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Mixed chimerism using a nonmyelosuppressive regimen in miniature swine
Mixed Chimerism Using a Nonmyelosuppressive Regimen in Miniature Swine Y. Fuchimoto, C.A. Huang, Z.L. Gleit, H. Kitamura, A. Griesemer, E. Melendy, R. Scheier-Dolberg, M.E. White-Scharf, and D.H. Sachs
M
IXED CHIMERISM has great clinical potential as a treatment for nonmalignant hematological diseases such as sickle cell disease and thalassemia, as well as for the attainment of tolerance to organ allografts. However, standard preparative regimens to achieve hematopoietic stem cell engraftment include myelosuppressive agents such as whole-body irradiation (WBI) to create “space” or sufficient immunosuppression. The toxicity of these conditioning regimens has limited the clinical applicability of this approach. Here, we report the establishment of mixed chimerism across both minor and major histocompatibility barriers in MGH miniature swine, by using high doses of peripheral blood stem cells (PBSC) without myelosuppressive conditioning.
MATERIAL AND METHODS Transplant donors and recipients were selected from our partially inbred, MHC-defined herd of MGH miniature swine. PBSC donors were 4 to 8 months old and weighed 30 to 50 kg. Recipients were 8 to 12 weeks old and weighed 5 to 15 kg. Donor swine were administered recombinant porcine stem cell factor (pSCF, 100 g/kg) subcutaneously and recombinant porcine interleukin 3 (pIL-3, 100 g/kg), both from BioTransplant (Boston, Mass, USA), with or without recombinant human granulocyte colony-stimulating factor (rhG-CSF, 10 g/kg). Collection of PBSCs was achieved by leukapheresis, beginning on day 5 of cytokine therapy and continuing daily for 3 to 6 days. PBSC, either fresh or frozen and quickly thawed, were administered on day 0. Recipients were conditioned by in vivo depletion of T cells with a new mutant diphtheria toxin antiswine CD3 conjugate (pCD3-CRM9, 0.05 mg/kg) and thymic irradiation (TI) with 700 cGy or 1000 cGy on day ⫺2, following which 100 to 200 ⫻ 108/kg PBSC were infused over 3 to 6 days from day 0. Cyclosporin A (Neoral) was administered through a gastric tube at 30 mg/kg/d in divided doses for 30 to 60 days. Donor cell chimerism in peripheral blood and thymus was monitored by flow cytometry, using donor-specific mAb specific for swine PAA (pig allelic antigen).
RESULTS
No toxicity was observed due to this regimen. The nadir white blood cell (WBC) count generally occurred on day 0 and was 2000 to 7000/L, and stabilization at more than 5000/L occurred within 2 to 4 days. The platelet level was generally maintained at more than 100,000/L, and platelet 0041-1345/01/$–see front matter PII S0041-1345(00)01934-5
infusion was never required. Engraftment of PBSC across a minor histocompatibility barrier was successful in both animals tested, which were treated with 700 cGy TI and 30 days of CyA. Stable multilineage chimerism in peripheral blood (lymphocyte lineage ⬎ 10%, myeloid lineage ⬎ 5%) could be detected for more than 300 days with no evidence of GvHD, even after cessation of CyA. Successful engraftment was also achieved across a single-haplotype full-MHC barrier with 700 cGy (1/2) or 1000 cGy (4/4) thymic irradiation. Three of these animals were treated with 30 days of CyA, and in two of them, grade II GvHD was observed following CyA cessation. GvHD was avoided in subsequent animals given single-haplotype–mismatched PBSC when the CyA regimen was extended to day 60 (2/2). Four surviving single-haplotype–mismatched animals have maintained chimerism for more than 200 days. DISCUSSION
