Durable Chimerism Research Articles

Bone marrow as stem cell source for allogeneic HLA-identical sibling transplantation following reduced-intensity preparative regimen

Objective Reduced-intensity conditioning regimens (RIC) and peripheral blood stem cells (PBSC) are increasingly used for allogeneic stem cell transplantation (allo-BMT). RIC has been shown to allow engraftment with minimal early transplant-related mortality (TRM). However, in the context of RIC, the use of bone marrow (BM) as stem cell source is still little evaluated. Patients and Methods In this report, we analyzed the outcome of 32 high-risk patients with hematological malignancies who received an HLA-identical sibling allo-BMT after RIC including fludarabine, busulfan, and anti-thymocyte globulin (ATG). Results Sustained neutrophil and platelet recovery occurred at a median of 13 days (range, 10–19) and 17 days (range, 0–45) respectively. Early and durable full donor chimerism could be established as soon as the first month after allo-BMT. Also, a sustained and early CD8 + T-cell recovery was observed, but the CD4 + T-cell compartment remained profoundly low. The cumulative incidences of grade II–IV acute GVHD and chronic GVHD were 26% (95% CI, 11–41%) and 31% (95% CI, 15–47%) respectively. The overall cumulative incidence of TRM was 28% (95% CI, 12–44%) occurring mainly in patients aged over 50. In this setting, GVHD showed a protective effect on disease progression or relapse with better progression-free survival for patients with GVHD as compared to patients without GVHD ( p = 0.03). Conclusions Collectively, these results confirm that the use of BM grafts for RIC is feasible with durable donor engraftment and no detrimental GVHD.

Stem cell activity of porcine c-kit+ hematopoietic cells.

A marker for hematopoietic stem cells (HSCs) of pigs, which are considered to be the most suitable donors for clinical xenotransplantation, has not yet been identified. In this study, we examined the HSC activity of porcine c-kit+ bone marrow cells (BMCs). The HSC activity of porcine c-kit+ BMCs was evaluated both in vitro using colony-forming unit (CFU) and cobblestone area-forming cell (CAFC) assays and in vivo in nonobese diabetic/severe combined immunodeficiency transgenic (NOD/SCID-Tg) mice carrying porcine cytokine transgenes. Purified c-kit+ BMCs were substantially enriched for both CFUs and CAFCs in vitro and their transplantation led to long-term porcine hematopoiesis in vivo in mice. Although porcine chimerism was detectable in the peripheral blood of NOD/SCID-Tg mice receiving porcine c-kit- BMCs at early time points after transplantation, the levels were markedly lower than those in mice receiving purified c-kit+ BMCs (0.2%+/-0.14% vs 7.7%+/-1.6% and 0.17%+/-0.17% vs 5.6%+/-2.1% at weeks 3 and 6, respectively). Importantly, all mouse recipients of porcine c-kit+ BMCs showed durable multilineage chimerism (>19 weeks), whereas no recipients of porcine c-kit- BMCs sustained long-term engraftment. Moreover, porcine HSCs that had engrafted for 19 weeks in the recipients of porcine c-kit+ BMCs gave rise to clonogenic progenitors in vitro and reconstituted porcine hematopoiesis in secondary recipients. The present study demonstrates that c-kit is an essential marker of both long-term-repopulating HSCs and progenitor cells with early engraftment capacity.

Low-dose total body irradiation (TBI) and fludarabine followed by hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors and postgrafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chimerism and sustained remissions in patients with hematological diseases

