Generation and Functional Properties Of FVIII-Specific Human T Regulatory Cells: A Novel Approach Using Hemophilia A patient’s T Cell Receptors

Antibody responses (inhibitors) against human factor VIII (FVIII) can obviate successful therapeutic efficacy of FVIII replacement therapy in hemophilia A patients. Novel efforts are necessary to overcome this problem: we propose that specific T regulatory cells (Tregs) can be used to prevent and reverse inhibitor formation. Tregs are the most important T subset for the maintenance of self-tolerance and homeostasis of our immune system. Clinical applications using Tregs are now considered a next-generation cellular therapy in a variety of autoimmune and inflammatory immune disorders such as type 1 diabetes, GVHD, and transplantation, and potentially as a treatment for antibody-mediated diseases such as hemophilia inhibitor formation. However, therapy with polyclonal Tregs is non-specific and a potential drawback is that it could be generally immunosuppressive. Therapy with antigen-specific Tregs (CARTs) would be highly preferable, as these have proved more potent in animal models of autoimmune diseases. We demonstrate herein the generation and functional properties of human T regulatory cells that specifically recognize FVIII. Tregs were generated that expressed the same T cell receptor (TCR) as that found on an HLA-DRB1*01:01-restricted T cell clone isolated from a hemophilia subject with a high-titer inhibitor. The T effector clone was isolated by MHC (DR0101) tetramer staining of this subject’s CD4 cells followed by single-cell sorting and expansion in cell culture. Nucleotide/amino acid sequences were identified to design retroviral FVIII-specific TCR constructs. To produce FVIII-specific human Foxp3+ Tregs, we transduced both expanded T effector cells and Tregs with a TCR expressed on an effector CD4 clone isolated from an inhibitor subject; these T cells recognize the same HLA-DRB1*01:01 restricted epitope as the “parent” effector T cell clone. Specific reactivity of the transduced cells bearing the designed TCR was validated by staining of the transduced T effector and Treg cells by DR0101 tetramers loaded with the same peptide as that recognized by the original effector T cell clone. Transduced effector T cells were expanded by stimulation with the expected FVIII peptide in vitro. Moreover, activation by this peptide in the presence of oligonucleotides mediated functional expansion and stabilization of transduced Tregs in ex vivo culture. Importantly, the expanded transduced Tregs were immunosuppressive only when stimulated by FVIII, even when FVIII-specific effector T cells were present in large excess. (Supported by NIH grant RO1 HL061883-15 to DWS and unrestricted research funding from Bayer, Pfizer and CSL Behring to KPP, and the intramural program of the NIAID to EMS).