It has generally been thought that WBI or myelosuppressive drugs were necessary to make “space” for the donor hematopoietic stem cell graft. However, it has recently been demonstrated that this requirement for space can be overcome by increasing the number of donor hematopoietic stem cells administered, the so-called “megadose” approach.1,2 In our miniature swine model, chimerism was established using this megadose approach without myelosuppressive therapy as has been done in rodents. Previously, the applicability of the megadose approach to large animals and human beings was considered limited, since it would require bone marrow harvests that would clearly not be feasible from living donors. However, the advent of PBSC technology has made it possible to obtain such megadose quantities From the Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA (Y.F., C.A.H., Z.L.G., H.K., A.G., E.M., R.S.-D., D.H.S.), and BioTransplant, Boston, Massachusetts, USA (M.E.W.-S.). Address reprint requests to David H. Sachs, MD, Transplantation Biology Research Center, Massachusetts General Hospital, MGH-East, Building 149-9019, 13th Street, Boston, MA 02129, USA. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
118
Transplantation Proceedings, 33, 118–119 (2001)
MIXED CHIMERISM IN MINIATURE SWINE
of stem cells from living donors by leukopheresis following stimulation with hematopoietic cytokines.3 An important component of the preparative regimen required to achieve engraftment without myelosuppression is effective depletion of mature T cells from the recipient. Although such depletion has been possible in rodents using antisera and mAbs, similar depletion in large animals and humans has been difficult to achieve. However, the use of a toxin-conjugated anti-CD3 mAb (pCD3-CRM9) has now enabled depletion to an extent sufficient to allow engraftment.4 In this report, we demonstrated that long-term mixed chimerism can be achieved in a miniature swine model across major and minor histocompatibility barriers using a
119
nonmyelosuppressive and minimally toxic conditioning regimen. Because of its low toxicity, this approach has potential for a wide range of clinical applications.5 REFERENCES 1. Stewart FM, Crittenden RB, Lowry P, et al: Blood 81:2566, 1993 2. Sykes M, Szot GL, Swenson K, et al: Nat Med 3:783, 1997 3. Colby C, Chang Q, Fuchimoto Y, et al: Transplantation 68:135, 2000 4. Huang CA, Yamada K, Murphy MC, et al: Transplantation 68:855, 1999 5. Fuchimoto Y, Huang CA, Yamada K, et al: J Clin Invest 105:1779, 2000
M
IXED CHIMERISM has great clinical potential as a treatment for nonmalignant hematological diseases such as sickle cell disease and thalassemia, as well as for the attainment of tolerance to organ allografts. However, standard preparative regimens to achieve hematopoietic stem cell engraftment include myelosuppressive agents such as whole-body irradiation (WBI) to create “space” or sufficient immunosuppression. The toxicity of these conditioning regimens has limited the clinical applicability of this approach. Here, we report the establishment of mixed chimerism across both minor and major histocompatibility barriers in MGH miniature swine, by using high doses of peripheral blood stem cells (PBSC) without myelosuppressive conditioning.
MATERIAL AND METHODS Transplant donors and recipients were selected from our partially inbred, MHC-defined herd of MGH miniature swine. PBSC donors were 4 to 8 months old and weighed 30 to 50 kg. Recipients were 8 to 12 weeks old and weighed 5 to 15 kg. Donor swine were administered recombinant porcine stem cell factor (pSCF, 100 g/kg) subcutaneously and recombinant porcine interleukin 3 (pIL-3, 100 g/kg), both from BioTransplant (Boston, Mass, USA), with or without recombinant human granulocyte colony-stimulating factor (rhG-CSF, 10 g/kg). Collection of PBSCs was achieved by leukapheresis, beginning on day 5 of cytokine therapy and continuing daily for 3 to 6 days. PBSC, either fresh or frozen and quickly thawed, were administered on day 0. Recipients were conditioned by in vivo depletion of T cells with a new mutant diphtheria toxin antiswine CD3 conjugate (pCD3-CRM9, 0.05 mg/kg) and thymic irradiation (TI) with 700 cGy or 1000 cGy on day ⫺2, following which 100 to 200 ⫻ 108/kg PBSC were infused over 3 to 6 days from day 0. Cyclosporin A (Neoral) was administered through a gastric tube at 30 mg/kg/d in divided doses for 30 to 60 days. Donor cell chimerism in peripheral blood and thymus was monitored by flow cytometry, using donor-specific mAb specific for swine PAA (pig allelic antigen).
