Olga Gurevitch

Transplantation of allogeneic or xenogeneic bone marrow within the donor stromal microenvironment

Successful engraftment of hematopoietic stem cells requires a supportive hematopoietic stromal microenvironment (HSM). Defects in the HSM associated with aplastic anemia, myelofibrosis, or caused by intensive ionizing radiation and chemotherapy generally result in failure of bone marrow (BM) engraftment. Transplantation of donor BM within donor HSM may therefore provide optimal conditions for allogeneic BM transplantation. We have transplanted donor hematopoietic cells together with their own HSM to improve acceptance of allogeneic or xenogeneic BM. The non-myeloablative treatment used induced tolerance to murine allografts and provided conditions for the life-long acceptance of allogeneic HSM. Allogeneic BM transplanted within its own HSM under the kidney capsule caused less graft-versus-host disease than BM transplanted i.v. Tolerance in mice to xenogeneic (rat) HSM was less complete. Ectopic ossicles were small and contained fewer hematopoietic cells. However, simultaneous transplantation of rat BM and HSM to preconditioned mice improved engraftment of rat BM compared with transplantation of BM alone. Donor hematopoietic cells survived longer on their own HSM than on HSM of recipients.

Reconstruction of Cartilage, Bone, and Hematopoietic Microenvironment with Demineralized Bone Matrix and Bone Marrow Cells

Highly specialized hard tissues, such as cartilage, bone, and stromal microenvironment supporting hematopoiesis, originate from a common type of mesenchymal progenitor cell (MPC). We hypothesized that MPCs present in bone marrow cell suspension and demineralized bone matrix (DBM) that possess natural conductive and inductive features might constitute a unit containing all the essential elements for purposive bone and cartilage induction. Using a rodent preclinical model, we found that implantation of a composite comprising DBM and MPCs into A) a damaged area of a joint; B) an ablated bone marrow cavity, and C) a calvarial defect resulted in the generation of A) a new osteochondral complex comprising articular cartilage and subchondral bone; B) trabecular bone and stromal microenvironment supporting hematopoiesis, and C) flat bone, respectively. The new tissue formation followed differentiation pathways controlled by site–specific physiological conditions, thus developing tissues that precisely met local demands.

Nonmyeloablative conditioning to induce bilateral tolerance after allogeneic bone marrow transplantation in mice

We recently described a new nonmyeloablative method to induce stable and specific transplantation tolerance to allogeneic tissues in adult mice. It included total lymphoid irradiation (TLI) of recipients with six fractions of 200 cGy each, inoculation with donor bone marrow (BM) cells and cyclophosphamide (Cy) for selective elimination or inactivation of residual donor-reactive cells of the host, and infusion with T-cell depleted donor BM cells after Cy. Here, we investigated the possibility to induce stable bilateral graft-vs-host and host-vs-graft transplantation tolerance using non-T-cell depleted allogeneic BM. Our results show that the dose of BM required for the induction of transplantation tolerance was inversely correlated with the intensity of the conditioning. Transfer of a low dose (3 x 10(6)) of total donor BM cells to recipients preconditioned with a less intensive regimen (two or three TLI fractions instead of six) diminished graft-vs-host disease (GVHD)-related mortality of recipients to 40% and converted 89% of the survivors into GVHD-free mixed hematopoietic chimeras that maintained donor skin allografts >180 days. A tenfold increase in the number of donor BM cells (3 x 10(7) instead of 3 x 10(6)) reduced the rate of GVHD-related mortality of recipients to 20% and resulted in bilateral transplantation tolerance in 100% of nonirradiated survivors.

Permanent and specific transplantation tolerance induced by a nonmyeloablative 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.

