Background: Even though hematopoietic stem cell transplantation can be curative in patients with severe\ncombined immunodeficiency, there is a need for additional strategies boosting T cell immunity in individuals\nsuffering from genetic disorders of lymphoid development. Here we show that image-guided intrathymic injection\nof hematopoietic stem and progenitor cells in NOD-scid IL2r�³null mice is feasible and facilitates the generation of\nfunctional T cells conferring protective immunity.\nMethods: Hematopoietic stem and progenitor cells were isolated from the bone marrow of healthy C57BL/6 mice\n(wild-type, Luciferase+, CD45.1+) and injected intravenously or intrathymically into both male and female, young or\naged NOD-scid IL2r�³null recipients. The in vivo fate of injected cells was analyzed by bioluminescence imaging and\nflow cytometry of thymus- and spleen-derived T cell populations. In addition to T cell reconstitution, we evaluated\nmice for evidence of immune dysregulation based on diabetes development and graft-versus-host disease. T cell\nimmunity following intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2r�³null mice\nwas assessed in a B cell lymphoma model.\nResults: Despite the small size of the thymic remnant in NOD-scid IL2r�³null mice, we were able to accomplish\nprecise intrathymic delivery of hematopoietic stem and progenitor cells by ultrasound-guided injection.\nThymic reconstitution following intrathymic injection of healthy allogeneic hematopoietic cells was most\neffective in young male recipients, indicating that even in the setting of severe immunodeficiency, sex and\nage are important variables for thymic function. Allogeneic T cells generated in intrathymically injected NOD-scid\nIL2r�³null mice displayed anti-lymphoma activity in vivo, but we found no evidence for severe auto/alloreactivity in\nT cell-producing NOD-scid IL2r�³null mice, suggesting that immune dysregulation is not a major concern.\nConclusions: Our findings suggest that intrathymic injection of donor hematopoietic stem and progenitor cells is a\nsafe and effective strategy to establish protective T cell immunity in a mouse model of severe combined\nimmunodeficiency.
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