RESULTS
No toxicity was observed due to this regimen. The nadir white blood cell (WBC) count generally occurred on day 0 and was 2000 to 7000/L, and stabilization at more than 5000/L occurred within 2 to 4 days. The platelet level was generally maintained at more than 100,000/L, and platelet 0041-1345/01/$–see front matter PII S0041-1345(00)01934-5
infusion was never required. Engraftment of PBSC across a minor histocompatibility barrier was successful in both animals tested, which were treated with 700 cGy TI and 30 days of CyA. Stable multilineage chimerism in peripheral blood (lymphocyte lineage ⬎ 10%, myeloid lineage ⬎ 5%) could be detected for more than 300 days with no evidence of GvHD, even after cessation of CyA. Successful engraftment was also achieved across a single-haplotype full-MHC barrier with 700 cGy (1/2) or 1000 cGy (4/4) thymic irradiation. Three of these animals were treated with 30 days of CyA, and in two of them, grade II GvHD was observed following CyA cessation. GvHD was avoided in subsequent animals given single-haplotype–mismatched PBSC when the CyA regimen was extended to day 60 (2/2). Four surviving single-haplotype–mismatched animals have maintained chimerism for more than 200 days. DISCUSSION
It has generally been thought that WBI or myelosuppressive drugs were necessary to make “space” for the donor hematopoietic stem cell graft. However, it has recently been demonstrated that this requirement for space can be overcome by increasing the number of donor hematopoietic stem cells administered, the so-called “megadose” approach.1,2 In our miniature swine model, chimerism was established using this megadose approach without myelosuppressive therapy as has been done in rodents. Previously, the applicability of the megadose approach to large animals and human beings was considered limited, since it would require bone marrow harvests that would clearly not be feasible from living donors. However, the advent of PBSC technology has made it possible to obtain such megadose quantities From the Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA (Y.F., C.A.H., Z.L.G., H.K., A.G., E.M., R.S.-D., D.H.S.), and BioTransplant, Boston, Massachusetts, USA (M.E.W.-S.). Address reprint requests to David H. Sachs, MD, Transplantation Biology Research Center, Massachusetts General Hospital, MGH-East, Building 149-9019, 13th Street, Boston, MA 02129, USA. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
118
Transplantation Proceedings, 33, 118–119 (2001)
MIXED CHIMERISM IN MINIATURE SWINE
of stem cells from living donors by leukopheresis following stimulation with hematopoietic cytokines.3 An important component of the preparative regimen required to achieve engraftment without myelosuppression is effective depletion of mature T cells from the recipient. Although such depletion has been possible in rodents using antisera and mAbs, similar depletion in large animals and humans has been difficult to achieve. However, the use of a toxin-conjugated anti-CD3 mAb (pCD3-CRM9) has now enabled depletion to an extent sufficient to allow engraftment.4 In this report, we demonstrated that long-term mixed chimerism can be achieved in a miniature swine model across major and minor histocompatibility barriers using a
119
nonmyelosuppressive and minimally toxic conditioning regimen. Because of its low toxicity, this approach has potential for a wide range of clinical applications.5 REFERENCES 1. Stewart FM, Crittenden RB, Lowry P, et al: Blood 81:2566, 1993 2. Sykes M, Szot GL, Swenson K, et al: Nat Med 3:783, 1997 3. Colby C, Chang Q, Fuchimoto Y, et al: Transplantation 68:135, 2000 4. Huang CA, Yamada K, Murphy MC, et al: Transplantation 68:855, 1999 5. Fuchimoto Y, Huang CA, Yamada K, et al: J Clin Invest 105:1779, 2000