Maria Carmina Castiello1, Marita Bosticardo2, Nicolò Sacchetti3, Enrica Calzoni4, Elena Fontana5, Yasuhiro Yamazaki2, Elena Draghici3, Cristina Corsino2, Ileana Bortolomai3, Lucia Sereni3, Hsin-Hui Yu2, Paolo Uva6, Rahul Palchaudhuri7, David T Scadden8, Anna Villa9, Luigi D Notarangelo10. 1. San Raffaele Telethon Institute for Gene Therapy SR-Tiget, IRCCS San Raffaele Scientific Institute, Milan, Cagliari, Italy; Institute of Genetic and Biomedical Research Milan Unit, National Research Council, Milan, Cagliari, Italy. 2. Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 3. San Raffaele Telethon Institute for Gene Therapy SR-Tiget, IRCCS San Raffaele Scientific Institute, Milan, Cagliari, Italy. 4. San Raffaele Telethon Institute for Gene Therapy SR-Tiget, IRCCS San Raffaele Scientific Institute, Milan, Cagliari, Italy; Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 5. Institute of Genetic and Biomedical Research Milan Unit, National Research Council, Milan, Cagliari, Italy; Human Genome Lab, Humanitas Clinical and Research Center, Milan, Cagliari, Italy. 6. CRS4, Science and Technology Park Polaris, Pula, Cagliari, Italy. 7. Department of Stem Cell and Regenerative Biology, Harvard University, Harvard Stem Cell Institute, Cambridge, Mass; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Mass; Magenta Therapeutics, Cambridge, Mass. 8. Department of Stem Cell and Regenerative Biology, Harvard University, Harvard Stem Cell Institute, Cambridge, Mass; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Mass. 9. San Raffaele Telethon Institute for Gene Therapy SR-Tiget, IRCCS San Raffaele Scientific Institute, Milan, Cagliari, Italy; Institute of Genetic and Biomedical Research Milan Unit, National Research Council, Milan, Cagliari, Italy. Electronic address: villa.anna@hsr.it. 10. Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. Electronic address: luigi.notarangelo2@nih.gov.
Abstract
BACKGROUND: Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation. OBJECTIVES: Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP). METHODS: Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray. RESULTS: Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery. CONCLUSIONS: Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis. Published by Elsevier Inc.
BACKGROUND: Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation. OBJECTIVES: Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP). METHODS: Rag1-KO and Rag1-F971Lmice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray. RESULTS: Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery. CONCLUSIONS: Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis. Published by Elsevier Inc.
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