Toxicities of high-dose conditioning regimens have limited the use of conventional unrelated donor hematopoietic cell transplantation (HCT) to younger, medically fit patients. Based on preclinical studies, an HCT approach has been developed for elderly or medically infirm patients with HLA-matched or mismatched unrelated donors. In this study, 52 patients with hematological diseases were included. Most (88%) had preceding unsuccessful conventional HCT or refractory/advanced disease. Patients were treated with fludarabine 30 mg/m(2)/d from days -4 to -2, 2 Gy total body irradiation on day 0, cyclosporine at 6.25 mg/kg twice daily from day -3, and mycophenolate mofetil at 15 mg/kg twice daily from day 0. Durable donor chimerism was attained in 88% of the patients. By day 28, a median of 100% of CD56(+) cells were of donor origin. Granulocyte and T-cell donor chimerism increased to medians of 100% on day 56 and day 180 (range, 55%-100%), respectively. Acute GVHD, grade II, was seen in 42% (CI, 29%-56%); grade III in 8% (CI, 0%-15%); and grade IV in 13% (CI, 4%-23%) of patients; it was fatal in 9%. The 100-day transplantation-related mortality was 11%. Complete remissions, including molecular remissions, were seen in 45% of patients with measurable disease before transplantation. Mortality from disease progression was 27% at one year. With a median follow-up of 19 months, 18 of the 52 patients (35%) were alive and 25% were in remission. HCT from HLA-matched or mismatched unrelated donors can be performed with a reduced intensity conditioning regimen in patients ineligible for conventional HCT.

Cutting edge: administration of anti-CD40 ligand and donor bone marrow leads to hemopoietic chimerism and donor-specific tolerance without cytoreductive conditioning.

Transplantation tolerance, defined as allograft acceptance by an immunocompetent recipient in the absence of long-term immunosuppression, has remained an elusive goal in clinical transplantation. Robust experimental tolerance induction strategies have in common methods to induce mixed hemopoietic chimerism. To date, however, chimerism induction across allogeneic barriers has required recipient conditioning with irradiation or cytoablative agents. In this paper we show that B6 recipients of fully allogeneic BALB/c skin grafts treated with repeated doses of donor bone marrow and anti-CD40 ligand (CD40L) develop durable (>300 days), readily detectable (6-12%) multilineage hemopoietic chimerism, indefinite allograft acceptance (>300 days), and donor-specific tolerance to secondary skin grafts. Analysis of the TCR repertoire of treated mice indicates that the underlying mechanisms of tolerance are in part mediated by deletion of donor-reactive T cells. These data demonstrate that durable hemopoietic chimerism and robust transplantation tolerance can be achieved without cytotoxic conditioning using a potentially clinically applicable regimen.

Development and analysis of transgenic mice expressing porcine hematopoietic cytokines: a model for achieving durable porcine hematopoietic chimerism across an extensive xenogeneic barrier.

The capacity of mixed hematopoietic chimerism to induce tolerance has not been demonstrated in discordant xenogeneic species combinations because of the difficulty in achieving lasting hematopoietic engraftment. In an effort to create a model of long-lasting disparate xenogeneic hematopoietic chimerism, we have developed transgenic (Tg) mice carrying porcine cytokines. Three lines of Tg mice were generated: one carrying porcine IL-3 and GM-CSF genes only (termed IL/GM) and the remaining two lines carrying in addition, the soluble SCF gene (termed IL/GM/sS) or membrane-bound SCF gene (termed IL/GM/mS). Sera from mice with IL/GM and IL/GM/sS transgenes markedly stimulated the proliferation of swine marrow cells in vitro. However, proliferation of swine marrow cells was not induced in cultures containing IL/GM/mS sera. Consistent with these observations, ELISA assays revealed detectable levels of porcine cytokines in the sera of IL/ GM and IL/GM/sS, but not in sera of IL/GM/mS Tg mice. Marrow stromal cells prepared from all three kinds of Tg mice, but not those from non-Tg littermates, were capable of supporting the growth of porcine hematopoietic cells in vitro. Immunodeficient Tg mice were generated by crossing Tg founders with C.B-17 SCID mice for five generations. All Tg immunodeficient mice showed improved porcine hematopoietic engraftment compared with non-Tg controls. These Tg mice provide a useful model system for studying porcine hematopoietic stem cells, and for evaluating the feasibility of donor-specific tolerance induction by mixed chimerism across highly disparate xenogeneic barriers.

