, n=3). These findings suggested that conditioning alone at the time of islet implant might induce long-term engraftment without further treatment. However, only chimeric animals accepted a second-set donor-specific graft, whereas all other groups rejected it. Our data indicates that stable allogeneic chimerism and islet indefinite survival can be achieved by the use of a nonmyeloablative protocol. The results of the conditioning-only experiments are consistent with the possibility of graft accommodation.

Chimerism analysis following allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning.

We have performed a prospective study to evaluate early chimerism and its kinetics after allogeneic peripheral blood stem cell transplantation among 68 patients who received a reduced-intensity conditioning (RIC) regimen with fludarabine plus melphalan (n=40) or busulphan (n=28). Chimerism was analyzed by polymerase chain reaction amplification of short tandem repeats in unfractionated (UF) and/or fractionated nucleated cells from bone marrow and peripheral blood (PB). All of the patients showed initial donor engraftment and no patient presented primary or secondary graft failure. In UF samples, the probability of achieving stable complete donor chimerism (CDC) in PB within the first 6 months was 70% on day +30, 85% on day +100 and 95% on day +180. CDC in granulocytes was observed in nearly all cases from day +30 onwards. CDC in T cells, however, differed among melphalan and busulphan recipients during the first 3 months (100 vs 0% on day +30 and 93 vs 20% on day +90, respectively). In multivariate analysis, the only significant variable associated with the achievement of early CDC was having received more than two lines of chemotherapy pretransplant (P<0.02). No correlation was found between the rate of achieving early CDC and the occurrence of acute graft-versus-host disease (GVHD) or disease progression post-transplant. In multivariate analysis, the only variable that influenced the incidence of disease progression post-transplant was the development of chronic extensive GVHD (P<0.05). In conclusion, a state of CDC is readily obtained within the first 6 months after our RIC protocols. Donor myeloid engraftment occurs rapidly in all cases, while early T-cell CDC is more common in more immunosuppressed hosts and, perhaps, in melphalan recipients.