The long-term success of organ transplantation is limited by complications resulting from consistent nonspecific immunosuppression. Induction of stable, donor-specific tolerance remains the main goal of transplantation immunology. In this article, a new, nonmyeloablative method is described for induction of transplantation tolerance to fully mismatched bone marrow cells (BMC), bone marrow stromal precursors, heart muscle, and skin allografts. The method is based on pretransplant conditioning with no postgraft immunosuppression, and consists of a short course (six daily fractions of 200 cGy) of total lymphoid irradiation (sTLI), followed by selective elimination of donor-specific alloreactive cells of the host escaping low-dose sTLI. Donor-specific alloreactive cells were activated by intravenous inoculation with a high dose of donor BMC (3 x 10(7) cells) 1 day after sTLI, and eliminated by a single intraperitoneal dose (200 mg/kg) of cyclophosphamide given 1 day after cell transfer. Infusion of a low number of T cell-depleted BMC (3 x 10(6) cells) after tolerogenic preconditioning converted recipients to stable mixed chimeras free of graft-versus-host disease. The same treatment provided long-lasting acceptance of heterotopically transplanted allografts of the heart muscle and of the stromal precursors to the hematopoietic microenvironment. This treatment also led to acceptance and life-long survival of full-thickness donor skin allografts. However, skin allografts survived only in mice that received donor T cell-depleted BMC after cyclophosphamide and had 20-50% donor cells in the blood. Our results suggest that after sTLI, additional selective clonal deletion of residual host cells induces a state of long-lasting specific tolerance to a wide variety of donor-derived tissues.

Repair of Bone Defect Using Bone Marrow Cells and Demineralized Bone Matrix Supplemented with Polymeric Materials

We present a novel, reverse thermo-responsive (RTR) polymeric osteogenic composite comprising demineralized bone matrix (DBM) and unmanipulated bone marrow cells (BMC) for repair of bone defects. The polymers investigated were low viscosity aqueous solutions at ambient temperature, which gel once they heat up and reach body temperature. Our goal to supplement DBM-BMC composite with RTR polymers displaying superior rheological properties, was to improve graft integrity and stability, during tissue regeneration. The osteogenic composite when implanted under kidney capsule of mice, proved to be biocompatible and biodegradable, with no residual polymer detected in the newly formed osteohematopoietic site. Implantation of the osteogenic composite into a large area of missing area of parietal bone of the skull of rats, resulted in an extensive remodeling of DBM particles, fully reconstituted hematopoietic microenvironment and well integrated normal flat bone within thirty days. The quality and shape of the newly created bone were comparable to the original bone and neither local or systemic inflammatory reactions nor fibrosis at the junction of the new and old calvarium could be documented. Furthermore, combined laser capture microdissection (LCM) technique and PCR analysis of male BMC in female rats confirmed the presence of male derived cells captured from the repaired/ regenerated flat bone defect. The use of active self sufficient osteogenic DBM-BMC composite supported by a viscous polymeric scaffold for purposive local hard tissue formation, may have a significant potential in enhancement of bone regeneration and repair following trauma, degenerative or inflamatory lesion, iatrogenic interventions and cosmetic indications.

Nonmyeloablative allogeneic bone marrow transplantation as immunotherapy for hematologic malignancies and metastatic solid tumors in preclinical models

OBJECTIVE We previously demonstrated that a combination of mild total lymphoid irradiation (TLI) with selective depletion of the hosts donor-reactive cells allows for stable and graft-vs-host disease (GVHD)-free engraftment of allogeneic bone marrow (BM). In this study, we investigated the efficacy of this nonmyeloablative strategy for BM transplantation (BMT) as immunotherapy for minimal residual disease. MATERIALS AND METHODS BALB/c mice inoculated with leukemia (BCL1) or breast carcinoma (4T1) cells were conditioned for BMT with TLI (200 cGy) followed by priming with donor (C57BL/6) BM cells on day 1, and by injection with 200 mg/kg cyclophosphamide on day 2. After conditioning (day 3), recipients were transplanted with BM cells from the same donor. Treated animals were monitored for 230 days for survival, development of leukemia/solid tumor, and GVHD. RESULTS BMT converted the mice to complete chimeras and prevented development of leukemia in 90% of recipients and locally growing breast carcinoma in 40% of the mice. Immunization of donors of the second BM with 4T1 cells prevented development of breast carcinoma in 80% of 4T1 inoculated mice. Fewer animals treated for malignancy by nonmyeloablative BMT died of GVHD than those treated by myeloablative BMT. However, late GVHD-related mortality in mice treated for leukemia was higher than after nonmyeloablative BMT to naive recipients (p < 0.00001). Infusion of host-type anti-donor immune lymphocytes 8 days after BMT improved the survival of recipients treated for leukemia without affecting engraftment and the graft-vs-leukemia potential of donor BM. CONCLUSIONS Effective eradication of malignant cells can be achieved following allogeneic BMT after nonmyeloablative conditioning.