Xenoreactions and their modulation with bone marrow transplantation to induce tolerance

There is a severe shortage of organs for transplantation that many conclude will never be solved by increasing organ donation. Xenotransplantation may offer a permanent solution to the donor organ shortage. The vigorous nature of immune responses to xenografts and our inability to control these responses using conventional immunosuppressive regimens limit the application of xenotransplantation to the clinic. Natural antibody-mediated hyperacute and cell-mediated delayed rejection constitute two forms of xenograft rejection reactions that have been the subject of intensive studies. Although these studies resulted in the design of molecular approaches, including phenotypic manipulation of the host and genetic modification of the donor, that have been effective in preventing hyperacute rejection in the preclinical pig-to-primate model, graft rejection remains a major limitation. The induction of donor-specific tolerance to xenografts remains a far-reaching goal of clinical transplantation and will probably be one key component in enabling xenotransplantation to become a clinical reality. The use of bone marrow transplantation to induce durable hematopoietic stem cell xenogeneic chimerism, however, holds great promise for achieving such a goal.

Induction of mixed allogeneic chimerism for leukemia

Hybrid (C56BL/6 × DBA) (BDF1; H-2 b/H-2 d) mice bearing the P815 leukemia (H-2 d) were grafted with a (CBA × C57BL/6)F1 (CBF1; H-2 k/H-2 b) cell suspension, comprising bone marrow cells (BMC; 25 × 10 6/mouse) and spleen cells (SC; 55 × 10 6/mouse) on day −4, then treated with cyclophosphamide (200 mg/kg) on day −2 and finally grafted once more with CBF1 cells (25 × 10 6 BMC + 7 × 10 6 SC) on day 0. Allogeneic cell graftings performed in this way induced durable mixed hematopoietic chimerism and significantly prolonged the survival of recipients, compared with that of leukemia-bearers grafted with syngeneic cells. The results obtained raise the possibility of using allogeneic hematopoietic tissue transplantation in combination with non-lethal cytoreductive therapy to induce a long-lasting graft-vs-leukemia effect.

Durable mixed allogeneic chimerism and tolerance by a nonlethal radiation-based cytoreductive approach.

For over 40 years, the association between hemopoietic chimerism and donor-specific tolerance for allografts has been recognized. However, toxicity associated with lethal conditioning has prevented the clinical application of bone marrow (BM) chimerism to induce tolerance. We previously demonstrated that engraftment could be achieved with less than total recipient myeloablation (700 cGy) and that the incidence of engraftment correlated with the dose of total body irradiation (TBI). Administration of cyclophosphamide (CyP) on Day +2 reduced the minimum TBI dose sufficient to permit engraftment to 500 cGy. In the current study, addition of antilymphocyte globulin (ALG) to the TBI/CyP-based conditioning approach reduced the radiation required for engraftment to < or = 300 cGy. B10 (H-2b) mice conditioned with ALG on day -3, 300 cGy of TBI with transplantation of B10.BR (H-2k) or BALB/c (H-2d) BM on day 0, and CyP on day +2 exhibited evidence of donor chimerism (49.6 +/- 3.7% and 38.2 +/- 2.4%, respectively) in 97% of recipients. ALG eliminated CD4+ and CD8+ cells and decreased NK1.1+ cells in the peripheral circulation at the time of transplantation. Moreover, T and NK cells in the host BM were significantly decreased compared with cells of recipients conditioned with TBI alone. CyP delayed repopulation of host thymocytes, providing time for the establishment of donor chimerism before production of mature T cells. Chimeric animals exhibited stable multilineage chimerism and donor-specific tolerance to skin grafts and in in vitro assays. This model may provide a clinically acceptable approach for the induction of donor-specific transplantation tolerance.

A nonlethal conditioning approach to achieve durable multilineage mixed chimerism and tolerance across major, minor, and hematopoietic histocompatibility barriers.

Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic (A+B-->A) bone marrow results in multilineage mixed allogeneic chimerism, donor-specific transplantation tolerance, superior immunocompetence and resistance to graft-vs-host disease. However, the morbidity and mortality associated with lethal irradiation would be a major limitation to the clinical application of chimerism to induce tolerance for solid organ grafts or treat other nonmalignant hematologic diseases. We report here that durable multilineage mixed allogeneic chimerism and donor-specific transplantation tolerance for skin and primarily vascularized allografts can be achieved across multiple histocompatibility barriers using a nonmyeloablative radiation-based approach. The percentage of B10 mouse recipients that engrafted directly correlated with the degree of disparity between donor and recipient and the dose of total body irradiation administered. Although the occurrence of engraftment following conditioning with doses of total body irradiation of > or = 600 cGy was similar for animals receiving bone marrow disparate at MHC or MHC, minor and hematopoietic (Hh-1) loci (67% vs 78%), the level of donor chimerism was significantly less when multiple histocompatibility barriers were present (94.6 +/- 3.8% vs 37.5 +/- 12.5%). Treatment of the recipient with cyclophosphamide 2 days following allogeneic bone marrow transplantation reduced the dose of radiation sufficient for reliable engraftment to only 500 cGy of total body irradiation, regardless of MHC and Hh-1 disparity. Donor chimerism was stable and present in all lineages, with production of lymphoid (T and B cell), NK, and myeloid (erythrocyte, platelet, granulocyte, and macrophage) cells. Mixed chimeras exhibited donor-specific tolerance in vitro, as assessed by mixed lymphocyte culture (MLR) and cytotoxicity (CML) assays, and in vivo to skin and primarily vascularized cardiac allografts. The observation that engraftment and tolerance can be achieved across multiple histocompatibility barriers using nonmyeloablative recipient conditioning may allow allogeneic bone marrow transplantation to be applied to nonmalignant disease states in which lethal conditioning cannot be justified, including the induction of donor-specific tolerance for solid organ transplantation and the treatment of hemoglobinopathies and enzyme deficiency states.

MIXED XENOGENEIC CHIMERISM AND TOLERANCE III

Previously, we reported that transplantation of rat bone marrow into lethally irradiated (950 rads) mouse recipients results in stable xenogeneic (rat-->mouse) chimerism and confers donor-specific transplantation tolerance for subsequent or simultaneous islet xenografts. For potential clinical application of chimerism to induce tolerance, it would be important to avoid the morbidity and mortality associated with lethal conditioning. Recently, we established a model to achieve durable multilineage xenogeneic chimerism using a nonlethal cytoreductive approach. We report here for the first time that donor-specific rat islet xenografts placed coincident with the bone marrow transplant are permanently accepted by nonlethally conditioned recipients. All recipients conditioned with 700 cGy of total body irradiation and transplanted with 40 x 10(6) F344 rat bone marrow cells repopulated as mixed donor/host chimeras. The chimeras exhibited permanent acceptance of donor islet xenografts, since donor-specific F344 (Rt1A1) rat islet xenografts were significantly prolonged (median survival time > 110 days), while MHC-disparate third-party WF (Rt1Au) rat islets were prolonged but rejected (median survival time = 33.2 days). The islets were functional to maintain normoglycemia and regulated in function to respond to an intraperitoneal glucose challenge. These data suggest that tolerance to donor-specific islet xenografts placed coincident with bone marrow transplantation can be achieved after nonlethal conditioning.

