Stable Mixed Hematopoietic Chimerism Research Articles

Current and future preparative regimens for bone marrow transplantation in thalassemia.

Preparative regimens for marrow allografts in thalassemia have two objectives. One is eradication of diseased marrow and the other suppression of host-versus-graft (HVG) reactions so that the allograft survives. A common regimen to accomplish these goals has combined high-dose busulfan with cyclophosphamide. Postgrafting immunosuppression with cyclosporine/methotrexate has been used for GVHD prevention. Some patients may die from regimen-related toxicity. Overall event-free survival is 75%. Occasional patients have become mixed donor/host hematopoietic chimeras and, yet, disease symptoms have abated. This has raised the possibility of developing safer and less toxic transplant programs that result in stable mixed hematopoietic chimerism. We have devised such a program in dogs consisting of a nonlethal dose of total body irradiation (200 cGy) before and a novel combination of mycophenolate mofetil and cyclosporine after transplant. Mixed donor/host chimerism (> or = 50% donor cells in all lineages) has persisted for > 80 weeks, even though immunosuppression was discontinued after five weeks.

CTLA4Ig REDUCES THE DOSE OF RADIATION REQUIRED TO ACHIEVE MIXED HEMATOPOIETIC CHIMERISM AND DONOR-SPECIFIC TRANSPLANTATION TOLERANCE IN RATS

317 Background: Various radiation-based conditioning regimens has been shown to produce stable mixed hematopoietic chimerism (MC) and donor-specific transplantation tolerance in adult animals, however the dose of radiation required to achieve MC may be too toxic for routine clinical use. The purpose of this study is to determine if blocking the costimulatory pathway involving in T-cell activation with CTLA4Ig will reduce the dose of radiation required to achieve MC in rats. Methods: Fully mismatched 4-6 week old ACI and Wistar Furth (WF) rats were used as donors and recipients, respectively. Recipients were administered tacrolimus (1 mg/kg, IM) from day -1 to day +9, human CTLA4Ig (2mg/kg, I.P.) on days 0, +2, +4, +6, and +8), and one dose of antilymphocyte globulin (ALS, 10 mg, I.P.) on day +10, and total body irradiation (TBI; from 300 to 1000 cGy) prior to bone marrow transplantation(BMT; on day 0) with 100 million of T-cell depleted bone marrow cells. Levels of donor chimerism were determined 28 days after marrow transplantation. Some chimeras underwent heterotopic heart transplants with donor-specific or third-party grafts 40 to 90 days after BMT. Graft survival was determined by daily palpation, and long term bone marrow engraftment was monitored by flow cytometry. Results: Recipient conditioning with radiation alone required 1000 cGy of TBI for successful allogeneic marrow engraftment. Addition of CTLA4Ig, ALS and FK506 to the conditioning regimen dramatically reduced the amount of TBI required: 100% (5/5) and 93% (13/14) of animals developed mixed chimerism at 400 cGy and 300 cGy, respectively, with mean donor chimerism = 15%. There was no BMT-related mortality, and recipients were free of graft-versus-host disease. Mixed chimeras (achieved at 300 cGy) permanently accepted (>200 days) donor-specific (ACI) heart grafts (n=10), yet rapidly rejected (< 10 days) third-party (Lewis) hearts (n=5). In addition, donor-specific (ACI) skin grafts (n=5) transplanted into recipients(WF) that had accepted the heart graft (ACI) for more than 100 days were accepted while third-party (Lewis) skin grafts (n=5) were promptly rejected(<10 days). Conclusion: A combination of tacrolimus, ALS, and costimulatory blockage with CTLA4Ig significantly reduces the dose of radiation required to achieve mixed chimerism and donor-specific transplantation tolerance in rats.

Mixed hematopoietic chimerism and transplantation tolerance.

Durable transplantation tolerance can be reliably achieved by inducing engraftment of hematopoietic cells in recipients initially depleted of T-lymphocytes. Engraftment of donor pluripotent hematopoietic stem cells (PPHSC) produces mixed hematopoietic chimeras in which both host and donor cells coexist and are tolerant of each other. The major mechanism of tolerance in these chimeras is central, intrathymic clonal deletion, which is induced and maintained by immigration of both host and donor marrow-derived cells to the host thymus, ensuring the ongoing central deletion of donor- and host-reactive cells. In this article, approaches developed in our laboratory to induce stable mixed hematopoietic chimerism and specific central deletional allogeneic and xenogeneic tolerance without toxic or myeloablative host conditioning are reviewed.

