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Mol Ther Methods Clin Dev. 2014 Jul 30;1. pii: 14030.

Ex Vivo Expanded Autologous Polyclonal Regulatory T Cells Suppress Inhibitor Formation in Hemophilia.

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Department of Pediatrics, University of Florida, Gainesville, FL 32610, USA.
Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston MA 02115. USA.
Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA.


Adoptive cell therapy utilizing ex vivo expanded polyclonal CD4+CD25+FOXP3+ regulatory T cells (Treg) is in use in clinical trials for the treatment of type 1 diabetes and prevention of graft vs host disease in bone marrow transplantation. Here we seek to evaluate this approach in the treatment of inherited protein deficiencies, i.e. hemophilia, which is often complicated by antibody formation against the therapeutic protein. Treg from mice that express GFP-marked FoxP3 were highly purified by two-step magnetic/flow sorting and ex vivo expanded 50- to 80-fold over a 2-week culture period upon stimulation with antibody-coated microbeads. FoxP3 expression was maintained in >80% of expanded Treg, which also expressed high levels of CD62L and CTLA-4. Transplanted Treg suppressed inhibitory antibody formation against coagulation factors VIII and IX in protein and gene therapies in strain-matched hemophilia A and B mice, including in mice with pre-existing antibodies. Although transplanted Treg became undetectable within two weeks, suppression persisted for >2 months. Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg. The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.


AAV vector; Regulatory T cells; cell therapy; hemophilia; tolerance

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