A minimal conditioning approach to achieve stable multilineage mouse plus rat chimerism

Transplantation of untreated rat bone marrow into lethally irradiated (950 cGy) mouse recipients results in durable xenogeneic (rat→mouse) chimerism and confers donor-specific transplantation tolerance for subsequent xenografts. The purpose of the present study was to characterize the minimal dose of total body irradiation (TBI) which would allow engraftment of rat bone marrow in mouse recipients. We report here that durable and stable lymphohaematopoietic cross-species chimerism can be achieved using a less than totally ablative radiation-based conditioning approach. The percentage of B10 mouse recipients which engrafted with rat bone marrow cells correlated with the dose of TBI. Engraftment of rat bone marrow stem cells occurred in all animals receiving 750 cGy prior to bone marrow transplantation, while no engraftment was detected at doses less than 650 cGy. Although most of the recipients were repopulated with mixed mouse and rat multilineage chimerism, some exhibited a predominance of rat cells. Although mixed xenogeneic rat/mouse chimeras prepared by lethal TBI produced only mouse derived RBC (red blood cells), chimeras prepared by sublethal conditioning produced both rat and mouse RBC. Only animals with detectable chimerism exhibited specific functional transplantation tolerance to donor xenoantigens, as assessed in vitro by mixed lymphocyte reaction assay. This model may offer an in vivo approach to study the role of species-specific growth factors in stem cell biology as well as the mechanisms for the induction of tolerance across species barriers.

Induction of transplantation tolerance by intraportal injection of allogeneic bone marrow cells: Possible implications for intrauterine bone marrow transplantation across major histocompatibility barriers

Abstract. Intraportal inoculation of C57BL/6 marrow cells into sublethally (400 rad) irradiated BALB/c recipients resulted in durable chimerism and the permanent acceptance of C57BL/6 skin allografts. Sublethally irradiated recipients of a similar number of marrow cells inoculated systemically did not develop chimerism or any significant prolongation of the survival of C57BL/6 skin allografts. Consequently, lethal graft-versus-host disease developed only in recipients of intraportal marrow allografts (80%). The intraportal injection of allogeneic C57BL/6 marrow cells into nonirradiated recipients resulted in significant, although not permanent, prolongation of skin allograft survival without durable chimerism, suggesting that the introduction of alloantigens intraportally may favor the induction of nonresponsiveness to alloantigens even across strong major histocompatibility barriers. The relevance of these findings is discussed regarding the intraportal inoculation of allogeneic bone marrow cells for the treatment of genetic disorders in utero through the induction of neonatal tolerance.

Separation of red blood cell-depleted mononuclear marrow cells by continuous-flow centrifugation using the IBM 2997 blood cell separator—Results of a preclinical study in a dog model

As an alternative technique to density-gradient separation, we studied the preparation of red blood cell (RBC)-depleted marrow mononuclear cells (MNC) by continuous-flow centrifugation (CFC) using the IBM 2997 cell separator in a dog model. Mean recoveries of nucleated and mononuclear cells obtained by this separation technique were 43.1% and 77.7% , respectively, and the mean residual RBC volume was 1.8 mL (1.2% of the RBC volume prior to separation). A mean of 91.4% of marrow progenitor cells were regained in the RBC-depleted fraction. Transplantation of the separated marrow MNC into seven lethally irradiated beagle littermates resulted in complete and durable hematopoietic chimerism. Our results indicate that separation of marrow MNC using an IBM 2997 cell separator is an effective and technically uncomplicated alternative to gradient medium separation.

The induction of long-term immunological tolerance

CBA mice were rendered tolerant by a prolonged course of antilymphocyte serum and the subsequent injection of allogeneic or xenogeneic lymphoid cells. Control mice received no other therapy. Experimental mice were grafted with allogeneic or xenogeneic lymph nodes prior to the administration of the cellular inocula. The survival pattern of tail-skin grafts was assessed in all mice. Those animals that were grafted with lymph node transplants failed to reject the skin grafts with the same rapidity or pattern as mice that were treated with ALS and cells alone. Furthermore, graft-v.-host disease was not noted in either the allogeneic or xenogeneic chimeras. The possible mechanisms of this durable chimerism are discussed. The experiments are still preliminary and others are in progress to evaluate the survival patterns of lymphoid cells in their heterotopic but syngeneic environment.