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

OBJECTIVES We previously demonstrated that allogeneic bone marrow transplantation (BMT) after low-dose total lymphoid irradiation (200 cGy) and depletion of donor-reactive cells with cyclophosphamide (Cy) converted recipients to graft-vs-host disease (GVHD)-free chimeras tolerant to donor skin grafts. BMT also generated strong graft-vs-leukemia (GVL) response. However, clinical application of the protocol was hampered by the requirement for a relatively high dose of Cy (200 mg/kg). In this study we have tried to minimize the Cy dose by a concomitant blockade of CD40-CD40L interaction. MATERIALS AND METHODS Mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (BM(1)) and skin graft on day 0, injected with Cy (200 mg/kg or less) on day 1, and transplanted with a second C57BL/6 BM cell inoculum (BM(2)) on day 2. CD40L-specific antibody (MR1) was given with BM(1), BM(2), and 2 days later. Treated animals were monitored for survival, chimerism, and skin allograft rejection. The GVL potential of transplanted cells was examined in mice inoculated with BCL1 leukemia cells before irradiation. RESULTS Blocking CD40-CD40L interaction with MR1 mAb allowed the reduction of a tolerance-generating Cy dose by 50%. Unfortunately, adding MR1 to the protocol reduced the GVL potential of the transplanted cells. Neither low-dose Cy nor antibodies alone could downregulate donor or recipient immune response. CONCLUSIONS CD40L-specific antibodies synergize with Cy to induce bilateral transplantation tolerance. Therefore, their use may be beneficial for safer allogeneic BMT for nonmalignant indications. However, due to MR1-associated reduction of GVL effects, MR1 should be considered with caution as conditioning for BMT for leukemia-bearing recipients.

Induction of Bilateral Transplantation Tolerance to Cellular and Perfused Allografts and Xenografts with Donor Hematopoietic Cells a

ABSTRACT: We have recently introduced a new approach for induction of transplantation tolerance to donor alloantigens using well‐tolerated non‐myeloablative conditioning across MHC and xenogeneic barriers in mice. Our regimen consists of no or very low doses of total lymphoid irradiation (TLI), previously shown to be tolerogenic because of profound yet non‐myeloablative immunosuppression, followed by deletion of donor‐reactive host lymphocytes activated in vivo with donor hematopoietic cells with a single dose of cyclophosphamide. Recipients of immunosuppressive regimen with lower intensity (e.g., no or one single dose of TLI) required a larger inoculum of donor bone marrow cells; the reverse was also true, both regimens resulting in stable mixed chimerism. A combination of low‐dose TLI followed by depletion of donor‐reactive host cells with cyclophosphamide resulted in consistent engraftment of even low numbers of T‐cell‐depleted donor‐derived hematopoietic cells, known to be much more difficult to engraft, with consistent induction of permanent and donor‐specific transplantation tolerance to donor skin allografts with signs of similar success across xenogeneic barriers.

Mixed chimerism following non-myeloablative stem cell transplantation (Nst) To induce transplantation tolerance to bone marrow and organ allografts

Abstract Durable engraftment of donor hematopoietic stem cells results in long lasting transplantation tolerance to donor alloantigens; mixed chimerism (MC) induced during fetal life or neonatally, results in life-long transplantation tolerance to donor alloantigens, using no pre- or post transplant immunosuppression. We showed previously that induction of MC in immunocompetent recipients conditioned by total lymphoid irradiation (TLI) results in permanent, specific transplantation tolerance to donor alloantigens with permanent engraftment of heart, pancreatic islets and full thickness skins. MC can be clinically induced by non-myeloablative stem cell transplantation. Host hematopoietic cells in MC may down-regulate donor anti-host alloreactivity, minimizing graft vs host disease, immunocompetent donor T lymphocytes may facilitate engraftment of donor hematopoietic cells, down-regulating residual host immunocompetent lymphocytes. Data from C57BL/6→ BALB/c chimeras show that MC with bilateral transplantation tolerance is inducible by deletion of host anti-donor alloreactive cells through activation-induced apoptosis, after stimulation of activated lymphocytes by donor alloantigens. Here we have induced host vs graft and graft vs host transplantation tolerance using NST, i.e. 1–6 fractions of TLI 200 cGy each, followed by deletion of BALB/c and C57BL/6 anti-alloreactive T lymphocytes by cytoxan 120–200 mg/kg as preparation for a 2 nd bone marrow transplantation 1 day later. Recipients accept donor skin allografts permanently (>200 days). Induction of permanent transplantation tolerance to donor alloantigens to gradually replace host with donor hematopoietic cells in hematologic, genetic and autoimmune diseases will be presented.