Stable Mixed Hematopoietic Chimerism in DLA-Identical Littermate Dogs Given Sublethal Total Body Irradiation Before and Pharmacological Immunosuppression After Marrow Transplantation

AbstractPostgrafting cyclosporine (CSP) given for 35 days resulted in establishment of stable marrow grafts from DLA-identical canine littermates after otherwise suboptimal, but nevertheless, lethal conditioning with 450 cGy of total body irradiation (TBI). We now asked whether sustained allografts could be achieved after sublethal TBI or without TBI. Five groups of recipients were studied. Dogs in group 1 were given 200 cGy TBI and postgrafting CSP, 15 mg/kg twice daily by mouth on days −1 to 35 posttransplant. Dogs in group 2 were given 200 cGy TBI and methotrexate (MTX), 0.4 mg/kg intravenously (IV) on days 1, 3, 6, and 11 along with CSP. Dogs in group 3 were given 200 cGy TBI and CSP along with mycophenolate mofetil (MMF ), 10mg/kg twice daily subcutaneously (SC) on days 0 to 27 after transplant, a novel immunosuppressive combination. Dogs in group 4 were given 100 cGy TBI and MMF/CSP. Dogs in group 5 were not given TBI and they received MMF/CSP posttransplant. Allografts were assessed by (Ca)n dinucleotide repeat polymorphism studies in cells from peripheral blood, lymph nodes, and marrow. Dogs in group 1 had transient mixed donor-host hematopoietic chimerism for no more than 4 weeks. Three of six dogs in group 2 had transient mixed chimerism for 3 to 11 weeks, and three have remained stable mixed chimeras for up to 60 weeks now. Four of five dogs in group 3 have remained stable mixed chimeras for 54 to 57 weeks now, while one lost the allograft after 12 weeks. All dogs in groups 4 and 5 rejected their allografts after 2 to 12 weeks. In summary, the establishment of stable mixed hematopoietic chimerism following nonmyelosuppressive and nontoxic conditioning programs has remained a difficult goal. Here we present evidence in a large random-bred animal species that this goal may be achievable with pharmacological immunosuppression postgrafting, capable of inhibiting both host-versus-graft (HVG) and graft-versus-host (GVH) reactions in the setting of DLA-identical grafts.

Mixed hematopoietic chimerism after allogeneic bone marrow transplantation: the impact of quantitative PCR analysis for prediction of relapse and graft rejection in children.

It still remains unclear whether patients with mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation (allo-BMT) have an increased risk of developing relapse or graft failure. To address this question, we monitored the individual dynamics of chimerism after allo-BMT in pediatric patients within a prospective case control study. The individual ratio of donor to recipient peripheral white cells was determined by quantification of genomic variable number of tandem repeats (VNTRs) with a polymerase chain reaction (PCR) approach. Within the study period from 1 January 1994 until 1 July 1996 we investigated 50 sequences of 46 pediatric patients after allo-BMT (32 with malignant, 18 with nonmalignant diseases). We found complete chimerism (CC) in 34/50 cases, MC in 12/50 follow-ups and 4/50 patients revealed autologous recovery (AC). Eight of 12 patients with MC showed increasing autologous patterns and subsequently relapsed or rejected their graft, 3/12 decreasing amounts of recipient DNA and turned to CC upon further follow-up. One patient of 12 who had severe combined immunodeficiency (SCID), attained engraftment with a stable MC pattern. Three patients of 34 with CC relapsed lacking a transitional MC interval. However, the time span between last CC confirmation and relapse in each of these three patients was 6 months or longer. We suggest that these patients also developed a stage of transitional MC but that the critical timepoint of molecular confirmation by PCR was missed as time intervals in the individual follow-up of these three patients were too long (> or = 6 months). In summary, the results demonstrate that the individual risk of developing relapse or graft failure is significantly enhanced in the MC situation (P < 0.0005). Therefore the quantitative analysis of MC at short time intervals might be of great value to identify high risk patients which will have a significantly/enhanced risk for relapse or graft rejection.

Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction

The influence of mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation remains unknown. Increasingly sensitive detection methods have shown that MC occurs frequently. We report a highly sensitive novel method to assess MC based on the polymerase chain reaction (PCR). Simple dinucleotide repeat sequences called microsatellites have been found to vary in their repeat number between individuals. We use this variation to type donor-recipient pairs following allogeneic BMT. A panel of seven microsatellites was used to distinguish between donor and recipient cells of 32 transplants. Informative microsatellites were subsequently used to assess MC after BMT in this group of patients. Seventeen of the 32 transplants involved a donor of opposite sex; hence, cytogenetics and Y chromosome-specific PCR were also used as an index of chimerism in these patients. MC was detected in bone marrow aspirates and peripheral blood in 18 of 32 patients (56%) by PCR. In several cases, only stored slide material was available for analysis but PCR of microsatellites or Y chromosomal material could be used successfully to assess the origin of cells in this archival material. Cytogenetic analysis was possible in 17 patients and MC was detected in three patients. Twelve patients received T-cell-depleted marrow and showed a high incidence of MC as revealed by PCR (greater than 80%). Twenty patients received unmanipulated marrow, and while the incidence of MC was lower (44%), this was a high percentage when compared with other studies. Once MC was detected, the percentages of recipient cells tended to increase. However, in patients exhibiting MC who subsequently relapsed, this increase was relatively sudden. The overall level of recipient cells in the group of MC patients who subsequently relapsed was higher than in those who exhibited stable MC. Thus, while the occurrence of MC was not indicative of a poor prognosis per se, sudden increases in the proportions of recipient cells may be a prelude to graft rejection or relapse.

Evaluation of Mixed Chimerism by In Vitro Amplification of Dinucleotide Repeat Sequences Using the Polymerase Chain Reaction

The influence of mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation remains unknown. Increasingly sensitive detection methods have shown that MC occurs frequently. We report a highly sensitive novel method to assess MC based on the polymerase chain reaction (PCR). Simple dinucleotide repeat sequences called microsatellites have been found to vary in their repeat number between individuals. We use this variation to type donor-recipient pairs following allogeneic BMT. A panel of seven microsatellites was used to distinguish between donor and recipient cells of 32 transplants. Informative microsatellites were subsequently used to assess MC after BMT in this group of patients. Seventeen of the 32 transplants involved a donor of opposite sex; hence, cytogenetics and Y chromosome-specific PCR were also used as an index of chimerism in these patients. MC was detected in bone marrow aspirates and peripheral blood in 18 of 32 patients (56%) by PCR. In several cases, only stored slide material was available for analysis but PCR of microsatellites or Y chromosomal material could be used successfully to assess the origin of cells in this archival material. Cytogenetic analysis was possible in 17 patients and MC was detected in three patients. Twelve patients received T-cell-depleted marrow and showed a high incidence of MC as revealed by PCR (greater than 80%). Twenty patients received unmanipulated marrow, and while the incidence of MC was lower (44%), this was a high percentage when compared with other studies. Once MC was detected, the percentages of recipient cells tended to increase. However, in patients exhibiting MC who subsequently relapsed, this increase was relatively sudden. The overall level of recipient cells in the group of MC patients who subsequently relapsed was higher than in those who exhibited stable MC. Thus, while the occurrence of MC was not indicative of a poor prognosis per se, sudden increases in the proportions of recipient cells may be a prelude to graft rejection or relapse.

USE OF DNA RESTRICTION FRAGMENT LENGTH POLYMORPHISMS TO DOCUMENT MARROW ENGRAFTMENT AND MIXED HEMATOPOIETIC CHIMERISM FOLLOWING BONE MARROW TRANSPLANTATION

We have studied the feasibility of using DNA restriction fragment-length polymorphisms (RFLP) to study marrow engraftment in 27 patients after allogeneic bone marrow transplantation, and have compared these results with those obtained using red blood cell antigens, cytogenetics, and immunoglobulin allotypes. Using highly polymorphic DNA probes, we have documented stable chronic mixed hematopoietic chimerism, have identified transient mixed chimeras, have excluded mixed chimerism with high probability in retrospective studies even when a pretransplant DNA sample was not available, have documented marrow engraftment in the early posttransplant period, and have studied the origin of leukemic cells in patients with recurrent disease. We have evaluated the advantages and disadvantages of several genetic markers and have developed tentative statements concerning the prognosis of patients with mixed chimerism. We conclude that DNA RFLP are powerful and practical genetic markers in bone marrow transplantation studies and that further studies of mixed hematopoietic chimerism are warranted.